ANDROGEN-DEPENDENT EXCITABILITY OF MOUSE VENTRAL HIPPOCAMPAL AFFERENTS TO NUCLEUS ACCUMBENS UNDERLIES SEX-SPECIFIC SUSCEPTIBILITY TO STRESS By Elizabeth Spencer Williams A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Pharmacology & Toxicology – Doctor of Philosophy 2019 ABSTRACT ANDROGEN-DEPENDENT EXCITABILITY OF MOUSE VENTRAL HIPPOCAMPAL AFFERENTS TO NUCLEUS ACCUMBENS UNDERLIES SEX-SPECIFIC SUSCEPTIBILITY TO STRESS By Elizabeth Spencer Williams Women experience major depressive disorder (MDD) nearly twice as often as men, but the neurophysiology of this sex difference in prevalence is not fully understood. In preclinical animal models of depression utilizing male mice, activity of glutamatergic afferents from ventral hippocampus (vHPC) to nucleus accumbens (NAc) regulates mood-related behavioral responses to stress, but the circuit-level mechanisms underlying stress responses in female mice are not as clear. Female mice, however, uniquely exhibit susceptibility to anhedonia, a common symptom of depression, following subchronic variable stress (SCVS). Using whole-cell patch clamp electrophysiology, we identify a lower excitability of male vHPC-NAc projections compared to those in female mice. We demonstrate that the lower excitability in male projections is adult testosterone- dependent, and that this accompanies resilience to anhedonia in males following stress: orchidectomy induces increased vHPC-NAc excitability and susceptibility to SCVS in male mice, and exogenous testosterone levels vHPC-NAc excitability and mitigates anhedonia susceptibility in ovariectomized female mice. We further demonstrate a causal link between vHPC-NAc excitability and SCVS susceptibility using designer receptors exclusively activated by designer drugs (DREADDs), with circuit activity inversely correlated to anhedonia resilience in both sexes. We also find that anhedonia following SCVS requires an extended increase in vHPC-NAc activity, as activation of vHPC-NAc only during behavioral assessment does not result in decreased sucrose preference in male mice. Using translating ribosome affinity purification (TRAP), we interrogate male and female vHPC-NAc circuit-specific gene expression and uncover differential expression of genes in multiple pathways that may regulate differing circuit excitability between the sexes, and ultimately may explain sex differences in SCVS susceptibility. Lastly, we investigate the role of ΔFosB, a transcription factor known to reduce HPC excitability, in vHPC-NAc neurons in male and female mice, and demonstrate that this molecule is a key influencer of chronic social defeat stress (CSDS) responses in male mice. This study highlights hormone-dependent differences in vHPC-NAc circuit excitability in adult mice, which are directly causative of resilience to SCVS-induced anhedonia, and identifies sex differences in vHPC-NAc gene transcription that may mediate sex-specific responses to stress and depression pathogenesis. To my family, for their unwavering love and support. iv ACKNOWLEDGEMENTS I am very grateful to have worked with many outstanding scientists and collaborators during my graduate research. All past and present members of the Robison and Mazei-Robison laboratories have been phenomenal colleagues and friends, and have provided the support and knowledge necessary to complete the current dissertation. The Pharmacology & Toxicology and Physiology Departments have provided an ideal work environment, and I would like to recognize the work of the staff of both departments in their support of students and research. I have been very fortunate to have worked with a brilliant group of scientists in the Robison laboratory. All students and postdoctoral fellows have been valuable influences on my scientific career, and have also been good friends. Ken Moon, our Laboratory Manager, helped to provide the tools and reagents necessary for the experiments presented in this document, as well as outstanding technical support for many of the techniques used in this thesis. I would like to specifically recognize Dr. Andrew Eagle, whose guidance and research were essential to much of the data presented in Chapter IV. Andrew is a thoughtful and careful scientist, and has become a great model for my own research goals. A fellow graduate student, Claire Manning, has also been an invaluable colleague and friend, and without her this dissertation would not have made for the story that it is. Claire’s behavioral work has paralleled my own physiology work beautifully, and truly makes the electrophysiology presented in Chapters III and IV shine. I cannot thank her enough for being such a wonderful friend and collaborator, and her v elegant work presented in Chapters III and IV has been critical in establishing the importance of our studies. I would also like to thank my thesis committee: Dr. Anne Dorrance, Dr. Greg Fink, Dr. Jim Galligan, and Dr. Bill Jackson. Their input and guidance throughout my research and their patience and availability for meetings was much appreciated. Dr. Cindy Arvidson and Mrs. Jonelle Golding of the MSU-CHM MD/PhD program have also been critical to my success in this long and arduous process, and I thank them sincerely for their guidance and support. I would also like to thank my mentor, Dr. A.J. Robison. His guidance and insight have been essential to my education and growth as a scientist. Moreover, he has been patient and understanding throughout my medical and graduate studies, and has always put my personal well-being and growth as a student first. His mentorship will undoubtedly shape the rest of my career, and his work on my behalf is forever appreciated. Dr. Michelle Mazei-Robison’s guidance throughout my research is also very much appreciated, and she is a great inspiration to me as a woman in research. Lastly, I would like to recognize the continuing love and support of my family. My parents, Anna Kalkman, Scott Kalkman, Danny Williams, and Robert Ingram, and my brother Daniel have given me the inspiration and support necessary to complete the current work and pursue my goals in research and medicine. I cannot thank them enough for putting up with me during my completion of two doctorate degrees, as well as for their humor and kindness in keeping my spirits up during challenging times. My husband, Tom, is the best friend and partner I could ask for in life and has been with me every step of the way in graduate and medical school. His love and support have been key in the vi completion of this dissertation, and I thank him for pushing me to be the best researcher and physician I can be. vii TABLE OF CONTENTS LIST OF FIGURES .......................................................................................................... ix I. INTRODUCTION ........................................................................................................... 1 General Introduction to the Brain and Disease ................................................................ 1 Major Depressive Disorder (MDD) and Preclinical Animal Models of Depression ........... 3 Epidemiology and Disease Burden ........................................................................... 4 MDD differences in men and women ........................................................................ 5 Preclinical Animal Models of Depression .................................................................. 7 Subchronic Variable Stress (SCVS) ........................................................................ 12 Sex differences in stress responses ....................................................................... 15 The Hippocampus .......................................................................................................... 19 Distinctive Dorsal and Ventral HPC Functions ........................................................ 21 Neurotransmitters of the HPC – Implications in Mood disorders ............................ 23 HPC Sex Differences in Stress and Depression ..................................................... 31 The Nucleus Accumbens ............................................................................................... 33 NAc Cell Types – Direct and Indirect Pathways ...................................................... 33 NAc function in stress models of depression .......................................................... 34 Sex differences in NAc ............................................................................................ 38 HPC to NAc connectivity ................................................................................................ 39 Ion Channels of the Hippocampus ................................................................................. 44 Leak Channels ........................................................................................................ 44 Potassium Channels ............................................................................................... 45 Sodium channels ..................................................................................................... 47 Calcium channels .................................................................................................... 49 HCN Channels ........................................................................................................ 51 Hormone Signaling in the Brain – Effects on Stress and Depression ............................ 52 II. MATERIALS AND METHODS .................................................................................... 60 Animals ........................................................................................................................... 60 Gonadectomies .............................................................................................................. 61 Intracranial Injections & Viral Vectors ............................................................................. 61 Subchronic Variable Stress ............................................................................................ 63 Chronic social defeat stress (CSDS) .............................................................................. 63 DREADD activation ........................................................................................................ 63 CRISPR Guide RNA design and testing ........................................................................ 64 Behavior ......................................................................................................................... 65 Electrophysiology ........................................................................................................... 66 Immunohistochemistry ................................................................................................... 68 Translating Ribosome Affinity Purification (TRAP) and cDNA library preparation ......... 69 Statistical Analysis .......................................................................................................... 70 viii III. ANDROGEN-DEPENDENT EXCITABILITY OF MOUSE VENTRAL HIPPOCAMPAL AFFERENTS TO NUCLEUS ACCUMBENS UNDERLIES SEX-SPECIFIC SUSCEPTIBILITY TO STRESS ..................................................................................... 72 Introduction ..................................................................................................................... 72 Results ........................................................................................................................... 74 Female mice are selectively susceptible to anhedonia following SCVS ................. 74 Female mice have increased vHPC-NAc neuronal excitability ............................... 77 Sex differences in baseline excitability are unique to the vHPC-NAc circuit ........... 85 SCVS does not affect basal vHPC-NAc excitability in female mice ........................ 89 Orchidectomy induces male susceptibility to SCVS-induced anhedonia ................ 89 Orchidectomy reduces excitability of vHPC-NAc neurons ...................................... 92 Orchidectomy reduces excitability of vHPC-BLA neurons .................................... 100 Androgen receptor antagonism increases male vHPC-NAc excitability ................ 106 Exogenous testosterone in female mice ameliorates susceptibility to SCVS ....... 109 Exogenous testosterone mitigates hyperexcitability in female vHPC-NAc neurons .............................................................................................................................. 114 vHPC-NAc excitability directly mediates SCVS-induced susceptibility to anhedonia .............................................................................................................................. 122 Sex-specific transcriptome interrogation of vHPC-NAc neurons ……………...…. 131 Discussion .................................................................................................................... 134 IV. VENTRAL HIPPOCAMPUS TO NUCLEUS ACCUMBENS ΔFOSB UNDERLIES RESILIENCE TO SOCIAL STRESS AND REGULATES CIRCUIT-SPECIFIC EXCITABILITY ............................................................................................................. 142 Introduction ................................................................................................................... 142 Results ......................................................................................................................... 143 Chronic social defeat stress and fluoxetine induce ΔFosB in ventral hippocampus .............................................................................................................................. 143 vHPC-NAc ΔFosB is necessary for resilience to social stress .............................. 145 ΔFosB regulates vHPC-NAc projection excitability ............................................... 152 Discussion .................................................................................................................... 162 V. SUMMARY, DISCUSSION AND FUTURE DIRECTIONS ....................................... 168 Summary ...................................................................................................................... 168 Discussion and Future Directions ................................................................................. 173 Resilience as an active process ............................................................................ 173 Gene expression changes in stress ...................................................................... 176 Intrinsic excitability and plasticity .......................................................................... 179 Final Summary ……………………………………………………………………………….183 REFERENCES ………………….……………………………………………………………184 ix LIST OF FIGURES Figure 1 | Chronic social defeat stress and social interaction test. ................................. 10 Figure 2 | Subchronic variable stress (SCVS) and associated behavioral assays. ........ 14 Figure 3 | Hypothalamic-pituitary-adrenal (HPA) axis and hippocampal negative feedback regulation. ................................................................................................ 16 Figure 4 | Information pathways of the hippocampus. .................................................... 20 Figure 5 | Depression and reward circuitry. .................................................................... 24 Figure 6 | Classical and nuanced direct and indirect pathways of NAc signaling. .......... 35 Figure 7 | Selective female susceptibility to SCVS in the measure of sucrose preference. .............................................................................................................. 75 Figure 8 | Additional behavioral assays of male vs. female SCVS. ................................ 76 Figure 9 Retrograde HSV-heF1α-Cre injection at NAc predominantly labels vHPC CA1 pyramidal cells. ....................................................................................................... 78 Figure 10 | Female vHPC-NAc projections are more excitable than male vHPC-NAc projections. .............................................................................................................. 79 Figure 11 | Membrane properties of male vs. female vHPC-NAc projections. ............... 80 Figure 12 | Spontaneous activity measures of male vs female vHPC-NAc projections. 82 Figure 13 | Spike frequency adaptation is elevated in male vHPC-NAc projections compared to female vHPC-NAc projections. ........................................................... 83 Figure 14 | AMPA to NMDA ratio of male and female vHPC-NAc projections. .............. 84 Figure 15 | Male and female vHPC-BLA projections do not differ in excitability. ............ 86 Figure 16 | Membrane properties of male vs. female vHPC-BLA projections. ............... 87 Figure 17 | Spontaneous activity measures of male vs female vHPC-BLA projections. 88 Figure 18 | Female control and post-SCVS vHPC-NAc projections do not differ in excitability. ............................................................................................................... 90 x Figure 19 | Membrane properties of control and post-SCVS female vHPC-NAc neurons ................................................................................................................................ 91 Figure 20 | Orchidectomy of male mice induces susceptibility to SCVS as measured by sucrose preference, but only with extended time following surgery. ....................... 93 Figure 21 | Additional behavioral assays of sham vs. orchidectomy SCVS. .................. 94 Figure 22 | Orchidectomy in male mice increases vHPC-NAc excitability. ..................... 95 Figure 23 | Membrane properties of sham vs. orchidectomy male vHPC-NAc projections. .............................................................................................................. 96 Figure 24 | Spontaneous activity measures of orchidectomy vs sham male vHPC-NAc neurons. .................................................................................................................. 97 Figure 25 | Spike frequency adaptation is impaired in male vHPC-NAc projections following orchidectomy. ........................................................................................... 98 Figure 26 | Sham and OVX female vHPC-NAc projections do not differ in excitability. . 99 Figure 27 | Membrane properties of sham vs. ovariectomy female vHPC-NAc projections. ............................................................................................................ 101 Figure 28 | Spontaneous activity measures of ovariectomy vs sham female vHPC-NAc neurons. ................................................................................................................ 102 Figure 29 | Spike frequency adaptation did not differ between female sham and OVX vHPC-NAc neurons. .............................................................................................. 103 Figure 30 | Orchidectomy increases the excitability of vHPC-BLA neurons in male mice. .............................................................................................................................. 104 Figure 31 | Membrane properties for orchidectomy and sham vHPC-BLA neurons. ... 105 Figure 32 | Female ovariectomy and sham vHPC-BLA neurons did not differ in excitability. ............................................................................................................. 107 Figure 33 | Membrane properties for female ovariectomy and sham vHPC-NAc neurons. .............................................................................................................................. 108 Figure 34 | Ventral HPC to NAc projections express androgen receptors (ARs). ........ 110 Figure 35 | Acute application of the AR antagonist cause flutamide causes an increase in vHPC-NAc neuronal excitability in male mice. .................................................. 111 xi Figure 36 | Membrane properties of male vehicle- and flutamide-treated vHPC-NAc projections. ............................................................................................................ 112 Figure 37 | Spontaneous activity measures of male vHPC-NAc projections treated with vehicle or flutamide. .............................................................................................. 113 Figure 38 | Female susceptibility to SCVS-induced anhedonia is ameliorated by adult testosterone. ......................................................................................................... 115 Figure 39 | Additional behavioral assays of female OVX + Blank and OVX + T SCVS. .............................................................................................................................. 116 Figure 40 | Female OVX vHPC-NAc excitability is decreased with chronic testosterone. .............................................................................................................................. 118 Figure 41 | Membrane properties of female OVX + Blank and OVX + T vHPC-NAc projections. ............................................................................................................ 119 Figure 42 | Spontaneous activity measures of female OVX + Blank and OVX + T vHPC- NAc projections. .................................................................................................... 120 Figure 43 | Spike frequency adaptation is enhanced by chronic testosterone in OVX female vHPC-NAc projections. .............................................................................. 121 Figure 44 | Viral DREADD proof-of concept electrophysiology. ................................... 123 Figure 45 | Long-term reduction of vHPC-NAc activity causes susceptibility to SCVS- induced anhedonia in male mice. .......................................................................... 125 Figure 46 | Additional behavioral assays of Gq-coupled (male) DREADD-expressing vHPC-NAc mice. ................................................................................................... 126 Figure 47 | Exposure to CNO without the presence of DREADD expression in vHPC- NAc neurons in male mice does not cause SCVS-induced anhedonia. ............... 127 Figure 48 | Inhibition of vHPC-NAc activity directly mediates resilience to SCVS-induced anhedonia. ............................................................................................................ 129 Figure 49 | Additional behavioral assays of Gi-coupled (female) DREADD-expressing vHPC-NAc mice. ................................................................................................... 130 Figure 50 | Translating ribosome affinity purification (TRAP) allows interrogation of circuit-specific vHPC-NAc transcriptome. .............................................................. 132 Figure 51 | TRAP strategy is efficient in pulldown of neuronal mRNA. ........................ 133 xii Figure 52 | Plot of transcript reads vs fold change in females vs males verifies efficacy of sex-specific vHPC-NAc TRAP and highlights most analysis-ready genes. ...... 135 Figure 53 | Ingenuity pathway analysis reveals many differentially regulated pathways in female vs male vHPC-NAc transcriptomes. .......................................................... 136 Figure 54 | Chronic social defeat stress and fluoxetine induce ΔFosB in ventral hippocampus. ........................................................................................................ 144 Figure 55 | ΔFosB is induced in vHPC-NAc projections by CSDS. .............................. 146 Figure 56 | vHPC ΔFosB is necessary for resilience to social stress. .......................... 148 Figure 57 | vHPC-NAc ΔFosB is necessary for resilience to social stress. .................. 150 Figure 58 | vHPC-BLA FosB KO is anxiolytic. .............................................................. 151 Figure 59 | ΔFosB regulates vHPC neuronal excitability. ............................................. 153 Figure 60 | ΔFosB expression in vHPC-NAc neurons is reduced in FosB circuit-specific KO mice. ................................................................................................................ 154 Figure 61 | ΔFosB regulates the cellular excitability of vHPC-NAc neurons in male mice. .............................................................................................................................. 156 Figure 62 | Membrane properties of male WT vs FosB KO vHPC-NAc projections. .... 157 Figure 63 | Spike frequency adaptation is decreased in male FosB KO vHPC-NAc projections. ............................................................................................................ 158 Figure 64 | ΔFosB regulates the cellular excitability of vHPC-NAc neurons in female mice. ...................................................................................................................... 159 Figure 65 | Membrane properties of female WT vs FosB KO vHPC-NAc projections. . 160 Figure 66 | Spike frequency adaptation is decreased in female FosB KO vHPC-NAc projections. ............................................................................................................ 161 Figure 67 | FosB KO does not affect the cellular excitability of vHPC-BLA neurons in male mice. ............................................................................................................. 164 Figure 68 | Membrane properties of male WT vs FosB KO vHPC-BLA projections. .... 165 Figure 69 | FosB KO does not affect the cellular excitability of vHPC-BLA neurons in female mice. .......................................................................................................... 166 xiii Figure 70 | Membrane properties of female WT vs FosB KO vHPC-BLA projections. . 167 xiv I. INTRODUCTION General Introduction to the Brain and Disease The nervous system allows animals to perceive information from their surroundings and integrate this information to learn, think, feel, and act. The functional unit of the nervous system is the neuron1, which individually and as a part of large network conveys information via the transmission of chemical and electrical signals. Neurons are extraordinarily diverse within the mammalian brain, with thousands of different classes of neurons intersecting functionally and structurally to drive behavior and cognition. Neuronal communication is accomplished through highly specialized areas of synaptic contact that function electrically via the flow of current or chemically via the release of neurotransmitters. The ultimate signaling mechanism of neurons and neuronal networks is the action potential: the generation of an electrical signal via the sufficient depolarization of the cell membrane following the movement of ions through selectively opened ion channels. Neurons have many forms of functional plasticity, including the ability to change the strength and number of synapses (synaptic plasticity) and to alter the conditions required for an action potential (intrinsic plasticity). Plasticity of individual neurons as well as that of circuits and other higher-level organizations is the foremost mechanism by which the brain grows and changes to accomplish complex tasks such as learning and memory; however, aberrancies in plasticity potentially contribute to disorders of the brain such as depression. Neurons and supporting cells are organized within the brain into many functionally distinct structures and circuits. One such structure, the hippocampus, is a highly- 1 organized region classically understood to mediate learning and memory. The hippocampus is stress-sensitive and plastic, and is thus a substrate in the development of mood disorders, including major depression. Specifically, the connectivity of the hippocampus to other stress- and reward-related regions, particularly the nucleus accumbens, is directly causal of depressive-like behaviors in preclinical animal stress models2. Additionally, the hippocampus has been characterized in depressed patients as being decreased in volume3, and fMRI studies have revealed less hippocampal activation in depressed patients when challenged with a recollection memory task4. Psychiatric disorders continue to rise in prominence each year as a significant social and financial burden, with depression recently highlighted by the World Health Organization as the number one cause of disability globally5. Surprisingly, women have roughly twice the lifetime risk of men for experiencing depression6, but the reasons for this remain unknown. Many genetic and environmental factors, such as inherited traits, early life adversity, and societal inequities, intersect at the level of the individual to produce depressive disorders. One of the most important of these factors is neurophysiology, which itself is influenced by countless external and internal forces, such as hormone status, stress exposure, age, and many others. The neurophysiology of depression is difficult to study in humans, as direct study of the structure and function of neurons and circuits requires invasive or terminal procedures. Accordingly, the field has relied on animal models of depression, which utilize stress and behavioral assays to assess depressive-like symptoms, most prominently in rodents. While previous work in animals has been instrumental in defining some basic physiological mechanisms of depression, the bulk of mood disorder research has been done in males and as such has 2 only just begun to explore the etiology of sex differences in depression incidence. As our current body of knowledge regarding sex differences in depression is lacking, more preclinical studies must investigate neurophysiology, hormonal signaling, and other factors that intersect to produce these differences. This introduction will outline the fundamental body of knowledge and relevant background studies that provide the foundation for this work. First, a review of current knowledge regarding major depression and preclinical stress models will provide significance and relevance to the experiments performed in this dissertation. Next, a summary of the anatomy and physiology of the major brain regions studied, the hippocampus and nucleus accumbens, will provide context for the focus of experiments on the connection between these two regions. Mechanisms of action potential generation with respect to intrinsic excitability and ion channel function in the hippocampus will then be outlined, followed by an introduction to the importance of neuronal steroid hormone signaling in the brain with respect to sex differences in stress and depression. Major Depressive Disorder (MDD) and Preclinical Animal Models of Depression Major depressive disorder (MDD), as currently characterized by the Diagnostic and Statistical Manual of Mental Disorders, 5th Edition (DSM-V), is a syndrome comprising a constellation of symptoms over at least a two-week period, with symptoms occurring for the majority of days and representing a departure from previous functional status7. One of the primary symptoms that must be present to warrant an MDD diagnosis is depressed mood and decreased interest or pleasure in most activities (anhedonia). Three or more of the following symptoms must also be present: fluctuations in weight and/or appetite, 3 sleep disruption (insomnia or hypersomnia), psychomotor disturbances, fatigue, feelings of worthlessness or guilt, difficulty in concentration, indecisiveness, and thoughts of death or suicidal ideation. The constellation of symptoms present in an individual must be disruptive to daily life, and may impair socialization or occupational functioning. Importantly, to be considered a diagnosis of MDD, the symptoms cannot be better explained by the effects of another condition (e.g. hypothyroidism) or substance (e.g. drugs of abuse). Major depressive symptoms can also be further complicated by the presence of a specifier; depression specifiers include anxiety, peripartum onset, or change in season. Epidemiology and Disease Burden Twelve-month prevalence of MDD in adults, according to the World Mental Health survey administered to nearly 90,000 persons across the globe, is estimated this decade at approximately 6%8. This survey also estimates that between one in five and one in six adults will experience MDD in their lifetime. Prevalence rates are similar in most countries regardless of economic status, with about 5.5% affected in countries classified as high- income and 5.9% in low-income countries. The difference between these country classifications is most apparent in availability and access to treatment, with about 50% of affected individuals in high-income countries receiving MDD treatment, compared to 10% or less of those in low-income countries9. In terms of disability-adjusted life years (i.e., number of years of life lost due to poor health), it is estimated that MDD is the second highest contributor to disease burden globally10. The economic burden of MDD is striking: over $200 billion in the United States 4 alone11, with 38% of these costs attributed to MDD itself. Most of the cost, however, is due to conditions commonly comorbid with MDD such as heart disease, diabetes mellitus, cancer, obesity, and others. Meta-analysis of longitudinal studies of depression comorbidities has shown that the relative risk of developing these and other conditions is increased in MDD patients12, a phenomenon attributed to the somatic effects of depression ranging from unhealthy lifestyle to complex physiological changes such as upregulation of stress-related immune and endocrine functions13. MDD differences in men and women One of the most striking statistics of MDD is that women are twice as likely to develop the disorder as men14. This sex difference does not seem to be due to frequency or length of major depressive episodes nor likelihood of recurrence in women, but due to a true difference in incidence15. Interestingly, when considering sex differences in MDD before puberty, boys are more likely to meet diagnostic criteria than girls16. The orientation of the difference in MDD prevalence begins to reverse at puberty, with female prevalence increasing to adulthood levels with each year of age post-puberty17. Symptom profiles also differ between men and women suffering depression: women are more likely to report “atypical” symptoms of increased appetite and sleep, and to experience more fatigue and pain18. Psychiatry has offered many explanations for the elevated risk and prevalence of depression in women. Some have posited that the sex difference may be artificial due to women being more likely to seek treatment, or to depression in men being more difficult to recognize due to diagnostic criteria being biased towards female-specific depression 5 symptoms. However, epidemiological studies of the recall of mental health disorders over the lifetime revealed no gender bias in the recall of depression19. Studies of sex-specific depression symptoms have also shown that the various internalizing (e.g. social withdrawal, somatic disturbances) and externalizing (e.g. substance abuse, attention problems) symptoms of depression do not differ significantly between men and women20. As such, the sex difference in depression prevalence is likely not an artefact of diagnostic bias; rather, the field must look to other physiological and environmental factors to explain this phenomenon. Sex differences in depression rates may arise in part due to specific biological and environmental risk factors experienced by men and women21. Twin studies suggest that MDD carries approximately 37% heritability22, and genome-wide association studies (GWAS) have uncovered a wide variety of potential heritable genetic loci that can contribute to depression pathogenesis23. Hormonal influences can also affect individual susceptibility to MDD, with evidence supporting hormone fluctuation as one factor that increases MDD risk in women24 (discussed further below). Differential activation of the HPA axis in men and women has also been implicated in the pathogenesis of MDD, as women experience hypoactivation of HPA hormone stress responses that, evolutionarily, may protect a fetus from the effects of maternal stress25. This hypoactivation, however, may deprive women of the possible protective effects of cortisol on depression-related changes to emotional brain circuitry26. Many environmental factors, especially during development, can also affect gender disparities in MDD development. For example, gender-based violence, such as domestic violence, rape, and sex trafficking, is more commonly experienced by women27. Early life adversity rates are generally equal in girls 6 and boys, but girls are more likely to be victims of child sexual abuse, which can contribute to myriad perturbations of neurological development28. Gender discrimination in the workplace, for example the wage gap between men and women, can also contribute to sex differences in MDD rates29. While not an exhaustive list, these biological and environmental risk factors, along with many others, begin to outline the complexity and heterogeneity of mood disorder etiology. Recognition of these factors, taken together with the neurophysiological bases for depression in this and other basic science studies, inform our understanding of individual variations in MDD experience. Preclinical Animal Models of Depression As mentioned above, it is difficult to study the basis of mood disorders in humans due to the invasive nature of many procedures that investigate the physiology