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Title
Eosinophilic rhinitis and nasal epithelial remodeling in mice episodically exposed to ozone
Creator
Ong, Chee Bing
Date
2014
Collection
Electronic Theses & Dissertations
Description
Ozone is an oxidant air pollutant in photochemical smog. Though nasal epithelial remodelling has been well-documented in laboratory animals repeatedly exposed to ozone, associated inflammatory responses have not been fully characterized. In this study, we investigated if the onset of ozone-induced nasal epithelial remodelling is related to temporal changes in granulocytic influx and cytokine gene expression. Mice exposed to 24-weekdays of ozone developed marked eosinophilic rhinitis with...
Show moreOzone is an oxidant air pollutant in photochemical smog. Though nasal epithelial remodelling has been well-documented in laboratory animals repeatedly exposed to ozone, associated inflammatory responses have not been fully characterized. In this study, we investigated if the onset of ozone-induced nasal epithelial remodelling is related to temporal changes in granulocytic influx and cytokine gene expression. Mice exposed to 24-weekdays of ozone developed marked eosinophilic rhinitis with epithelial hyperplasia, mucous cell metaplasia and hyalinosis. Repeated subacute ozone exposures in mice induced an eosinophilic rhinitis with epithelial remodelling that resembled the pathology of human allergic/non-allergic rhinitis. Ozone-induced eosinophilic rhinitis was associated with an initial T helper cell 1 (Th1), followed by Th2-inflammatory response. Based on these findings, a study was conducted to investigate the role of lymphocytes (hypothesized cellular sources of Th1- and Th2-cytokines) in the development of eosinophilic rhinitis and associated nasal epithelial remodelling. In Rag2 x common gamma chain (γc) - deficient [RAG2(-/-) x γc(-/-)] mice, which lack T- and B- lymphocytes as well as NK cells, ozone nasal epithelial remodelling and eosinophilic rhinitis did not develop, hence supporting the hypothesis that lymphocytes are a crucial component to ozone induced eosinophilic rhinitis and associated epithelial changes in mice. These results suggest that chronic exposure to air pollutants, like ozone, may contribute to rising prevalence of allergic and non-allergic rhinitis.
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Title
Effects of cannabinoid receptor deletion on bone marrow-derived dendritic cell subtype development, maturation and antigen loading on MHC class I
Creator
Suaŕez-Martińez, José E.
Date
2016
Collection
Electronic Theses & Dissertations
Description
Cannabis is the most frequently consumed illicit drug in the world. Mammals express at least two types of cannabinoid receptors (CBRs), cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). Previous studies demonstrated that peptide pulsed bone marrow DCs (BMDCs) from CB1-/-CB2-/- mice induced a CD8+ T cell response in the absence lipopolysaccharide (LPS)-induced DC maturation. DCs are professional antigen presenting cells indispensible in linking the innate and adaptive immune...
Show moreCannabis is the most frequently consumed illicit drug in the world. Mammals express at least two types of cannabinoid receptors (CBRs), cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2). Previous studies demonstrated that peptide pulsed bone marrow DCs (BMDCs) from CB1-/-CB2-/- mice induced a CD8+ T cell response in the absence lipopolysaccharide (LPS)-induced DC maturation. DCs are professional antigen presenting cells indispensible in linking the innate and adaptive immune response. Each distinct DC subset has its unique set of surface markers and capabilities to respond to environmental stimuli and process antigen. The objective of the present study is to characterize the role of CBRs on the development and function of DC subsets from mouse bone marrow (BM). Our results demonstrate CB1-/-CB2-/- mice have a higher percent of BMDCs in freshly isolated BM and after 24 hours in culture in comparison to WT. Freshly isolatedBM cells isolated from CB1-/-CB2-/- mice elicited a CD8+ T cell response in the absence of LPS stimulation. Interestingly, there were no differences in MHC I or antigen-bound MHC I complexes on the surface of DCs that can account for this exacerbated activity. CD83, a DC marker implicated in maturation and stimulation of T cells, had a lower expression in DCs from CB1-/-CB2-/- mice. Taken together the results from this investigation suggest that CBRs are involved in DC bone marrow development and maturation.