of the brain. Therefore, we must rely on animal models, which are most effective when they exhibit high validity30. To study a complex human disease like MDD, the model’s behavioral phenotype must resemble the clinical symptom(s) (face validity). These symptoms must also be responsive to clinically effective treatments (e.g. antidepressants) and non- responsive to clinically ineffective treatments (predictive validity). The model phenotype must also have similar physiological bases (construct validity) and triggers (etiological validity) to the human disorder. As MDD symptoms vary from case to case, and often symptoms may be opposite in some individuals (e.g. insomnia vs. hypersomnia), establishing reliable animal models has been challenging. As depression also occurs episodically, it is difficult to create a model by manipulating genetics as this causes congenital abnormalities of behavior that do not fully resemble the human condition. Many 7 traditional genetic models based on theories of depression pathophysiology exist, such as the corticotropin-releasing hormone receptor knockout model31. These are problematic, however, as they often focus on only one potentially involved molecule, when many molecular pathways are known to be involved in depression pathogenesis. Neuronal activity-based optogenetic techniques have also been used to model depression, and are advantageous as they are able to consider circuit-level mechanisms of psychiatric disease32. Optogenetic techniques have demonstrated face and construct validities, but are relatively expensive and difficult to achieve. As such, depression research has largely focused on stress models, many of which have achieved high levels of face, construct, etiological, and in many cases, predictive validity. Stress, along with other environmental factors, can be very influential on the highly plastic circuitry of the brain. A stressful circumstance can activate many systems and circuits in the brain that attempt to maintain the animal’s homeostasis, such as the hypothalamic-pituitary-adrenal (HPA) system. Stress responses in the brain target many limbic brain regions, including the hippocampus and nucleus accumbens, as well as many others such as the prefrontal cortex and amygdala. Changes in activity or connectivity in the brain in response to an initial stressful scenario can alter many functional processes (e.g. learning, memory), resulting in an adaptation that better prepares the animal for future stressful events33. However, when stress is chronic or traumatic, these changes can also coalesce to cause maladaptation to stress with accompanying susceptibility to depression or anxiety, as well as other somatic disorders such as heart disease. Vulnerability to stress and subsequent maladaptation vary from individual to individual, with some individuals maintaining physiological and psychological function even in the 8 face of stress, while others may suffer any number of pathologies in response to the same stress. Those that do not experience maladaptive stress response are considered resilient, and those that develop depression or other conditions in response to stress are susceptible. Gene expression profiling of resilient and susceptible rodents has revealed that resilience may be an active mechanism (i.e., not simply due to the “absence” of susceptibility): more genes are uniquely regulated in resilient individuals compared to non-stressed controls than are in susceptible individuals34. Two major stress paradigms modeling human depression, chronic social defeat stress (CSDS) and subchronic variable stress (SCVS), are discussed below. Many life events, such as divorce, moving to a new city, or suffering an illness represent significant stressors and as such can precipitate the onset or recurrence of depression35. Many life stressors in humans involve social factors (e.g. job loss leading to loss of income) that can lead to loss of social rank or loss of control over one’s life circumstances. To mimic these social stressors in humans, rodent models of depression have utilized social defeat to study environmental perturbations that a mouse or rat might face. These perturbations may cause loss of control of the social situation, and in susceptible rodents, lead to the development of depression-like behaviors. CSDS (Figure 1, left) utilizes a resident-intruder paradigm, in which a male mouse (intruder) is placed into the home cage of another male mouse (resident), usually of a different strain (e.g. a small brown C57BL/6 mouse is placed into the home cage of a larger, white-coated CD-1 mouse). The intruder (experimental) mouse is quickly attacked by the resident (aggressor); bouts of aggressive attack on the experimental mouse are allowed to continue for up to 10 minutes of physical stress. The experimental mouse is 9 Figure 1 | Chronic social defeat stress and social interaction test. Resident aggressor mice (e.g. CD-1) are singly housed in cages for several days prior to testing to establish territory. The experimental intruder mouse (e.g. C57BL/6) is placed into the home cage of the aggressor and the two mice interact for 5-10 minutes (Physical Stress, left). Typically, the experimental mouse is quickly attacked by the aggressor mouse, and these attacks continue throughout the duration of the physical stress period. The experimental mouse is then moved to the other side of a perforated divider where it is safe from further attack, but can still see, hear, and smell the aggressor (Sensory Stress, middle). The experimental mouse is housed here for 24 hours. The cycle then repeats for 10 or more days with the experimental mouse exposed to a novel aggressor mouse each day. Social interaction (SI) testing (right) is used following the chronic stress to evaluate social withdrawal as a measure of stress susceptibility. Susceptible mice exhibit social withdrawal and spend less time in the interaction zone (red) with the social target, and more time in the corners (yellow), withdrawn and isolated. 10 then moved to the other side of a perforated divider to be protected from further attack (Figure 1, middle). The divider, however, allows for the experimental mouse to see, smell, and hear the aggressor mouse; this separated co-habitation lasts 24 hours as a form of sensory stress. This combination of physical and sensory stress repeats for 10 or more days, with the experimental mouse exposed to a novel aggressor each day36. Evaluation of susceptibility to CSDS is typically achieved through the social interaction (SI) test (Figure 1, right), in which the experimental mouse is placed into an arena containing a novel aggressor mouse (social target) in a caged enclosure. The experimental mouse is free to roam the arena and interact with the social target. Social withdrawal behavior is quantified by calculation of a social interaction ratio: the time spent in the interaction zone (red) near the social target over the time spent away from the social target in the rest of the arena and in the corners (yellow). The time spent in the corners by the experimental mouse is also indicative of social withdrawal, as susceptible mice are more likely to “hide” in the corners, the most isolated portions of the arena and furthest away from the social target. In contrast, resilient mice are eager to interact with the social target, as this interaction is rewarding. In addition to exhibiting social withdrawal, susceptible mice also show many other depression-like behaviors34. Compared to non-stressed controls, susceptible mice show a significant decrease in body weight. They also demonstrate a reduction in sucrose preference, which is a measure of anhedonia (loss of interest in activities one previously found rewarding)37. Additionally, only susceptible mice demonstrate conditioned place preference (CPP) for low-dose cocaine, which is indicative of sensitization to drug reward. While only susceptible mice demonstrate these changes in reward-related behavior, both 11 resilient and susceptible groups experience anxiety, as measured by elevated corticosterone responses to forced swim test (FST) as well as decreased open arm time in the elevated plus maze (EPM). The predictive validity of CSDS has been verified by experiments utilizing standard human antidepressant treatments38,39. Treatment of defeated (susceptible) animals with one of the two pharmacologically distinct antidepressants imipramine and fluoxetine reduced the social withdrawal behavior caused by CSDS. Importantly, chronic administration of the antidepressant was required, as an acute injection with the drug did not reverse the withdrawal behavior38. Interestingly, this study also demonstrated that the reversal of social withdrawal is specific to antidepressant drugs, as chlordiazepoxide (used to treat anxiety, but not depression) did not alleviate the stress-induced withdrawal behavior. The relationship between CSDS and the vHPC-NAc circuit is discussed in an upcoming section of this introduction entitled “vHPC-NAc Connectivity.” Subchronic Variable Stress (SCVS) One of the major limitations of CSDS is that, in the resident-intruder paradigm, it relies on the intrasexual aggression between two conspecific males. As a male mouse will not attack a female intruder due primarily to her status as a potential mate, and intrasexual aggression does not typically occur in females of commonly used laboratory mouse species, it is difficult to include the study of female subjects in traditional social defeat experiments. As such, depression research has suffered a lack of the study of depression in females until recently40. One solution to this issue is to use a stress model that does not rely upon aggression between individuals. One such paradigm is subchronic 12 variable stress (SCVS), in which only female subjects are subsequently susceptible to depression-like behaviors. SCVS comprises six days of alternating stressors: foot shock, tail suspension, and restraint (Figure 2, top). Following stress, mice are then assessed for depression-like changes in behavior using a variety of assays: sucrose preference in two-bottle choice task, SI, EPM, novelty-suppressed feeding (NSF) test, and splash test (Figure 2, bottom). In one of the first studies to utilize SCVS as an assessment of sex differences in stress outcomes, Hodes et al discovered that only female mice are susceptible to this subchronic battery of stressors41. Specifically, female mice spent less time grooming in the splash test following SCVS than male mice or unstressed female mice, indicating a reduced motivation for self-care. Female mice also displayed increased latency to eat in a novel environment, indicating an increase in anxiety induced by the stress of a novel environment. Sucrose preference, a key measure of anhedonia (loss of motivation to consume a rewarding sucrose solution), was also reduced exclusively in stressed female mice. This study also highlighted an increased corticosterone in stressed female mice only, indicating dysregulation of the HPA axis. Female mice did not differ from male mice or unstressed control females in EPM measures, indicating no change in exploratory behavior induced by anxiety. SCVS represents a useful stress model of depression that is distinct in that only females exhibit depression-like behaviors following its stress battery. This sex difference and its etiology are further discussed throughout this introduction. 13 Figure 2 | Subchronic variable stress (SCVS) and associated behavioral assays. SCVS comprises a 6-day battery of repeated stressors (top): foot shock, tail suspension, and restraint stress. Each stress session typically lasts 1 hour per day. Following the stress period, a variety of behavioral assays are used to assess stress susceptibility (bottom): splash test, sucrose preference, elevated plus maze (EPM), novelty suppressed feeding (NSF), and social interaction (SI, not pictured). Splash test measures grooming behavior, with susceptible animals showing reduced grooming time when sprayed with a sticky solution. Sucrose preference indicates anhedonic response with two-bottle choice task, with susceptible animals showing no preference for sucrose drinking solution. EPM measures anxiety response, with “anxious” animals spending less time in open arms and more time in closed arms of the apparatus. Novelty suppressed feeding represents a more subtle measure of anxiety, with “anxious” animals exhibiting increased latency to eat (following overnight food deprivation) in a novel environment. 14 Sex differences in stress responses It is clear from the female-specific susceptibility in the SCVS paradigm that sex differences in stress response exist. These differences may account for the disproportionate number of women diagnosed with depression compared to men. Studies of stress responses in animal models of depression have revealed myriad sex differences, including differences in cellular and hormonal responses, brain circuits, synaptic and intrinsic plasticity, cognition, and emotional processing. The HPA axis has long been recognized as a central component of stress responses in humans, and in particular, of the pathophysiology of anxiety and depression. Corticotropin-releasing factor (CRF) is the hormonal initiator of the HPA response. Neurons in the paraventricular nucleus of the hypothalamus (PVN) release CRF in response to stress, which reaches the pituitary and stimulates the release of adrenocorticotropic hormone (ACTH) into the systemic circulation, stimulating glucocorticoid release from the adrenal glands (Figure 3); the PVN is known to receive negative feedback regulation from the HPC and other limbic structures42. CRF also acts at the level of individual neurons via CRF receptors43, and is released from the emotion- regulating limbic regions such as the bed nucleus of the stria terminalis (BNST) and central nucleus of the amygdala (CeA)44 and can act throughout the brain as a neuromodulator. Indeed, CRF is elevated in cerebrospinal fluid of depressed humans45 and antidepressant pharmacotherapies are known to decrease these levels46,47. CRF1 receptor expression is also disturbed in many areas of the brain in depressed patients, including an increase in PVN48 and possible compensatory decreases in cortical regions49. Generally, HPA axis hormones (e.g. corticosterone) are known to be elevated 15 Figure 3 | Hypothalamic-pituitary-adrenal (HPA) axis and hippocampal negative feedback regulation. The hypothalamic-pituitary-adrenal (HPA) axis is activated by stress and integrates stress responses. Anxiety responses via activation of the amygdala can exacerbate the stress response through its projections to the paraventricular nucleus of the hypothalamus (PVN). The hippocampus (HPC) is important for the evaluation of stress and is a site of glucocorticoid receptor (GR)-mediated negative feedback to the PVN. Release of the neuropeptides corticotrophin-releasing hormone (CRH) and arginine vasopressin (AVP) from the hypothalamus promotes the release of adrenocorticotrophin (ACTH) from the pituitary. ACTH then stimulates the release of glucocorticoids from the adrenal cortices. These hormones circulate to the body and brain and bind to intracellular steroid hormone receptors. 16 in female rodents compared to males at baseline50. Female rodents, when stressed, release more ACTH and corticosterone than males and elevated levels also persist longer51,52. These sex differences in rodents appear to reflect increased activation of the HPA system by CRF in females. PVN expression of CRF appears to be elevated in females at baseline, particularly when estrogen levels are high (i.e. proestrus stage). Furthermore, early life and footshock stresses increase CRF in female rodents only53,54. Sex differences in epigenetic regulation of CRF have also recently been demonstrated, with elevated methylation of Crf gene globally in the brain of female rats, but only in BNST and CeA in male rats55. Particularly interesting to the current dissertation study is the differential expression of CRF1 receptors in the CA1 region of the hippocampus. Female rats, especially with elevated estrogen levels, have an elevated CRF1 receptor expression on pyramidal cells of the CA1 region56; however, males have higher CRF1 receptor expression on inhibitory interneurons in the DG. The same study demonstrated that CRF1 receptor expression does increase in CA1 pyramidal cells in male and female rodents following chronic immobilization stress (CIS), but male levels do not even reach the female baseline expression level. The higher CRF1 expression level on CA1 pyramidal cells in the hippocampus represents one mechanism by which females may be more sensitive to stress. Collectively, these studies indicate that increased CRF signaling in females can differentially influence the HPA axis and central neuromodulation, contributing to observed sex differences in stress outcomes57. In addition to the vHPC-NAc circuit discussed below and investigated in the current work, a variety of other circuits are implicated in mediating sex differences in stress responses. The connection between the medial prefrontal cortex (mPFC) and amygdala 17 is of particular interest, as the amygdala regulates responses to negative and traumatic stimuli58 and the mPFC regulates a wide range of functions including cognition and determination of affect59. Together, these brain regions play a significant role in generating behavioral responses to emotionally relevant contexts60. Indeed, mood disorders including MDD have been correlated with overactivity in the amygdala61, as well as decreased mPFC activity62. Regarding stress models of depression, chronic restraint stress in rodents causes restructuring and reorganization of dendrites in pyramidal cells of mPFC, which is posited to decrease the region’s ability to suppress HPA activation63. Chronic stress has also been found to cause hyperexcitability64 and increased dendritic arborization65 in rat basolateral amygdala (BLA) neurons. As such, the mPFC-amygdala circuit is of interest in the exploration of sex differences in stress response. Shansky et al demonstrated sensitivity of BLA-projecting mPFC neurons to stress in estrogen-treated, ovariectomized female rats, with only this group experiencing an increase in dendritic arborization in these cells66. Preclinical animal studies of depression have also highlighted sex differences in locus coeruleus (LC) norepinephrine (NE) circuitry67. Dendritic architecture of LC neurons in female rats has been shown to be more complex than in males, with LC dendrites receiving significantly more synaptic input68. Moreover, tonic and phasic firing patterns are linked to changes in arousal, and, while not significantly different at baseline, male and female changes in LC neuron firing were found to differ following swim stress in rats69. Specifically, female rat LC NE neurons were found to have a greater increase in firing than those of male rats, indicating heightened sensitivity to stress. Taken together, these findings identify the LC NE circuitry as a key substrate in the mediation of sex differences 18 in arousal, which may contribute to differences in male and female stress response and development of psychiatric disorders such as MDD. However, the remainder of this introduction will focus on the HPC, NAc, and their connectivity in the context of stress with special attention to sex differences. The Hippocampus The hippocampus is a medial temporal region of the brain long-known to be necessary for memory70,71 and spatial reasoning72. The hippocampus, named due to its resemblance to a seahorse, is a long, C-shaped, bilateral brain region that is typically described along a dorsal-ventral (septotemporal) axis in rodents (corresponding to posterior-anterior axis in humans). In rodents, the hippocampus spans the distance between the neocortex and diencephalon and curves ventrally adjacent to the temporal lobe. The hippocampal formation is made up of four regions: the dentate gyrus (DG), the hippocampus proper (CA1, CA2, and CA3), the subicular complex, and the entorhinal cortex (Figure 4). The primary inputs to the hippocampus extend from the entorhinal cortex to the DG via the perforant pathway (blue)73. The body of the hippocampus, or the hippocampus proper, shares efferent and afferent connections with many cortical and subcortical regions (green)74. The hippocampus proper is made up of the cornu ammonis (CA) regions, with the CA3 receiving input from DG granule cells via mossy fiber tracts. CA3 pyramidal cell axons (Schaffer collaterals) then project to the CA1 region, which then projects to the entorhinal cortex and the subiculum, as well as many other brain regions, including the nucleus accumbens (NAc) and basolateral amygdala (BLA) via the fornix. The CA2 region is small and lies between CA3 and CA1, and receives some input from 19 Figure 4 | Information pathways of the hippocampus. The red arrows represent the direct internal hippocampal pathway of information flow from the entorhinal cortex (EC) Layer III to CA1 to the subiculum (SUB). The green arrows represent the primary output projections of the hippocampus: SUB sends fibers back to EC or to the fimbria via the alveus (white matter outer layer of hippocampus). The main output region of the hippocampus is the CA1, which send fibers through alveus. The alveus fibers merge to form the fimbria, which eventually becomes the fornix through which the hippocampus sends projections to outside regions such as the nucleus accumbens (NAc). The blue arrows represent the canonical trisynaptic loop: the EC sends fibers to the dentate gyrus (DG) via the perforant pathway (synapse 1), the DG then projects to CA3 via the mossy fiber tracts (synapse 2), and the CA3 then sends fibers to the CA1 via Schaffer collaterals (synapse 3). The subiculum is also included in this circuit as it receives input from CA1. 20 the entorhinal cortex perforant pathway. The entorhinal cortex à DG à CA3 à CA1 series of synaptic transmission is known as the trisynaptic loop, first described by Santiago Ramon y Cajal75 in his seminal Golgi staining neuroanatomy work. Other internal hippocampal pathways of information flow have also been observed, for example EC à CA1 à SUB à EC (red). Distinctive Dorsal and Ventral HPC Functions As mentioned above, the hippocampus is classically recognized as a key memory center, as exemplified by the case of H.M., who experienced severe amnesia after bilateral medial temporal lobectomy (including removal of both hippocampi) in a radical attempt to cure his intractable epilepsy70. The hippocampus was later identified as critical for the processing of spatial memory in navigation tasks, as “place cells” within the hippocampus fire at specific locations within an experimental arena76. Furthermore, rats with bilateral hippocampal lesions are incapable of navigation relying only upon spatial cues in the Morris water maze72. Functional imaging studies in humans also confirmed the importance of the hippocampus in visual and verbal information coding and memory77,78. Intrahippocampal connectivity remains largely conserved along the dorsal-ventral (septotemporal) plane79, but connections to (afferent) and from (efferent) the hippocampal formation differ, suggesting the dorsal and ventral hippocampus (dHPC and vHPC, respectively) have distinct functions. Another key difference between these two subdivisions of the hippocampus is their intrinsic excitability: vHPC CA1 neurons are hyperexcitable compared to their dHPC counterparts80. This is likely due to differential ion 21 channel expression and unbalanced excitatory and inhibitory inputs81, further discussed later in this introduction. As the connections within the entorhinal cortex, the major informational relay that sends projections to the hippocampus proper, are topographically organized, dHPC receives information from sensory cortical regions (visual, auditory, somatosensory) that is distinct from vHPC input. Lesions of dHPC, but not vHPC, result in impaired maze learning82 proportional to the severity of the lesion83. These and other related studies demonstrate the unique necessity of the dHPC for spatial learning, further supported by the majority of “place cells” being located in dHPC84. The vHPC has more extensive efferent connections with major forebrain structures than dHPC,including the hypothalamus and the amygdala74,85, and as discussed extensively in this work, the NAc. Due to its associations with these and other regions, the vHPC distinguishes itself from the dHPC in its involvement in mediating stress responses, emotion, and anxiety- and depression-related behaviors. While the vHPC does have some role in spatial learning and memory in unison with dHPC function86, the vHPC is uniquely suited to influence emotional processing, physiological and behavioral stress responses, and motivation. Strict lesions of the vHPC reduce fear and anxiety behaviors87,88 and increase conditioned place preference for food88. Ventral HPC function regarding the mediation of stress outcomes and depression is discussed in more detail throughout this introduction. Anterograde and retrograde tracing studies have demonstrated extremely diverse targets of CA1 projections89, with, as alluded to above, projection areas differing based on location of cell bodies along the CA1 septotemporal axis. The temporal (ventral) CA1 has unique projections compared to the septal (dorsal) and splenial (middle) CA1 22 divisions, with outputs to the subiculum/parasubicular area, entorhinal cortex, anterior olfactory nucleus, and olfactory bulb. Many subcortical regions also receive projections from the ventral CA1, including the amygdala, hypothalamus, lateral septal nucleus, and NAc. Interestingly, the anterograde labeling studies in question did not highlight the labeling of any commissural axons, indicating that the ventral CA1 region likely sends its projections to the ipsilateral hemisphere. Van Groen and Wyss89 describe a great number of projection axons traveling to the septum via the fornix, where they split into three groups to project to the lateral septal nucleus and the nearby NAc, a number of cortical regions, and the hypothalamus. Neurotransmitters of the HPC – Implications in Mood disorders A wide range of neurotransmitters (NTs) and their receptors can be found in the hippocampus, with the most prominent being glutamate, γ-amino butyric acid (GABA), norepinephrine (NE), serotonin (5-HT), and acetylcholine (ACh). Synaptic transmission in the hippocampus happens in the classically understood sense: upon sufficient influx of calcium generated by the propagation of an AP down the axon, NTs are released from the presynaptic terminal and diffuse through the synaptic cleft to reach receptors on the postsynaptic membrane. In addition to postsynaptic receptors, presynaptic autoreceptors for many NTs (e.g. ACh, GABA, and 5-HT) can also serve to regulate the activity of NTs via feedback modulation90. A diagram of the diverse circuitry implicated in depression, including inputs to and from the HPC and reward circuitry, can be found in Figure 5. The main excitatory neurotransmitter in the brain is the amino acid glutamate, and the majority of the brain functions as a “glutamatergic excitatory machine”91, as evidenced 23 24 Figure 5 | Depression and reward circuitry. A simplified summary of neural circuits in that have been implicated in the pathophysiology of depression. The glutamatergic projections of the hippocampus (HPC) and prefrontal cortex (PFC) are prominently featured, and many subcortical regions that mediate reward, involved: nucleus accumbens (NAc), amygdala, and hypothalamus. This diagram also shows the innervation of many tegmental area (VTA) regions by monoaminergic projections and (dopaminergic structures: noradrenergic projections (LC) and serotonergic projections from the dorsal raphe (DR). The NAc also sends GABAergic projections to the hypothalamus, and GABAergic interneurons are found within the HPC. This diagram, while not exhaustive, highlights some of the major circuits involved in depression and referenced throughout this dissertation. amygdala, PFC) locus coeruleus fear, and motivation are also from the ventral from the input to NAc, other by the fact that up to 85% of synapses in the neocortex are excitatory92. Receptors for glutamate can be either ionotropic or metabotropic, with the ionotropic receptors mediating fast excitatory neurotransmission while the metabotropic receptors permitting more prolonged stimuli93. The ionotropic glutamate receptors (iGluRs) are classified by their responses to synthetic agonists N-methyl-D-aspartate (NMDA) or α-amino-3- hydroxy-5-methyl-4-isoxazole propionate (AMPA), or the naturally occurring kainate; all three iGluRs are found throughout the hippocampus. AMPA receptors are formed by different combinations of four different subunits (GluA1, 2, 3, and 4), and can be further diversified by alternative splicing (in all subunits) and editing of introns in their primary transcripts (specific to GluA2). AMPA receptors are primarily permeable to monovalent cations (i.e. Na+ and K+) and Ca2+, but become impermeable to Ca2+ if they contain the edited form of the GluA2 subunit (containing an arginine in place of a glutamine at the Q/R editing site). The arginine in the edited form of the GluA2 subunit presents a positively-charged side chain in the channel and hence blocks the passage of the larger, divalent Ca2+. Furthermore, all AMPA receptor subunits can undergo alternative splicing to form “flip” or “flop” isoforms. Flip isoform-containing receptors allow more inward current to pass than those that contain only flop isoform subunits. Kainate receptors are tetramers formed by combinations of subunits GluA5, 6, 7 and KA1, and 2. GluA5 and 6, much like the GluA2 subunit of AMPA receptors, are subject to editing, and the GluA5, 6 and 7 subunits can undergo alternative splicing to alter their conductances. As the sequences of these subunits are similar to AMPA receptor subunits, kainate receptors are permeable to cations. Editing of GluR5 or 6 accordingly changes the Ca2+ permeability of a given kainate receptor. 25 NMDA receptors represent heteromeric complexes formed by combinations of GluN1, GluN2B, GluN2A-D, and GluN3A and B subunits that bind glycine and glutamate. NMDA receptors are blocked by Mg2+ at voltages near the resting membrane potential of the neuron, and, when the Mg2+ block is released by AMPA-mediated depolarization, they are highly permeable to monovalent cations and Ca2+. Glycine binding at the GluN1 subunit potentiates the NMDA response by increasing frequency of its channel opening; glycine binding is required for full activation of the NMDA receptor by glutamate binding at the GluN2A-D subunits. When synaptic glutamate diffuses to the postsynaptic membrane of a neuron at rest, it binds to AMPA and NMDA receptors. Inward current through AMPA receptors, the major driver of excitatory postsynaptic current (EPSC), causes an evoked postsynaptic excitatory potential (EPSP). Depolarizing currents, if strong enough, allow the release of the Mg2+ blockade of NMDA channels and further membrane depolarization. The further membrane depolarization allows for unblocking of additional NMDA receptors. Ca2+ entry through unblocked NMDA channels increases intracellular Ca2+ concentrations, leading to an increase in the function of Ca2+-dependent processes. One such process is long-term potentiation (LTP), which represents a persistent increase in synaptic strength94. First described by Bliss and Lomo in the rabbit hippocampus95, LTP is a process that occurs frequently at the excitatory glutamatergic synapses of dendritic spines of postsynaptic CA1 pyramidal cells and, in part, mediates the plasticity required for learning and memory. In brief, CA1 LTP strengthens synapses by increasing the depolarization at the postsynaptic cell caused by a given presynaptic release of glutamate (i.e., the postsynaptic cell can reach the threshold for AP generation 26 with a lesser stimulus; verified by the increase in response to glutamate receptor agonists following LTP96). This is accomplished by increased postsynaptic Ca2+ influx via NMDA channels, which activates adenylyl cyclase (AC). AC acts to produce cyclic AMP (cAMP), which in turn activates protein kinase A (PKA). PKA phosphorylates a variety of postsynaptic mediators, including Ca2+/calmodulin-dependent kinase II (CaMKII). CaMKII drives LTP by facilitating AMPA receptor trafficking to the postsynaptic membrane and subsequent exocytosis97, as well as phosphorylating AMPAR GluA1 subunits, increasing AMPAR conductance98. It is important to note that LTP at other synapses (e.g., not CA3- CA1) may involve many different or additional mechanisms, including presynaptic components99. CA1 LTP and its importance in mediating stress responses in preclinical models of MDD is further discussed in later sections of this introduction. Chronic stress, used in preclinical animal models to mimic human depression pathologies, has been associated with atrophy of HPC pyramidal neuron apical dendrites, the main site of excitatory glutamatergic synapses, as well as reductions in number and size of dendritic spines100,101. Chronic stress impairs LTP102 at synapses from Schaffer collateral projections from CA3 to CA1 and reduce GluA1 transcripts in CA1 pyramidal neurons103. Schmidt et al further investigated the behavioral phenotypes following chronic social stress, and found that susceptible mice displayed a decreased GluA1/GluA2 AMPA subunit ratio in CA1 compared to resilient mice; susceptibility was predictable based on performance in a short-term memory, a CA1 AMPAR-mediated task. CA1 synapses from EC are also affected by stress: chronic unpredictable stress (CUS) has been shown to significantly reduce AMPAR-mediated excitation at these synapses, an effect that resulted in sucrose preference reduction104. This study further demonstrated that the 27 change in excitation at EC-CA1 synapses as well as the sucrose preference reduction were reversed by chronic fluoxetine treatment. Additionally, Chourbaji et al showed that GluA1 knockout mice exhibit a depression-like phenotype as indicated by increased learned helplessness105. These studies suggest that glutamatergic transmission at the level of CA1 pyramidal neurons is directly influenced by chronic stress, and that changes in excitatory signaling at these neurons can influence depression-like behaviors in mouse models of depression. Excitatory output from the HPC in the form of glutamatergic afferents to areas such as the NAc also have great influence over stress responses and the development of depression-like behaviors; this output is further discussed below with focus on vHPC connectivity to NAc. In addition to the ionotropic glutamate receptors discussed above, glutamate neurotransmission can also act through metabotropic receptors (mGluRs). There are eight members of this family of receptors (mGluR1-8), and belong to the G-protein coupled receptor (GPCR) class. Like other GPCRs, mGluRs comprise seven transmembrane segments and initiate intracellular signaling through their coupled G- proteins. These receptors are further grouped based on their G-protein mediators: mGluR1 and 5 are coupled to stimulatory Gq proteins, and the other family members are coupled to inhibitory Gi/o proteins. The Gq-coupled mGluRs are, in the hippocampus, mainly found at postsynaptic membranes, and the Gi/o are largely found presynaptically106. Metabotropic GluRs can modulate neuronal excitability by modulating ion channels. For example, hippocampal mGluR5 can inhibit afterhyperpolarization current mediated by K+ channels, resulting in reduced K+ efflux and impaired spike frequency 28 adaptation107. These receptors can also modulate synaptic transmission: Gi/o-coupled mGluRs in the CA1 region can mediate Ca2+ influx, particularly when paired with a backpropagating AP108. Although the role of mGluRs in mediating mood disorders is not fully understood, there is evidence that their expression may be related to depression pathogenesis. For example, Wieronska et al demonstrated changes in mGluR5 expression in rat hippocampus following chronic mild stress in rats, with levels increased in CA1 and decreased in CA3109. Metabotropic GluRs can also modulate the release of glutamate via negative feedback mechanisms at the presynaptic membrane, and it is hypothesized that changes in this feedback and subsequent perturbations in synaptic availability of glutamate may be involved in mood disorder pathogenesis. Indeed, mGluR antagonists exhibit antidepressant effects in the forced swim and tail suspension despair models of depression in mice110. Accordingly, mGluRs represent interesting potential mediators of cellular excitability and glutamatergic synaptic transmission in the context of mood disorders and stress models of depression. While glutamate represents the main excitatory neurotransmitter in the brain, the “excitatory machine” formed by glutamatergic transmission must be regulated to allow for normal neurologic function. Much of this regulation is accomplished by inhibitory GABAergic neurotransmission, which comprises the majority of synaptic connections that are not excitatory. In the CNS, two classes of GABA receptors can be found: GABAA and GABAB. GABAA receptors are ligand-gated ion channels: receptor activation results in the opening of a chloride-selective pore111. Increased chloride (Cl-) conductance causes a decrease in membrane potential towards the chloride reversal potential (@ -75 mV), inhibiting depolarization and the generation of APs. GABAB receptors are metabotropic 29 GPCRs that are coupled to K+ channels. Stimulation of GABAB receptors results in the opening of additional K+ channels, driving the membrane potential closer to that of the equilibrium potential of K+ (@ -84mV) and, much like the increased Cl- conductance caused by GABAA activation, inhibits depolarization and AP generation. Both GABAA and GABAB receptors are found in the HPC, and deficiency of GABAergic neurotransmission has been hypothesized as a mechanism of depression pathogenesis112. In the hippocampus, mice lacking specific functional GABAA receptor subunits are deficient in object location memory tasks113 as well as more sensitive to trace fear conditioning114. GABAA receptor-mediated currents are also decreased in DG granule cells following stress, an effect reversed by treatment with the antidepressant escitalopram115. As excitatory-inhibitory imbalance in HPC has been implicated in mediating stress outcomes in rodents116, dysfunction of GABA circuitry must be considered when investigating the etiology of mood disorders and depression-like behaviors in preclinical models. Glutamatergic and GABAergic neurotransmission as summarized above are also accompanied and modulated by other types of neurotransmission. Nicotinic acetylcholine (ACh) receptor subunit mRNA, for example, is found in all neuronal types of the CA1 region, though currents mediated by these ligand-gated ion channels are detectable in interneurons in the strata radiatum and oriens, but not in pyramidal cells117. Both muscarinic and nicotinic (mACh and nACh) presynaptic receptors may mediate the release of glutamate118,119, leading to the enhancement of synaptic transmission. The CA1 is also innervated by serotonergic (from dorsal raphe120), noradrenergic (from locus coeruleus120), and dopaminergic (from midbrain121) projections, all of which can affect the excitability of CA1 pyramidal cells as well as their synaptic plasticity. Through β- 30 adrenergic, D1, and 5-HT4 receptors, respectively, these monoamines are known to increase pyramidal cell excitability through cAMP-mediated mechanisms122-124. Serotonergic signaling via the 5-HT2 receptors at pyramidal neurons, however, decrease cellular excitability122. Inhibitory neurons in the CA1 are activated by α-adrenergic125 and 5-HT3 126 signaling, which may have downstream effects on the excitability of their connected pyramidal neurons. NE also facilitates LTP induction127, and dopamine D1/D5 receptors induce a slow, long-lasting potentiation of field EPSPs associated with LTP128, both in CA1. While not an exhaustive account of the complex neurotransmission of the HPC, the above discussion highlights the many NT systems that can affect excitatory transmission within and from this critical brain region. HPC Sex Differences in Stress and Depression Many systems in the brain converge to affect the complex function of cognition and emotional processing, both of which are impaired in MDD and other mood disorders. The HPC has been recognized as a central substrate in the pathophysiology of a wide range of mood disorders, including MDD129-131, bipolar disorder132, and post-traumatic stress disorder (PTSD)133. Many of these studies have identified varying degrees of hippocampal atrophy in patients with MDD134, with an inverse correlation between depression duration and hippocampus volume135. This atrophy is likely due to decreased neurogenesis in the DG136, as well as shrinking and retraction of dendrites and possible loss of glial support137, and these stress-induced processes are often prevented by antidepressant treatment138. HPC abnormalities in MDD also extend to sex differences. For instance, women that do not respond to the antidepressant fluoxetine have smaller 31 hippocampal volumes than those women that do respond to fluoxetine treatment3. Critically, this difference in hippocampus size with respect to antidepressant response was not observed in men. The differences in hippocampus in women that may contribute to increased susceptibility to depression are also mirrored by sex differences in preclinical models. Woolley et al demonstrated a fluctuation of dendritic spine density in the CA1 region corresponding to changes in estrogen levels throughout the rodent estrous cycle: spine density was found to decrease in late proestrus to estrus, then return to early proestrus levels over a period of days139. This difference was exclusive to CA1, as spine densities in CA3 and DG did not vacillate over the estrous cycle. Further studies determined that the effects of estradiol on the spine density in CA1 were dependent upon glutamate neurotransmission via NMDA receptors (NMDARs), as density did not fluctuate when rats were treated with competitive NMDAR antagonists140. Changes in CA1 spine density were further investigated by Shors et al, who found that male rat CA1 spine density increased following just one bout of tail shock stress, while female rat CA1 spine density decreased following the same stressor141. This group later showed that the opposite effects of stress on CA1 spine density were, like the estradiol-dependent changes, also NMDAR-dependent: NMDAR antagonism prevented both the increase in spine density in female rats and the decrease in spine density in male rats following stress142. The effects of steroid hormones (i.e. estradiol and testosterone and derivatives) on stress outcomes and MDD with focus on the HPC in human and preclinical rodent models are further discussed in later sections of this introduction. 32 The Nucleus Accumbens The nucleus accumbens (NAc), along with the olfactory tubercle, are parts of the basal forebrain that form the ventral striatum, a portion of the basal ganglia. It contains two substructures: the core and shell. The NAc shell functions in the processing of reward (i.e. “liking” of a pleasurable stimulus)143, as well as mediating the cognitive process of motivational salience (i.e. “wanting” of a reward or associated cues that motivates an animal to attain a goal)144. The NAc core acts to process reward-related motor function145. As such, the NAc is widely recognized as a key mediator of reward-related behaviors, and has also gained attention over recent decades for its involvement in the pathophysiology of depression146,147. In stress models of depression, molecular and synaptic adaptations to physical or psychological stress are dysregulated in NAc, ultimately leading to pathological behaviors148. NAc Cell Types – Direct and Indirect Pathways Both NAc core and shell substructures primarily contain GABAergic medium spiny neurons (MSNs – 95% of all NAc neurons), which primarily express either D1-type or D2- type dopamine receptors (D1-MSNs and D2-MSNs, respectively), with a subset of MSNs containing both149. The remaining population of cells comprise interneurons, either cholinergic or GABAergic150. Classically, D1-MSNs are thought to inhibit the dopaminergic ventral mesencephalon (i.e. the ventral tegmental area, VTA and substantia nigra, SN) via the “direct pathway,” resulting in disinhibition of the thalamus and the overall effect of enhancing motivated behavior. D2-MSNs, on the other hand, act through the “indirect pathway” via the ventral pallidum which usually inhibits the ventral 33 mesencephalon, resulting in an overall inhibition of the thalamus and in turn, inhibition of motivated behavior (Figure 6). This classical understanding of NAc MSN signaling is supported by the promotion of drug-seeking behavior by D1-MSN activation and converse inhibition of seeking by D2-MSNs151. However, optogenetic studies have recently demonstrated that the direct and indirect NAc pathways are not as well-differentiated by dopamine receptor MSN types in NAc as they are in dorsal striatum152. This work shows that many D1-MSNs are involved in the indirect pathway via innervation of ventral pallidum neurons that project to the ventral mesencephalon, and that many D2-MSNs project to ventral pallidum neurons that innervate the thalamus as part of the direct pathway. As such, signaling of the NAc is more nuanced than previously thought, and further study of the dynamics of this system may reveal more specialized mental health treatments. NAc function in stress models of depression The NAc is a key substrate in the expression of depression-related behaviors in mouse models, as well as in mediating resilience to stress. Berton et al demonstrated an increase in c-Fos (a marker of neuronal activation) in the NAc and VTA of mice following exposure to a social target, an effect that was exaggerated in defeated subjects38. This group also highlighted an increase in brain-derived neurotrophic factor (BDNF) expression in the NAc following CSDS; BDNF is known to increase dopamine release in this region through TrkB receptor activation on terminals of dopaminergic projections153. The functional knockout of BDNF in the VTA, including in those projections to the NAc, induced the antidepressant effect of mitigating social withdrawal behavior following 34 Figure 6 | Classical and nuanced direct and indirect pathways of NAc signaling. The classical distinction of “direct” and “indirect” pathways of motivated behavior drive being mediated by D1-MSNs and D2-MSNs, respectively, is shown on the left. The classic direct pathway (blue) was understood to involve direct inhibition of the dopaminergic areas of the mesencephalon (VTA and SN, collectively VM) by GABAergic D1-MSNs, resulting in disinhibition of the thalamus. The classic indirect pathway (red) was thought to involve the inhibition of the VP, which in turn inhibits the VM, resulting in inhibition of the thalamus. However, recent optogenetic studies have demonstrated mixed populations of D1-MSNs and D2-MSNs participating in the direct and indirect innervation pathways that result in the initiation and fine-tuning of motivated behavior. NAc: nucleus accumbens; VTA: ventral tegmental area; SN: substantia nigra; VM: ventral mesencephalon; VP: ventral pallidum. 35 CSDS. In experiments investigating epigenetic changes in the NAc following stress, Wilkinson et al demonstrated widespread and persistent changes in methylation of histones, as well as differences in the binding of cAMP response element binding protein (CREB)154. Interestingly, these changes were largely reversed by treatment with the antidepressant imipramine, and the changes observed in animals resilient to CSDS mirrored those induced by the antidepressant treatment. To this end, Covington et al showed that increases in repressive histone methylation in NAc of mice following CSDS are paralleled by decreases in histone deacetylase 2 (HDAC2) levels, and that inhibition of HDAC function produces antidepressant effects in social interaction, sucrose preference, and forced swim tests in defeated mice155. Much of the focus of the role of the NAc in mediating resilience to stress has been on the transcription factor, ΔFosB. This transcription factor is a member of the Fos family, and is a truncated splice variant of the full-length FosB protein. Fos family proteins form heterodimers with Jun family proteins to form activator protein-1 (AP-1) complexes, which bind to corresponding consensus sequences in the promoters of a variety of genes to affect their transcription156. Fos family proteins are induced in a number of brain regions following acute stress157, but this induction is transient due to the instability of Fos proteins. Chronic stress, however, causes a unique induction of ΔFosB, particularly in the NAc157,158. ΔFosB’s induction by chronic stimuli is unique as it is more stable than its other Fos family relatives: while other Fos family members are mostly degraded within 8 hours of translation, ΔFosB has a half-life in vivo of 8 days159. This is due to the truncation of degron domains present in the full-length form of FosB, as well as phosphorylation at its N-terminus160. This stability affords ΔFosB the ability to accumulate with repeated 36 exposures to stress, and this accumulation is observed in both D1- and D2-MSNs but not interneurons nor structural support cells of the NAc161. This study by Lobo et al also observed the striking phenomenon of differential cell subtype-specific induction of ΔFosB in animals susceptible or resilient to CSDS: susceptible mice exhibited a modest induction in D2-MSNs, while resilient mice exhibited a higher degree of induction in D1-MSNs. Indeed, mice overexpressing ΔFosB in D1-MSNs via inducible transgenic mouse line or with non-cell type-specific virally-induced overexpression in NAc are resilient to CSDS158,162. Conversely, mice expressing ΔJunD in the NAc - a dominant negative form of ΔFosB’s AP-1 complex binding partner JunD - demonstrate heightened susceptibility to even subchronic exposure to social defeat stress158. Additionally, while ΔFosB is induced in the NAc in response to fluoxetine, this treatment’s antidepressant effects are blocked by viral overexpression of ΔJunD. An important gene target of ΔFosB is the aforementioned AMPA receptor subunit GluA2: ΔFosB overexpression in the NAc is accompanied by a specific induction of GluA2163. Mice with viral-mediated overexpression of GluA2 in the NAc demonstrate resilience to CSDS158, a particularly interesting effect due to the Ca2+-impermeable nature of AMPARs containing this subunit. Reduced glutamatergic response in NAc due to the increased induction of GluA2 represents an important mechanism of resilience. Glutamatergic inputs to the NAc, including those originating in vHPC, are discussed further below with regard to stress outcomes and depression. 37 Sex differences in NAc There is mounting evidence regarding sex differences in the NAc in the context of motivated behavior and the dysfunction thereof. For example, female rats are more susceptible to addiction (a NAc-dependent process) than male rats, as they are faster to acquire drug-self administration and are more likely to relapse164. Female rats are also quicker at acquiring CPP for cocaine, and this CPP was achieved at half the dose or less required for male rats165. These differences may be attributable to differences in development of the male and female brain conferred by the actions of ovarian hormones166. Gonadal steroid hormones can also influence the density and activity of dopamine (DA) terminals in the NAc, as many DA neurons in the ventral tegmental area (VTA) contain estrogen and androgen receptors and are also regulated by glia and other afferents that may be affected by hormone signaling167. Structural sex differences are also apparent in the NAc. For example, Forlano and Woolley discovered that distal dendritic spine density in NAc MSNs is higher in female rats168, indicating that excitatory synaptic connections in the NAc likely differ between the sexes, which may in part explain sex differences observed in drug addiction and depression. Indeed, Wissman et al demonstrated sex-dependent variability in the morphology and distribution of dendritic spine synapses in NAc169. Brancato et al have also recently demonstrated sex-specific effects on glutamatergic synapses in the NAc170, which are implicated in the development of mood disorders171,172. This group investigated pre- and postsynaptic plasticity of glutamatergic synapses in the NAc shell in male and female mice by evaluating the expression of VGLUT transporters, which essentially represent vesicles in glutamatergic nerve 38 terminals. They found an interaction between stress and sex in the expression of VGLUT1 in the NAc shell, with a significant decrease of VGLUT1 labeling only in female mice exposed to SCVS, but no post-stress sex differences in PSD95, a postsynaptic marker, nor sex differences in MSN spine density or morphology. Overall, this study suggests sex differences in presynaptic glutamate neurotransmission in the NAc shell, which supports aberrations of excitatory signaling in this brain region as potential mediator of disparities in MDD prevalence in men and women. Excitatory neurotransmission to NAc originating in the vHPC is discussed in the following section, with focus on this circuit’s contribution to stress outcomes and depression. HPC to NAc connectivity As mentioned above, the NAc is a key mediator of motivated behaviors, and is thought to influence the seeking of reward by integrating both dopaminergic and glutamatergic inputs. The dopaminergic input to the NAc via the mesolimbic pathway mediates reinforcement of incentive salience (i.e. “wanting”) of a reward173,174, while the glutamatergic input to NAc and midbrain dopamine neurons encodes information regarding reward-related environmental stimuli175. The excitatory input into the NAc specifically is thought to mediate the integration of context and cues with goal-directed behavioral output176. Dopaminergic and glutamatergic signaling in the NAc are thought to coincide to affect synaptic plasticity, and this plasticity allows for processing of environmental information to prioritize the salience of some cues over others (i.e. in the case of drug reward processing)177,178. Dysfunction of the NAc’s capacity to properly integrate these inputs, then, leads to perturbations in reward processing that can result in 39 a change in motivated behavior. Accordingly, an increase in the NAc’s drive of motivated behavior may lead to addiction, and a weakening of this drive may lead to anhedonic behaviors such as those found in MDD and other mood disorders. In a functional magnetic resonance imaging (fMRI) study, Kahn and Shohamy demonstrated low-frequency changes in signal that correlate to resting-state functional connectivity between the vHPC, NAc, and VTA in humans179. In rodents, Britt et al elegantly profiled three different glutamatergic projections to the NAc: those from BLA, PFC, and vHPC180. This group demonstrated that projections from the vHPC are concentrated mainly at the medial NAc shell, and that projections from the BLA and PFC are mixed between the shell and the core in their destinations. Furthermore, their retrograde labeling studies indicated that the majority of all cells projecting to the medial shell are located in the vHPC, with fewer also coming from the BLA and PFC. Next, they characterized pathway-specific NAc strength of synapses originating at each of these areas using optogenetic stimulation, and demonstrated that the amplitude of optically evoked EPSCs are largest after stimulation of vHPC input to the NAc shell. Ventral HPC to NAc synapses were also shown to be unique in their NMDAR-mediated passage of inward current, possibly due to NMDARs of these synapses being less sensitive to blockade by Mg2+, a known phenomenon at thalamocortical synapses181. The authors suggested that this may be the primary mechanism of the exaggerated vHPC-NAc EPSC, and posit that this could also be the mechanism by which this pathway is able to uniquely induce a stable depolarized state in NAc MSNs, increasing their likelihood of activity182. This phenomenon was first described by O’Donnell and Grace, who showed that this depolarized state induced by vHPC input into the NAc was necessary for the induction of 40 spike firing by inputs from the PFC (i.e., vHPC inputs are capable of “gating” the response of the NAc to other excitatory inputs). Britt et al in the same study also found that cocaine selectively potentiates synaptic responses at vHPC-NAc inputs at the medial NAc shell, as measured by an increase in AMPA to NMDA ratio. This was accompanied by attenuation of cocaine-induced locomotion with vHPC-NAc optical inhibition and an increase with optical stimulation. This study highlights the importance of vHPC-NAc input in integration of dopaminergic reward signaling, as well as the unique properties of vHPC- NAc synapses at the medial NAc shell. Bagot et al in 2015 made crucial contributions to our understanding of the function of vHPC-NAc projections with respect to stress outcomes2. In male mice, this group demonstrated opposite regulation of vHPC-NAc and mPFC-NAc activity by CSDS by first examining immediate early gene (IEG) expression in vHPC and mPFC. Expression of the IEGs Arc and Egr1 was reduced in vHPC of resilient mice, but the expression of Arc was reduced in mPFC of susceptible mice. Next, optically induced activation of glutamatergic projections from vHPC and mPFC to NAc was accomplished using glutamatergic neuron- specific expression of channelrhodopsin (ChR2) in either region and illuminating projection nerve terminals in NAc. Paired pulse ratio (PPR) of EPSCs was assessed to examine the vesicle release probabilities suggestive of alterations in presynaptic glutamate release, and PPR was elevated in vHPC-NAc in resilient mice but decreased in mPFC-NAc in resilient mice. These findings suggest that glutamate release from vHPC- NAc projections is reduced in resilient mice, but oppositely, glutamate release from mPFC-NAc projections is increased in resilient mice. Next, the group used optically- induced LTD at vHPC-NAc synapses to investigate whether weakening the connectivity 41 between these two regions would affect stress outcomes. Following CSDS, LTD was induced shortly before assessing SI ratio, and the weakening of vHPC-NAc connectivity with this strategy resulted in an increase in social interaction to levels observed in resilient mice. LTD induced at mPFC-NAc inputs did not affect social interaction. Acute optical stimulation of vHPC-NAc projections, however, reduced SI ratio following CSDS and increased immobility in the FST, thus promoting susceptibility to stress. The opposite was found in acute stimulation of mPFC-NAc projections, which increased SI ratio following CSDS in a pro-resilient fashion. This seminal study, while only examining male mice, highlights the importance of glutamatergic inputs to the NAc in regulating susceptibility to stress, and demonstrates that increasing vHPC-NAc activity is pro-depressive in male mice. In another recent study of the vHPC-NAc connection, LeGates et al demonstrated that these synapses display activity-dependent plasticity, and that their strength directly regulates reward behavior183. This elegant study characterized the nature of vHPC-NAc synapses, and showed similar LTP induction at D1-MSNs and D2-MSNs by electrical and light-induced optogenetic stimulation of vHPC projections, and that the plasticity was accomplished through postsynaptic mechanisms (i.e., there was no change in paired- pulse ratio following high-frequency stimulation to induce LTP). The induction of LTP was blocked by AP5 (a selective NMDAR antagonist), BAPTA (a Ca2+ chelator), KN62 (a CaMKII inhibitor), and Rp-cAMPs (a blocker of PKA activation by cAMP), indicating that the induction of LTP at vHPC-NAc synapses is NMDAR-, Ca2+-, CaMKII- and PKA- dependent, much like the classical LTP induced at CA3-CA1 synapses in the HPC discussed above184. Furthermore, LTP induction was not blocked by D1R nor D2R 42 antagonists, suggesting that plasticity at vHPC-NAc synapses occurs in a dopamine- independent fashion. With respect to motivated behavior, high frequency, but not low frequency, optical stimulation of vHPC-NAc synapses was sufficient to induce CPP of the stimulation-paired chamber. In contrast, interruption of vHPC-NAc plasticity using optical inhibition was sufficient to abolish CPP of a chamber containing the natural reward of a social target. Pertinent to the current work was this group’s investigation of vHPC-NAc plasticity with respect to stress: chronic multimodal stress (CMS) was sufficient to weaken synapses at D1R-MSNs but not D2R-MSNs. This weakening of synapses by stress was accompanied by a deficit in LTP induction at D1R-MSN synapses, as well as an abolishment of the CPP induced by optical high-frequency stimulation mentioned above. The link between stress-induced alterations at vHPC-NAc synapses and MDD was further strengthened by the mitigation of these alterations by the antidepressant fluoxetine. Chronic, but not acute, fluoxetine was sufficient to restore pre-stress sucrose preference levels, as well as the optically-induced CPP of the light stimulation-paired chamber. Furthermore, chronic fluoxetine rescued the decrease in synaptic strength as measured by AMPA to NMDA ratio caused by chronic stress in D1R-MSNs, and accordingly was sufficient to ameliorate the accompanying deficit in LTP induction. This study is critical in our understanding of the effects of chronic stress on vHPC-NAc synapses and the plasticity thereof, and suggests the key role of the connection between these two regions in mediating reward- and stress-related behaviors. 43 Ion Channels of the Hippocampus As alluded to above, changes in excitability and activity of vHPC-NAc neurons, driven by changes in current passage by ion channels, is implicated in stress responses and depression pathogenesis. Current in all excitable cells, including neurons, exists in the form of ions that pass across the cell membrane with the aid of ion channels. Ion channels are specialized proteins that, according to their genetic coding and post- translational modification, can comprise specialized structures to drive specific ion permeabilities and cellular excitability states. The heterogeneity of ion channels conferred through subunit diversity allows different neuronal subtypes, for example those in different hippocampal regions, to have specific membrane properties and excitabilities that are malleable through changes in gene transcription, translation, and modification. Pharmacology studies paired with the study of single ion channel types in cell culture have allowed for the identification of distinct currents generated by specific ion channels; many of the major ion channel types found in the hippocampus and the currents they facilitate are discussed below, followed by an overview of intrinsic excitability and the plasticity thereof. Leak Channels At rest, neuronal cell membranes are more permeable to potassium (K+) than other ions, which causes the cell’s resting membrane potential (RMP) to lie near potassium’s equilibrium potential. Resting membrane potential is established by the Goldman- Hodgkin-Katz relationship, which takes into account charges, concentrations, and membrane permeability of all relevant ions, including potassium185. The basis for this high 44 permeability to potassium is the presence of leak channels, which are constitutively open, potassium-selective channels of the KCNK gene family186. KCNK family channels comprise two α subunits that, across the family, differ in their biophysical and pharmacological properties. The α subunits each contain four transmembrane segments having two pore loops. Accordingly, density of K+ leak channels and differential expression of subunits can dramatically affect neuronal intrinsic excitability via modulation of RMP. For example, TASK-3 subunit expression is higher in the excitable CA1 pyramidal neurons than in the less excitable stratum oriens interneurons187. In CA1 neurons and many others, the density of K+ leak channels directly regulates the RMP and membrane resistance. Potassium Channels Potassium channels comprise three major classes: tandem pore domain, calcium- activated, and voltage-gated. All are tetramers that form a pore-loop structure that allows for potassium-selective permeability. The tandem pore domain potassium channels, or “leak” channels, were discussed above. Calcium-activated potassium channels in neurons, such as the pyramidal cells of the hippocampus, are activated by calcium entry through voltage-gated calcium channels. These channels are involved in the regulation of neuronal excitability, particularly through the mechanisms of interspike interval maintenance and spike frequency adaptation188. Two types of calcium-activated potassium channels in the HPC are large-conductance (BK channels) or small- conductance (SK) channels. Activation of BK channels underlies repolarization in HPC pyramidal cells, as well as formation of fast afterhyperpolarization (AHP)189. SK channel 45 activation causes formation of the slow AHP following AP bursts190, which limits the frequency of repeated APs (spike frequency adaptation). Adaptation of this kind is a mechanism by which a neuron is protected from the effects of repetitive activity and contributes to normal neuronal function and neurotransmission. Voltage-gated potassium channels comprise four α subunits, a pore, and a voltage sensing domain. Subunit variation and combination give rise to a wide range of channels differing in voltage dependence, pharmacology, and kinetic and gating properties. KCNA, B, C, and D genes code for the canonical voltage-gated potassium channels KV1, 2, 3, and 4. Localization of different voltage-gated potassium channels in different neuronal compartments, e.g. axons, soma, and dendrites, confers distinct membrane electrical properties to each part of the neuron191. The transiently active (A-type) channels are a subset of voltage-gated potassium channels that regulate the passage of transient outward current subject to rapid inactivation. In hippocampal CA1 pyramidal neurons, A-type channels are predominantly found in dendrites and are responsible for preventing AP initiation and backpropagation of APs, as well as the reduction of the magnitude of synaptic events192 and synaptic integration. This influence of A-type channels on synaptic integration and long-term potentiation is characterized by the inactivation of the channel. When an EPSP is present in a CA1 neuron, the dendritic membrane is depolarized and A-type channels are inactivated. If these channels are unable to diminish the generation of a dendritic spike, the summation of the EPSP and the dendritic spike will result in an increased calcium influx, which can affect many downstream processes such as long-term potentiation191. A-type channels are also present in presynaptic neurons, where they may act to filter low- 46 frequency signals (by which they are only transiently inactivated). Higher-frequency signals result in longer lasting A-type channel inactivation, resulting in AP prolongation accompanied by increased calcium influx and neurotransmitter release193. In the context of stress-related conditions such as depression, there is also evidence that corticotropin- releasing hormone (CRH, released in response to stress) increases CA1 pyramidal cell excitability via the modulation of A-type and delayed rectifier potassium channels194, the channels primarily responsible for the repolarization of the neuronal membrane following an AP. Sodium channels Throughout the body, sodium (Na+) channels exist as a single α subunit containing four repeat homologous domains that often associate with other proteins (sometimes referred to as β subunits). These domains comprise six transmembrane segments (S1- S6) and a pore-forming region. In voltage-gated Na+ channels (the predominant class in neurons responsible for the rising phase of APs), the S4 segment of the channel acts as the voltage sensor by way of positively-charged amino acids every third position in the primary structure of the channel195. When a neuronal membrane is depolarized (e.g. by excitatory input activity), Na+ channels activate quickly by the movement of the S4 segment to the extracellular side of the neuron, which opens the channel’s pore. The S5 and S6 segments form the narrow portion of the channel, allowing for its selectivity for Na+. Na+ channels can exist in resting (closed), active (open), or inactive states. The transition between states are referred to as activation, inactivation, and recovery. Activation represents the transition between resting (closed) and active (open) states, 47 inactivation between open and inactivated states, and recovery between inactivated and resting states. Inactivation is accomplished by the segment of the channel linking the third and fourth domains, and essentially plugs the pore following prolonged activation (i.e. when enough Na+ has entered the neuron to increase the membrane potential at which the “plug” moves to the inactivating conformation). This ceases the rise of the membrane potential, and the channel remains inactivated for a period of time known as the refractory period. The decrease in voltage (the falling phase of the AP) repolarizes the membrane, and sodium channels are “deinactivated” back to the resting (closed, but not inactivated) state and are able to participate in the generation of another AP. The role of the sodium channel in generating APs makes obvious its connection to cellular excitability, but there is scarce research available regarding sodium channel changes in the context of mood disorders. One interesting association between sodium channels and mood disorder pathogenesis is the observation that lamotrigine, an anticonvulsant drug that blocks sodium channels and subsequently blocks glutamate release196, is effective in the treatment of bipolar disorder197. This effectiveness is thought to in part be due to the prevention of epilepiform burst activity in the hippocampus. Other anticonvulsants, such as carbamazepine and valproate, are also used to treat bipolar disorder, but primarily affect the manic stage, and have been associated with exacerbation of the depressive stage. Lamotrigine, on the other hand, is more effective at treating the depressive stages of bipolar disorder, and, while not mitigating manic symptoms, has been shown to delay the onset of manic episodes198. The overall effectiveness of lamotrigine in treating bipolar disorder, however, is contested, with some studies demonstrating that the drug has little clinical efficacy199. Further research into the 48 role of sodium channels in the pathogenesis of mood disorders, particularly with respect to their effects on cellular excitability in reward-related circuits such as vHPC-NAc, is needed to determine how these channels may be targeted for novel antidepressant treatments. Calcium channels The maintenance of intracellular calcium (Ca2+) is important for normal neuronal function. Neurons possess various mechanisms to maintain low cytoplasmic Ca2+ concentrations, including Ca2+-sequestration in organelles and the employment of Ca2+ buffering molecules. The equilibrium potential of Ca2+ is very high (»140 mV), and as such, Ca2+ is the source of depolarizing current. Ca2+ is also involved in a variety of other cellular processes, including activation of Ca2+-activated K+ channels, initiation of second messenger signaling (e.g. in LTP, discussed above), apoptosis, and stimulating the release of neurotransmitters. The release of neurotransmitters at the synapse is triggered when an AP reaches a nerve terminal, causing the elevation of intracellular Ca2+ via the opening of voltage-gated Ca2+ channels. These channels are formed as a complex of α1, α2d, β1-4 and γ subunits. The ion conducting pore is formed by the α1 subunit, and the other subunits of the complex participate in the other functions of the channel such as gating200. Ca2+ stimulates the release of neurotransmitters by facilitating the interaction of synaptotagmins, phospholipids, and SNARE proteins, which mechanically influence the fusion of neurotransmitter vesicles with the presynaptic membrane201. Three families of voltage-gated Ca2+ channels have been described (Cav1, 2, and 3), with the α subunits of each being coded by various CACN genes202. Cav1 and Cav2 families comprise the high- 49 voltage activated (HVA) channels, with the Cav1 family inactivating slowly (500 ms) and the Cav2 family inactivating quickly (50 ms) at positive membrane potentials. The current produced by the Cav1 channels is the L-type Ca2+ current, which is blocked by dihydropyridines. Cav2 channels are more diverse based on alternative splicing, and are blocked by a variety of toxins specific to their subfamilies. Cav2 channels produce the P- type, Q-type, N-type, and R-type currents. Cav3 channels, unlike other voltage-gated Ca2+ channels, activate at hyperpolarized potentials and produce the T-type current. Disrupted intracellular calcium homeostasis has been implicated in a variety of psychiatric disorders, including bipolar disorder and MDD. For instance, GWAS studies have uncovered an association of a variation of the 1C α-subunit of the L-type voltage- gated calcium channel encoded by the gene CACNA1C with bipolar disorder and unipolar depression (i.e., MDD)203. Isoform-specific knockout studies have suggested that Cav1.2 may contribute to spatial memory formation, and that Cav1.3 contributes to consolidation of fear memory, and both may be involved in the expression of drug sensitization in rodent models204. Further support for the contribution of intracellular calcium homeostasis in mood disorder pathogenesis is provided by the actions of the NMDAR antagonist ketamine. NMDARs (described above) are antagonized by ketamine to prevent excessive Ca2+ influx, and modulate the firing rate of glutamatergic neurons205. In rodent models of depression, ketamine has potent antidepressant actions206, with ventral hippocampal activity being implicated as a key mediator of the drug’s antidepressant efficacy207. In addition, ketamine induces ΔFosB in vHPC CA3 neurons, and this is critical for some aspects of its antidepressant function207. Voltage-gated calcium channel blockers also 50 display antidepressant effects. For example, the dihydropyridine agent nifedipine, which blocks the aforementioned L-type calcium current, reduces immobility time in the forced swim test and the tail suspension test in mice208. However, the