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Title
PERTURBATION OF ASTROCYTIC KEAP1-NRF2-ARE PATHWAY AND GLUTAMATE TRANSPORTER EXPRESSION IN SPINAL ASTROCYTIC DEGENERATION
Creator
Wiwatratana, Duanghathai
Date
2021
Collection
Electronic Theses & Dissertations
Description
Several cellular mechanisms are known to be involved in methylmercury (MeHg) induced central nervous system (CNS) toxicity, including the dysregulation of intracellular Ca2+, redox, and glutamate homeostasis. However, the factors that make particular neurons susceptible to MeHg toxicity, and the latency period of neurological signs and symptoms, have not yet been clearly delineated. For example, the spinal dorsal root ganglia (DRG) is the primary target of MeHg. Mercury (Hg) granules are...
Show moreSeveral cellular mechanisms are known to be involved in methylmercury (MeHg) induced central nervous system (CNS) toxicity, including the dysregulation of intracellular Ca2+, redox, and glutamate homeostasis. However, the factors that make particular neurons susceptible to MeHg toxicity, and the latency period of neurological signs and symptoms, have not yet been clearly delineated. For example, the spinal dorsal root ganglia (DRG) is the primary target of MeHg. Mercury (Hg) granules are first detected in spinal cord motor neurons (SMNs) in the non-symptomatic phase, whereas Hg granules are detected in glia later, following with neurological symptoms (Møller-Madsen, 1991). This finding suggested that the latent period (non-symptomatic phase) is associated with Hg accumulation in neurons, while the symptomatic phase occurs following Hg accumulation in glia, and the susceptibility is not associated with Hg granule accumulation in cells (Møller-Madsen, 1991). Astrocytes generally provide glutathione (GSH) for neurons to detoxify toxic insult. In the spinal cord, MeHg might perturb the antioxidant pathway, Keap1-Nrf2-ARE pathway in the spinal cord astrocytes (SCAs) consequently contribute to DRG or SMN susceptibility to MeHg toxicity. In this study, the comparative responses of different SCAs maturity to a non-toxic MeHg concentration (0.1 μM) suggested that the fully mature SCAs (Day in vitro 30; DIV30), were more susceptible to MeHg than SCAs on DIV14. The perturbation of the Keap1-Nrf2-ARE pathway in SCAs (DIV 30) during exposure to sub-toxic MeHg concentration (0.50 μM) caused a biphasic increase in antioxidant genes such as Keap1, Nrf2, Gclc, Abcc1 mRNAs expression. The concomitant increase of glutamate transporter Slc7a11 encoded for the system Xc-, and Slc1a3 encoded for EAAT1, and Slc1a2 encoded for EAAT2 expression during MeHg exposure might suggest the cooperative expression or function of these glutamate transporters. This concomitant expression was further demonstrated in studies using Nrf2-knockout (Nrf2-KO) derived SCAs. The increase of basal Slc7a11 mRNA, was concurrent to the increase of basal Slc1a3 and Slc1a2 mRNA expressions in Nrf2-KO derived SCA. The function of time of MeHg exposure indicated that Nrf2-KO derived SCAs were more susceptible to MeHg than the wild-type (WT)-derived SCAs. The pronounced susceptibility of Nrf2-KO derived SCAs was mainly due to the loss of GSH) metabolism and transport genes Gclc, GPx1, GPx4, and Abcc1 mRNAs in this genotype. MeHg significantly reduced these mRNA expressions in both genotypes. However, not all Nrf2-ARE regulated genes were affected by MeHg in similar ways in these genotypes. For example, MeHg induced the increase of Slc7a11 mRNA expression in WT-derived SCAs, but it appears to cause the reduction of this mRNA expression in Nrf2 KO-derived SCAs. Administration of antioxidant N-acetyl-L-cystine (NAC) in pretreatment (NP), co-treatment (CO), and post-treatment of MeHg (MP) prevented the reduction of SCAs metabolic functions for over 160h. The mechanism of NAC action in preventing MeHg induced SCAs degeneration is primarily due to its thiol antioxidant property.In conclusion, this study suggests that age and genetic predisposition contribute to SCAs susceptibility to MeHg toxicity. The dysregulation of the antioxidant pathways and glutamate homeostasis in SCAs potentially contributes to SMNs or DRG susceptible to MeHg.
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Title
Neuromuscular transmission in a naturally occurring mouse mutant of the ß subunit of the neuronal calcium channel
Creator
Molina Campos, Elizabeth
Date
2011
Collection
Electronic Theses & Dissertations
Description
Entry of Ca2+ through voltage gated calcium channels (VGCCs) into nerve terminals is a necessary step coupling the action potential to release of acetylcholine (ACh). VGCCs are heteromultimeric complexes of α1, α2δ, and β subunits, and sometimes γ subunits. The specific α1-β combination assembled determines the channel properties. The mouse mutant lethargic (lh) has severe neurological defects due to a...
Show moreEntry of Ca2+ through voltage gated calcium channels (VGCCs) into nerve terminals is a necessary step coupling the action potential to release of acetylcholine (ACh). VGCCs are heteromultimeric complexes of α1, α2δ, and β subunits, and sometimes γ subunits. The specific α1-β combination assembled determines the channel properties. The mouse mutant lethargic (lh) has severe neurological defects due to a mutation that deletes α1 subunit interaction domain of the β4 subunit. β4 normally associates with the α1A subunit of the P/Q-type VGCCs, and has a major role in stabilizing the final α1A subunit conformation and targeting it to the cell membrane. Loss of the β4 subunit could alter the channel characteristics and localization of α1A. The overall goal of this dissertation was to test the hypothesis that disruption of the β4 subunit affects the function of the α1A subunit of the P/Q-type VGCCs. Electrophysiological recordings were performed at neuromuscular junctions (NMJs) of adult lh and wild type (wt) mice. The quantal content and phrenic nerve evoked release showed a significant decrease in lh with respect to wt. The frequency of spontaneous release of ACh also decreased significantly, although the reduction was only evident when Ca2+ was replaced by Sr2+ or Ba2+ as charge carriers. The amplitude of spontaneous release was not affected by this mutation, implying that each vesicle contains approximately the same amount of ACh in wt and lh mice. These results are due to a significantly slower process of neurotransmitter vesicles release, as confirmed by FM1-43 staining method.There are specific VGCCs antagonists that can be used to determine the contribution of the different types of VGCCs in nerve-stimulated ACh release from motor nerve terminals. ω-agatoxin IVA and SNX-482, specific antagonists for P/Q- and R-type VGCCs respectively, significantly reduced the quantal content in adult lh mice. Immunolabeling of VGCC subunits revealed an increase in α1E, β1 and β3, but no apparent change in the levels of α1A at adult lh neuromuscular junctions. Therefore, lh animals control ACh release by P/Q- and R-type VGCCs. The studies of this dissertation provide evidence for: 1) decreased nerve-evoked ACh release in lh mice, 2) slowed vesicle release process in lh mice, 3) increased level of β1 and β3, compensating for the lack of β4 subunit, and 4) P/Q- and R-type VGCC involvement in release of ACh from motor nerve terminals.
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Title
Characterization of two large gene families in the sea lamprey
Creator
Chang, Steven
Date
2013
Collection
Electronic Theses & Dissertations
Description
ABSTRACTCHARACTERIZATION OF TWO LARGE GENE FAMILIES IN THE SEA LAMPREYBySteven ChangThis dissertation employed molecular biology and bioinformatics to examine two large gene families in the sea lamprey, Petromyzon marinus. An integrative approach was used to define these gene families in order to ensure the validity of the size and members of each gene family. There are two chapters: Chapter 1 examines chemosensory gene expression in a specialized part of the olfactory system and Chapter 2...