nondihydropyridine calcium channel blockers diltiazem and verapamil increase immobility time in the forced swim test and block the effects of tricyclic and atypical antidepressants209. These studies together may muddy the interpretation of calcium’s role in depression and related behaviors in rodents, but calcium homeostasis and calcium-mediated currents are clearly implicated in mood disorder pathogenesis. HCN Channels Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are nonselective voltage-gated cation channels found in excitable cells throughout the body, particularly in the heart and brain. They consist of four subunits of six membrane- spanning domains each (S1-6), with S4 acting as the voltage sensor, S5 and 6 creating the pore region, and the C-terminus acting as the cyclic nucleotide binding domain. Cyclic nucleotides (cAMP, cGMP, and cCMP) bind to the C-terminus to lower the threshold potential of the channel. In the brain, HCN channels influence neuronal response to synaptic input, and are expressed on dendrites in the HPC210. HCN channels mediate the Ih non-selective cation current, are activated in states of neuronal hyperpolarization, and are critical regulators of neuronal excitability211. Trafficking of HCN channels to dendrites in the CA1 is thought to correspond to neuronal activity, with increased cell surface expression of HCN channels and subsequent increases in Ih current in response to increased glutamatergic input. This phenomenon could represent a fast response of 51 neuronal intrinsic plasticity in response to a change in the activity of inputs. This consideration is especially interesting due to the HCN channel’s unique open state at hyperpolarized potentials less than -40 mV, and the observation that these channels do not inactivate, so they are often active when the cell is at rest212. These properties allow HCN channels to stabilize neuronal excitability by reacting to excitatory and inhibitory inputs. HCN channels have gained recent attention as possible mediators of depressive behavior213. In a transgenic mouse lines with various knockouts (HCN1, HCN2, and auxiliary subunit TRIP8b) that cause attenuation of the Ih current in HPC pyramidal cells, mice displayed resilience to a wide range of tests measuring behavioral dispair214. As HCN channels are expressed in CA1 pyramidal cell dendrites with increasing density with distance from the soma, they are thought to act to integrate inputs and modulate the cell’s response by attenuating Ca2+ signaling215,216. In this sense, they function to regulate intrinsic excitability independent of synaptic mechanisms and provide a homeostatic feedback mechanism that prevents CA1 hyperexcitability217. This regulation by HCN channels of CA1 excitability may play a part in modulating vHPC-NAc circuit activity, thereby affecting stress outcomes and development of depressive behaviors. Hormone Signaling in the Brain – Effects on Stress and Depression Hormones are chemical messengers that are secreted by endocrine glands and travel to a distant effector sites via the bloodstream to enact regulation of physiological processes. The primary hormones of the ovary, estrogen and progesterone, are synthesized and secreted to affect many actions throughout the body, including in the 52 brain. Release of these hormones by the ovary is regulated by the hypothalamus and pituitary via various feedback loops of the HPA axis. Neurons of the hypothalamus secrete gonadotropin-releasing hormone (GnRH), which signals the pituitary to secrete follicle-stimulating hormone (FSH). FSH signals the maturation of the ovarian follicle, which releases estrogens (e.g. estradiol) and inhibin, which feed back to the hypothalamus and pituitary to inhibit further FSH secretion. Estrogens also positively feed back to the hypothalamus, resulting in pulses of GnRH secretion, stimulating luteinizing hormone (LH) secretion from the pituitary. This LH release stimulates ovulation, and the resultant ruptured follicle (corpus luteum) secretes progesterone. Progesterone then feeds back to the hypothalamus and pituitary to inhibit the secretion of GnRH, FSH, and LH. The corpus luteum matures, progesterone secretion decreases, and this negative feedback diminishes to allow for the initiation of a new reproductive cycle218. Estrogen signaling in the brain is primarily accomplished via intracellular estrogen receptors (ERs), which, when bound to estrogen, can translocate to the cell nucleus and bind to hormone response elements in target genes to affect their transcription; these events typically occur over hours219. Intracellular ERs are most densely expressed in the BNST, ventromedial nucleus of the hypothalamus, amygdala, various midbrain structures, and the pituitary220, and more sparsely expressed in the HPC and cortex221. Progesterone receptors (PRs) are generally expressed in the same pattern as ERs222. In situ hybridization and immunocytochemical studies suggest that most cells in the HPC that express intracellular ERs are interneurons223,224. Progesterone, estrogen, and their derivatives can also have non-genomic effects on neuronal physiology, including on cellular excitability225. Progesterone metabolites 53 have been demonstrated to allosterically bind GABAA receptors in the HPC, causing potentiated chloride conductance and dampened excitability226. Estrogens (primarily estradiol) can also act rapidly in a non-genomic fashion to affect excitability of CA1 pyramidal cells. For example, Wong and Moss demonstrated prolonged EPSPs in CA1 pyramidal cells of ovariectomized female rats that were “primed” with estrogen injections for two days prior to acute slice preparation and recording227. This group also demonstrated that EPSCs were potentiated via the actions of second messenger cascades (e.g. cAMP signaling)228,229. These non-genomic effects make ovarian-derived hormones, especially estradiol, interesting potential mediators of sex differences in stress outcomes, particularly in the context of circuit-level excitability. Estrogens also modulate synaptic plasticity in the hippocampus. Warren et al demonstrated that LTP is enhanced during proestrus in rats230, and Cordoba Montoya and Carrer showed that LTP induction in CA1 is facilitated by estradiol treatment in ovariectomized rats231. Furthermore, Joels and Karst showed that the priming of ovariectomized rats with a combined treatment of estrogen and progesterone enhances voltage-gated calcium channel conductances in CA1 pyramidal neurons232. These studies complement well the finding by Gould et al that estradiol has a potent effect on CA1 dendritic spine density233, and, taken together, highlight important effects of estrogens on CA1 excitatory neurotransmission. Whether through transcriptional or non- genomic effects, ovarian hormones clearly play a role in regulating HPC physiology through modulation of excitability and synaptic transmission. As discussed previously, animal models of stress have revealed changes in the structure and function of the HPC234. Male and female rodents react differently to stress 54 in HPC-dependent learning tasks such as Morris water maze, with the most robust differences being identified when females are in proestrus235. Furthermore, studies of ovariectomized rats have shown that stress enhances performance in the radial arm maze (another HPC-dependent measurement of spatial memory and learning) in animals with estrogen replacement, but not in those without236. Stress effects on CA1 dendritic spine density (discussed previously) also appear to be estrogen-dependent: a single stressor can induce an increase in spine density in male rats but a decrease in female rats in proestrus141, when spine density is usually the highest for females139. Estrogen and stress can interact to cause sex differences in other regions as well. For example, the amygdala-dependent process of extinction of a conditioned fear response is impaired in estrogen-treated female rats237. There is also evidence that estrogen can enhance stress effects in the mPFC, as proestrus or estrogen-replaced ovariectomized female rats demonstrated more deficiencies in mPFC-dependent working memory tasks238. Overall, estrogens have a clear effect on stress outcomes and brain function, and are likely key mediators of sex differences in depression. Testosterone has also been implicated in mediating stress outcomes and depression development. In humans, hypogonadism leading to lower testosterone levels in men is associated with a higher prevalence of MDD239,240, and treatment with testosterone has been shown to alleviate depression symptoms in hypogonadal240 and eugonadal men241,242. There is also evidence that low-dose testosterone treatment can improve depression scores and augment the effectiveness of traditional depression pharmacotherapies in treatment-resistant women243. 55 Testosterone is metabolized to a variety of different effectors. For example, 5α- reductase converts testosterone into dihydrotestosterone (DHT), which is a more potent agonist at ARs and has a longer half-life than testosterone. DHT can then be converted by aldo-keto reductases to 3α-diol or 3β-diol. The former of these metabolites has a low affinity for AR but acts in the brain as an agonist at the GABAA receptor244, and the latter acts primarily through ERβ in the brain to affect gene transcription245. Like estrogens, androgens can also act through genomic and non-genomic mechanisms. Androgens also diffuse across the cellular membrane to interact with intracellular receptors and diffuse into the nucleus to affect gene transcription via androgen receptor binding to DNA at hormone response elements246. Androgens and estrogens, as alluded to above, can also cause more rapid, non-genomic effects through activation of cell membrane-bound receptors247. The rapid effects of androgens in the brain include modulation of neuronal excitability and plasticity. There is evidence that androgens acutely decrease neuronal excitability248, but some studies indicate increased excitability with acute application of testosterone by measure of field potentials249. These studies, however, assess general excitability in various brain regions, including the HPC CA1, and do not address specific subpopulations of neurons (e.g. those in the vHPC-NAc circuit). The current work will help to clarify hormonal effects on the physiology of the vHPC-NAc circuit, and the observed effects of testosterone on the HPC are discussed further below. Testosterone has potent effects on anxiety- and depression-related behaviors in animal models. Orchidectomy of adult male rodents, for example, causes an increase in anxiety behaviors in marble burying, EPM and open field tests250-252, as well as behavioral despair in the forced swim test following chronic mild unpredictable stress (CMS), which 56 was accompanied by a decrease in hippocampal cell proliferation and neurogenesis253. Testosterone relieves these anxiety behaviors in intact adult male rodents254 as well as in the orchidectomized adult males of studies cited above. Testosterone enhances hippocampal neurogenesis induced by the antidepressant imipramine255. Particularly relevant to the current work, testosterone prevents anhedonia in aged adult male rats exposed to CMS if administered weeks before stress exposure256. Interestingly, this mitigation of anhedonia as measured by sucrose preference was not observed if testosterone was administered following stress exposure, suggesting the necessity of testosterone’s actions before and during stress in male resilience. Further investigation of testosterone’s actions with respect to behavior following stress revealed that the prevention of anhedonia and behavioral despair by testosterone was likely mediated by aromatization to estradiol, as gonadectomized rats treated with DHT (insensitive to aromatase) still exhibited these depressive behaviors, but those treated with estradiol did not257. However, other studies did implicate androgen metabolites in the mediation of stress resilience, as treatment with DHT or 3α-diol in intact adult male rodents did indeed decrease behavioral despair in the forced swim test258. The effects of testosterone treatment in female rodents with respect to stress outcomes is even less clear. Testosterone, DHT, or 3α-diol treatment in intact female mice reduces anxiety behaviors in open field and EPM tests259 and depression behavior in the forced swim test258. In contrast, however, other groups have observed no amelioration of anxiety (EPM) or depression (sucrose preference, NSF) behaviors following administration of testosterone in ovariectomized female rats255. The difference between these observations may lie in the ovariectomy, as the studies demonstrating 57 benefits of testosterone treatment in female animals did not include gonad removal. This could suggest that higher doses of testosterone are needed to induce anxiolytic or antidepressant effects in females, as maintaining the ovaries would accomplish this. As alluded to above, the protective effects of androgens against stress-induced changes in behavior may in part be mediated through the HPC. Indeed, the volume of the hippocampal formation, its neuronal soma sizes, and extent of dendritic branching increase with androgen treatment in the perinatal period260,261. The protective effects of neonatal androgens are associated with HPC neurogenesis and spine density, as male pups treated with the AR antagonist flutamide demonstrated depressive behaviors in forced swim and sucrose preference tests that correlated with decreased microtubule associated protein-2 (MAP-2) labeling in the CA1 and DG and decreased spine density in CA1262. Additionally, testosterone in orchidectomized adult male rodents may offer protection against oxidative damage and consequent changes in morphology263, and its metabolites in intact female adults appears to reduce HPC cell death induced by adrenalectomy264. Testosterone and DHT also reverse orchidectomy-induced reduction of spine synapse density in males (independently of ER-mediated effects), while offering partial protection to females with contribution of the aromatization to estrogens265. These morphological changes in response to steroid hormones may relate to HPC neuronal excitability. Teyler et al observed field potential differences in HPC slices from rat brains in response to treatment with testosterone and estradiol: male rat slices showed increased excitability in response to estradiol but not testosterone, and female rat slices showed increased excitability in response to testosterone in diestrus but decreased excitability in proestrus266. Thus, there is a pressing need to uncover additional effects of 58 steroid hormone manipulation on HPC physiology and effects on stress outcomes. The work described in this thesis addresses this need by providing novel cellular and molecular circuit-based mechanisms for sex differences in vulnerability to depression. 59 II. MATERIALS AND METHODS Animals All experiments were approved by the Institutional Animal Care and Use Committee (IACUC) at Michigan State University and performed in accordance with AAALAC and NIH guidelines. All behavioral experiments were performed on group housed C57BL6/J 8-12 week old male and female mice purchased from Jackson Labs at 8 weeks of age, or transgenic mice bred in our colony (Cre-inducible RosaeGFP-L10a). All electrophysiology experiments were performed on 9-13 week old male and female RosaeGFP-L10a transgenic mice. The floxed FosB mouse strain (FosBfl/fl)267 was a gift from the laboratory of Dr. Eric Nestler at the Icahn School of Medicine at Mount Sinai, and the Rosa26eGFP-L10a strain268 was a gift from the laboratory of Dr. Gina Leinninger at Michigan State University. Unless otherwise stated, all mice were group housed in a 12:12 h light/dark cycle with ad libitum food and water. RosaeGFP-L10a genotyping primers: Mutant forward: 5’ – TCTACAAATGTGGTAGATCCAGGC – 3’ Wild type forward: 5’ – GAGGGGAGTGTTGCAATACC – 3 Common reverse: 5’ – CAGATGACTACCTATCCTCCC – 3’ FosBfl/fl genotyping primers: FB loxPu sequence: 5’ – GCTGAAGGAGATGGGTAACAG – 3’ LIPz sequence: 5’ – AAGCCTGGTGTGATGGTGA – 3’ LNEo1 sequence: 5’ – AGAGCGAGGGAAGCGTCTACCTA – 3’ 60 Gonadectomies Eight-week old male and female mice were anesthetized with a mixture of ketamine and xylazine 0.9/0.1 mg/kg and either testes or ovaries were removed according to previously published protocols269. In electrophysiology experiments, gonadectomy occurred one week following intracranial injection surgery. In hormone replacement studies, either empty 1.5 cm Silastic pellets or filled with 0.6 cm of testosterone (Sigma- Aldrich; 1001774366) were placed subcutaneously between the scapulae at the time of ovariectomy. Mice then either recovered for 10 days (electrophysiology) or 28 days (behavior). Intracranial Injections & Viral Vectors Male and female mice were anesthetized with a mixture of ketamine and xylazine 0.9/0.1 mg/kg and injected with 0.5uL/hemisphere of virus with Hamilton syringes. Herpes simplex retrograde Cre viral vector (HSV-heF1α-Cre) was purchased from Massachusetts General Hospital Viral Core, and adeno-associated viral vectors encoding DREADDs were purchased from Addgene (Watertown, Massachusetts, USA). For ΔJunD experiments, viral vectors (AAV2-CMV-GFP or AAV2-CMV-ΔJunD-GFP) were bilaterally infused at two dHPC sites (10° angle; Bregma -2.2 AP, ±2.0 ML, -2.1 and -1.9; 0.3 µL per DV site) or two vHPC sites (5° angle; Bregma -3.6 AP, ±3.2 ML, -4.8 & -3.0 DV; 0.3 µL per DV site). Two coordinates were used to ensure viral transduction throughout each structure. Experimental procedures began at least 4 weeks after surgery. For vHPC-NAc induction of L10-GFP expression and vHPC-NAc FosB KO (where applicable), the retrograde Cre viral vector (HSV-hEF1α-Cre; 0.5 µL each hemisphere) was injected 61 bilaterally into NAc (Angle: 10° AP:+1.6, ML:±1.5, DV:-4.4) or BLA (Angle: 0° AP:-1.3, ML:+3.4, DV:-4.5) of RosaeGFP-L10a mice. For electrophysiology of ΔFosB-expressing vHPC neurons, viral vectors (HSV-IE4/5-ΔFosB-CMV-GFP; or HSV-CMV-GFP as control; 0.5 µL each hemisphere) were bilaterally injected into vHPC (Angle: 3° AP:-3.4, ±3.2, DV:-4.8) and experimental procedures commenced 2-4 d following surgery. For DREADD experiments, either rAAV2-hSyn-DIO-hM3D-mCherry (44361-AAV2 lot v30428; 0.5 µL) or rAAV2-hSyn-DIO-hM4D-mCherry (44362-AAV2 lot v30430; 0.5 µL) was injected bilaterally into vHPC (Angle: 3° AP:-3.4, ML:±3.2, DV:-4.8) and HSV-hEF1α- Cre (0.5 µL) was injected bilaterally into NAc (Angle: 10° AP:+1.6, ML:±1.5, DV:-4.4). Electrophysiology and immunohistochemistry experimental procedures in RosaeGFP-L10a mice began at least three weeks following HSV-hEF1α-Cre injection. In DREADD SCVS experiments, stress procedures began at least three weeks following intracranial injection of rAAV2-hSyn-DIO-hM3D-mCherry (male mice) or rAAV2-hSyn-DIO-hM4D-mCherry (female mice) and HSV-hEF1α-Cre. For dual virus CRISPR/Cas9 FosB gene silencing experiments, Cas9-expressing retrograde vector (HSV-hEf1a-LS1L-myc-Cas9; 0.5 uL) was infused into NAc (+1.6 AP, ±1.5 ML, -4.4 DV relative to bregma, 10° angle) or BLA (-1.6 AP, ±3.4 ML, -4.5 DV relative to bregma, 0° angle). After 3 wks, control viral vector (HSV-IE4/5-TB-eYFP-CMV-IRES- Cre) or FosB gRNA (HSV-IE4/5-TB-FosB gRNA-CMV-eYFP-IRES-CRE) were infused into the ventral CA1 region of vHPC (vCA1; -3.4 AP, ±3.2 ML, -4.8 DV relative to bregma, 3° angle; 0.5 uL). Experimental procedures commenced at least 2 weeks following vHPC surgeries. 62 Subchronic Variable Stress SCVS was performed according to previously published protocols41,170. Briefly, group-housed mice were exposed to a stressor every day for six days under white light conditions. Stressors were administered daily in the following order on consecutive days: group foot shock of 10 mice with 100 random foot shocks over an hour at 0.45mA (Shock Box H13-15, Coulbourn Instruments, Holliston, MA), one hour tail suspension, and one hour restraint stress; sequence repeated once for six days total stress. Restraint tubes were manufactured in-house by drilling air holes in 50mL falcon tubes. Chronic social defeat stress (CSDS) CSDS was performed as previously described38,270-272. In brief, mice were placed into the home cage of an aggressive retired breeder CD1 mouse containing a perforated plexiglass divider placed between the walls of the cage. The experimental mice were allowed to physically interact with the CD1 for 10 min. Following the aggressive encounter, the mice were placed into the other side of the divider from the CD1 aggressor mouse allowing sensory, but not physical, contact for 24 hours. This protocol was repeated daily for 10 d with a new aggressor every day. Behavioral testing began the day following the final day of stress. DREADD activation Clozapine N-oxide (CNO; Fischer Scientific NC1044836) was diluted in vehicle solution: 5%DMSO and 95% 0.9% saline. CNO or vehicle was administered i.p. 63 throughout DREADD SCVS experiments at 0.3mg/kg daily an hour prior to the stressor or behavior. CRISPR Guide RNA design and testing Guide RNAs (gRNAs) targeting exon 2 of the FosB gene were designed using e- CRISP software (www.e-CRISP.org). The top four sequences were: gRNA1: TACACCGGGAGCCGGAGTCG gRNA2: TTACGATCTAAAACTTACCT gRNA3: TCAACATCCGCTAAGGAAGA gRNA4: CCGTCTTCCTTAGCGGATGT This gRNA was most effective and was selected for all in vivo work described in the current manuscript; also referred to as AJR4 as it was the fourth gRNA produced for our lab). Each gRNA was tested by transfection in a mammalian expression plasmid also containing Cas9. Briefly, Neuro2a cells (N2a, American Type Culture Collection) were cultured in EMEM (ATCC) supplemented with 10% heat-inactivated fetal bovine serum (ATCC) in a 5% CO2 humidified atmosphere at 37° C. Cells were plated into 12-well plates, and 24 h later (when cells were ~30% confluent) cells were transiently transfected using Effectene (Qiagen) with a total of 200 ng DNA per well. Cells were transfected with empty vector, Cas9 alone, or Cas9 with a gRNA to be tested. Cells were then serum starved for 24 h, then re-fed for 4 h to induce FosB gene expression. Cells were pelleted, samples were run on gradient polyacrylamide gels and transferred to PVDF membranes, and Western blot was performed using rabbit anti-FosB antibody (2251; 1:500; Cell 64 Signaling) and HRP conjugated anti-rabbit secondary at (PI-1000; 1:40,000; Vector). Signal was detected on film and quantified using ImageJ software. Behavior All behavioral tests were performed under red light conditions after one hour habituation, except sucrose preference. Splash test: Splash test was performed according to previously published protocols41,170. Mice were sprayed twice on their backs with a 10% sucrose solution and placed in an empty cage and videotaped for 5 minutes. The time spent autogrooming was hand-scored by blind video observers. After splash tests, animals were singly housed for the remainder of behavioral tests. Novelty suppressed feeding: After an overnight fast, mice were placed into a corner of a bare novel arena measuring 38 x 38cm with a single pellet of chow at the center of the arena, and videotaped up to 10 minutes. The videos were then scored by blind observer for the latency to feed. Feeding was defined as using forepaws during mastication. Sucrose Preference: Mice were given two bottles in the home cage from which they could drink freely. During the first day, the two bottles contained water and allowed habituation to the bottles. On the following day, one bottle was replaced with a 1% sucrose solution. Mice were then allowed to drink from the bottles over two days, and consumption of each bottle (weight and volume) was recorded daily. The bottles were switched daily to ameliorate side bias. Elevated Plus Maze: EPM was performed as previously described273. In brief, mice were placed with heads in the center of the maze parallel to the open arms and allowed to roam for 5 minutes while video recorded. The amount of time spent in the open arms and distance travelled were quantified by Anymaze software (CleverSys). Animals 65 that fell or jumped from the arena were excluded from analyses. Social Interaction: SI was performed as previously described272. Briefly, mice were placed into the corner of a 38 x 38cm arena with a mesh cage at one end. After 3 minutes of habituation to the arena, a novel conspecific mouse (CD1 strain) was placed into the mesh cage, and the experimental mouse was again allowed to move around the arena for 3 minutes. Time spent within a 5cm radius of the conspecific mouse and in the opposing corners was measured for both sessions. SI ratio was calculated as a ratio of the time spent in the interaction radius while the conspecific mouse is present to the time spent in the same location with empty mesh cage. Electrophysiology Ex vivo acute brain slices were prepared from 9-13 week old male or female L10- GFP transgenic mice. Animals were anesthetized with isofluorane and transcardially perfused with ice-cold sucrose artificial cerebrospinal fluid (sucrose aCSF, in mM: 234 sucrose, 11 D-glucose, 26 NaHCO3, 2.5 KCl, 1.25 NaH2PO4, 10 MgSO4, 0.5 CaCl2). Animals were decapitated and brains rapidly removed and placed in oxygenated (95% O2, 5% CO2) slurried sucrose aCSF for 15 seconds. Brains were blocked and transferred to a vibratome slicing chamber (Leica; Germany) containing aCSF (in mM: 126 mM NaCl, 2.5 KCl, 1.25 NaH2PO4, 2 MgCl, 2 CaCl2, 26 NaHCO3, 10 glucose). Coronal slices (250 µm) were obtained and transferred to an incubation chamber containing oxygenated aCSF. Slices were incubated at 34C for 30 minutes and then at room temperature for a minimum of 30 min before recording. Whole-cell patch clamp recordings were made with slices held in a submersion chamber perfused at 2 mL/min with oxygenated aCSF held 66 at 30(± 2) C using a single inline heater (Warner Instruments; Hamden, CT). Borosilicate glass electrodes with a tip resistance of 3-6 MW were filled with internal solution (in mM: 115 potassium gluconate, 20 KCl, 1.5 MgCl, 10 phosphocreatine-Tris, 2 Mg-ATP, 0.5 Na3-GTP; pH 7.2-7.4; 280-290 mOsm). L10-GFP+ cells representing vHPC-NAc or vHPC-BLA projections were visualized in the ventral CA1 region of the hippocampus with an upright epifluorescent microscope (BX51WI Olympus; Japan). Pyramidal cells were distinguished from other cell types by their morphology and location in the cell body layer of the CA1 region. Recordings were made from projection cells using a Multiclamp 700B amplifier and Digidata 1440A digitizer (Molecular Devices; San Jose, CA). Membrane properties and cell excitability data were sampled (10 kHz), filtered (10 kHz), and stored on a PC for analysis. Membrane capacitance, membrane resistance, and access resistance were automatically calculated by pClamp 10 software (Molecular Devices; San Jose, CA). Any cell with a resting membrane potential more positive than -60mV or access resistance >25 MW were omitted from analyses. Resting membrane potential was measured automatically by the Multiclamp 700B Commander while injecting no current (I=0); this value was recorded immediately after breaking into a cell. Rheobase measurements were taken by administering 250ms, +5pA steps starting from 0pA with 250ms between current injections. The first current level issued to elicit a spike was recorded as rheobase for each cell. Action potential (AP, or spike) numbers were measured using a +25pA increasing current step protocol. The number of spikes at each step from 0 to 300 pA was manually counted. Input resistance was determined as the slope of the line best fit to the I-V plot generated by the input-output current clamp protocol, with the minimum current step being that which generated a voltage of 67 approximately -120 mV. Sag ratio was determined using peak and steady-state potentials obtained at the first current step that reached lower than -120 mV and was calculated as steady-state/peak. Spontaneous event frequency and amplitude were determined using Minianalysis software (Synaptosoft, Inc.) by manual selection of events from the first 60- 120 seconds of gap free voltage clamp recording from each cell. For DREADD validation, projection cells were identified as described above and recordings were obtained in regular aCSF. After initial baseline recordings were collected, aCSF + Clozapine-N-Oxide (CNO, 1 µM) was washed onto the slice. Approximately 8 minutes were allowed for the solution to reach the bath from the reservoir and for the DREADDs to bind CNO before excitability was measured again. For flutamide studies, vehicle vHPC-NAc recordings were collected with the slice washed in aCSF + vehicle (DMSO, 0.001%). The slices were incubated as described above, now with the addition of 0.001% DMSO in the aCSF. Treatment recordings were collected from slices incubated and bathed in aCSF plus flutamide (100 nM) and picrotoxin (100 µM) in DMSO. Picrotoxin was included due to the ability of flutamide to affect GABAergic transmission274. Immunohistochemistry Animals were deeply anesthetized with chloral hydrate and transcardially perfused with cold phosphate buffered saline for 9 minutes followed by 9 minutes of 10% formalin at a rate of 3 mL/min. Brains were fixed overnight in 10% formalin and subsequently switched to 30% sucrose. Brains were sliced to 35 µm sections on a frozen SM2010R microtome (Leica; Wetzlar, Germany) and stained using an antigen retrieval step (for AR staining only), 0.05% sodium borohydride (Sigma-Aldrich; 452882-25g lot:SHBK0324) in 68 PBS, and the following primary antibodies: Mouse anti-FosB (ab11959; 1:1000; Abcam), Goat anti-GFP (Abcam; ab5450 1:1000) and Rabbit anti-AR (Abcam; ab52615 1:1000); and secondary antibodies: Alexa-Fluor 488 anti-goat IgG (Jackson; 705-545-147 1:200) and Cy3 or Cy5 anti-rabbit IgG (Jackson; 711-165-152 1:200). Fluorescent images were visualized on an Olympus FluoView 1000 filter-based laser scanning confocal microscope. Translating Ribosome Affinity Purification (TRAP) and cDNA library preparation Three weeks following injection of retrograde HSV-Cre into NAc, Cre-dependent L10-GFP-expressing mice (Rosa26eGFP/L10a) were sacrificed and brains were immediately dissected into 1 mm coronal sections. Transduced tissue from ventral hippocampi (vHPC) of male and female mice was collected using 14-gauge biopsy punches guided by a fluorescent dissecting microscope (Leica) and stored at -80° C until processing (n = 4/biological replicate, 3-4 mice pooled per replicate). Polyribosome-associated RNA was affinity purified as previously described275. Briefly, tissue was homogenized in ice-cold tissue-lysis buffer (20 mM HEPES [pH 7.4], 150 mM KCl, 10 mM MgCl2, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide, protease inhibitors, and recombinant RNase inhibitors) using a motor-driven Teflon glass homogenizer. Homogenates were centrifuged for 10 minutes at 2000 g (4° C), supernatant was supplemented with 1% NP- 40 (AG Scientific, #P1505) and 30 mM DHPC (Avanti Polar Lipids, #850306P), and centrifuged again for 10 minutes at 20000 g (4°C). Supernatant was collected and incubated with Streptavidin MyOne T1 Dynabeads (Invitrogen, #65601) that were coated with anti-GFP antibodies (Memorial Sloan-Kettering Monoclonal Antibody Facility; clone 69 names: Htz-GFP-19F7 and Htz-GFP-19C8, 50 ug per antibody per sample) using recombinant biotinylated Protein L (Thermo Fisher Scientific,
# 29997) for 16-18 hours on a rotator (4° C) in low salt buffer (20 mM HEPES [pH 7.4], 350 mM KCl, 1% NP-40, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide). Beads were isolated and washed with high salt buffer (20 mM HEPES [pH 7.4], 350 mM KCl, 1% NP-40, 0.5 mM dithiothreitol, 100 µg/ml cycloheximide) and RNA was purified using the RNeasy Micro Kit (Qiagen, #74004). To increase yield, each RNA sample was initially passed through the Qiagen MinElute™ column 3 times. Following purification, RNA was quantified using a Qubit fluorometer (Invitrogen) and RNA quality was analyzed using a 4200 Agilent Tapestation (Agilent Technologies). cDNA libraries from 5 ng total RNA were prepared using the SMARTer® Stranded Total RNA-Seq Kit (Takara Bio USA, #635005), according to manufacturer’s instructions. cDNA libraries were pooled following Qubit measurement and TapeStation analysis, with a final concentration ~5-10 nM. Sequencing was performed at the Icahn School of Medicine at Mount Sinai Genomics Core Facility (icahn.mssm.edu/research/genomics/core-facility). Statistical Analysis Statistical analysis was performed with PRISM software 7.0 (Graphpad). Alpha criterion was set to 0.05 for all experiments. Behavior 2-way ANOVAs were followed by Sidak post hoc comparisons between groups. Spike number in electrophysiology experiments was analyzed by mixed 2-way ANOVAs with current as the within factor followed by Holm-Sidak corrected post hoc comparisons between groups. For all other electrophysiological measures: rheobase, spike amplitude, spike half-width, sEPSC 70 amplitude, sEPSC frequency, and other cellular properties data were analyzed by independent samples t-tests between groups. All error bars throughout this work represent the mean +/- SEM. 71 III. ANDROGEN-DEPENDENT EXCITABILITY OF MOUSE VENTRAL HIPPOCAMPAL AFFERENTS TO NUCLEUS ACCUMBENS UNDERLIES SEX-SPECIFIC SUSCEPTIBILITY TO STRESS The experiments in the following chapter are the subject of a manuscript currently in revision at Nature Neuroscience. Claire E. Manning performed the stress, behavior, and microscopy. Elizabeth S. Williams performed the electrophysiology experiments and performed stress and behavior protocols with the coauthor. Introduction Psychiatric disorders of affect, such as major depression disproportionately affect women6,276, but studies investigating depression-related behaviors in animal models that include both female and male subjects are unfortunately lacking. There are sex differences in brain regions that regulate anhedonia following some stress paradigms in rodent models of depression, including subchronic variable stress (SCVS)170, chronic mild stress277 and Peromyscus californicus social defeat stress278. However, the physiological mechanisms underlying sex differences in stress responses and depression-related behaviors remain unclear. As previously introduced, numerous studies have demonstrated the importance of the nucleus accumbens (NAc) in regulating depression- like behaviors after stress34,158,279,280, and glutamatergic inputs to NAc from areas such as prefrontal cortex and ventral hippocampus (vHPC) are critical in regulation of these stress responses281,282. Critically, the vHPC is essential for social and affective memories283-285, and, as discussed in the previous chapter, excitatory vHPC afferents to the NAc directly regulate male behavioral responses to chronic social defeat stress2, 72 making this circuit a potential candidate mediating sex differences in mood-related disorders. Unfortunately, most circuit-specific animal model studies investigating depression- related behaviors have not included female subjects. This gap in research exists despite sex differences in several depression-related brain regions, including the hippocampus. For example, hippocampal spine morphology differs between male and female rats prior to stress, and can exhibit sex-specific responses to stressful events141, and male and female rats differ in hippocampal LTP and hippocampus-dependent contextual learning286. Direct sex differences in responses to stress are also well-documented, and one key model in mice is subchronic variable stress (SCVS), after which only female mice exhibit anhedonic responses41,170. To this end, gonadal steroid hormone receptors have been identified in the NAc as modulators of female susceptibility to SCVS287. However, whether vHPC-NAc activity in male and female mice is regulated by gonadal hormones has not been tested. In this chapter, we demonstrated testosterone-dependent differences in adult male and female vHPC-NAc excitability as well as corresponding changes in susceptibility to SCVS. Importantly, we established a causal link between vHPC-NAc physiology and SCVS-induced anhedonia using a circuit-specific viral DREADD system to artificially manipulate vHPC-NAc excitability. We showed that stress- induced anhedonia is dependent upon long-term adaptation of vHPC-NAc projections, as activation of this circuit prior to behavioral assessment, and not simply during behavior, was necessary for induction of anhedonia. We also investigated circuit-specific transcriptional genomic differences between male and female vHPC-NAc neurons using translating ribosome affinity purification (TRAP), which identified many sex differences in 73 gene transcription that will inform future study of male and female circuit-level physiology. This chapter introduces a novel, circuit-specific physiological difference between male and female mice that drives corresponding sex differences in depression-related stress outcomes that may enhance future understanding of depression susceptibility and potential treatments in men and women. Results Female mice are selectively susceptible to anhedonia following SCVS We observed that female mice are selectively vulnerable to SCVS-induced anhedonia, in agreement with prior studies utilizing this protocol41,170,288. Following the 6- day SCVS battery of stressors (foot shock, tail suspension, and restraint; Figure 2 and Figure 7, top), female mice showed a significant reduction in free-choice preference for sucrose solution over water alone (Fig 7, bottom). As previously discussed, reduction of sucrose preference is an indicator of anhedonia34,289,290, thus SCVS models this aspect of female-specific depression vulnerability. We also used a variety of other assays to evaluate anxiety, reduced self-care, and social withdrawal behaviors in the same male and female mice (Figure 8a-e), but we did not observe the robust, sex-specific vulnerability to stress in any measure other than sucrose preference. Therefore, we focused on sucrose preference and anhedonia for the remainder of the current chapter, though we report all other behaviors measured throughout. 