Show moreABSTRACTCHARACTERIZATION OF TWO LARGE GENE FAMILIES IN THE SEA LAMPREYBySteven ChangThis dissertation employed molecular biology and bioinformatics to examine two large gene families in the sea lamprey, Petromyzon marinus. An integrative approach was used to define these gene families in order to ensure the validity of the size and members of each gene family. There are two chapters: Chapter 1 examines chemosensory gene expression in a specialized part of the olfactory system and Chapter 2 studies the expression of detoxification genes in the liver and gills in response to the lampricide, 3-trifluoromethyl-4-nitrophenol (TFM).CHEMORECEPTOR GENESFor this dissertation, I will restrict chemoreception to the detection of chemical signals in the nose (note: chemoreception includes taste), and is accomplished by detection of odorants in the environment by specialized sensory cells in the main olfactory epithelium (MOE). In certain tetrapods, a second sensory epithelium is also found in the nose, called the vomeronasal organ (VNO). Canonically, each epithelium represents the start of different olfactory pathways, which govern different behavioral responses. Each epithelium expresses different classes of chemoreceptor (CR) genes; the MOE expresses odorant receptors (ORs) and trace amine-associated receptors (TAARs), while the VNO expresses ORs, vomeronasal type-1 and type-2 receptors (V1Rs and V2Rs). The sea lamprey olfactory organ has one nostril and so has one nasal capsule, which is divided into two spatially distinct regions: the main olfactory epithelium (MOE) and the accessory olfactory organ (AOO). The MOE has been well studied in lampreys but the function of the AOO has eluded description for over 100 years. Based on other research and due to its proximity to the MOE, we hypothesized that the AOO represents an ancestral VNO. If this AOO is indeed an ancestral VNO, we expect a different connectivity to the central nervous system than from the MOE, and would expect expression of pheromone receptors (V1Rs and V2Rs). CR expression in the MOE and AOO of sea lamprey were examined. The differential expression of CR genes between the two epithelia was determined and the connectivity of the main and accessory epithelia was determined using neural tract tracing. Quantitative PCR confirmed and quantified the differential expression of specific genes in the main and accessory olfactory epithelia.CYTOCHROME P450 GENESThe second gene family to be explored is the cytochrome P450 family. P450 genes encode for steroidogenic or detoxification enzymes that are inducible by a substrate. As part of the strategy for controlling sea lamprey populations TFM is applied to streams. Very little is known at the molecular level of how TFM works to kill sea lamprey larvae, but based on responses by other organisms to xenobiotic substances, our hypothesis is that P450 genes are induced by exposure to TFM. P450 genes were predicted from the sea lamprey genome and larvae were exposed to TFM and gill and liver tissues were harvested over an 8-hour time course. Expression was confirmed using high-throughput sequencing and quantitative PCR. The immediate goal was to determine which P450 genes are induced by exposure to TFM. Alternatively, we generated a list of predicted Phase II detoxification enzymes in the event that P450 genes showed no difference in expression. The long-term goal is to use that knowledge to design more efficient and specific lampricides.
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Title
Peripartum plasticity in the serotonergic dorsal raphe : implications for postpartum socioemotional behavior and physiology
Creator
Holschbach, Mary A.
Date
2015
Collection
Electronic Theses & Dissertations
Description
Postpartum rats are highly maternal and show high aggression and low anxiety compared to nulliparous rats. To promote these dramatic changes in behavior, new mothers experience equally dramatic endocrine changes that elicit widespread neural plasticity. This neural plasticity includes cell birth and death in several regions of the peripartum forebrain, but such plasticity has never been reported in the dorsal raphe (DR), a midbrain site that provides most of the forebrain’s serotonin. Because...
Show morePostpartum rats are highly maternal and show high aggression and low anxiety compared to nulliparous rats. To promote these dramatic changes in behavior, new mothers experience equally dramatic endocrine changes that elicit widespread neural plasticity. This neural plasticity includes cell birth and death in several regions of the peripartum forebrain, but such plasticity has never been reported in the dorsal raphe (DR), a midbrain site that provides most of the forebrain’s serotonin. Because 1) postpartum lesions of the dorsal raphe reduce offspring development and survival, 2) serotonin affects postpartum social behaviors including caregiving and aggression, and 3) serotonin modulates anxiety in nulliparous males and females, I hypothesized that motherhood alters DR plasticity and serotonin synthesis/metabolism to support postpartum changes in socioemotional behaviors. To test this hypothesis, I examined effects of reproductive state and maternal experience on DR cell proliferation, newborn cell survival, cell death, and many aspects of the serotonin synthesis/metabolism pathway, then tested postpartum social and emotional behavior after lesioning the serotonergic DR. I discovered that although an equal number of cells are born in the DR of virgin, pregnant, and postpartum rats, fewer cells survived into the late postpartum period compared to cells surviving into the early postpartum period. These late postpartum females also had the highest levels of cell death within the DR. Next, I determined that interacting with the litter reduced cell survival and increased cell death in the DR of late postpartum rats. These effects were not due to high maternal corticosterone because adrenalectomized and sham-operated postpartum rats had equivalent DR cell survival. DR newborn cell survival and cell death were related to changes in serotonin synthesis and metabolism because late postpartum rats also had lower levels of serotonin’s precursor (5-HTP) and metabolite (5-HIAA) than early postpartum rats. To begin to test the functional significance of these changes in neuroplasticity and neurochemical function, I performed serotonin-specific DR lesions using a saporin-conjugated toxin targeting the serotonin transporter. Lesioning the DR altered numerous postpartum behaviors. During undisturbed observations, lesioned animals actively nursed pups (in kyphosis) more and licked pups less. Lesioning the DR did not greatly affect anxiety-like behavior, but did reduce maternal aggression. These data demonstrate that the DR is a site of significant peripartum plasticity, and that, along with this plasticity, there are concurrent changes in local serotonin synthesis and metabolism. These neurochemical changes may guide postpartum behavioral adaptations because lesioning the DR of new mothers had numerous effects on postpartum social behaviors. Taken together, these data suggest that the DR is an integral part of the maternal neural network that guides the initiation, modulation, and regression of postpartum behaviors.
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Title
Role of serum- and glucocorticoid-inducible kinase 1 (SGK1) in morphine and cocaine reward behaviors
Creator
Fallon, Barbara Leahy
Date
2014
Collection
Electronic Theses & Dissertations
Description
The mechanisms by which drugs of abuse, such as morphine and cocaine, induce long-term neuroadaptations that underlie addiction have not yet been fully defined. One promising brain region for study is the dopamine-rich ventral tegmental area (VTA), known to play a critical role in reward. Given recent evidence that chronic drug exposure increases VTA expression of serum- and glucocorticoid-inducible kinase 1 (SGK1), we wanted to investigate whether chronic drug exposure also increases SGK1...
Show moreThe mechanisms by which drugs of abuse, such as morphine and cocaine, induce long-term neuroadaptations that underlie addiction have not yet been fully defined. One promising brain region for study is the dopamine-rich ventral tegmental area (VTA), known to play a critical role in reward. Given recent evidence that chronic drug exposure increases VTA expression of serum- and glucocorticoid-inducible kinase 1 (SGK1), we wanted to investigate whether chronic drug exposure also increases SGK1 kinase activity and if altering VTA SGK1 activity affects drug reward behaviors. We found that chronic, but not acute, morphine or cocaine administration increased phosphorylation of SGK1 at S78 as well as phosphorylation of an SGK1 substrate, NDRG. To then investigate whether VTA SGK1 activity influences drug reward behaviors, we generated herpes simplex virus (HSV) constructs for local, short-term overexpression of SGK1 mutants. We found VTA expression of catalytically inactive SGK1-K127Q significantly lowered voluntary morphine consumption compared to constitutively active SGK1-S422D. We next examined cocaine locomotor activity and conditioned place preference (CPP) behaviors. Both HSV-GFP and HSV-K127Q mice exhibited locomotor sensitization to cocaine and robust CPP; however, there were no significant differences between the two groups. Given the complexity in these results, future studies will be needed to more fully understand the role of VTA SGK1 activity in reward behavior, including the specific role of S78 phosphorylation. We hope that such studies may identify molecular mechanisms underlying drug dependence that might serve as novel targets for therapeutic intervention in addiction.