74 SCVS Tail Suspension Footshock Restraint Tail Suspension Footshock Restraint Behavior Assessment Sucrose Preference Day: 1 5 10 100 Control SCVS * f e c n e r e e r P e s o r c u S % 80 60 40 Male Figure 7 | Selective female susceptibility to SCVS in the measure of sucrose preference. Experimental time course of stress and behavior assessment, including sucrose preference (top). Only female mice displayed reduced sucrose preference following SCVS as measured by two bottle choice task (bottom). Two-way ANOVA: Group: Male vs Female F(1,112) = 1.109, p = 0.2946; Trial: Control vs Stress F(1,112) = 1.212, p = 0.2734; Group X Trial F(1,112) = 6.518, p = 0.0120. Sidak’s multiple comparisons: Male Control vs Stress Mean Diff = -2.406, 95% CI = -7.763 to 2.951; Female Control vs Stress Mean Diff = 6.054, 95% *CI = 0.7882 to 11.32; n = 30/group. Female 75 a i g n m o o r G e m T % i c ) m c ( d e v o M e c n a t s D i 2000 1500 1000 500 0 80 60 40 20 0 M F d ) s ( d e e F o t y c n e a L t 600 400 200 0 b ) % i ( e m T m A n e p O r 20 15 10 5 0 M Control SCVS F e o i t a R I S 5.0 4.5 4.0 2.5 2.0 1.5 1.0 0.5 0.0 M F M F M F Figure 8 | Additional behavioral assays of male vs. female SCVS. All behavioral assays other than sucrose preference did not reflect sex differences in SCVS as there was no interaction of stress and sex in any of the following measures: (a) Percent time grooming in splash test. Two-way ANOVA: Group: Male vs Female F(1,73) = 6.223, p = 0.0149; Trial: Control vs Stress F(1,73) = 1.481, p = 0.2276; Group X Trial F(1,73) = 0.1272, p = 0.07223 (no interaction). (b) Percent open arm time in EPM. Two- way ANOVA: Group: Male vs Female F(1,111) = 0.9638, p = 0.3284; Trial: Control vs Stress F(1,111) = 0.0319, p = 0.8585; Group X Trial F(1,111) = 1.358, p = 0.2465 (no interaction). (c) Distance moved (cm) in EPM. Two-way ANOVA: Group: Male vs Female F(1,112) = 2.112, p = 0.1489; Trial: Control vs Stress F(1,112) = 4.352e-6, p = 0.9983; Group X Trial F(1,112) = 0.2906, p = 0.5909 (no interaction). (d) Latency to feed (s) in novelty suppressed feeding test. Two-way ANOVA: Group: Male vs Female F(1,107) = 10.23, p = 0.0018; Trial: Control vs Stress F(1,107) = 2.92, p = 0.0904; Group X Trial F(1,107) = 0.5162, p = 0.4741 (no interaction). (e) SI Ratio in social interaction test. Two- way ANOVA: Group: Male vs Female F(1,115) = 0.8692, p = 0.3531; Trial: Control vs Stress F(1,115) = 3.712, p = 0.0565; Group X Trial F(1,115) = 3.093, p = 0.0813 (no interaction). 76 Female mice have increased vHPC-NAc neuronal excitability The sex difference in susceptibility to SCVS suggests an underlying difference in physiology between male and female mice. Glutamatergic vHPC-NAc projections have been linked to stress susceptibility and reward in several elegant studies2,282. To explore the vHPC-NAc circuit and its role in mediating anhedonic responses in mice following stress, we utilized whole cell patch clamp electrophysiology and the circuit-specific retrograde HSV-Cre labeling strategy291 used in the previous chapter to record from vHPC-NAc cells in both male and female mice. As described above, transgenic mice expressing the Cre-inducible L10-GFP275 were injected in NAc with a retrograde herpes simplex virus (HSV) expressing Cre recombinase (Figure 9, left), leading to GFP expression in all neurons projecting to or within NAc. After 21 days to allow full retrograde expression, brain slices containing vHPC were prepared for whole-cell recordings from GFP-positive vHPC neurons projecting to NAc. In hippocampus, only pyramidal neurons of the ventral CA1/subiculum region were found to project to NAc using this method (Figure 9, right). We found female vHPC-NAc neurons to be more excitable than those of male mice, as indicated by an elevated number of action potentials elicited by increasing depolarizing current injections (Figure 10 top, example traces for each group below). No differences in resting membrane potential (VM, Figure 11a) or membrane resistance (RM, Figure 11b) were observed between male and female vHPC-NAc neurons. Despite the apparent heightened excitability of female vHPC-NAc neurons, input resistance (Rin) was significantly lower in these neurons compared to male vHPC-NAc neurons (Figure 11c). Membrane capacitance was significantly higher in female vHPC-NAc neurons (Figure 11d), again despite the female neurons’ exaggerated response to injected current 77 HSV- heF1α-Cre injection retro transport Day 1 Retrograde Cre NAc (or BLA) Record vHPC Day 21+ HPC Slice for recording (right) DG CA3 CA1 vHPC-NAc projections VTA Sub Figure 9 | Retrograde HSV-heF1α-Cre injection at NAc predominantly labels vHPC CA1 pyramidal cells. Schematic depicting retrograde Cre viral vector strategy and electrophysiology time course (left) and L10-GFP vHPC stained with anti-GFP demonstrating vHPC-NAc projections (right). Hippocampal projections to NAc arise from the ventral CA1 region and subiculum. Other areas appearing with projections to NAc as labeled by GFP staining include BLA and VTA. 78 * * ** ** ** ** ** ** ** 200 100 0 150 250 100 Current Step (pA) 200 300 25 20 r e b m u N P A 15 10 5 0 50 Male V m 0 5 100 ms Female V m 0 5 100 ms 200pA Figure 10 | Female vHPC-NAc projections are more excitable than male vHPC-NAc projections. Action potential (AP) number across sequential depolarizing current steps (25-300 pA) for male (n = 20 cells from n = 6 animals) and female (n = 19 cells from n = 5 animals) vHPC-NAc projections. Females showed significantly higher AP number at all steps ≥ 125 pA (top). Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: Male vs Female [25 pA t(444) = 0.1142, p = 0.9091], [50 pA t(444) = 0.4545, p = 0.8773], [75 pA t(444) = 1.147, p = 0.5816], [100 pA t(444) = 2.322, p = 0.0801], [125 pA t(444) = 3.012, *p = 0.0163], [150 pA t(444) = 2.901, *p = 0.0194], [175 pA t(444) = 3.46, **p = 0.0047], [200 pA t(444) = 3.41, **p = 0.0050], [225 pA t(444) = 3.76, **p = 0.0019], [250 pA t(444) = 3.879, **p = 0.0015], [275 pA t(444) = 3.762, **p = 0.0019], [300 pA t(444) = 3.826, **p = 0.0016]. Inset: sum of AP number across all current steps. Two-tailed t-test t(37)=2.96, **p = 0.0053. Representative traces for male and female vHPC-NAc groups, 200 pA step (bottom). 79 a ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 b ) Ω M ( e c n a t s s e R i 250 200 150 100 50 0 Rheobase Resting Membrane Potential M F e ) A p ( e s a b o e h R 150 100 50 0 Membrane Resistance c ) Ω M ( e c n a t s s e R i F M f ) V m ( e g a t l o V -20 -30 -40 -50 -60 600 400 200 0 Voltage Threshold * Input Resistance ** F M g k a e P S S / 1.1 1.0 0.9 0.8 0.7 d ) F p ( e c n a t i c a p a C 150 100 50 0 Sag Ratio Membrane Capacitance * M F M F M F M F Figure 11 | Membrane properties of male vs. female vHPC-NAc projections. (a) Resting membrane potential (mV) [two-tailed t-test t(37) = 0.7643, p = 0.4495] and (b) membrane resistance (MW) [two-tailed t-test: t(37) = 0.5434, p = 0.5901] did not differ between male and female vHPC-NAc projections. (c) Input resistance (MW) was significantly lower in female vHPC-NAc cells. Two-tailed Mann-Whitney U-test: U = 96, sum of ranks M = 494, F = 286, **p = 0.0075. (d) Membrane capacitance (pF) was significantly higher in female vHPC-NAc cells. Two-tailed t-test: t(37) = 2.687, *p = 0.0107. (e) Rheobase (pA) did not differ between male and female vHPC-NAc cells. Two-tailed t- test: t(37) = 1.293, p = 0.2042. (f) Action potential voltage threshold (mV) was lower in female vHPC-NAc cells. Two-tailed t-test: t(37) 2.202, *p = 0.0340. (g) IH as measured by sag ratio did not differ between male and female vHPC-NAc cells. Two-tailed Mann- Whitney U-test: U = 163, sum of ranks M = 373, F = 407, p = 0.4609. 80 compared to male neurons. Rheobase was not significantly different between male and female vHPC-NAc neurons (Figure 11e). Action potential threshold voltage was significantly lower in female neurons (Figure 11f), also indicating a heightened excitability in vHPC-NAc projections in female mice. There was no difference in sag ratio between male and female vHPC-NAc neurons (Figure 11g), indicating no difference in after- hyperpolarization current (Ih) in this circuit between sexes. Frequency of spontaneous excitatory postsynaptic currents (sEPSCs) did not differ between male and female vHPC- NAc neurons (Figure 12a), but sEPSC amplitude was significantly decreased in female mice (Figure 12b), suggesting that increased excitability in these neurons could be a homeostatic response to decreased excitatory input over time via modulation of postsynaptic effectors in females. Female vHPC-NAc neurons also demonstrate a dramatically lowered spike frequency adaptation compared to male neurons of the same circuit, as indicated by less increase in the interspike interval over the course of a depolarizing current injection step (Figure 13a), but no difference was observed between male and female neurons in the measure of latency to first spike in the same current step (Figure 13b). There was also no difference between male and female vHPC-NAc AMPA:NMDA ratio (Figure 14), indicating that there is no difference in the strength of synapses at the level of the vHPC in this circuit. Taken together, these results indicate robust sex differences in the neurophysiology of vHPC-NAc neurons, which may explain sex differences in stress responses to SCVS and disparities in depression prevalence between men and women. 81 sEPSC Amplitude ** c M F a 20 15 10 5 0 ) z H ( y c n e u q e r F sEPSC Frequency b ) A p ( e d u t i l p m A 25 20 15 10 5 0 sEPSC Traces A p 0 2 500ms -80mV -80mV M F M F Figure 12 | Spontaneous activity measures of male vs female vHPC-NAc projections. (a) sESPC frequency did not differ between male and female vHPC-NAc cells [two-tailed Mann-Whitney U-test: U = 125, sum of ranks M = 315, F = 426, p = 0.1089], but sEPSC amplitude (b) was lower in female cells. Two-tailed t-test: t(37) = 2.938, **p = 0.0057. (c) Example sEPSC traces for male (M) and female (F) vHPC-NAc cell gap-free recordings at -80 mV holding potential. 82 a ) s m ( l a v r e n t I i e k p s r e t n I 80 60 40 20 0 Male Female *** b ) s m ( y c n e a L t 60 40 20 0 83 1st Pair Last Pair M F Figure 13 | Spike frequency adaptation is elevated in male vHPC-NAc projections compared to female vHPC-NAc projections. Interspike interval increases over the course of 500 ms current injection as measured by the time (ms) between the 1st pair of spikes and last pair of spikes. (a) Males had more spike frequency adaptation as measured by the interspike interval increase; interspike interval of the last pair of spikes was significantly longer in male vHPC-NAc projections than in female projections. One-way ANOVA: F(3,74) = 50.87, p < 0.0001. Sidak’s multiple comparisons: Male Last Pair vs Female Last Pair mean difference 16.52, 95% ***CI = 6.094 to 26.94, Adj. p = 0.0005. (b) Latency to 1st spike (ms) from beginning of current injection did not differ between male and female vHPC-NAc projection neurons. Two-tailed t-test: t(37) = 0.4442, p = 0.6595. Interspike intervals and latency to 1st spike were evaluated at the rheobase current for each cell. o i t a R e d u t i l p m A 4 3 2 1 0 F M A p 0 0 2 50 ms +40 mV -70 mV +40 mV -70 mV Figure 14 | AMPA to NMDA ratio of male and female vHPC-NAc projections. Evoked AMPA and NMDA response amplitudes were used to calculate AMPA:NMDA as an indicator of synaptic strength. AMPA-mediated current amplitudes were evaluated at -70 mV holding potential, and NMDA-mediated current amplitudes were evaluated at +40 mV holding potential (amplitudes measured 50 ms following stimulation). AMPA:NMDA did not differ between male and female vHPC-NAc projections (left). Two-tailed t-test: t(37) = 1.576, p = 0.1238. Example traces of AMPA and NMDA evoked responses (right). M F 84 Sex differences in baseline excitability are unique to the vHPC-NAc circuit To investigate whether the observed excitability differences were specific to the vHPC-NAc circuit, we injected retrograde HSV-Cre into basolateral amygdala (BLA) of a separate cohort of male and female mice. Whole-cell recordings obtained from vHPC- BLA projections revealed no significant difference in excitability of female vHPC-NAc neurons compared to male (Figure 15 top, example traces for each group below). There were also no differences between male and female vHPC-BLA neuron membrane properties in any measure (VM, RM, Rin, CM, and rheobase; Figure 16a-e). No difference was observed between sexes in AP voltage threshold (Figure 16f), supporting similar male and female excitability in this circuit. Sag ratio was observed to be lower in female vHPC-BLA neurons (Figure 16g), indicating a possible sex difference in after- hyperpolarization current in this circuit. Spontaneous EPSCs did not differ in frequency or amplitude between male and female vHPC-BLA projections (Figure 17a-c). These findings, contrasted with the above vHPC-NAc circuit observations, demonstrate the specificity of the vHPC-NAc difference in excitability between male and female mice. Overall, our electrophysiology experiments demonstrate that female vHPC-NAc, but not vHPC-BLA, neurons have increased excitability compared to males, suggesting a circuit-specific physiological difference that could underlie the sex differences in susceptibility to the anhedonia following stress. Next, we investigated the effects of adult hormone manipulation on susceptibility to SCVS and corresponding changes in vHPC- NAc projection physiology. 85 200 100 0 100 250 Current Step (pA) 150 200 300 r e b m u N P A 25 20 15 10 5 0 50 Male V m 0 5 100 ms Female V m 0 5 100 ms 200pA Figure 15 | Male and female vHPC-BLA projections do not differ in excitability. AP number across sequential depolarizing steps for male (n = 22 cells from n = 7 animals) and female (n = 12 cells from n = 4 animals) vHPC-BLA projections. Male and female AP number did not differ at any current step (top). Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: [25 pA t(384) = 0.745, p = 0.9993], [50 pA t(384) = 2.523, p = 0.1352], [75 pA t(384) = 2.781, p = 0.0661], [100 pA t(384) = 2.434, p = 0.1699], [125 pA t(384) = 2.3, p = 0.2343], [150 pA t(384) = 1.823, p = 0.5765], [175 pA t(384) = 1.396, p = 0.8828], [200 pA t(384) = 1.356, p = 0.9019], [225 pA t(384) = 1.321, p = 0.9169], [250 pA t(384) = 1.272, p = 0.9356], [275 pA t(384) = 1.346, p = 0.9063], [300 pA t(384) = 1.108, p = 0.9766]. Inset: sum of AP number across all current steps. Two- tailed t-test: t(32) = 1.861, p = 0.0720. Representative traces for male and female vHPC- BLA groups, 200 pA step (bottom). 86 a ) V m ( e g a t l o V Potential Resting Membrane -55 -60 -65 -70 -75 -80 -85 M F b ) Membrane Resistance 200 M ( e c n a t s s e R i 150 100 50 0 M F c Input Resistance 1500 ) M ( e c n a t s s e R i 1000 500 0 M F Rheobase f Voltage Threshold -20 e 150 ) A p ( e s a b o e h R 100 50 0 ) V m ( e g a t l o V -30 -40 -50 -60 Capacitance M F d ) F p ( e c n a t i c a p a C 200 150 100 50 0 Sag Ratio ** g 1.1 k a e P S S / 1.0 0.9 0.8 0.7 M F M F M F Figure 16 | Membrane properties of male vs. female vHPC-BLA projections. vHPC-BLA projection cells did not differ between male and female groups in any of the following measures: (a) resting membrane potential (mV). Two-tailed t-test: t(32) = 0.9449, p = 0.3518; (b) membrane resistance (MW). Two-tailed t-test: t(32) = 1.24, p = 0.2238; (c) input resistance (MW). Two-tailed t-test: t(32) = 0.3264, p = 0.7463; (d) capacitance (pF). Two-tailed t-test: t(32) = 0.7014, p = 0.4881; (e) rheobase (pA). Two- tailed t-test: t(32) = 1.852, p = 0.0733; and (f) voltage threshold (mV). Two-tailed t-test: t(32) = 1.399, p = 0.1713. (g) IH as measured by sag ratio was lower in female vHPC-BLA cells. Two-tailed t-test: t(32) = 2.41, **p = 0.0219. 87 sEPSC Amplitude c M F a ) z H ( y c n e u q e r F 20 15 10 5 0 sEPSC Frequency b ) A p ( e d u t i l p m A 30 20 10 0 sEPSC Traces A p 0 2 500ms -80mV -80mV M F M F Figure 17 | Spontaneous activity measures of male vs female vHPC-BLA projections. Spontaneous ESPC frequency [two-tailed t-test: t(32) = 1.205, p = 0.2373] (a) nor amplitude [two-tailed t-test: t(32) = 0.6158, p = 0.5425] (b) did not differ between male and female vHPC-BLA cells. (c) Example sEPSC traces for male (top) and female (bottom) vHPC-BLA gap-free cell recordings at -80 mV holding potential. 88 SCVS does not affect basal vHPC-NAc excitability in female mice To investigate whether SCVS affects the excitability of vHPC-NAc neurons in female mice, we injected 7-week-old female C57/Bl6 mice with retrograde Cre virus at the NAc (as previously described). Following two weeks for recovery, mice were then subjected to the same SCVS protocol used above and immediately sacrificed within two days of completion of stress for whole-cell patch clamp recordings. We found no difference in the excitability of vHPC-NAc projections in these mice, as indicated by no change in spike number at any current injection step (Figure 18) nor an increase in the total number of spikes across all steps (Figure 18, inset). SCVS also did not cause a change in any membrane property measured (VM, RM, Rin, CM, and rheobase; Figure 19a- e). These data indicate that SCVS does not cause a change in the physiology of female vHPC-NAc neurons when recordings were taken immediately following stress. Orchidectomy induces male susceptibility to SCVS-induced anhedonia To investigate the role of adult sex hormones in susceptibility to SCVS, male mice were orchidectomized (ORCH), allowed to recover for 10 or 28 days, and exposed to SCVS (Figure 20a-b). Ten days of recovery resulted in main effects of ORCH and stress without interaction between the two (Figure 20a), suggesting that SCVS this close to the stressful surgery (sham or ORCH) was sufficient to render male mice susceptible to SCVS-induced anhedonia. In contrast, twenty-eight days following ORCH, male mice displayed reduced sucrose preference following SCVS, while those undergoing sham surgeries remained resilient to decrease in sucrose preference 89 r e b m u N e k p S i 25 20 15 10 5 0 50 200 100 0 150 100 250 Current Step (pA) 200 300 Control SCVS 200pA A p 0 5 200ms Figure 18 | Female control and post-SCVS vHPC-NAc projections do not differ in excitability. Action potential (AP) number across sequential depolarizing current steps (25-300 pA) for female control (n = 15 cells from n = 4 animals) and female SCVS (n = 27 cells from n = 7 animals) vHPC-NAc projections. Groups did not differ in number of spikes at any step (top). Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: Male vs Female [25 pA t(456) = 0.6533, p = 0.9998], [50 pA t(456) = 0.5444, p > 0.9999], [75 pA t(456) = 0.7017, p = 0.9996], [100 pA t(456) = 0.5565, p > 0.9999], [125 pA t(456) =0.1936, p > 0.9999], [150 pA t(456) = 0.0242, p > 0.9999], [175 pA t(456) = 0.04839, p > 0.9999], [200 pA t(456) = 0.1573, p > 0.9999], [225 pA t(456) = 0.03629, p > 0.9999], [250 pA t(456) = 0.2783, p > 0.9999], [275 pA t(456) = 0.06049, p > 0.9999], [300 pA t(456) = 0.121, p > 0.9999]. Inset: sum of AP number across all current steps. Two-tailed t-test t(29) = 0.607, p = 0.5486. Representative traces for control and SCVS female vHPC- NAc groups, 200 pA step (bottom). 90 Membrane Resistance c ) Ω M ( e c n a t s s e R i 600 400 200 Input Resistance 800 600 400 200 0 SCVS Control e 0 Control SCVS Rheobase b ) Ω M ( e c n a t s s e R i SCVS Control d Capacitance Resting Membrane Potential a ) V m ( l a i t t n e o P -85 -80 -75 -70 -65 -60 -55 ) F p ( e c n a t i c a p a C 150 100 50 80 60 40 20 ) A p ( e s a b o e h R 0 Control SCVS 0 Control SCVS Figure 19 | Membrane properties of control and post-SCVS female vHPC-NAc neurons. Control and SCVS female vHPC-NAc neurons did not differ in any of the following measures: (a) resting membrane potential (mV; two-tailed Mann-Whitney U-test: U = 151, sum of ranks control = 374, SCVS = 529, p = 0.1806); (b) membrane resistance (MW; Mann-Whitney U-test: U = 177, sum of ranks control = 348, SCVS = 555, p = 0.5161); (c) input resistance (MW; t(40) = 0.903, p = 0.3719); (d) membrane capacitance (pF; t(40) = 0.3628, p = 0.7187); and (e) rheobase (pA; t(40) = 0.7795, p = 0.4402). 91 (Figure 20b), with an interaction between the two variables. There were no differences between sham and ORCH groups in any other behavioral measure at either time point (Figure 21a-d). These data thus suggest that long-term reduction in androgen signaling is sufficient for SCVS-induced anhedonia in male mice. Orchidectomy reduces excitability of vHPC-NAc neurons To determine whether the sex difference we observed in vHPC-NAc excitability is also dependent on adult sex hormones, we compared vHPC-NAc excitability in ORCH vs. sham male and OVX vs. sham female mice at 10 days post-gonadectomy. Retrograde Cre was injected into NAc, 10 days following intracranial injection gonads were removed, and 10 days following gonadectomy, vHPC-NAc activity was recorded (Figure 22a). ORCH significantly increased the excitability of male vHPC-NAc neurons compared to that of sham controls (Figure 22b top, example traces for each group below). ORCH had no effect on VM, RM, Rin, or CM (Figure 23a-d), but did increase rheobase (Figure 23e), with no change in AP voltage threshold (Figure 23f). There was no observed difference between sham and ORCH animals in sag ratio (Figure 23g), and ORCH did not affect sEPSC frequency or amplitude (Fig 24a-c). Orchidectomy did cause a decrease in spike frequency adaptation, as indicated by a smaller interspike interval duration over the course of a depolarizing current injection (Figure 25a). This was accompanied by an increase in the latency to first spike in the same current step (Figure 25b). In contrast, ovariectomy (OVX) was performed using the same experimental timeline as the above ORCH experiment. OVX had no effect on the excitability of female vHPC-NAc neurons compared to those neurons in sham-operated mice (Figure 26 top, 92 a 100 OVX or ORCH SCVS Behavior Assessment Sucrose Preference b OVX or ORCH SCVS Behavior Assessment Sucrose Preference Day 1 Day 10 Day 19 Day 1 Day 28 Day 37 Short Time Course 100 Control SCVS Long Time Course ** e c n e r e f e r P e s o r c u S % 80 60 40 Sham Orch Sham Orch Figure 20 | Orchidectomy of male mice induces susceptibility to SCVS as measured by sucrose preference, but only with extended time following surgery. (a, top) Schematic depicting experimental time course of surgery, stress, and measurement of sucrose preference for short time course orchidectomy experiment. (a, bottom) Short time course sham vs orchidectomized male mice showed no interaction between surgery and stress. Two-way ANOVA: Group: Sham vs ORCH F(1,73) = 21.78, p <0.0001; Trial: Control vs Stress F(1,73) = 0.0462; Group X Trial F(1,73) = 2.116, p = 0.1500 (no interaction); n = 20 control sham, 18 control ORCH, 20 SCVS sham, 18 SCVS ORCH. (b, top) Schematic depicting experimental time course of surgery, stress, and measurement of sucrose preference for long time course orchidectomy experiment. (b, bottom) Orchidectomized male mice showed significant reduction in sucrose preference compared to non-stress controls, while sham male sucrose preference was unaffected by SCVS. Two-way ANOVA: Group: Sham vs ORCH F(1,34) = 24.76, p < 0.0001; Trial: Control vs Stress F(1,34) = 12.74, p = 0.0011; Group X Trial F(1,34) = 38.33, p < 0.0001. Sidak’s multiple comparisons: Orch Control vs Stress Mean Difference = 15.59, 95% **CI = 10.84 to 20.35; n = 10 control sham, 8 control ORCH, 10 SCVS sham, 9 SCVS ORCH. e c n e r e f e r P e s o r c u S % 80 60 40 93 Control SCVS Sham Orch c ) s ( d e e F o t y c n e t a L d Sham Orch ) s ( d e e F o t y c n e a L t Sham Orch 600 400 200 0 600 400 200 0 a i g n m o o r G e m T % i b i g n m o o r G e m T % i 80 60 40 20 0 80 60 40 20 0 Sham Orch Figure 21 | Additional behavioral assays of sham vs. orchidectomy SCVS. All behavioral assays other than sucrose preference did not reflect an interaction of hormone status and stress in any of the following measures: Percent time grooming in splash test in the 10-day [two-way ANOVA: Group: Sham vs ORCH F(1,73) = 1.244, p = 0.2684; Trial: Control vs Stress F(1,73) = 20.28, p < 0.0001; Group X Trial F(1,73) = 0.2295, p = 0.6333 (no interaction)] (a) and 28-day [two-way ANOVA: Group: Sham vs ORCH F(1,30) = 0.0934, p = 0.1460; Trial: Control vs Stress F(1,30) = 1.007, p = 0.3237] (b) time courses; and latency to feed (s) in novelty suppressed feeding test in the 10-day [two-way ANOVA: Group: Sham vs ORCH F(1,73) = 6.339, p = 0.0140; Trial: Control vs Stress F(1,73) = 6.258, p = 0.0146; Group X Trial F(1,73) = 0.06081 (no interaction)] (c) and 28-day (two-way ANOVA: Group: Sham vs ORCH F(1,32) = 0.45, p = 0.5072; Trial: Control vs Stress F(1,32) = 3.28, p = 0.0795; Group X Trial F(1,32) = 0.0534, p = 0.8187 (no interaction)] (d) time courses. 94 HPC Retrograde Cre HSV- heFα-Cre injection ORCH vHPC recording area NAc Day 1 Day 8 Record vHPC Day 21+ a b 25 20 15 10 5 0 r e b m u N e k p S i * * ** ** ** * M - Sham * * * * * ** M - ORCH 200 100 0 V m 0 5 100 ms V m 0 5 100 ms 200pA 50 250 100 Current Step (pA) 150 200 300 Figure 22 | Orchidectomy in male mice increases vHPC-NAc excitability. (a) Schematic depicting retrograde Cre viral strategy for labeling vHPC-NAc projections (left) and experimental time course for surgery and recording (right). (b, left) Action potential number across sequential depolarizing current steps (25-300 pA) for sham male (n = 11 cells from n = 3 animals) and orchiectomized male (n = 31 cells from n = 6 animals) vHPC-NAc projections. Orchiectomized male projections showed significantly higher AP number at all steps > 25 pA. Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: [25 pA t(360) = 1.614, p = 0.1073], [50 pA t(360) = 3.27, *p = 0.0102], [75 pA t(360) = 3.621, **p = 0.0040], [100 pA t(360) = 3.449, **p = 0.0063], [125 pA t(360) = 3.506, **p = 0.0056], [150 pA t(360) = 3.28, *p = 0.0102], [175 pA t(360) = 2.868, *p = 0.0302], [200 pA t(360) = 2.862, *p = 0.0302], [225 pA t(360) = 2.776, *p = 0.0302], [250 pA t(360) = 2.489, *p = 0.0457], [275 pA t(360) = 2.536, p = 0.0457], [300 pA t(360) = 2.514, *p = 0.0457]. Inset: sum of AP number across all current steps. Two-tailed t-test: t(30) = 3.131, **p = 0.0039. (b, right) Representative traces for sham and ORCH vHPC- NAc projections, 200 pA step. 95 a ) V m ( e g a t l o V Potential Resting Membrane -55 -60 -65 -70 -75 -80 -85 ORCH SHAM e 0 ORCH Rheobase * SHAM f ) V m ( e g a t l o V -30 -35 -40 -45 -50 -55 -60 150 100 50 ) A p ( e s a b o e h R 0 c 600 400 200 ) Ω M ( e c n a t s s e R i b ) Ω M ( e c n a t s s e R i Membrane Resistance 400 300 200 100 Input Resistance Capacitance d 150 100 50 ) F p ( e c n a t i c a p a C 0 ORCH Voltage Threshold SHAM g 0 ORCH SHAM Sag Ratio k a e P S S / 1.05 1.00 0.95 0.90 0.85 0.80 ORCH SHAM ORCH SHAM ORCH SHAM Figure 23 | Membrane properties of sham vs. orchidectomy male vHPC-NAc projections. vHPC-NAc projection cells did not differ between male sham and orchidectomy groups in any of the following measures: (a) resting membrane potential (mV). Two-tailed t-test: t(30) = 1.077, p = 0.2902; (b) membrane resistance (MW). Two-tailed Mann-Whitney U- test: U = 67.5, sum of ranks ORCH = 394.5, Sham = 133.5, p = 0.0573; (c) input resistance (MW). Two-tailed t-test: t(30) = 1.713, p = 0.0970; and (d) membrane capacitance (pF). Two-tailed t-test: t(30) = 1.098, p = 0.2786. (e) Orchidectomy male vHPC-NAc projection cells showed a decreased rheobase (pA) compared to sham projections. Two-tailed t-test: t(30) = 2.648, *p = 0.0128. (f) Voltage threshold (mV) [two- tailed t-test: t(30) = 0.7637, p = 0.4510] and (g) sag ratio [two-tailed t-test: t(30) = 0.74, p = 0.4651] did not differ between sham and orchidectomy groups. 96 a 30 20 10 ) z H ( y c n e u q e r F b sEPSC Frequency ) A p ( e d u t i l p m A sEPSC Amplitude 30 20 10 c ORCH SHAM 0 ORCH SHAM 0 ORCH SHAM sEPSC Traces A p 0 2 500ms -80mV -80mV Figure 24 | Spontaneous activity measures of orchidectomy vs sham male vHPC- NAc neurons. (a) Spontaneous EPSC frequency (Hz) [two-tailed t-test: t(30) = 0.9992, p = 0.3257] and (b) amplitude (pA) [two-tailed t-test: t(30) = 0.4635, p = 0.6464] did not differ between sham and orchidectomy groups. (c) Example sEPSC traces for ORCH (top) and sham (bottom) vHPC-NAc recordings. 97 b ORCH Sham ** 1st Pair Last Pair ) s m ( y c n e a L t 80 60 40 20 0 80 60 40 20 0 a ) s m ( l a v r e n t I i e k p s r e n t I * ORCH SHAM Figure 25 | Spike frequency adaptation is impaired in male vHPC-NAc projections following orchidectomy. Interspike interval increases over the course of 500 ms current injection as measured by the time (ms) between the 1st pair of spikes and last pair of spikes in male sham and ORCH vHPC-NAc neurons. (a) ORCH vHPC-NAc neurons had less spike frequency adaptation as measured by the interspike interval increase over current step; interspike interval of the last pair of spikes was significantly shorter in ORCH vHPC-NAc projections than in sham projections. One-way ANOVA: F(3, 60) = 65.91, p < 0.0001. Sidak’s multiple comparisons: ORCH Last Pair vs Sham Last Pair mean difference -13.25, 95% **CI = - 23.05 to -3.439, Adj. p = 0.0039. (b) Latency to 1st spike (ms) from beginning of current injection was also decreased in ORCH vHPC-NAc neurons compared to sham. Two- tailed t-test: t(30) = 2.587, *p = 0.0148. Interspike intervals and latency to 1st spike were evaluated at the rheobase current for each cell. 98 r e b m u N e k p S i 25 20 15 10 5 0 50 200 100 0 SHAM OVX 250 100 Current Step (pA) 200 150 300 F - Sham F - OVX V m 0 5 100 ms V m 0 5 100 ms 200pA Figure 26 | Sham and OVX female vHPC-NAc projections do not differ in excitability. Sham (n = 16 cells from n = 4 animals) and OVX (n = 15 cells from n = 4 animals) female vHPC-NAc neurons did not differ in excitability as measured by number of spikes per 25 pA current injection step from 25-300 pA (top); Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: [25 pA t(348) = 0.5113, p = 0.9997], [50 pA t(348) = 0.6087, p = 0.9996], [75 pA t(348) = 0.8684, p = 0.9971], [100 pA t(348) = 0.2069, p = 0.9998], [125 pA t(348) = 0.3165, p = 0.9998], [150 pA t(348) = 0.0244, p = 0.9998], [175 pA t(348) = 0.1461, p = 0.9998], [200 pA t(348) = 0.0730, p = 0.9998]. [225 pA t(348) = 0.3003, p = 0.9998], [250 t(348) = 0.5275, p = 0.9997], [275 pA t(348) = 0.4951, p = 0.9997], [300 pA t(348) = 0.7061, p = 0.9993]. Inset: total number of spikes summed over all current injection steps did not differ between sham and OVX groups. Two-tailed t-test: t(29) = 0.2763, p = 0.7843. Representative traces for sham and OVX vHPC-NAc projections, 200 pA step (bottom). 99 example traces for each group below). OVX also did not affect membrane properties (Figure 27a-f), sag ratio (Figure 27g) nor sEPSC frequency or amplitude (Figure 28a-c). There was also no change in spike frequency adaptation following OVX compared to sham-operated controls (Figure 29a), and no change in the latency to first spike at the same current step (Figure 29b). Taken together with the above ORCH experiments, these findings demonstrate the importance of testes-derived mediators (e.g. androgen hormones) in the time-dependent regulation of vHPC-NAc excitability in male mice. This points to male gonadal hormone reduction of vHPC-NAc activity as the possible mechanism for the established sex difference in SCVS-induced anhedonia, and may work to elucidate neurophysiological differences between men and women that lead to differential MDD diagnoses. Orchidectomy reduces excitability of vHPC-BLA neurons To determine whether vHPC-BLA excitability is also dependent on adult sex hormones, we compared ORCH vs. sham male and OVX vs. sham female mice at 10 days post-gonadectomy. Retrograde Cre was injected into BLA, 10 days following intracranial injection gonads were removed, and 10 days following gonadectomy, vHPC- BLA activity was recorded. ORCH increased the excitability of male vHPC-BLA neurons compared to that of sham controls as indicated by an increase in the total number of spikes over all current injection steps (Figure 30, inset), but did not cause significant differences in spike number at any individual current step (Figure 30). ORCH had no effect on resting membrane potential (Figure 31a), but did increase membrane resistance (Figure 31b). Input resistance did not differ between groups (Figure 31c), but 100 a ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 -85 Resting Membrane Potential OVX SHAM e ) A p ( e s a b o e h R 150 100 50 0 Membrane Resistance c Input Resistance 600 ) M ( e c n a t s s e R i 400 200 0 OVX Voltage Threshold OVX SHAM SHAM g k a e P S S / 1.1 1.0 0.9 0.8 0.7 OVX SHAM f ) V m ( e g a t l o V -30 -35 -40 -45 -50 -55 b ) M ( e c n a t s s e R i 250 200 150 100 50 0 Rheobase OVX SHAM Capacitance OVX SHAM d ) F p ( e c n a t i c a p a C 150 100 50 0 Sag Ratio OVX SHAM Figure 27 | Membrane properties of sham vs. ovariectomy female vHPC-NAc projections. Sham and OVX groups did not differ over any of the following measures: (a) resting membrane potential (mV). Two-tailed t-test: t(29) = 0.0709, p = 0.9439; (b) membrane resistance (MW). Two-tailed t-test: t(29) = 0.708, p = 0.4846; (c) input resistance (MW). Two-tailed t-test: t(29) = 0.7262, p = 0.4761; (d) membrane capacitance (pF). Two-tailed t-test: t(29) = 0.0903, p = 0.9287; (e) rheobase (pA). Two-tailed t-test: t(29) = 0.0605, p = 0.9522; (f) voltage threshold (mV). Two-tailed t-test: t(29) =0.0075, p = 0.9941; (g) sag ratio. Two-tailed t-test: t(29) = 0.2533, p = 0.8018. 101 a ) z H ( y c n e u q e r F 30 20 10 0 sEPSC Frequency OVX SHAM sEPSC Amplitude sEPSC Traces c OVX SHAM -80mV -80mV OVX SHAM A p 0 2 500ms b ) A p ( e d u t i l p m A 30 20 10 0 Figure 28 | Spontaneous activity measures of ovariectomy vs sham female vHPC- NAc neurons. Female OVX and sham vHPC-NAc neurons did not differ in the measures of (a) sEPSC frequency (Hz) [two-tailed t-test: t(29) = 0.1247, p = 0.9017] and (b) sEPSC amplitude [two-tailed t-test: t(29) = 1.326, p = 0.1954]. (c) Example sEPSC traces for OVX (top) and sham (bottom) vHPC-NAc cell gap-free recordings at -80 mV holding potential. 102 OVX Sham a ) s m ( l a v r e n t I i e k p s r e n t I 80 60 40 20 0 b ) s m ( y c n e t a L 60 40 20 0 OVX SHAM 1st Pair Last Pair Figure 29 | Spike frequency adaptation did not differ between female sham and OVX vHPC-NAc neurons. Interspike interval increases over the course of 500 ms current injection as measured by the time (ms) between the 1st pair of spikes and last pair of spikes in female sham and OVX vHPC-NAc neurons. (a) Spike frequency adaptation of vHPC-NAc neurons did not differ between sham and OVX groups. One-way ANOVA F(3,58) = 122.6, p <0.0001. Sidak’s multiple comparisons: OVX last pair vs sham last pair mean difference = -1.622, 95% CI = -7.839 to 4.596. (b) Latency to 1st spike following start of current injection did not differ between OVX and sham groups. Two-tailed t-test: t(29) = 0.04007, p = 0.9683. Interspike interval and latency to 1st spike were evaluated at rheobase current step for each cell. 103 * 250 150 50 150 100 250 Current Step (pA) 200 30 25 20 15 10 5 0 50 r e b m u N e k p S i M - Sham M - ORCH 200pA 300 A p 0 5 200ms Figure 30 | Orchidectomy increases the excitability of vHPC-BLA neurons in male mice. Action potential (AP) number across sequential depolarizing current steps (25-300 pA) for sham male (n = 19 cells from n = 4 animals) and orchiectomized male (n = 11 cells from n = 3 animals) vHPC-BLA projections. Orchiectomized male projections showed significantly higher AP number at all steps > 25 pA. Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: [25 pA t(348) = 0.8312, p = 0.9981], [50 pA t(348) = 1.698, p = 0.6791], [75 pA t(348) = 1.848, p = 0.5560], [100 pA t(348) = 1.367, p = 0.8969], [125 pA t(348) = 1.048, p = 0.9850], [150 pA t(348) = 0.7479, p = 0.9993], [175 pA t(348) = 1.198, p = 0.9577], [200 pA t(348) = 1.746, p = 0.6405], [225 pA t(348) = 1.944, p = 0.4782], [250 pA t(348) = 2.086, p = 0.3692], [275 pA t(348) = 2.36, p = 0.2038], [300 pA t(348) = 2.77, p = 0.0686]. Inset: sum of AP number across all current steps. Two-tailed t-test: t(28) = 2.298, *p = 0.0039. Representative traces below for sham and ORCH vHPC- NAc projections, 200 pA step. 104 a ) V m ( e g a t l o V Resting Membrane Potential -50 -60 -70 -80 Membrane Resistance ** 300 200 100 b ) Ω M ( e c n a t s s e R i c ) Ω M ( e c n a t s s e R i -90 ORCH d ) F p ( e c n a t i c a p a C SHAM Capacitance ** 200 150 100 50 0 ORCH e ) A p ( e s a b o e h R SHAM 200 150 100 50 Input Resistance 500 400 300 200 100 0 ORCH SHAM Rheobase 0 ORCH SHAM 0 ORCH SHAM Figure 31 | Membrane properties of orchidectomy vs sham vHPC-BLA neurons. (a) Orchidectomy (ORCH) and sham vHPC-BLA neurons did not differ in the measure of resting membrane potential (mV; two-tailed t-test: t(28) = 1.194, p = 0.2425). (b) Orchidectomy vHPC-BLA neurons had a higher membrane resistance compared to sham neurons (MW; two-tailed Mann-Whitney U-test: U = 42, sum of ranks ORCH = 233, Sham = 232, **p = 0.0060). (c) ORCH and Sham vHPC-BLA neurons did not differ in the measure of input resistance (MW; two-tailed t-test t(28) = 0.7965, p = 0.4324). (d) ORCH vHPC-BLA neurons had a lower membrane capacitance than sham neurons (pF; two- tailed t-test: t(28) = 3.261, **p = 0.0029). (e) ORCH and Sham vHPC-BLA neurons did not differ in the measure of rheobase (pA; two-tailed t-test: t(28) = 0.5956, p = 0.5562). 105 ORCH did decrease membrane capacitance (Figure 31d). There was no significant difference between groups in the measure of rheobase (Figure 31e). Ovariectomy (OVX) was performed using the same experimental timeline as the above vHPC-BLA ORCH experiment. OVX had no effect on the excitability of female vHPC-BLA neurons compared to those neurons in sham-operated mice (Figure 32 top, example traces for each group below). OVX also did not affect resting membrane potential (Figure 33a) nor membrane resistance (Figure 33b), but OVX projections did have an increased input resistance compared to sham projections (Figure 33c). OVX did not cause a change in membrane capacitance (Figure 33d) nor rheobase (Figure 33e). These gonadectomy experiments demonstrated that androgen hormones may also affect vHPC-BLA projections, indicating that the effect of decreasing the availability of these hormones may affect the physiology of the HPC globally. Although not as robust as an effect as vHPC-NAc projections, ORCH did increase the excitability of vHPC-BLA projections while OVX did not affect their physiology. These results may indicate a broader role for androgen hormones in the regulation of HPC physiology, and may inform future studies of sex differences in other hippocampal circuits. Androgen receptor antagonism increases male vHPC-NAc excitability We next questioned whether androgen receptors (ARs) are directly involved in the regulation of vHPC-NAc physiology. To verify that ARs are present on vHPC-NAc projection neurons, we used double-label immunofluorescence to stain for AR and GFP in vHPC of L10-GFP reporter mice injected with retrograde HSV-Cre in NAc. As 106 250 150 50 150 250 100 Current Step (pA) 200 300 A p 0 5 200ms r e b m u N e k p S i 25 20 15 10 5 0 50 F - Sham F - OVX 200pA Figure 32 | Female ovariectomy and sham vHPC-BLA neurons did not differ in excitability. Action potential (AP) number across sequential depolarizing current steps (25-300 pA) for sham male (n = 13 cells from n = 4 animals) and ovariectomy (OVX) female (n = 11 cells from n = 3 animals) vHPC-BLA projections. OVX female projections did not differ in AP number across steps. Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: [25 pA t(264) = 0.4336, p > 0.9999], [50 pA t(264) = 0.6906, p = 0.9997], [75 pA t(264) = 0.4367, p > 0.9999], [100 pA t(246) = 0.2168, p > 0.9999], [125 pA t(246) = 0.1084, p > 0.9999], [150 pA t(246) = 0.3159, p > 0.9999], [175 pA t(246) = 0.4429, p > 0.9999], [200 pA t(246) = 0.5915, p > 0.9999], [225 pA t(246) = 0.6411, p = 0.9999], [250 pA t(246) = 0.7711, p = 0.9991], [275 pA t(246) = 0.8362, p = 0.9980], [300 pA t(246) = 0.9539, p = 0.9933]. Inset: sum of AP number across all current steps. Two-tailed Mann- Whitney U -test: U = 65, sum of ranks OVX = 169, Sham = 131, p = 0.7330. Representative traces below for sham and OVX vHPC-NAc projections, 200 pA step. 107 Membrane Resistance c ) Ω M ( e c n a t s s e R i 300 200 100 b ) Ω M ( e c n a t s s e R i Resting Membrane Potential a ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 -85 800 600 400 200 0 Input Resistance * OVX SHAM OVX SHAM d 0 OVX Capacitance SHAM e Rheobase ) F p ( e c n a t i c a p a C 200 150 100 50 0 OVX ) A p ( e s a b o e h R 100 80 60 40 20 0 SHAM OVX SHAM Figure 33 | Membrane properties for female ovariectomy and sham vHPC-NAc neurons. OVX and sham vHPC-NAc neurons from female mice did not differ in the measures of (a) resting membrane potential (mV; two-tailed t-test: t(22) = 0.07113, p = 0.9439); or (b) membrane resistance (MW; two-tailed t-test: t(22) = 0.2517, p = 0.8036). (c) OVX projections compared to sham had higher input resistance (MW; two-tailed t-test: t(22) = 2.738, *p = 0.0120). OVX and sham projections did not differ in the measures of (d) membrane capacitance (pF; two-tailed t-test: t(22) = 0.09835, p = 0.9225); or (e) rheobase (pA; two-tailed t-test: t(22) = 1.435, p = 0.1654). 108 predicted, vHPC-NAc projection neurons, as well as many of the surrounding pyramidal cells in vHPC CA1, do indeed express AR (Figure 34). To test whether ARs are directly involved in the regulation of vHPC-NAc neuronal excitability, the AR antagonist flutamide was used in conjunction with slice electrophysiology to determine effects on projection physiology. Either vehicle (DMSO) or flutamide + picrotoxin-spiked aCSF solutions were used in the slice incubation chamber as well as the main bath to record from vHPC-NAc cells in L10-GFP mice injected with retrograde Cre virus at NAc. Those cells exposed to flutamide + picrotoxin aCSF were found to have increased excitability compared to those treated with vehicle aCSF, as indicated by a significantly elevated total number of spikes across all current injections (Figure 35), although no significant differences were revealed at any current injections. Vehicle- and flutamide + picrotoxin-treated male vHPC-NAc neurons did not differ in any membrane properties (Figure 36a-f), nor sag ratio (Figure 36g). There were also no observed differences in sEPSC frequency (Figure 37a) nor amplitude (Figure 37b). These data support AR activation, possibly through androgen stimulation as suggested by our orchidectomy findings, as a potential direct regulator of excitability of male vHPC-NAc neurons that could mediate resilience to anhedonia following SCVS. Exogenous testosterone in female mice ameliorates susceptibility to SCVS As the above studies implicate circulating androgens in the mediation of stress resilience in male mice, we next sought to determine whether exogenous testosterone could protect female mice against the anhedonic effects of SCVS. To this end, we implanted 6mm Silastic blank or testosterone pellets in adult female mice 109 L10-GFP AR Merge Figure 34 | Ventral HPC to NAc projections express androgen receptors (ARs). Representative AR staining of vHPC slices from a male L10-GFP mouse; top left panel shows L10-GFP staining, top right panel shows AR staining, and the bottom panel shows a merged image demonstrating AR expression on vHPC-NAc projection cells. All images at 100x magnification. L10-GFP expression was induced in vHPC-NAc projections using same retrograde cre viral strategy described in previous figures. 110 V m 0 5 100 ms V m 0 5 100 ms 200pA * 200 100 0 Veh Flut 25 20 15 10 5 0 r e b m u N e k p S i 50 150 250 100 Current Step (pA) 200 300 Figure 35 | Acute application of the AR antagonist flutamide causes a slight increase in vHPC-NAc neuronal excitability in male mice. Representative traces for vehicle- and flutamide-treated vHPC-NAc neurons, 200 pA step (top). Action potential number across sequential depolarizing current steps (25-300 pA) for vehicle- (n = 20 cells from n = 7 animals) and flutamide-treated (n = 16 cells from n = 6 animals) vHPC-NAc projections. Two-way repeated measures ANOVA: [25 pA t(408) = 1.626, p = 0.4664], [50 pA t(408) = 2.573, p = 0.1182], [75 pA t(408) = 2.471, p = 0.1426], [100 pA t(408) = 2.151, p = 0.2782], [125 pA t(408) = 1.652, p = 0.4664], [150 pA t(408) = 1.511, p = 0.4664], [175 pA t(408) = 1.498, p = 0.4664], [200 pA t(408) = 1.652, p = 0.4664], [225 pA t(408) = 1.639, p = 0.4664], [250 pA t(408) = 1.882, p = 0.3542], [275 pA t(408) = 2.036, p = 0.3231], [300 pA t(408) = 1.972, 0.3328]. Total number of spikes for flutamide-treated cells vs vehicle-treated cells (inset) was increased; two-tailed t-test: t(34) = 2.119, *p = 0.0415. 111 c b Membrane Resistance 400 Resting Membrane Potential Input Resistance a ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 -85 d ) F p ( e c n a t i c a p a C 150 100 50 0 Sag Ratio Capacitance Veh Flut ) Ω M ( e c n a t s s e R i 300 200 100 0 Rheobase Veh Flut f ) V m ( e g a t l o V Veh Flut Veh Flut e ) A p ( e s a b o e h R 150 100 50 0 ) Ω M ( e c n a t s s e R i 600 400 200 0 Veh Flut g k a e P S S / 1.05 1.00 0.95 0.90 0.85 0.80 Voltage Threshold -30 -35 -40 -45 -50 -55 -60 Veh Flut Veh Flut Figure 36 | Membrane properties of male vehicle- and flutamide-treated vHPC-NAc projections. vHPC-NAc projection cells did not differ between male vehicle (DMSO) and flutamide- treated groups in any of the following measures: (a) resting membrane potential (mV). Two-tailed t-test: t(34) = 1.521, p = 0.1375; (b) membrane resistance (MW). Two-tailed t- test: t(34) = 1.702, p = 0.0979; (c) input resistance (MW). Two-tailed t-test: t(34) = 0.1268, p = 0.8998; (d) membrane capacitance (pF). Two-tailed t-test: t(34) = 1.317, p = 0.1966; (e) rheobase (pA). Two-tailed t-test: t(34) = 1.562, p = 0.1274; (f) voltage threshold (mV). Two-tailed t-test: t(34) = 0.3899, p = 0.6990; and (g) sag ratio. Two-tailed t-test: t(34) = 0.705, p = 0.4856. 112 b sEPSC Frequency ) A p ( e d u t i l p m A 30 20 10 0 c Veh Flut sEPSC Traces A p 0 2 500ms -80mV -80mV Veh Flut Veh Flut Figure 37 | Spontaneous activity measures of male vHPC-NAc projections treated with vehicle or flutamide. (a) Spontaneous EPSC frequency (Hz) [two-tailed Mann-Whitney U-test: U = 121, sum of ranks Veh = 409, Flut = 257, p = 0.2334] and (b) amplitude [two-tailed t-test: t(34) = 0.6132, p = 0.5438] did not differ between Flut and Veh groups. (c) Example sEPSC traces for vehicle (top) and flutamide (bottom) vHPC-NAc recordings. a ) z H ( y c n e u q e r F 30 20 10 0 sEPSC Amplitude 113 simultaneously with ovariectomy (OVX). Following OVX and implantation, 28 days were allowed for recovery and hormone equilibration. Mice were then subjected to the 6-day SCVS battery, and behavioral assessment began immediately following the last day of SCVS (Fig 38, top). There was a significant interaction between stress and hormone status, with female mice implanted with blank pellets having reduced sucrose preference after SCVS and those implanted with testosterone pellets showing no effect of stress on sucrose preference, indicating resilience similar to that of male mice (Figure 38, bottom). There was a main effect of stress on % time grooming in the splash test, with stress- exposed mice spending less time grooming (Figure 39a). In the EPM test, a main effect of stress was observed in the distance moved (Figure 39b), and there was an interaction between stress and sex in the % open arm time (Figure 39c) There was a main effect of testosterone on the NSF test, with testosterone-implanted mice demonstrating less latency to feed (Figure 39d). There were no main effects of stress nor testosterone nor an interaction between variables in the assessment of SI ratio (Figure 39e). These data suggest a direct protective effect of testosterone, or possibly its metabolic derivatives, against SCVS-induced anhedonia in female mice. Exogenous testosterone mitigates hyperexcitability in female vHPC-NAc neurons We next questioned whether exogenous testosterone could also attenuate the hyperexcitability of female vHPC-NAc neurons. To this end, we injected L10-GFP female mice with retrograde HSV-Cre in NAc, and after one week of recovery, subjected the mice to OVX and pellet implantation in a single operation. After allowing two more weeks for recovery and hormone equilibration, we then performed whole-cell patch clamp 114 OVX and pelleting SCVS 100 Day 1 Day 28 **** Behavior Assessment Sucrose Preference Day 37 Control SCVS f e c n e r e e r P e s o r c u S % 80 60 40 Blank +T Figure 38 | Female susceptibility to SCVS-induced anhedonia is ameliorated by adult testosterone. Schematic depicting experimental implantation surgery, ovariectomy, SCVS, and behavior assessment (top). OVX females with blank pellet implants maintained a reduction in sucrose preference following SCVS, while OVX female mice exposed to testosterone showed no significant reduction in sucrose preference following SCVS, indicating resilience (bottom). Two-way ANOVA: Group OVX + Blank vs OVX + T F(1,34) = 26.00, p = 0.0580; Trial: Control vs Stress F(1,34) = 20.79, p < 0.0001; Group X Trial F(1,34) = 3.849, p < 0.0001; Sidak’s multiple comparisons: OVX + Blank mean difference 11.78, 95% ****CI = 5.965 to 17.6; OVX + T mean difference 4.693, 95% CI = -1.441 to 10.83. time course of pellet 115 a i g n m o o r G e m T i % 80 60 40 20 0 +Bl d ) s ( d e e F o t y c n e a L t +T 600 400 200 0 c i e m T A O % 20 15 10 5 0 b ) m c ( e c n a t s D i 2000 1500 1000 500 0 +Bl e Control SCVS +T 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 o i t a R I S +Bl +T +Bl +T +Bl +T Figure 39 | Additional behavioral assays of female OVX + Blank and OVX + T SCVS. All behavioral assays other than sucrose preference were unaffected by hormone status with SCVS in OVX + Blank and OVX + T groups, with no interaction of stress and hormone status in (a) Percent time grooming in splash test. Two-way ANOVA: Group OVX + Bl vs OVX + T F(1,35) = 0.0884, p = 0.7680; Trial: Control vs Stress F(1,35) = 5.449, p = 0.0254; Group X Trial F(1,35) = 3.598, p = 0.0661; (b) Distance moved (cm) in EPM. Two- way ANOVA: Group: OVX + Bl vs OVX + T F(1,34) = 0.6634, p = 0.4210; Trial: Control vs Stress F(1,34) = 5.778, p = 0.0218; Group X Trial F(1,34) = 3.132, p = 0.0857; (c) Percent open arm (OA) time in EPM did show a significant interaction between stress and hormone status, but no differences between stress and control in either the OVX + Bl or OVX + T group with Sidak’s multiple comparisons test. Two-way ANOVA: Group: OVX + Bl vs OVX + T F(1,34) = 1.78, p = 0.1910; Trial: Control vs Stress F(1,34) = 0.0865, p = 0.7705; Group X Trial F(1,34) = 7.131, p = 0.0114; Sidak’s multiple comparisons: OVX + Bl mean difference 3.05, 95% CI = -1.07 to 7.17; OVX + T mean difference -3.805, 95% CI = -8.175 to 0.5647. There was also no interaction between hormone status and stress in the measures of (d) latency to feed (s) in novelty suppressed feeding test. Two-way ANOVA: Group: OVX + Bl vs OVX + T F(1,33) = 36.41, p < 0.0001; Trial: Control vs Stress F(1,33) = 0.4705, p = 0.4975; Group X Trial F(1,33) = 1.291, p = 0.2641; (e) SI Ratio in social interaction test. Two-way ANOVA: Group: OVX + Bl vs OVX + T F(1,35) = 0.3507, p = 0.5575; Trial: Control vs Stress F(1,35) = 0.4839, p = 0.4913; Group X Trial F(1,35) = 0.681, p = 0.4148. 116 electrophysiology on vHPC-NAc projections. We found that OVX mice implanted with testosterone pellets exhibited significantly decreased excitability in vHPC-NAc neurons compared to those with blank pellets (Figure 40 bottom, example traces for each group above). No differences were observed between testosterone- and blank-pelleted OVX mice in the measures of VM, RM, Rin, or CM (Figure 41a-d). Mice implanted with testosterone pellets had an increased rheobase compared to those implanted with blank pellets (Figure 41e), supporting the decrease in excitability with testosterone treatment noted above. However, no difference was observed between testosterone- and blank- pelleted OVX mice in the measure of AP voltage threshold (Figure 41f), which was observed in female WT vHPC-NAc neurons compared to male WT projections. Sag ratio was no different between testosterone and blank pellet groups (Figure 41g), indicating similar afterhyperpolarization current in both groups. There were also no observed differences in sEPSC amplitude nor frequency between OVX + Blank and OVX + T groups (Figure 42a-c), suggesting no immediate pre- nor postsynaptic changes with testosterone treatment. Testosterone treatment, however, did enhance spike frequency adaptation of vHPC-NAc neurons in OVX mice, as indicated by a greater increase in interspike interval over the course of a depolarizing current step (Figure 43a). This enhancement of spike frequency adaptation was accompanied by an increase in latency to first spike in the same current step (Figure 43b). Overall, these results suggest that exogenous testosterone, or its metabolic derivatives, reduces the excitability profile of female vHPC- NAc neurons and, as noted in the SCVS experiments above, drives resilience to SCVS- induced anhedonia in female mice. 117 V m 0 5 100 ms V m 0 5 100 ms 200pA ** ** ** ** ** ** ** ** ** ** ** 200 100 0 150 250 100 Current Step (pA) 200 300 Blank +T 25 20 15 10 5 0 50 r e b m u N e k p S i Figure 40 | Female OVX vHPC-NAc excitability is decreased with chronic testosterone. Representative traces for OVX + BLANK and OVX + T vHPC-NAc projections, 200 pA step (top). Action potential number across sequential depolarizing current steps (25-300 pA) for OVX + BLANK (n = 11 cells from n = 3 animals) and OVX + T (n = 19 cells from n = 5 animals) vHPC-NAc projections. OVX + T projections showed significantly lower AP number at all steps > 50 pA. Two-way repeated measures ANOVA, Holm-Sidak multiple comparisons: [25 pA t(420) = 0.165, p = 0.8690], [50 pA t(420) = 1.762, p = 0.1514], [75 pA t(420) = 3.036, **p = 0.0076], [100 pA t(420) = 3.687, **p = 0.0026], [125 pA t(420) = 3.847, **p = 0.0017], [150 pA t(420) = 3.739, **p = 0.0023], [175 pA t(420) = 3.579, **p = 0.0035], [200 pA t(420) = 3.453, **p = 0.0049], [225 pA t(420) = 3.309, **p = 0.0061], [250 pA t(420) = 3.375, **p = 0.0056], [275 pA t(420) = 3.187, **p = 0.0062], [300 pA t(420) = 3.249, **p = 0.0062]. Inset: sum of AP number across all current steps. Two-tailed t-test: t(35) = 3.254, **p = 0.0025. 118 Resting Membrane Potential Membrane Resistance c ) Ω M ( e c n a t s s e R i 300 200 100 Input Resistance 800 600 400 200 b ) Ω M ( e c n a t s s e R i a ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 d ) F p ( e c n a t i c a p a C 150 100 50 0 Sag Ratio Membrane Capacitance +BL +T +BL +T +BL +T e ) A p ( e s a b o e h R 200 150 100 50 0 Rheobase * +BL +T +T 0 +BL f ) V m ( e g a t l o V -30 -35 -40 -45 -50 0 +BL +T g Voltage Threshold k a e P S S / +BL +T 1.1 1.0 0.9 0.8 0.7 Figure 41 | Membrane properties of female OVX + Blank and OVX + T vHPC-NAc projections. vHPC-NAc projection cells did not differ between female OVX + Blank and OVX + T groups in any of the following measures: (a) resting membrane potential (mV). Two-tailed t-test: t(34) = 0.6576, p = 0.5152; (b) membrane resistance (MW). Two-tailed Mann- Whitney U-test: U = 107, sum of ranks OVX + Bl = 349, OVX + T = 317, p =0.0949; (c) input resistance (MW). Two-tailed t-test: t(34) = 0.6972, p = 0.4904; and (d) capacitance (pF). Two-tailed t-test: t(34) = 0.5770, p = 0.5672. Female OVX + T vHPC-NAc projection cells showed an increased rheobase (pA) (e) compared to OVX + Blank projections. Two- tailed t-test: t(34) = 2.643, *p = 0.0123. (f) Voltage threshold (mV) [two-tailed t-test: t(34) = 0.7842, p = 0.4384] and (g) sag ratio [two-tailed t-test: t(34) = 0.4344, p = 0.6668] did not differ between OVX + Blank and OVX + T groups. 119 a ) z H ( y c n e u q e r F 30 20 10 0 sEPSC Frequency b A p ( e d u t i l ) p m A +BL +T sEPSC Traces sEPSC Amplitude c +BL +T +BL +T A p 0 2 500ms 30 20 10 0 -80mV -80mV Figure 42 | Spontaneous activity measures of female OVX + Blank and OVX + T vHPC-NAc projections. (a) Spontaneous EPSC frequency (Hz) [two-tailed t-test: t(34) = 0.4026, p = 0.6898] and (b) amplitude [two-tailed Mann-Whitney U-test: U = 123, sum of ranks OVX + Bl = 372, OVX + T = 294, p = 0.2231] did not differ between OVX + Blank and OVX + T groups. (c) Example sEPSC traces for OVX + Blank (top) and OVX + T (bottom) vHPC-NAc recordings. 120 *** OVX + Blank OVX + T 1st Pair Last Pair 80 60 40 20 0 b ) s m ( y c n e t a L 80 60 40 20 0 a ) s m ( l a v r e n t i I e k p s r e t n I * + Bl + T Figure 43 | Spike frequency adaptation is enhanced by chronic testosterone in OVX female vHPC-NAc projections. Interspike interval increases over the course of 500 ms current injection as measured by the time (ms) between the 1st pair of spikes and last pair of spikes in female OVX + Blank and OVX + T vHPC-NAc neurons. (a) OVX + T vHPC-NAc neurons had greater spike frequency adaptation as measured by the interspike interval increase over current step; interspike interval of the last pair of spikes was significantly longer in OVX + T vHPC-NAc projections than in OVX + Bl projections. One-way ANOVA F(3,70) = 77.7, p < 0.0001; Sidak’s multiple comparisons: OVX + T 1st pair vs OVX + Bl 1st pair mean difference = 4.714, 95% CI = -2.952 to 12.38, OVX + T last pair vs OVX + Bl last pair mean difference = 12.69, 95% ***CI = 5.026 to 20.36, adjusted p = 0.0003. (b) Latency to 1st spike (ms) from beginning of current injection was also increased in OVX + T vHPC-NAc neurons compared to OVX + Bl. Two-tailed Mann-Whitney U-test: U = 105.5, sum of ranks OVX + Bl = 276.5, OVX + T = 426.5, *p = 0.0465. 121 vHPC-NAc excitability directly mediates SCVS-induced susceptibility to anhedonia The above experiments parallel susceptibility to stress and vHPC-NAc excitability, but do not directly link the physiology of this circuit to stress outcomes. To determine whether vHPC-NAc neuronal excitability is directly causative of susceptibility to SCVS- induced anhedonia, we utilized an intersecting viral DREADD strategy. In wild-type mice, retrograde HSV-Cre was injected into NAc, and a Cre-dependent AAV- mCherry-hM4Dq (Gq-coupled DREADD in male mice) or AAV-mCherry-hM3Di (Gi-coupled DREADD in female mice) was injected into vHPC (Figure 44a, top). Using this strategy, only vHPC- NAc neurons infected with both viruses expressed the respective DREADD and were to be affected by systemic clozapine-N-oxide (CNO) administration. We verified Gq- and Gi- coupled DREADD activation with CNO using slice electrophysiology in vHPC guided by the mCherry tag and recording before and after CNO wash-on (timeline Figure 44a, bottom). We demonstrated that mCherry+, Gq-coupled DREADD-expressing vHPC-NAc neurons in males had elevated excitability following CNO wash-on, indicated by an increased number of spikes at 150 pA injected current, as well as a decrease in rheobase (representative cell shown in Figure 44b). Likewise, female vHPC-NAc neurons expressing the Gi-coupled DREADD showed diminished excitability following CNO wash- on, indicated by a decrease in the number of spikes at 150 pA injected current, as well as an increase in rheobase (representative cell shown in Figure 44c). Together, these recordings verify the efficacy of DREADD activation using the designer drug CNO. Three weeks after the second viral injection surgery male mice expressing the excitatory Gq-coupled DREADD in vHPC-NAc projections were exposed to SCVS 122 Figure 44 | Viral DREADD proof-of concept electrophysiology. (a, top) Schematic depicting intersecting viral DREADD strategy for circuit-specific manipulation of vHPC-NAc excitability and experimental time course. AAV encoding Cre- inducible DREADD (either Gq- or Gi-coupled) was injected in vHPC, and retrograde HSV- Cre was injected in NAc, causing circuit-specific receptor expression. (a, bottom) Schematic depicting time course for DREADD recordings. Cells were first recorded in regular aCSF, then CNO-containing aCSF was washed on and the same cell was recorded 8-10 minutes later. (b and c) Whole-cell slice electrophysiology demonstrating CNO activating Gq- (b) and Gi-coupled (c) receptors. Example traces before (grey) and after (yellow) CNO application along with rheobase at time of each recording. One cell is represented each in (b) and (c). 123 followed by intraperitoneal CNO or saline injection every day of behavioral testing (timeline Figure 45a, top). Interestingly, CNO failed to directly evoke changes in sucrose preference (Figure 45a, bottom), suggesting that a short-term activation of the vHPC-NAc circuit is not sufficient to induce anhedonia following stress. A second group of male mice expressing Gq-coupled DREADD in vHPC-NAc neurons was exposed to SCVS with CNO or saline administration throughout both stress and behavior assessment (Figure 45b, top). Long-term CNO treatment in male mice caused a dramatic decrease in sucrose preference even in the control-handled group, as well as a further SCVS-induced decrease in sucrose preference (Figure 45b, bottom). There was also a significant interaction between stress and CNO treatment in social interaction assessment in the short CNO administration, with SI ratio being decreased following SCVS in the CNO- treated group (Figure 46a). There was no interaction between stress and CNO treatment in EPM open arm time for the short CNO administration experiment (Figure 46b). For the long CNO administration group, SI assessment did show a main effect of stress on reduction of SI ratio, but no interaction between stress and CNO treatment variables (Figure 46c). EPM open arm time for this experiment remained unaffected by stress and CNO treatment with no interaction between the two (Figure 46d). Importantly, we also performed a control experiment with a separate cohort of male mice that did not express any DREADDs in the brain, but did receive CNO or saline IP injections during SCVS and behavior assessment (Figure 47, top). Similar to male mice with no viral modifications or drug treatments, these mice were not affected by SCVS in the measure of sucrose preference (Figure 47, bottom): there was not a main effect of stress nor CNO treatment, with no interaction between the two. These data suggest that short-term activation of the 124 100 e c n e r e f e r P e s o r c u S % 80 60 40 30 CNO Control SCVS ** 100 e c n e r e f e r P e s o r c u S % 80 60 40 a AAV-Gq injection CNO: Behavior Only HSV- hEF1α- CRE injection SCVS Behavior Assessment Sucrose Preference b CNO: Stress + Behavior AAV-Gq injection HSV- hEF1α- CRE injection SCVS Behavior Assessment Sucrose Preference Day: 1 10 24 30 CNO Day: 1 10 24 VEH CNO VEH CNO Figure 45 | Long-term reduction of vHPC-NAc activity causes susceptibility to SCVS-induced anhedonia in male mice. (a, top) Experimental timeline of DREADD surgery, retrograde Cre surgery, SCVS, and subsequent behavioral assessment for short (behavior-only) DREADD experiment for male mice. (a, bottom) Male mice expressing excitatory Gq-coupled DREADD in vHPC- NAc projections and exposed to CNO only during behavior assessment did not show any change in sucrose preference following SCVS. Two-way ANOVA: Group: Saline vs CNO F(1,36) = 0.9478, p = 0.3368; Trial: Control vs Stress F(1,36) = 0.001178, p = 0.9728; Group X Trial F(1,36) = 0.03545, p = 0.8517 (no interaction); n = 10 control vehicle, 10 control CNO, 10 SCVS vehicle, 10 SCVS CNO. (b, top) Experimental timeline for long (CNO during both SCVS and behavior assessment) DREADD experiment for male mice. (b, bottom) Male mice expressing excitatory Gq-coupled DREADD in vHPC-NAc projections and exposed to CNO during both SCVS and behavior assessment experienced an overall reduction in sucrose preference and a further reduction in sucrose preference following SCVS. Two-way ANOVA: Group: Saline vs CNO F(1,36) = 133.6, p < 0.0001; Trial: Control vs Stress F(1,36) = 9.481, p = 0.0040; Group X Trial F(1,36) = 5.086, p = 0.0303. Sidak’s multiple comparisons: VEH Control vs Stress mean difference = 1.94, 95% CI = -5.83 to 9.71; CNO Control vs Stress mean difference = 12.56, 95% **CI = 4.79 to 20.33; n = 9 control vehicle, 10 control CNO, 9 SCVS vehicle, 10 SCVS CNO. 125 a b 4 3 2 1 0 20 15 10 5 o i t a R I S e m T i A O % c * o i t a R I S VEH CNO d e m T i A O % 4 3 2 1 0 20 15 10 5 VEH CNO 0 0 VEH CNO VEH CNO Figure 46 | Additional behavioral assays of Gq-coupled (male) DREADD- expressing vHPC-NAc mice. For the short CNO administration Gq DREADD vHPC-NAc activation, there was also an interaction between SCVS and male vHPC-NAc activation in (a) SI ratio in social interaction test, with CNO injections reducing social interaction after stress [two-way ANOVA: Group: Vehicle vs CNO F(1,35) = 0.1444, p = 0.7063; Trial: Control vs Stress F(1,35) = 0.2276, p = 0.1387; Group X Trial F(1,35) = 4.212, *p = 0.0477; Sidak’s multiple comparisons: Vehicle control vs stress mean difference = -0.05422, 95% CI = -0.3922 to 0.2838, CNO control vs stress mean difference = 0.3601, 95% *CI = 0.03111 to 0.6891] but (b) percent open arm time in EPM remained unaffected [two-way ANOVA: Group: Veh vs Control F(1,33) = 1.097, p = 0.3025; Trial: Control vs Stress F(1,33) = 0.2781, p = 0.6015; Group X Trial F(1,33) = 0.59, p = 0.4479 (no interaction)]. No interaction was observed between SCVS and long CNO administration Gq DREADD vHPC-NAc activation in any of the following measures (c) SI ratio in social interaction test [two-way ANOVA: Group: Vehicle vs CNO F(1,36) = 0.5818, p = 0.4506; Trial: Control vs Stress F(1,36) = 5.372, p = 0.0263; Group X Trial F(1,36) = 0.0530, p = 0.8192 (no interaction)] and (d) percent open arm (OA) time in EPM [two-way ANOVA: Group: Vehicle vs Control F(1,34) = 0.3007, p = 0.3213; Trial: Control vs Stress F(1,34) = 0.5462, p = 0.4650; Group X Trial F(1,34) = 0.161, p = 0.6908 (no interaction)]. 126 No DREADD, CNO: Stress + Behavior SCVS Sucrose Preference Day 1 100 Control SCVS 6 CNO Figure 47 | Exposure to CNO without the presence of DREADD expression in vHPC-NAc neurons in male mice does not cause SCVS-induced anhedonia. There was no interaction between stress and CNO exposure in male mice when DREADD expression was not present in male vHPC-NAc neurons [two way ANOVA: Group: Vehicle vs CNO F(1,35) = 0.7583, p = 0.3898; Trial: Control vs Stress F(1,35) = 0.5333, p = 0.4701; Group X Trial F(1,35) = 0.7462, p = 0.3935 (no interaction)]. 127 e c n e r e f e r P e s o r c u S % 80 60 40 VEH CNO vHPC-NAc circuit using Gq-coupled DREADDs does not cause anhedonia in stressed or non-stressed male mice. However, our findings demonstrate that a prolonged increase in the excitability of this circuit induces anhedonia even in the absence of stress, and if these changes in excitability are paired with stress, the anhedonic response in male mice is enhanced. To complement the study of activation of the vHPC-NAc circuit in male mice in the context of stress, female mice expressing the inhibitory Gi-coupled DREADD in vHPC- NAc projections were exposed to SCVS while CNO or saline was administered intraperitoneally each day of stress and throughout behavior assessment (Figure 48, top). Saline-treated female mice showed a decrease in sucrose preference following SCVS with an interaction between stress and CNO treatment that reached p=0.0518, while CNO-treated female mice showed a mitigation of this susceptibility to anhedonia following stress (Figure 48, bottom). There was no significant interaction between stress and CNO treatment in the measures of SI ratio (Figure 49a) or EPM open arm time (Figure 49b), but there was a possible anxiolytic effect of CNO treatment in the EPM test, with the main effect indicating an increase in the time spent in the open arms of the maze in CNO- treated animals. Our findings in Gi-coupled DREADD-expressing female mice demonstrate that the inactivation of this circuit, in contrast to the activating experiments in male mice, can alleviate stress-induced anhedonia. Taken together with the above DREADD experiments in male mice, these data indicate that prolonged, but not acute, alterations in vHPC-NAc neuronal excitability can elicit or relieve anhedonic behavioral effect of SCVS according to whether they are activating or inactivating. 128 CNO: Stress + Behavior AAV-Gi injection HSV- hEF1α- CRE injection SCVS Behavior Assessment Sucrose Preference Day: 1 10 24 100 #p=0.0518 30 CNO Control SCVS f e c n e r e e r P e s o r c u S % 80 60 40 VEH Figure 48 | Inhibition of vHPC-NAc activity directly mediates resilience to SCVS- induced anhedonia. (Top) Experimental timeline for female mice. (Bottom) Female mice expressing inhibitory Gi-coupled DREADD in vHPC-NAc projections and exposed only to vehicle (saline) had decreased sucrose preference following SCVS (much like that of non-modified females), but those exposed to activating CNO showed no change in sucrose preference following SCVS, indicating resilience to SCVS-induced anhedonia. Two-way ANOVA: Group: Saline vs CNO F(1,34) = 0.2236, p = 0.6393; Trial: Control vs Stress F(1,34) = 1.041, p = 0.3147; Group X Trial F(1,34) = 4.064, #p = 0.0518; n = 10 control vehicle, 8 control CNO, 10 SCVS vehicle, 10 SCVS CNO. CNO 129 Control SCVS a o i t a R I S 4 3 2 1 0 b i e m T A O % 20 15 10 5 0 130 VEH CNO VEH CNO Figure 49 | Additional behavioral assays of Gi-coupled (female) DREADD- expressing vHPC-NAc mice. Female mice expressing inhibitory Gi-coupled DREADD in vHPC-NAc projections had no interaction between CNO treatment and stress in the measures of (a) SI ratio [two-way ANOVA: Group: Vehicle vs CNO F(1,36) = 0.7445, p = 0.3939; Trial: Control vs Stress F(1,36) = 0.0876, p = 0.7689; Group X Trial F(1,36) = 1.832, p = 0.1844 (no interaction)]; and (b) percent open arm time in EPM [two-way ANOVA: Group: Vehicle vs CNO F(1,34) = 4.658, p = 0.0381; Trial: Control vs Stress F(1,34) = 3.642, p = 0.0648; Group X Trial F(1,34) = 0.0797, p = 0.7794 (no interaction)]. There was a main effect of stress on the percent OA time in EPM, with mice exposed to SCVS spending less time in the open arms. Sex-specific transcriptome interrogation of vHPC-NAc neurons The above findings regarding the excitability of vHPC-NAc neurons in the context of stress responses and the role of androgens in the regulation of this excitability introduces a new physiologic mechanism that works to explain why women are more likely to suffer from depression than men. However, the mechanisms by which androgens may mediate lower excitability in this circuit to cause the observed sex differences are unclear. To investigate potential mechanisms by which the excitability of male and female vHPC-NAc neurons differ and to begin to dissect how androgens affect the physiology of this circuit, we utilized translating ribosome affinity purification (TRAP; Figure 50) to interrogate circuit-specific gene expression. Using the retrograde Cre strategy described in the above experiments, vHPC-NAc neurons of male and female mice were transfected to express a GFP tag on the L10 ribosomal subunit (L10-GFP) and bilateral ventral hippocampus punches were collected and pooled (2 hippocampus punches per animal, 4 animals per pooled sample). Using this technique, actively translating mRNA from this circuit was purified and used to prepare cDNA libraries for sequencing (Figure 50). RNA sequencing (RNAseq) revealed enrichment of neuron-specific mRNA (e.g. Vamp2, Figure 51a) in the pulldown samples compared to input, while genes specific to glia (e.g. Slc14a1 and Bcas1) were depleted (Figure 51b-c). Vamp2 encodes synaptobrevin 2, a protein member of the complex responsible for synaptic vesicle docking and fusion to the presynaptic membrane for neurotransmitter release292. Slc14a1 is a urea transporter gene enriched in astrocytes, and Bcas1 is a gene related to myelination in oligodendrocytes293. This enrichment of neuron-specific genes and depletion of glia- related genes indicated that our pulldown of vHPC-NAc neurons was efficient. Moreover, 131 Figure 50 | Translating ribosome affinity purification (TRAP) allows interrogation of circuit-specific vHPC-NAc transcriptome. Schematic depicting retrograde L10-GFP labeling strategy and general TRAP and sequencing steps: 1. Retrograde Cre virus (the same as used in electrophysiology experiments in this work) is injected into the NAc. Cre virus is transported via nerve terminals in the NAc to cell bodies of origin in other brain regions to induce expression of a GFP tag ribosomal subunit L10 (L10-GFP). 2. The vHPC, containing cell bodies of vHPC-NAc projection cells expressing L10-GFP, is excised for processing. 3. Tissue is processed and L10-GFP ribosomes, bound to translating messenger RNA (mRNA), are immunoprecipitated using magnetic beads bound to anti-GFP antibodies. 4. Bound mRNA is extracted from antibodies, and mRNA is prepared for sequencing. 5. RNA sequencing (RNAseq) is used to compare transcriptomes from vHPC-NAc projections in groups of interest (e.g. male vs female mice). 132 a b c +3 +2 Vamp2 +4 +5 (kb) chr11:69,093,000 M F Input +20 +30 Slc14a1 +10 0 (kb) chr18:78,095,000 M F Input 0 +1 ch11:69,088,000 d e h c i r n e n o r u e N 100 0 100 0 0.1 0 100 0 +50 +40 ch18:78,145,000 d e h c i r n e e t y c o r t s A 2 0 2 0 2 0 +90 +72 +54 +36 +18 0 (kb) ch2:170,430,000 Bcas1 chr2:170,340,000 2 0 2 0 2 0 M F Input d e h c i r n e e t y c o r d n e d o g i l O Figure 51 | TRAP strategy is efficient in pulldown of neuronal mRNA. (a) RNA sequencing reads for neuron-specific gene vesicle-associated membrane protein 2 (VAMP2) in male (blue) and female (red) TRAP samples compared to input (black). Gene depletion was also verified for genes related to glia, as these genes were expected not to be enriched in the neuron-specific TRAP pulldown. (b) Astrocyte-related gene Slc14a1 was depleted in male and female TRAP samples compared to input. (c) Oligodendrocyte-related gene Bcas1 depleted in male and female TRAP samples compared to input. Enrichment of neuron-related genes but depletion of glia-related genes in TRAP samples verifies that the TRAP pulldowns were neuron-specific. 133 we observed that more transcripts were higher than were lower in abundance in male mice compared to female mice (Figure 52), suggesting the possibility of an active mechanism in this circuit driving resilience to stress-induced anhedonia. Importantly, we also observed the largest difference in transcript abundance between the sexes in Y- linked genes (many orders of magnitude more abundant in males, see inset Figure 52), indicating that our strategy for uncovering sex differences in gene expression in the vHPC-NAc circuit was valid. Sequencing of TRAP mRNA from vHPC-NAc neurons revealed many differentially regulated genes between female and male vHPC-NAc neurons (Appendix 1), a variety of which are highlighted in Figure 53a. Many of these genes represent potential mediators of the baseline sex difference in excitability that we observed in this circuit. Moreover, Ingenuity pathway analysis (IPA, Qiagen; Hilden, Germany) applied to these TRAP RNAseq data reveals many cellular pathways that could be involved in regulating hippocampal excitability (Figure 53b). These analyses demonstrate successful enrichment of vHPC-NAc mRNA and reveal clear sex differences in the transcriptome of this circuit, and point to several potential mechanisms for the excitability differences we observed between male and female mice. These transcriptomics results uncovered myriad paths for the future study of the mechanisms of stress response in rodent models of depression, and may help to elucidate molecular mediators of the difference in MDD between men and women. Discussion In this chapter, we utilized behavior assessment, whole-cell slice electrophysiology, dual-viral DREADD manipulation of circuit excitability, and circuit- 134 Downregulation of Y 40 30 20 10 -15 -10 -5 <0.0001 0.0001-0.01 0.01-0.05 >0.05 ±2 log2foldchange p=0.05 -4 -2 log2FoldChange 0 2 4 ) j d a p ( g o L - 5 4 3 2 1 0 Figure 52 | Plot of transcript reads vs fold change in female and male samples verifies efficacy of sex-specific vHPC-NAc TRAP and highlights most analysis- ready genes. Volcano plot showing distribution of transcript enrichment in female vs. male vHPC-NAc TRAP. Inset shows Y-linked genes: Uty [-log(padj)=32.662]; Eif2s3y [-log(padj)=13.517]; Kdm5d [-log(padj)=19.380]; Ddx3y [-log(padj)=24.487]. X-linked genes that were greatly significantly enriched in female mice were omitted for clarity of illustration. Genes above dashed line (indicating p = 0.05) are those that were significantly downregulated in females compared to males in vHPC-NAc projections, with the Y-linked genes being the extreme examples. Complete RNAseq results can be found in Appendix A, including a list of differentially expressed transcripts in female vs male vHPC-NAc neurons. 135 b Sirtuin Sig. Neuregulin Sig. GABA-R Sig. cAMP Sig. CREB Sig. GnRH Sig. nNOS Sig. Neurotrophin Sig. Percentage of Pathway 0 10 20 30 40 50 60 70 80 90 100 160 64 61 136 148 109 y a w h t a P n i s e n e G # l 31 t a o 59 T a ADCY8 ADCY1 CACNA1C CACNA1D Gene Name log2foldchange -0.395351265 -0.640754251 -0.277446531 -0.308260359 -0.374359973 -1.545788205 0.221996784 0.210360400 KCNN3 SVIL CAMK2A NOS1 p value 6.58E-06 0.018048473 0.035693124 0.008390456 0.020067456 1.30E-06 0.025480145 0.009662824 Downregulated Upregulated Figure 53 | Ingenuity pathway analysis reveals many differentially regulated pathways in female vs male vHPC-NAc transcriptomes. (a) Table listing selected significantly differentially regulated genes. Log2foldchange listed for female vHPC-NAc neurons compared to male vHPC-NAc neurons. Selection of these genes was made based upon relevance to mood disorder pathophysiology or cellular excitability as discussed in the text. (b) Selected pathways affected by sex as determined by Ingenuity Pathway Analysis. Down- and upregulated gene numbers (female vs male) represented as a percentage of the total number of genes within the given pathway. Many other differentially regulated pathways were identified; this list was selected based on clear involvement in neuronal function. 136 specific transcriptome interrogation to uncover the potenital neurophysiological mechanism for sex differences in responses to stress, with focus on stress-induced anhedonia. We found that susceptibility to anhedonia following SCVS parallels an exaggerated vHPC-NAc excitability profile in female mice. We demonstrated that androgens or androgen derivatives underlie the observed sex differences in behavior and vHPC-NAc excitability. We also demonstrated a time-dependent relationship between vHPC-NAc circuit physiology and stress susceptibility to anhedonia. Increasing the excitability of male vHPC-NAc neurons was sufficient to precipitate SCVS-induced anhedonia, but only when this excitability was increased over a prolonged period. This time dependence in behavioral changes suggests a necessary long-term adaptation to changes in circuit physiology. Complementing the male mouse studies, we demonstrated that decreasing the excitability of vHPC-NAc neurons over a prolonged period is sufficient to confer resilience to SCVS-induced anhedonia in female mice. As there is an enigmatic sex difference in human depression diagnosis, including the variable of sex in preclinical models of mood disorders is critical. As previously discussed, women are approximately twice as likely as men to be diagnosed with depression across the lifespan294, and these diagnoses often correspond to female- specific changes to the reproductive cycle such as puberty, menstruation, postpartum, and menopause295,296. Meta-analyses demonstrate that adult (18-60yrs) but not aged (>60yrs) males with low testosterone are more likely to experience depression symptoms. These symptoms, including anhedonia, have been shown to be significantly reduced by testosterone treatment297. However, exploring the molecular and physiological etiologies of sex differences in depression has been difficult, in part due to the fact that many 137 preclinical behavioral models of depression utilize intrasexual aggression, leaving female subjects understudied34,38,298. Recently, new stress models, including SCVS41,170, have attempted to address this inequality of sexes in animal subjects299-302. In the current chapter, we employed SCVS in both male and female subjects to explore sex differences in stress outcomes. Previous work has identified the importance of the NAc in the regulation of SCVS- induced behaviors. The NAc, for example, is the site of extensive transcriptional and epigenetic changes in response to stress, and many studies have implicated steroid hormones and their receptors in regulating these processes41,287,303. Recent work has also demonstrated that stress-induced changes in female rodents may be presynaptic, either at glutamatergic inputs onto NAc medium spiny neurons or in other reward-related circuits170,304. This complements previous work in male animals that has shown that the strength of vHPC-NAc synapses underlies susceptibility to CSDS2. Additionally, circulating steroid hormones are known to affect vCA1 structure and function305-307 as well as social and hippocampal-dependent learning308,309. Human studies have also demonstrated a decreased hippocampal volume in patients with MDD310. Together, these works implicate vHPC as a key mediator of sex differences in stress outcomes in preclinical models and depression development in humans, particularly through its excitatory projections to the NAc. In this chapter, we showed that vHPC afferents to NAc, but not BLA, are more excitable in female mice, and that testosterone supplementation is sufficient to decrease the excitability of the vHPC-NAc circuit and confer resilience to SCVS in female mouse subjects. As discussed above, vHPC-NAc projections and the strength of the connectivity 138 between these regions have been shown to modulate social behaviors311,312. Here, we demonstrated vHPC-NAc projection-specific regulation of the likewise positively-valenced measure of sucrose preference, a well-validated measure of anhedonia34,313,314. We confirmed that reduced sucrose preference following SCVS is female-specific and showed that this susceptibility correlated with higher vHPC-NAc, but not vHPC-BLA, excitability. This sex difference was found to be dependent on gonad hormones, as orchidectomy in male mice increased vHPC-NAc excitability and induced anhedonia after SCVS, and testosterone supplementation in female mice decreased vHPC-NAc excitability and prevented SCVS-induced anhedonia. It is important to note, however, that our hormone manipulations were systemic and may therefore act in a non-cell autonomous manner, perhaps exerting their effects indirectly at the level of adjacent circuitry or possibly via the actions of glial support cells. However, we showed using immunohistochemistry that vHPC-NAc projection cells do indeed express AR, and bath application of the AR antagonist flutamide increases vHPC-NAc excitability. Therefore, we hypothesize that direct activation of androgen receptors mediates these effects, potentially via changes in the vHPC-NAc cells themselves. The baseline sex differences we demonstrated in vHPC-NAc excitability and behavioral responses to stress support work in males that implicates the synaptic strength of vHPC-NAc connectivity as a key regulator of behavioral outcomes following chronic stress2,282, as elevated excitability in female vHPC-NAc projections could drive changes in synaptic strength leading to the observed disparate female susceptibility to SCVS. To demonstrate a definitive link between vHPC-NAc excitability and susceptibility to SCVS- induced anhedonia, we utilized virus-mediated DREADD expression in male and female 139 mice to artificially increase or decrease projection excitability, respectively. We found that a prolonged (10 day), but not short (during behavior assessment only) systemic administration of CNO in males with excitatory DREADD expression in vHPC-NAc induced anhedonia following SCVS as indicated by decreased sucrose preference. We hypothesize that these prolonged changes in excitability in vHPC-NAc projections may lead to various neuroadaptations, including changes in gene expression and synaptic strength, to drive differential responses to stress. This is supported by our finding of sex differences in circuit-specific gene expression, as well as literature suggesting that long- term alterations in the strength of vHPC-NAc synapses drive changes in motivated behaviors in various pathologies, including mood disorders and drug addiction2,282. There are obvious differences in sex chromosome gene expression between males and females, but sex differences also occur in somatic chromosome genes in the human brain315. Studies utilizing preclinical models suggest that sex differences in somatic gene expression in the HPC may be influenced through the collective actions of cell-autonomous feedback mechanisms316, epigenetics317, and gonadal hormone effects318. Our unbiased approach to interrogate the vHPC-NAc transcriptome revealed myriad genes and pathways of interest. Our IPA analyses demonstrated sex differences in genes of the cAMP signaling pathway, and indeed PKA signaling has recently been shown to be required in females, but not males, for synaptic potentiation316. Our TRAP data also showed that female mice have reduced ADCY1 and ADCY8 transcription compared to male mice in vHPC-NAc neurons, perhaps indicating reduced phosphorylation of PKA and a failure to potentiate synaptic responses with repeated stress. As previously mentioned, the strength of vHPC-NAc MSN synapses has recently 140 been shown to regulate reward behaviors282, and the necessity of long-term adaptation following stress to change behavior may explain the differences in anhedonia with different time courses of hormone or vHPC-NAc activity manipulation that we observed. The cAMP signaling pathway and many others that we identified represent exciting subjects for future studies of vHPC-NAc transcriptome with respect to sex differences in stress responses and depression. The evidence presented in this chapter suggests a baseline sex difference in excitability of a specific brain circuit driven by adult androgens that is causative of stress- induced anhedonia. This discovery may work to explain the observation that women are more than twice as likely as men to experience depression, and elucidates critical knowledge important for our understanding of these sex differences. Our circuit-specific transcriptome studies also provide novel gene expression data that will potentially drive future research necessary to validate molecular targets for sex-specific depression treatments. 141 IV. VENTRAL HIPPOCAMPUS TO NUCLEUS ACCUMBENS ΔFOSB UNDERLIES RESILIENCE TO SOCIAL STRESS AND REGULATES CIRCUIT-SPECIFIC EXCITABILITY The experiments in the following chapter are the subject of a manuscript currently in revision at Nature Communications. Dr. Andrew L. Eagle and Claire E. Manning performed the stress, behavior, and microscopy. Elizabeth S. Williams performed the electrophysiology experiments individually and stress protocols with the coauthors. Introduction The ventral hippocampus (vHPC), due to its extensive efferent connections with major forebrain structures such as the hypothalamus, amygdala, and nucleus accumbens (NAc)74,85, is a key component of the regulation of an animal’s emotional response to stress. As introduced above, perturbations in these responses to stress can cause psychiatric conditions such as depression319,320. The connectivity of vHPC and NAc mediates reward behavior321 and susceptibility to social withdrawal following chronic social defeat stress322, but the field yet lacks a full understanding of the role of the vHPC in the regulation of regions such as the NAc in the pathogenesis of depressive and anxiety disorders. It is known that stress affects gene expression in vHPC neurons323-325, but the mechanisms of circuit-specific changes in gene expression and activity of vHPC outputs in response to stress require further investigation. The transcription factor ΔFosB is uniquely stable326,327, and induced by chronic neuronal activity throughout the brain328. In the NAc, ΔFosB mediates stress resilience150,158,329 and influences synapses and dendritic morphology of NAc medium spiny neurons330. Dorsal hippocampus (dHPC) ΔFosB also is also necessary for 142 learning331, and decreases excitability of CA1 pyramidal neurons in this region332. ΔFosB is also induced throughout the HPC by stress and antidepressants272,329,333,334, and vHPC CA3 ΔFosB mediates the protective antidepressant effects of ketamine334. These studies suggest that ΔFosB plays a key role in regulating vHPC function, likely through long-term changes in gene-expression. These changes in gene expression that result in altered vHPC physiology may in turn mediate the development of psychopathologies such as MDD. In the following chapter, circuit-specific CRISPR gene editing is used to investigate the role of ΔFosB in vHPC-NAc projections in resilience to social withdrawal following CSDS. We show that ΔFosB uniquely regulates the excitability of these projections, identifying this molecule as a prime candidate in the mediation of resilience to stress through its effects on vHPC-NAc physiology. Results Chronic social defeat stress and fluoxetine induce ΔFosB in ventral hippocampus To investigate the induction of ΔFosB in vHPC by stress, male C57Bl6/J mice were exposed to CSDS (Figure 1), a well-validated chronic stress model that causes a variety of depressive-like behaviors in susceptible mice, including anhedonia and social withdrawal38,270,271. CSDS increased the number of ΔFosB+ DG neurons in vHPC (Figure 54a, b). Additionally, in accordance with our previous investigation of dHPC272,333, we found that ΔFosB was also induced in all subregions of the vHPC following chronic exposure to the 143 b s l l e C + B s o F ) l o r t n o C % ( # 250 200 150 100 50 0 Control Defeat DG ** CA1 CA3 **** *** Sal Fluox Sal Fluox Sal Fluox a l o r t n o c s s e r t s d 2 m m / s l l e C + B s o F 3000 2000 1000 0 c e n i l a S e n i t e x o u F l Figure 54 | Chronic social defeat stress and fluoxetine induce ΔFosB in ventral hippocampus. (a) Representative microscopy of coronal slices stained for ΔFosB in vHPC from control and stressed mice, quantified in (b). Two-tailed t-test: t(18) = 1.77, #p = 0.0936 (n = 12 control, n = 8 stress). (c) Representative microscopy of coronal slices stained for ΔFosB in vHPC dentate gyrus (DG), CA1, and CA3 from mice treated with chronic saline or fluoxetine, quantified in (d). Two-tailed t-test: DG t(10) = 3.908, **p = 0.0029; CA1 t(10) = 7.951, ****p < 0.0001; CA3 t(10) = 5.674, p = 0.0002; (n = 4 saline, n = 8 fluoxetine). All images at 4X magnification. 144 antidepressant fluoxetine (Figure 54c, d). Considering this finding of its induction in vHPC by chronic stress and antidepressant treatment, vHPC ΔFosB may play a role in mediating resilience to stress, as it is known to do in the NAc158. As previously mentioned, the activity of vHPC-NAc projections mediates resilience to CSDS322 and ΔFosB decreases excitability of dHPC CA1 neurons332. Therefore, we investigated the role of vHPC-NAc ΔFosB in stress outcomes following CSDS. To label vHPC-NAc projections, we used a transgenic mouse line expressing Cre-dependent GFP (Rosa26eGFP-L10a), and injected a persistent, retrograde expressing viral vector carrying Cre recombinase (HSV-hEf1ɑ-Cre) into the NAc (Figure 55a, left). Three weeks following injection, GFP expression was observed in the CA1 subregion of the vHPC (vCA1) and in other areas from which NAc projections originate (e.g. ventral subiculum and VTA; Figure 55a, right). Labeled mice were then exposed to CSDS and vHPC sections were prepared for immunohistochemistry (IHC) and stained for GFP and ΔFosB. We found that CSDS induced ΔFosB in vHPC-NAc projections (Figure 55b,c), validating its consideration as a regulatory molecule in this circuit in the context of stress. vHPC-NAc ΔFosB is necessary for resilience to social stress The above induction studies demonstrate that ΔFosB is induced in vHPC in response to stress and antidepressants, and this includes that in the vHPC-NAc circuit in response to CSDS. To investigate ΔFosB’s role in resilience to stress in the hippocampus, we utilized ΔJunD, an inhibitor of ΔFosB’s transcriptional capacity158. ΔJunD, a dominant negative form of ΔFosB’s AP-1 complex binding partner, was virally overexpressed in either vHPC or dHPC (Fig. 7a). Mice were then exposed to a subthreshold microdefeat, 145 Figure 55 | ΔFosB is induced in vHPC-NAc projections by CSDS. (a) Schematic depicting viral injection strategy for labeling vHPC-NAc projections (left) and representative coronal section containing labeled NAc projections (right). (b) Representative microscopy of vHPC CA1 coronal sections (40X) showing immunofluorescent labeling of NAc-projecting neurons expressing L10-GFP (green, left), ΔFosB (red, middle) and merged images demonstrating projection-specific ΔFosB expression (right). Defeat-stressed mice (bottom; n=109 cells) showed an increase ΔFosB signal in GFP-positive cells compared to control-handled mice (top; n=147 cells), as indicated by white arrows and quantified in (c). Two-tailed t-test: t(254) = 2.847, **p = 0.0048. All images at 40X magnification; single data points omitted in (c) for clarity. 146 GFPFosBMergeControlDefeat0.00.51.01.52.0FosB Intensity**baControlDefeatHPCNAcRetrograde CreSlice for stain-ingCA3CA1DGVTASubvHPC-NAc projectionsc which causes a social withdrawal only in mice sensitized to stress270,271. The microdefeat paradigm, similar to CSDS, utilized CD-1 aggressors and placed the experimental mouse in the aggressor’s home cage for 5 minutes. During this time, the experimental mouse is subjected to attack from the aggressor, and then placed into its own cage to rest for 15 minutes. The stress then repeats twice more with novel aggressors, with a rest period between bouts. After a 24-hour incubation period, the experimental mice were evaluated with the standard social interaction (SI) test. Mice overexpressing ΔJunD in vHPC, but not dHPC, spent less time interacting with a social target and as such had a reduced SI ratio (Figure 56b). These results suggest that ΔFosB in vHPC, but not dHPC, is necessary for resilience to social stress. This experiment investigated general HPC ΔFosB in the context of stress, but the importance of ΔFosB function in vHPC-NAc still remained unclear. To target ΔFosB in only vHPC-NAc neurons, we utilized a dual viral CRISPR system with two constructs (Cas9 and gRNA) packaged in two separate viral vectors (Figure 57a). With this strategy, a viral vector encoding retrogradely expressing Cas9 endonuclease (HSV-hEf1a-LS1L- Cas9) was injected into the NAc. Following three weeks for retrograde expression and recovery, a viral vector encoding guide RNA (gRNA) specific for exon 1 of FosB (HSV- IE4/5-TB-gRNA-eYFP-CMV-IRES-Cre) or a control virus (Scr gRNA; HSV-IE4/5-TB- eYFP-CMV-IRES-Cre) was injected into the vHPC. Only neurons expressing both viruses (i.e. vHPC-NAc projections) had the Cas9 and FosB gRNA necessary to edit the FosB gene and cause reduction of ΔFosB expression. This dual viral CRISPR approach 147 AAV-ΔJunD-GFP HPC o i t a R I S 2.0 1.5 1.0 0.5 0.0 2.0 1.5 1.0 0.5 0.0 vHPC * GFP ΔJunD NAc dHPC GFP ΔJunD a b o i t a R I S Figure 56 | vHPC ΔFosB is necessary for resilience to social stress. (a) Schematic depicting viral injection strategy for AAV-ΔJunD-GFP (pictured) or AAV- GFP in vHPC. (b) Inhibition of ΔFosB function in vHPC with ΔJunD reduced SI ratio following social defeat stress when injected in vHPC (left; two-tailed t-test: t(35) = 2.632, *p = 0.0125; n=19 GFP, n=18 ΔJunD), but not dHPC (right; two-tailed t-test: t(16) = 0.4163, p = 0.6827; n=9/group). 148 was found to decrease the expression of ΔFosB in co-labeled GFP and Cas9 vHPC cells (i.e. vHPC-NAc projections; Figure 57b). This novel circuit-specific gene editing tool produced silencing of the FosB gene (FosB KO) leading to reliable reduction of ΔFosB protein expression in vHPC-NAc projections. This allowed us to interrogate the circuit-specific role of ΔFosB in the regulation of stress responses. Adult mice with FosB KO in the vHPC-NAc circuit (Figure 57a) were exposed to CSDS and assessed for social withdrawal behavior using the SI test. FosB KO in vHPC-NAc increased social withdrawal following CSDS (Figure 57c), and caused a small decrease in locomotor activity (data not shown). These findings suggest that ΔFosB regulates locomotor activity and stress resilience through its activity in vHPC-NAc neurons, consistent with previous findings335,336. FosB KO in vHPC-NAc neurons did not, however, affect baseline anxiety measures in the EPM (Figure 57d,e), behavior not typically associated vHPC-NAc function. These findings suggest that ΔFosB expression in vHPC-NAc projections is necessary for resilience to stress, and in the absence of normal levels of ΔFosB expression in this circuit, mice are sensitized specifically to stress-induced social withdrawal behavior. As vHPC sends projections to other stress-related brain regions (e.g. the BLA337- 339), it was also critical to investigate ΔFosB’s role in other circuits in the context of stress. To highlight the circuit-specificity of ΔFosB in mediating stress outcomes, we also investigated FosB KO in the vHPC-BLA circuit using the same dual viral CRISPR strategy described above (Figure 58a). FosB KO in vHPC-BLA did induce social withdrawal as measured with the SI test, (Figure 58b), however, it reduced anxiety behaviors in the EPM (Figure 58c,d), with no change in locomotor activity (data not shown). Decreased anxiety 149 a b A N R g B s o F l o r t n o C c HPC Cas9 gRNA NAc Cas9 FosB Merge 1.5 1.0 0.5 ) d e z i l a m r o N ( y t i s n e t n I B s o F 0.0 Con EPM OA Entries Social Interaction ** d 40 30 20 10 i e m T A O % EPM %OA Time e 60 s e i r t n E 40 20 4 3 2 1 o i t a R I S *** FosB 0 Control FosB KO 0 Control FosB KO 0 Control FosB KO Figure 57 | vHPC-NAc ΔFosB is necessary for resilience to social stress. (a) Schematic of dual-vector CRISPR strategy to silence FosB in vHPC-NAc neurons: retrograde Cre-dependent Cas9 viral vector (Cas9; yellow) is injected into NAc while local vector expressing Cre and FosB guide RNA (gRNA; red) is injected into vHPC. FosB silencing (FosB KO) occurs only in co-transduced vHPC-NAc neurons (orange). (b, left) Representative images of Cas9 (red, left), FosB (cyan, middle), and merged (right) from vHPC of mice with control (no gRNA) or FosB KO vectors (FosB gRNA). Intensity of FosB staining quantified in (b, right). Two-tailed Mann-Whitney U-test: U = 5423, sum of ranks Control = 23483, FosB = 12563, ***p < 0.001 (n = 149 control cells, n = 119 FosB gRNA cells). (c) FosB silencing in vHPC-NAc neurons reduces SI ratio following CSDS. Two- tailed t-test: t(13) = 3.055, **p = 0.0092 (n=7 control, n=9 FosB KO). FosB silencing in vHPC-NAc does not affect anxiety-like behavior in the elevated plus maze (EPM) % open arm time [two-tailed t-test: t(13) = 0.1441, p = 0.8877] (d) or open arm entries [two-tailed t-test: t(13) = 2.082, p = 0.0576] (e). 150 HPC Cas9 gRNA a b o i t a R I S 4 3 2 1 Social Interaction c 40 30 20 10 i e m T A O % 0 Control FosB KO 0 Control FosB KO Control FosB KO Figure 58 | vHPC-BLA FosB KO is anxiolytic. (a) Schematic of dual-vector CRISPR strategy to silence FosB in vHPC-BLA neurons (same as vHPC-NAc KO strategy in Figure 57). (b) FosB silencing in vHPC-BLA does not affect SI ratio following CSDS. Two-tailed t-test: t(19) = 0.8723, p = 0.3939 (n=11 control, n=10 FosB KO). (c) FosB silencing in vHPC-BLA decreases anxiety-like behavior in the EPM, increasing open arm time [two-tailed t-test: t(11) = 3.241, **p = 0.0079] (c) and entries [two-tailed t-test: t(11) = 3.956, **p = 0.0022). BLA EPM %OA Time ** d EPM OA Entries ** s e i r t n E 60 40 20 0 151 has been demonstrated after vHPC lesions320,339,340, which suggests that ΔFosB in vHPC- BLA projections is necessary for the expression of anxiety behavior. These data, along with the above vHPC-NAc ΔFosB findings, suggest that vHPC FosB regulates stress responses in a circuit-specific fashion. Furthermore, these circuit-specific studies work to clarify the role of different vHPC circuits in stress-related disorders, namely depression and anxiety. ΔFosB regulates vHPC-NAc projection excitability As previous studies have demonstrated a link between vHPC-NAc activity and CSDS susceptibility322, we next examined how ΔFosB affected the physiological properties of the vHPC and its projections. To determine ΔFosB’s effect on the excitability of vHPC neurons in general, viral-mediated overexpression of ΔFosB was utilized in this region (Figure 59a). Overexpression of ΔFosB reduced the excitability of vCA1 neurons, indicated by a decrease in the number of spikes elicited with increasing current injection (Figure 59b) and an increase in rheobase (Figure 59c). These findings suggest that ΔFosB expression reduces the excitability of vHPC neurons, leading us to question whether this was true of vHPC projections to the NAc, as stress-induced increases in ΔFosB could influence the decreased activity in this circuit known to drive resilience to CSDS322. To investigate whether ΔFosB regulates excitability in vHPC-NAc projection neurons specifically, we crossed floxed FosB mice (FosBfl/fl)267 with the Cre-dependent GFP-L10a (Rosa26eGFP-L10a) mice described above to generate floxed FosB/GFP-L10a mice (FosB KO) and utilized non-floxed GFP-L10a mice (WT) as controls (Figure 60a). 152 Figure 59 | ΔFosB regulates vHPC neuronal excitability. (a) Schematic depicting viral injection of HSV vector carrying ΔFosB and GFP (pictured) or GFP. (b) Viral overexpression of ΔFosB reduces the number of spikes in vCA1 pyramidal neurons with increasing current injection. Two-way ANOVA, Holm-Sidak multiple comparisons: [25 pA t(312) = 0.03107, p = 0.9957], [50 pA t(312) = 0.08191, p = 0.9957], [75 pA t(312) = 0.5253, p = 0.9359], [100 pA t(312) = 1.376, p = 0.5253], [125 pA t(312) = 2.152, p = 0.1507], [150 pA t(312) = 2.776, *p = 0.0345], [175 pA t(312) = 3.127, *p = 0.0135], [200 pA t(312) = 3.293, **p = 0.0088], [225 pA t(312) = 3.446, **p = 0.0058], [250 pA t(312) = 3.765, **p = 0.0020], [275 pA t(312) = 3.793, **p = 0.0020], [300 pA t(312) = 3.881, **p = 0.0015]. Inset: total spikes for all steps (two-tailed t-test: t(26) = 2.746, *p = 0.0108; n = 17 GFP cells, n = 11 ΔFosB cells) (c) Representative traces from 200 pA depolarizing current injection in ventral vHPC CA1 neurons expressing GFP or ΔFosB-GFP. (d) ΔFosB overexpression in vHPC caused a trend toward an increase in rheobase in pyramidal neurons. Two-tailed t-test, t(26) = 1.905, #p = 0.0680. 153 255075100125150175200225250275300051015202520 mV200 msGFPΔFosB0100200*Current Step (pA)Spike Number************0204060Rheobase#HPCNAcHSV-ΔFosB-GFPabcd Figure 60 | ΔFosB expression in vHPC-NAc neurons is reduced in FosB circuit- specific KO mice. (a) Schematic depicting strategy for FosB knockout in neurons projecting to NAc. Floxed FosB mice (FosBfl/fl) were crossed with mice expressing Cre-dependent GFP-L10 ribosomal fusion protein (Rosa26eGFP-L10a) to generate floxed FosB/GFP-L10 mice (KO) and non-floxed GFP-L10 controls (WT). Retrograde Cre vector was injected in NAc to drive GFP-L10 expression and splice out exons 2 and 3 in FosB in NAc-projecting neurons. (b) Representative coronal vHPC microscopy (20X) showing NAc-projecting neurons expressing GFP (green, top), ΔFosB (red, middle), and merge (bottom). White arrows indicate co-labeled L10-GFP and ΔFosB-expressing cells. KO significantly reduces the % of co-labeling of GFP and ΔFosB in vHPC-NAc neurons. Two-tailed Mann- Whitney U-test: U = 2, sum of ranks WT = 55 and KO = 23, **p = 0.0087 (n = 6 mice/group). 154 HPCNAcRetrograde CreEx 2&3LoxPLoxPFosB GeneLoxPLoxPStopL10GFPLoxPLoxPL10GFPX+WTKOGFPΔFosBMergeWTKO0510152025**% Co-labeled Cells ab Retrograde Cre HSV was injected into the NAc to allow GFP-L10a expression in NAc- projecting neurons of all mice, and knock out of FosB expression in NAc-projecting neurons of KO mice (Figure 60b). Whole-cell ex vivo slice electrophysiology from L10- GFP+ vCA1-NAc neurons revealed that FosB KO causes elevated excitability in NAc- projecting vHPC neurons in male mice as indicated by an increase in the sum of the total number of spikes elicited at all current steps (Figure 61). FosB KO did not affect any membrane properties (Figure 62a-f), except for an increase in membrane capacitance (Figure 62d). FosB KO in male vHPC-NAc projections did cause an increase in sag ratio, indicating a decrease in Ih current (Figure 62g). Interestingly, FosB KO in male vHPC-NAc neurons also caused a decrease in spike frequency adaptation as indicated by lessened increase in the time between spikes over the span of a depolarizing current step (Figure 63a), but did not change the latency to first spike at the same current (Figure 63b). The same experiments performed with female WT and KO mice revealed a similar, but exaggerated, increase in vHPC-NAc excitability, as indicated by a significantly elevated number of spikes at nearly all current steps as well as an increase in the total number of spikes at all steps (Figure 64). FosB KO in female vHPC-NAc neurons did cause an increase in input resistance (Figure 65c), but did not affect any other membrane properties (Figure 65a-f) nor sag ratio (Figure 65g). Like male mice, female FosB KO mice also showed a decrease in spike frequency adaptation in vHPC-NAc projections (Figure 66a) but no change in latency to first spike (Figure 66b). As reduced activity in excitatory vHPC projections to NAc MSNs has been shown to induce resilience to CSDS and increased activity conversely induces susceptibility322, and our circuit-specific studies above demonstrate that vHPC-NAc ΔFosB is necessary for CSDS resilience, these 155 * 200 100 0 25 50 75 175 125 150 100 250 Current Step (pA) 200 225 275 300 r e b m u N e k p S i 25 20 15 10 5 0 WT - M FosBfl/fl - M V m 0 5 200 ms Figure 61 | ΔFosB regulates the cellular excitability of vHPC-NAc neurons in male mice. (Top) KO of FosB increases the number of spikes in vHPC-NAc neurons across increasing depolarizing current injections. Two-way ANOVA, Holm-Sidak multiple comparisons: [25 pA t(540) = 0.7411, p = 0.4589], [50 pA t(540) = 1.352, p = 0.4495], [75 pA t(540) = 1.418, p = 0.4495], [100 pA t(540) = 1.758, p = 0.3910], [125 pA t(540) = 1.589, p = 0.4495], [150 pA t(540) = 0.4495, p = 1.573], [175 pA t(540) = 2.086, p = 0.2347], [200 pA t(540) = 2.158, p = 0.2253], [225 pA t(540) = 2.481, p = 0.1264], [250 pA t(540)= 2.527, p = 0.1224], [275 pA t(540) = 2.592, p = 0.1113], [300 pA t(540) = 2.456, p = 0.1264]. Inset: total spikes for all steps increased with FosB KO. Two-tailed t-test: t(45) = 2.095, *p = 0.0418 (n = 20 WT cells from n = 5 animals, n = 27 KO cells from n = 8 animals). (Bottom) Representative voltage traces from 200 pA depolarizing current step vHPC-NAc projections in WT and KO male mice. 156 c 600 ) Input Resistance Ω M ( e c n a t s s e R i g k a e P S S / 400 200 0 1.05 1.00 0.95 0.90 0.85 0.80 FloxFosB W T Sag Ratio * a ) V m ( e g a t l o V Potential Resting Membrane -55 -60 -65 -70 -75 -80 -85 ) Ω M ( e c n a t s s e R i b 300 200 100 0 FloxFosB W T Rheobase Membrane Resistance FloxFosB W T Voltage Threshold e 150 ) A p ( e s a b o e h R 100 50 0 f ) V m ( e g a t l o V -60 -40 -20 0 d ) F p ( e c n a t i c a p a C 150 100 50 0 Capacitance ** FloxFosB W T FloxFosB W T FloxFosB W T FloxFosB W T Figure 62 | Membrane properties of male WT vs FosB KO vHPC-NAc projections. WT and FosB KO (FloxFosB) vHPC-NAc projections did not differ in the following membrane properties: (a) Resting membrane potential (mV) [two-tailed Mann-Whitney U- test: U = 198, sum of ranks WT = 552, KO = 576, p = 0.1233]; (b) membrane resistance (MW) [two-tailed t-test: t(45) = 1.228, p = 0.2262]; and (c) input resistance (MW) [two- tailed t-test: t(45) = 0.9708, p = 0.3369]. FosB KO vHPC-NAc projections had a higher membrane capacitance than WT projections [two-tailed t-test: t(45) = 3.112, **p = 0.0032). WT and FosB KO vHPC-NAc projections did not differ in the measures of (e) rheobase (pA) [two-tailed t-test: t(45) = 0.7551, p = 0.4541] and (f) voltage threshold (mV) [two-tailed t-test: t(45) = 0.0004458, p = 0.9996]. (g) FosB KO vHPC-NAc projections had a higher sag ratio than WT projections [two-tailed t-test: t(45) = 2.592, *p = 0.0128], indicating less Ih current. 157 * 1st Pair Last Pair 80 60 40 20 0 b 100 ) s m ( y c n e a L t 80 60 40 20 0 W T a ) s m ( l a v r e n t I i e k p s r e n t I FloxFosB Figure 63 | Spike frequency adaptation is decreased in male FosB KO vHPC-NAc projections. Interspike interval increases over the course of 500 ms current injection as measured by the time (ms) between the 1st pair of spikes and last pair of spikes. (a) Male FosB KO (FloxFosB) had less spike frequency adaptation as measured by the interspike interval increase; interspike interval of the last pair of spikes was significantly longer in male WT vHPC-NAc projections than in KO projections. One-way ANOVA: F(3, 90) = 46.32; mean difference WT last pair vs. FloxFosB last pair = 12.78, 95% CI = 2.047 to 23.51, *p = 0.0128. (b) Latency to 1st spike (ms) from beginning of current injection did not differ between male WT and KO vHPC-NAc projection neurons. Two-tailed Mann-Whitney U- test: U = 181, sum of ranks WT = 391 and KO = 737, p = 0.0554. Interspike intervals and latency to 1st spike were evaluated at the rheobase current for each cell. 158 r e b m u N e k p S i 25 20 15 10 5 0 * ** ** ** ** * * * ** 300 200 100 0 ** ** ** 25 50 75 275 300 125 150 100 250 Current Step (pA) 200 225 175 V m 0 5 200 ms WT - F FosBfl/fl - F Figure 64 | ΔFosB regulates the cellular excitability of vHPC-NAc neurons in female mice. (Top) KO of FosB increases the number of spikes in vHPC-NAc neurons across increasing depolarizing current injections. Two-way ANOVA, Holm-Sidak multiple comparisons: [25 pA t(372) = 1.143, p = 0.2538], [50 pA t(372) = 3.214, **p = 0.0099], [75 pA t(372) = 3.472, **p = 0.0069], [100 pA t(372) = 3.333, **p = 0.0098], [125 pA t(372) = 3.209, **p = 0.0099], [150 pA t(372) = 3.099, *p = 0.0104], [175 pA t(372) = 3.045, *p = 0.0104], [200 pA t(372) = 3.067, *p = 0.0104], [225 pA t(372) = 3.052, *p = 0.0104], [250 pA t(372)= 3.292, **p = 0.0098], [275 pA t(372) = 3.343, **p = 0.0098], [300 pA t(372) = 3.348, **p = 0.0098]. Inset: total spikes for all steps increased with FosB KO. Two-tailed t-test: t(31) = 3.338, **p = 0.0022 (n = 19 WT cells from n = 4 animals, n = 14 KO cells from n = 4 animals). (Bottom) Representative voltage traces from 200 pA depolarizing current step vHPC-NAc projections in WT and KO female mice. 159 c Membrane Resistance ) Ω M ( e c n a t s s e R i 400 300 200 100 0 b 400 ) 300 200 100 Ω M ( e c n a t s s e R i 0 FloxFosB W T Voltage Threshold g k a e P S S / 1.05 1.00 0.95 0.90 0.85 0.80 d ) F p ( e c n a t i c a p a C 150 100 50 0 Capacitance FloxFosB W T Input Resistance ** FloxFosB W T Sag Ratio a ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 -85 e ) A p ( e s a b o e h R 150 100 50 0 Resting Membrane Potential FloxFosB W T Rheobase f ) V m ( e g a t l o V -60 -40 -20 0 FloxFosB W T FloxFosB W T FloxFosB W T Figure 65 | Membrane properties of female WT vs FosB KO vHPC-NAc projections. WT and FosB KO (FloxFosB) vHPC-NAc projections did not differ in the following membrane properties: (a) Resting membrane potential (mV) [two-tailed t-test: t(31) = 0.8337, p = 0.4108] and (b) membrane resistance (MW) [two-tailed t-test: t(31) = 0.4226, p = 0.6755]. (c) Input resistance (MW) was increased in KO vHPC-NAc projections [two- tailed t-test: t(31) = 2.816, **p = 0.0084]. WT and FosB KO vHPC-NAc projections did not differ in the measures of (d) membrane capacitance (pF) [two-tailed t-test: t(31) = 1.953, p = 0.0599]; (e) rheobase (pA) [two-tailed t-test: t(31) = 0.1287, p = 0.8984]; (f) voltage threshold (mV) [two-tailed t-test: t(31) = 1.187, p = 0.2442]; and (g) sag ratio [two-tailed t-test: t(31) = 0.4986, p = 0.6216]. 160 **** 1st Pair Last Pair 80 60 40 20 0 b ) s m ( y c n e a L t 80 60 40 20 0 W T a ) s m ( l a v r e t n I i e k p s r e n t I FloxFosB Figure 66 | Spike frequency adaptation is decreased in female FosB KO vHPC- NAc projections. Interspike interval increases over the course of 500 ms current injection as measured by the time (ms) between the 1st pair of spikes and last pair of spikes. (a) Female FosB KO (FloxFosB) had less spike frequency adaptation as measured by the interspike interval increase; interspike interval of the last pair of spikes was significantly longer in female WT vHPC-NAc projections than in KO projections. One-way ANOVA: F(3, 62) = 140.3; mean difference WT last pair vs. FloxFosB last pair = 8.132, 95% CI = 3.63 to 12.63, ****p < 0.0001. (b) Latency to 1st spike (ms) from beginning of current injection did not differ between female WT and KO vHPC-NAc projection neurons. Two-tailed t-test: t(31) = 1.541, p = 0.1334. Interspike intervals and latency to 1st spike were evaluated at the rheobase current for each cell. 161 electrophysiology studies support stress-induced ΔFosB as a regulator of excitability in this circuit to drive resilience to social withdrawal following CSDS. To demonstrate the uniqueness of ΔFosB in the vHPC-NAc in the regulation of cellular physiology, we also utilized the same FosB KO strategy in the vHPC-BLA circuit by instead injecting retrograde Cre HSV in BLA in floxed FosB/L10-GFP (KO) or L10-GFP (WT) mice. Whole-cell patch clamp recordings in ex vivo slices of vHPC-BLA projections of male mice showed that FosB KO did not cause the same elevated excitability in this circuit as it did in vHPC-NAc projections (Figure 67). FosB KO did cause an increase in membrane resistance (Figure 68b), but did not affect any other membrane properties (Figure 68a-e). In female vHPC-BLA neurons, FosB KO also did not cause a change in excitability (Figure 69) nor did it affect any membrane properties (Figure 70a-e). Although we did demonstrate the role of ΔFosB in vHPC-BLA projections in the expression of anxiety behaviors, these studies highlight the unique role of ΔFosB in the regulation of cellular excitability in the vHPC-NAc neurons, which may relate its induction in vHPC and NAc to its role in mediating stress responses. Discussion The above studies demonstrate that ΔFosB is induced by stress in glutamatergic vHPC-NAc neurons, and that ΔFosB in this circuit is uniquely necessary for resilience to stress. FosB and its gene products, including ΔFosB, also appear to have dramatic effects on the excitability of vHPC-NAc projections, with knockout of the FosB gene causing an increase in this circuit’s responsiveness to current. The mechanism of ΔFosB’s effects on cellular excitability still remains to be studied, but with the circuit-specific KO’s robust 162 effect on the interspike interval duration, it is possible that the increase in excitability is achieved through the alteration of ion channels at the cell’s membrane. A change in the membrane’s passage of current would explain this phenomenon, but would be expected to be accompanied by changes in one or more membrane properties. The only membrane property changed in male vHPC-NAc neurons with FosB KO was capacitance, which may relate to a change in the size of cells following KO. FosB KO in female vHPC-NAc projections, however, caused an exaggerated increase in cellular excitability when juxtaposed with male projections. This increase in cellular responsiveness to current was indeed accompanied by an increase in membrane input resistance, which may directly explain the differences observed in excitability and spike frequency adaptation. The differences in the magnitude of male and female vHPC-NAc neuron response to FosB KO point to possible sex-specific FosB regulatory capacities, particularly with regard to the influence of the cell membrane’s expression of ion channels. Further investigation regarding ΔFosB’s mechanism of modulating cellular excitability in the context of stress will be required, but the current study highlights its importance in the vHPC-NAc circuit in mediating stress resilience, possibly through its effects on cellular excitability. 163 r e b m u N e k p S i 25 20 15 10 5 0 25 50 75 125 150 175 100 250 Current Step (pA) 225 200 275 300 WT - M FosBfl/fl - M A p 0 5 200 ms Figure 67 | FosB KO does not affect the cellular excitability of vHPC-BLA neurons in male mice. (Top) KO of FosB does not change the number of spikes in vHPC-BLA neurons across increasing depolarizing current injections. Two-way ANOVA, Holm-Sidak multiple comparisons: [25 pA t(348) = 0.6193, p > 0.9999], [50 pA t(348) = 0.9014, p = 0.9959], [75 pA t(348) = 1.271, p = 0.9358], [100 pA t(348) = 1.446, p = 0.8558], [125 pA t(348) = 1.122, p = 0.9742], [150 pA t(348) = 0.6161, p > 0.9999], [175 pA t(348) = 0.3664, p > 0.9999], [200 pA t(348) = 0.3145, p > 0.9999], [225 pA t(348) = 0.3697, p > 0.9999], [250 pA t(348)= 0.3437, p > 0.9999], [275 pA t(348) = 0.1394, p > 0.9999], [300 pA t(348) = 0.0843, p > 0.9999] (n = 22 WT cells from n = 5 animals, n = 12 KO cells from n = 4 animals). (Bottom) Representative voltage traces from 200 pA depolarizing current step vHPC-BLA projections in WT and KO male mice. 164 Membrane Resistance * FloxFosB W T e 150 c ) Ω M ( e c n a t s s e R i 1500 1000 500 0 Input Resistance FloxFosB W T Rheobase b ) Ω M ( e c n a t s s e R i 250 200 150 100 50 0 Capacitance Resting Membrane Potential a ) V m ( e g a t l o V -50 -60 -70 -80 -90 FloxFosB W T d 200 ) F p ( e c n a t i c a p a C 150 100 50 0 FloxFosB W T FloxFosB W T Figure 68 | Membrane properties of male WT vs FosB KO vHPC-BLA projections. WT and FosB KO (FloxFosB) vHPC-BLA projections did not differ in (a) Resting membrane potential (mV) [two-tailed t-test: t(32) = 0.001299, p = 0.9990]. (b) KO vHPC- BLA projections had increased membrane resistance (MW) [two-tailed t-test: t(32) = 2.066, *p = 0.0470]. WT and KO vHPC-BLA projections did not differ in any of the following measures: (c) input resistance (MW) [two-tailed t-test: t(32) = 1.229, p = 0.2282]; (d) membrane capacitance (pF) [two-tailed t-test: t(32) = 1.763, p = 0.0874); and (e) rheobase (pA) [two-tailed t-test: t(32) = 1.893, p = 0.0674]. ) A p ( e s a b o e h R 100 50 0 165 r e b m u N e k p S i 25 20 15 10 5 0 25 50 75 275 300 125 150 100 250 Current Step (pA) 175 200 225 WT - F FosBfl/fl - F A p 0 5 200 ms Figure 69 | FosB KO does not affect the cellular excitability of vHPC-BLA neurons in female mice. (Top) KO of FosB does not change the number of spikes in vHPC-BLA neurons across increasing depolarizing current injections. Two-way ANOVA, Holm-Sidak multiple comparisons: [25 pA t(264) = 0.5072, p > 0.9999], [50 pA t(264) = 1.739, p = 0.6470], [75 pA t(264) = 1.392, p = 0.8850], [100 pA t(348) = 1.446, p = 0.8558], [125 pA t(348) = 1.122, p = 0.9742], [150 pA t(348) = 0.6161, p > 0.9999], [175 pA t(348) = 0.3664, p > 0.9999], [200 pA t(348) = 0.3145, p > 0.9999], [225 pA t(348) = 0.3697, p > 0.9999], [250 pA t(348)= 0.3437, p > 0.9999], [275 pA t(348) = 0.1394, p > 0.9999], [300 pA t(348) = 0.0843, p > 0.9999] (n = 22 WT cells from n = 5 animals, n = 12 KO cells from n = 4 animals). (Bottom) Representative voltage traces from 200 pA depolarizing current step vHPC-BLA projections in WT and KO male mice. 166 a Resting Membrane Potential ) V m ( e g a t l o V -55 -60 -65 -70 -75 -80 Membrane Resistance FloxFosB W T e 150 c ) Ω M ( e c n a t s s e R i 800 600 400 200 0 Input Resistance FloxFosB W T Rheobase b ) Ω M ( e c n a t s s e R i 200 150 100 50 0 FloxFosB W T d 150 ) F p ( e c n a t i c a p a C 100 50 0 Capacitance FloxFosB W T ) A p ( e s a b o e h R 100 50 0 FloxFosB W T Figure 70 | Membrane properties of female WT vs FosB KO vHPC-BLA projections. WT and FosB KO (FloxFosB) vHPC-BLA projections did not differ in any of the following measures: (a) resting membrane potential (mV) [two-tailed t-test: t(23) = 0.9614, p = 0.3463]; (b) membrane resistance (MW) [two-tailed t-test: t(23) = 0.1493, p = 0.8826]; (c) input resistance (MW) [two-tailed t-test: t(23) = 0.3648, p = 0.7186]; (d) membrane capacitance (pF) [two-tailed t-test: t(23) = 0.6822, p = 0.5019); and (e) rheobase (pA) [two-tailed t-test: t(23) = 0.3739, p = 0.7119]. 167 V. SUMMARY, DISCUSSION AND FUTURE DIRECTIONS Summary This dissertation investigated the circuit-specific underpinnings of sex differences in stress susceptibility in preclinical models of depression, with focus on the excitatory vHPC-NAc circuit due to its recent discovery as a key mediator of stress outcomes. First, we verified that only female mice were susceptible to SCVS-induced anhedonia as measured by sucrose preference. We next found that vHPC-NAc projections in female mice are more excitable at baseline than those derived from male mice, and that this difference in excitability is possibly due to differential adaptation of male and female cells in response to fluctuating inputs. We discovered that male resilience to SCVS-induced anhedonia as well as the lower excitability in male vHPC-NAc neurons was dependent on the presence of testes-derived hormones, as orchidectomy caused male mice to be susceptible to anhedonia following stress and caused an increase in the excitability of vHPC-NAc projections. This change in physiology was time-dependent, as an extended incubation was necessary to cause the observed change in sucrose preference. In contrast, we found that ovariectomy did not affect the excitability of female vHPC-NAc neurons. The dramatic changes observed in male mice in SCVS-induced anhedonia and vHPC-NAc excitability paired with the absence of these changes in female mice following ovariectomy suggest that androgens in males may confer an active resilience by regulating the physiology of this circuit. In contrast to the vHPC-NAc circuit, vHPC-BLA neurons were not found to differ in excitability in male and female mice. These projections in male mice did show a slight increase in excitability following orchidectomy, but this change was not as robust as in 168 the vHPC-NAc circuit. Ovariectomy, like in the vHPC-NAc experiments, did not affect vHPC-BLA physiology. These findings suggest a potential role for androgens in male mice in affecting the global physiology of glutamatergic neurons in the vHPC, but the lack of differences in excitability between male and female vHPC-BLA projections highlights the unique nature of the difference in baseline excitability between male and female vHPC-NAc projections. We also demonstrated the effectiveness of chronic subcutaneous testosterone administration in ovariectomized female mice in the amelioration of SCVS-induced anhedonia and in the mitigation of vHPC-NAc hyperexcitability. Exogenous testosterone pellets implanted four weeks prior to stress were sufficient to increase sucrose preference in female ovariectomy mice following SCVS. Testosterone pellets also caused a robust decrease in the excitability of vHPC-NAc projections in ovariectomy female mice. These findings support the critical role of androgens in the regulation of vHPC-NAc excitability and consequent resilience to stress-induced anhedonia. To further support this role, we also investigated the effects of the AR antagonist flutamide on the physiology of vHPC- NAc neurons in male mice. Acute flutamide administration caused a small but significant increase in excitability of these projections, as indicated by an increase in the total number of spikes across all current steps, but not an increase in spike number at any individual step. These data, taken together with the above findings, make a strong case for androgens in the regulation of vHPC-NAc physiology to cause the relatively lower excitability in male projections at baseline and confer resilience to stress-induced anhedonia in male mice. 169 These behavior and electrophysiology experiments paralleled one another to suggest that vHPC-NAc excitability may be responsible for male resilience to anhedonia following SCVS, but they did not establish a causal link. To accomplish this, we employed circuit-specific DREADD expression to artificially change the excitability of vHPC-NAc neurons with the application of the designer drug CNO. When a stimulatory Gq-coupled DREADD was expressed in male vHPC-NAc neurons, we found that long-term, but not short-term, CNO administration induced a substantial decrease in sucrose preference. This decrease was observed in control-handled male mice, with a further decrease in sucrose preference observed in CNO-treated male mice exposed to SCVS. This drastic effect on male susceptibility to anhedonia, even before animals were exposed to stress, demonstrates the key role of vHPC-NAc projections in regulating reward behaviors as well as stress-induced anhedonia. In parallel to the male studies, a Gi-coupled DREADD was expressed in female vHPC-NAc neurons, and the administration of CNO during SCVS and behavioral assessment was found to induce resilience to anhedonia following stress, a departure from previously observed female-only susceptibility to anhedonia in this paradigm. We also demonstrated that CNO administration in the absence of DREADD expression in male mice did not affect SCVS outcomes, which is an important consideration as the use of CNO has come under recent scrutiny341 as it is peripherally metabolized to clozapine, which could have unpredictable effects on the circuit or brain as a whole. Overall, our DREADD studies demonstrate a causal link between the excitability of vHPC-NAc neurons in male and female mice and stress-induced changes in behavior, with emphasis on the circuit’s control of stress-induced anhedonia. 170 With the causal link between vHPC-NAc excitability and stress outcomes now established in male and female mice, we then sought to interrogate the circuit-specific mechanisms that may be responsible for regulating projection physiology. To accomplish this, we employed circuit-specific TRAP followed by RNA sequencing to investigate the vHPC-NAc transcriptome in male and female mice. Using the convention of comparing male transcript levels to that of females, we found more downregulated than upregulated genes in the vHPC-NAc circuit in females. This suggests that male mice have more significantly upregulated genes in this circuit; this may represent an additional protective property or properties conferred by active gene expression present in male vHPC-NAc neurons and primes the animal for stress resilience. Hundreds of genes were found to have differential expression in male and female vHPC-NAc neurons, and using pathway analysis, we identified many avenues for future research into the molecular mechanisms of active resilience in male mice that may work to explain the known sex differences in SCVS-induced anhedonia, as well as potential sex differences in other preclinical models of depression and human MDD. The concept of active resilience in males and selected pathways of interest are discussed further below. Although the sex-specific TRAP sequencing data did not reveal a difference in FosB expression between male and female vHPC-NAc neurons, stress and antidepressant induction of ΔFosB in hippocampus272 and its role as a resilience factor in other brain regions158,342 drove us to investigate FosB gene products in this circuit. Specifically, we evaluated ΔFosB as a potential regulator of vHPC-NAc physiology such that it could contribute to active resilience by attenuating the excitability of these projections. First, we showed that CSDS as well as the antidepressant fluoxetine induce 171 ΔFosB globally in the ventral hippocampus, verifying its potential as a regulator of cell physiology in this region. We further investigated ΔFosB induction in a circuit-specific fashion, and showed that it is indeed induced by CSDS in vHPC-NAc projections as indicated by co-labeling of L10-GFP and FosB in immunohistochemistry experiments. This discovery, along with the aforementioned reduction in dHPC cellular excitability with overexpression of ΔFosB, made this molecule an excellent candidate for the regulation of projection physiology and the consequential change in stress outcomes. To evaluate ΔFosB’s role in stress resilience, we began by globally overexpressing ΔJunD, an inhibitor of ΔFosB function, in the vHPC or dHPC of male mice. We then assessed behavioral phenotype following subchronic social stress and found that silencing ΔFosB’s activity in ventral, but not dorsal, HPC was sufficient to induce susceptibility as indicated by a decreased SI ratio. Next, we investigated ΔFosB’s circuit- specific role in stress, and found that knocking out FosB expression in vHPC-NAc neurons in male mice using our novel dual-viral CRISPR strategy also caused a reduction in SI ratio following CSDS. We also demonstrated that FosB KO in vHPC-BLA neurons was anxiolytic, suggesting that expression of FosB gene products may play a role in anxiogenesis, though it had no effect on social interaction score after stress. These experiments identified circuit-specific roles for ΔFosB in regulating stress outcomes, emphasizing that vHPC-NAc ΔFosB function is necessary for resilience to social stress. Next, we investigated ΔFosB’s capacity for regulation of vHPC pyramidal cell excitability. We virally overexpressed ΔFosB in vHPC and found a dramatic decrease in the number of spikes elicited at each increasing current step, and a trend towards an increase in rheobase, indicating that ΔFosB can mediate an overall reduction in cellular 172 excitability. We then studied the circuit-specific role of FosB expression in regulating cellular excitability using a circuit-specific KO strategy in male and female mice. We found that FosB KO in both male and female vHPC-NAc neurons did indeed increase cellular excitability compared to control projections. FosB KO in both male and female vHPC-NAc projections also caused a decrease in spike frequency adaptation, suggesting that FosB gene products, including ΔFosB, may regulate cellular excitability via homeostatic regulation of intrinsic excitability in response to sustained activity of inputs. We also demonstrated that FosB KO does not significantly alter cellular physiology in vHPC-BLA projections in male or female mice, emphasizing the specificity of ΔFosB’s attenuation of cellular excitability in the vHPC-NAc projections. Discussion and Future Directions Resilience as an active process As mentioned above, the idea of resilience as an active process, particularly in male mice, has gained recent attention due to a variety of studies that show converging evidence to that effect. From a broad perspective, active approaches in humans have been utilized in psychology and psychiatry to promote resilience to stress. These include active coping mechanisms such as cognitive reappraisal (i.e. reframing of adverse events in a positive light) and coping self-efficacy (e.g. mastering the skills necessary to manage a given stressor in increasingly challenging scenarios), as well as basic active approaches such as improving diet, exercise, and sleep343. Active coping mechanism equivalents have also been observed in rodent stress models of depression; for example, during social defeat stress, mice that display less submissive posturing during aggressive 173 attacks exhibit less social avoidance later in the social interaction test344. On a deeper level, neurobiological mechanisms of active resilience have also been described. Resilience to social stress has been associated with active genetic and epigenetic changes in distinct brain regions that are not observed in susceptible animals34,154. These changes are discussed below, with implications for the findings presented in this dissertation. As mentioned previously, c-Fos and FosB gene products, including ΔFosB, are induced by chronic stress in various brain regions. One of these regions is the mPFC, where resilient mice show an increase in the expression of these immediate early genes345,346, suggesting that the increase in neuronal activity in this region is an active adaptation that promotes resilience. This was confirmed by optogenetic work demonstrating that non-cell type-specific stimulation of mPFC neurons promotes resilience347. Later studies determined that glutamatergic projections of the mPFC are responsible: specific optogenetic stimulation of glutamatergic neurons is pro-resilient171. ΔFosB in also preferentially induced in the NAc of resilient animals, where it is thought to promote resilience through induction of GluA2 and causing subsequent decrease in AMPAR Ca2+ conductivity158. Our studies demonstrated that ΔFosB is also induced in vHPC-NAc projections, and that FosB expression in this circuit is necessary for resilience to social stress. Therefore, the induction of vHPC-NAc ΔFosB, like its induction in the mPFC and NAc, may represent an active mechanism of resilience. In future studies, the experiments conducted in this dissertation regarding FosB and its gene products in vHPC-NAc circuit will need to be repeated in female mice. Indeed, the increase in excitability that we observed in vHPC-NAc projections of male mice was also observed in 174 vHPC-NAc neurons of female mice when FosB was knocked out. More experiments will need to be performed to determine the effects of this change in projection physiology on stress outcomes in female mice, possibly using social stress models that facilitate the incorporation of female subjects. One preclinical stress model that could be utilized to assess social stress outcomes in female mice in the presence of vHPC-NAc FosB KO is the vicarious social defeat model348. This model could also be used to directly compare the effects of social stress, rather than SCVS, on the vHPC-NAc circuit and to determine whether gonadal hormone manipulation modulates social stress outcomes in the same manner as we observed in SCVS. The vicarious social defeat stress model utilizes adult male or female C57/Bl6 mice that vicariously experience the social defeat of a male conspecific by a CD1 aggressor. This “emotional” or “psychological” stress is achieved by keeping the experimental mouse on the opposite side of a perforated divider during the attack sessions. The witnessing of aggression is repeated for 10 consecutive days with the experimental mouse housed on the other side of a perforated divider from a different aggressor overnight following each stress session. The witness or vicarious nature of this paradigm avoids the difficulty of achieving aggressive behavior towards experimental female mice, and allows for the use of both sexes in experimental design. Females that experience vicarious social defeat experience many of the depression-like measures of face validity seen with traditional CSDS in male mice: decreased social interaction in the SI test, anhedonia as measured by sucrose preference, increased immobility in TST, and increased serum corticosterone levels. This model also exhibits predictive validity: the susceptible phenotype in females exposed to vicarious defeat stress is mitigated by the 175 newly-minted antidepressant ketamine. Unlike CSDS, however, susceptibility in the vicarious defeat model is ameliorated by the benzodiazepine chlordiazepoxide, suggesting that an anxiety component may be involved. As social interaction engages reward circuitry and is a positively-valenced rewarding stimulus349, much like consumption of the sucrose solution in the assessment of hedonic behavior, the use of vicarious defeat stress may indeed induce ΔFosB in vHPC-NAc neurons. Indeed, studies of emotional stress in male mice have shown ΔFosB induction in the HPC350, indicating that this type of stress may have similar effects to CSDS on this region. Future studies could investigate whether this is the case for female mice, and could dissect specific HPC projections, such as vHPC-NAc, in the context of the active resilience mechanism of ΔFosB induction. I predict that FosB KO in this circuit would induce susceptibility in both male and female mice in the vicarious defeat paradigm, and that this susceptibility would be dependent upon an increase in vHPC-NAc excitability due to attenuated ΔFosB expression. The vicarious defeat model could also prove valuable in the assessment of social stress in the presence of hormone manipulation. In this scenario, I would predict that testosterone manipulations (removal or replacement) would change stress outcomes. Testosterone may provide an active protection against the effects of emotional stress in the vicarious defeat model, much like it did in our SCVS experiments presented in Chapter III, through attenuation of vHPC-NAc hyperexcitability. Gene expression changes in stress Adaptation of the activity and connectivity of brain regions like HPC and NAc in response to stress require corresponding changes in gene expression and epigenetics279. 176 Seminal experiments investigating gene transcription differences in resilient and susceptible male mouse populations following CSDS have demonstrated that gene regulation may be a mechanism of active resilience. For example, in the NAc, Krishnan et al found that although many of the same genes were upregulated in resilient and susceptible populations, the resilient group had a significantly higher number of genes upregulated that did not overlap with those upregulated in the susceptible group34. The same was true of differentially regulated genes in the VTA: the resilient group had a higher number of upregulated genes than the susceptible group. In both regions, there was also a higher number of downregulated genes in the resilient group than in the susceptible group, indicating that downregulation of certain genes may also represent an active mechanism of resilience. Interestingly, this group found several upregulated K+ channels in the VTA of resilient mice, and demonstrated that overexpression of these channels in the VTA of susceptible mice reduced neuronal excitability and reversed the susceptible phenotype in these animals. In RNAseq studies, Hodes et al also described different transcriptional profiles in the NAc of male and female mice following SCVS. This group found that 17% more genes were differentially regulated by stress in male mice than in female mice, with 40% of these being upregulated and 60% downregulated. In contrast, in female mice, 60% of the differentially regulated genes in the NAc were upregulated and 40% downregulated, with only 3% overlap of differentially regulated genes between the sexes, only 0.5% oppositely regulated between the sexes, and only a single gene upregulated in both sexes. These findings demonstrate that different molecular mechanisms mediate the behavioral phenotypes of male and female mice following SCVS, and, since males are not 177 susceptible to this stress modality, may point to genetic mechanisms of active resilience in males. This group also described an epigenetic change that is altered in NAc by SCVS in mice as well as in MDD in humans – the upregulation of DNA methyltransferase 3a (Dnmt3a) in both male and female mice. Female mice had higher levels of Dnmt3a, which can lead to transcriptional silencing of genes by adding a sterically hindering methyl group to transcription factor binding sites on DNA, and has been linked to social stress susceptibility351. The group also showed that KO of Dnmt3a in NAc in females shifted their gene regulation patterns to closer resemble that of males, indicating that differential epigenetic control of gene expression may also explain sex differences in stress susceptibility. In our interrogation of actively translated genes in vHPC-NAc projections of male and female mice, we found that Dnmt3a was significantly downregulated in these cells in females compared to males. While this may not correspond to the increase that Hodes et al showed within the NAc in female mice, epigenetic influences may cause the differential excitability that we observed between male and female vHPC-NAc projections. For instance, we found other epigenetic regulators, such as histone deacetylases 2 and 5 (Hdac2 and Hdac5), were upregulated in female vHPC-NAc projections compared to males. Histone deacetylases remove positively charged acetyl groups from histones, decreasing their DNA binding and opening up chromatin for facilitation of gene transcription. Indeed, Covington et al demonstrated increased NAc histone acetylation following CSDS, suggesting that epigenetic-mediated silencing of gene transcription may offer beneficial neuronal adaptations following stress155. This was confirmed by their demonstration of the antidepressant effects of histone deacetylase inhibition. Future studies could focus on epigenetic changes in vHPC-NAc neurons of male and female 178 mice, as there are clearly differences in the transcription of epigenetic regulators in these projections between the sexes as shown in our RNAseq findings following TRAP. An additional experiment that will be crucial to answering questions about sex differences in gene regulation in the context of stress outcomes will be to perform vHPC-NAc TRAP on stressed male and female mice. This experiment may help to reveal those genes up- or downregulated in male vHPC-NAc neurons that confer resilience to this stress paradigm. Circuit-specific TRAP experiments could also be performed in the presence of gonadectomy to investigate whether gonadal steroid hormones affect the transcriptional profiles of vHPC-NAc neurons in male and female mice. Intrinsic excitability and plasticity Intrinsic plasticity encompasses the ability of a neuron’s electrical properties and excitability to change with persistent autonomous or synaptic activity. It is defined by the malleability of ion channels, in terms of their expression levels or changes in their physical properties. Intrinsic plasticity is thought to affect many processes, including integration of synaptic inputs and action potential generation. The function of intrinsic plasticity, unlike its more widely studied counterpart, synaptic plasticity, is ill-defined; however, it is thought to be involved in many processes linked to the hippocampus, including learning352,353, memory354, and homeostatic regulation of neuronal excitability355,356. The integration of synaptic input within a neuron to generate an action potential is dependent upon the number and makeup of the neuron’s ion channels. Voltage- and calcium-gated channels in dendrites accomplish this synaptic integration, while channels of the cell body participate in action potential generation. In vitro studies have 179 demonstrated that the electrical features conferred upon a neuron by ion channels, i.e. a neuron’s intrinsic excitability, are plastic and are subject to modification by various internal and external factors357. Three major mechanisms of intrinsic plasticity have been described: long term plasticity of intrinsic excitability (LTP-IE)358, homeostatic plasticity of intrinsic excitability359, and plasticity of dendritic synaptic input integration360. Bliss and Lomo, in their seminal work describing the phenomenon of synaptic long- term potentiation (LTP), also recognized that the postsynaptic neuron itself was more likely to fire, or more excitable, following the exposure to excitatory postsynaptic potentials (EPSPs) generated by the LTP protocol95. This occurrence, known as E-S potentiation, was further described in pyramidal cells following the induction of LTP356. Specifically, pyramidal cells of the CA1 region of the hippocampus have an increased number of action potentials in response to injected current, as well as a decreased AP threshold, following LTP induction. Chavez-Noriega et al postulated that in the CA1, this phenomenon could be the result of an increase in the excitation to inhibition ratio at a given neuron, possibly through a reduction in inhibition via GABAA signaling. The induction of LTP, however, is not strictly necessary to achieve LTP-IE, as the phenomenon has been observed following injection of repeated depolarizing current without the presence of synaptic stimulation in neurons of the visual cortex361 and cerebellum362. Other mechanisms are also possible to achieve LTP-IE, for example, the activation of metabotropic glutamate receptors (mGluRs) in the hippocampus causes a long-term increase in the excitability of CA1 pyramidal neurons by way of the suppression of afterhyperpolarization potentials (AHPs)363. 180 Homeostatic plasticity of intrinsic excitability, in contrast to LTP-IE, represents a reactivity of neurons to a change in the activity of inputs that tends to stabilize excitability to a given baseline level. This concept is critical, as neurons usually must function for a long period of an animal’s life and must be able to adapt to changes in input as a result of processes critical to the animal’s survival and propagation (e.g. learning and development). The homeostasis achieved allows the neuron to maintain functionality, either decreasing excitability in the face of increased input or vice versa. O’Leary et al. demonstrated a shift in cultured hippocampal pyramidal neurons towards decreased excitability after being exposed for days to elevated KCl concentrations, as evidenced by heightened rheobase and a rightward shift in the frequency-current input relationship without a change in AP threshold voltage364. Complementarily, Desai et al showed that cultured cortical pyramidal neurons, when deprived of activity for days, were more sensitive to current injection with a leftward shift in the frequency-current relationship365. In both cases, the homeostatic plasticity of intrinsic excitability was dependent upon functionality of ion channels; regulating voltage-gated ion channel conductances allowed the neurons to maintain a homeostatic, baseline excitability. Although neither the membrane nor input resistance of vHPC-NAc neurons differed between male and female mice in a direction that would explain the change in passage of current such that excitability would change, the voltage threshold of female vHPC-NAc projections was more negative than that of male projections. It is possible that channels responsible for resting membrane properties are unaltered in female vHPC-NAc neurons, but those that are opened in response to activity, such as voltage-gated Na+ channels, may explain the hyperexcitability of female projections. Indeed, many voltage-gated channels were 181 differentially regulated between male and female vHPC-NAc projections, but the directionality of regulation is variable across these channels. Validation of differentially regulated genes will need to be conducted in order to tease out mechanisms by which the voltage threshold of female projections differs. Quantitative PCR (qPCR) is one method of validation that could be used to investigate these channels, and indeed additional TRAP samples for male and female vHPC-NAc neurons have been generated for this purpose. It is possible that differentially regulated ion channels act in concert to cause a less negative voltage threshold in male vHPC-NAc projections, conferring an active mechanism of resilience in males through regulation of excitability. Dendritic integration of synaptic inputs also exhibits plasticity. This concept represents focused changes in synaptic integration, as opposed to widespread changes in intrinsic excitability of the neuron that leads to differential AP generation. Plasticity of dendritic integration has been observed in hippocampal CA1 pyramidal neurons, where the linearity of summation of EPSPs following LTP induction at a given dendrite was increased based on proximity to the cell body366. In other words, the closer the dendrite was to the cell body, the longer it exhibited increased excitability when detecting the coincidence of inputs, resulting in a summed EPSP with an amplitude closer to what it would be if the expected individual EPSPs were added. This is thought to be due to changes in A-type potassium conductance367,368. This phenomenon may be due to the effect of AP backpropagation from the cell body and axon hillock, where APs are initiated, to dendrites. Backpropagation causes an increase in calcium influx locally in dendrites, which was shown to facilitate spikes in the apical dendrites of CA1 pyramidal neurons in a CaMKII-dependent fashion369. As previously discussed, CA1 pyramidal cell dendritic 182 spine density varies over the estrous cycle in female rodents139, a process that is estradiol- and NMDAR-dependent140. Future experiments will need to assess female vHPC-NAc excitability over the estrous cycle. If there are differences in the physiology of these projections in proestrus and diestrus phases, it may indicate that dendritic spine density is fluctuating, affecting the intrinsic excitability of these cells. Overall, the above mechanisms of intrinsic excitability and the plasticity thereof may prove to be critical in mediating the observed differences between male and female stress outcomes, as the activity of circuits such as vHPC-NAc in these mice may be key in mediating stress susceptibility. Final Summary As described throughout this dissertation, ventral hippocampal circuits are key regulators of the integration of emotion and behavior. 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