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Title
Optogenetic analysis of excitatory and inhibitory neurotransmission in the enteric nervous system
Creator
Perez-Medina, Alberto L.
Date
2019
Collection
Electronic Theses & Dissertations
Description
The enteric nervous system (ENS) is embedded within the gastrointestinal (GI) tract and controls GI function. Impaired ENS function leads to altered patterns of motility and secretion, causing GI disease. For instance, functional gastrointestinal disorders (FGID) are caused by poorly understood alterations in the structure and function of nerves, smooth muscle cells (SMC), and other cell types in the GI tract. It is estimated that these disorders comprise about 41% of the total GI...
Show moreThe enteric nervous system (ENS) is embedded within the gastrointestinal (GI) tract and controls GI function. Impaired ENS function leads to altered patterns of motility and secretion, causing GI disease. For instance, functional gastrointestinal disorders (FGID) are caused by poorly understood alterations in the structure and function of nerves, smooth muscle cells (SMC), and other cell types in the GI tract. It is estimated that these disorders comprise about 41% of the total GI complications in the United States. Also, altered patterns of motility that occur in the GI muscles is a hallmark characteristic of FGIDs. Although the ENS is fairly understood, further elucidation of the enteric circuitry that governs GI motility would help to understand the pathophysiology of FGID. For that reason, identifying the contributions of classes of enteric neurons that control GI motility and secretion could aid in the identification of novel therapeutic targets for the treatment of FGIDs. A widely used method to study neural control of GI motility is sharp-electrode electrophysiological recordings from the smooth muscle or enteric neurons. Conventional, intracellular electrophysiological recordings have relied on electrical stimulation of enteric neurons which will activate all neurons in an ex vivo preparation of the ENS, and does not allow cell-specific activation of individual subpopulations of myenteric neurons. To overcome this problem, we used immunohistochemical methods to identify subpopulations of myenteric neurons and the optogenetically activated protein channelrhodopsin-2 (ChR2) that can be selectively expressed in subsets of enteric neurons. In Chapter 3, immunohistochemical studies of the mouse enteric nervous system are performed using the purinergic neuronal marker, vesicular nucleotide transporter (VNUT) along with markers for specific subsets of myenteric neurons and nerve fibers (e.g., neuronal nitric oxide synthase, choline acetyltransferase, calretinin, calbindin, and tyrosine hydroxylase),. Chapter 4 compares electrical and optogenetic electrophysiology recordings from myenteric neurons of mice that express ChR2 in nNOS neurons. The studies described in Chapter 5 use ChAT-ChR2-YFP-BAC transgenic mice which have eYFP tagged ChR2 expressed in cholinergic neurons. Optogenetics was used to isolate the cholinergic component of the ENS. The findings discussed in this dissertation provides evidence of a more sophisticated enteric circuitry of GI motility. (1) Purinergic neurons are likely a separate subpopulation of enteric neurons. VNUT is only expressed in the form of punctate varicosities at the nerve fibers and is not endogenously expressed in the soma of enteric neurons. VNUT also does not appear to colocalize with other neuronal immunoreactive markers within the myenteric plexus (mp), the tertiary plexus (tp), or circular smooth muscle layer of all tested tissue preps. (2) BLS of ChR2 expressed in nNOS neurons induced a purinergic/nitrergic biphasic IJP, suggesting that nNOS IMNs co-releases a purine as a neurotransmitter. Ectopic expression of ChR2 in non-nNOS neurons, however, could explain the biphasic IJP responses during electrophysiology recordings. Hence, the existence of separate subset populations of IMN populations (e.g., nNOS only and purinergic only IMNs) can't be ruled out. (3) BLS of ChR2 expressed in ChAT positive neurons induced EJPs and IJP responses. Inhibition of the nicotinic ACh receptor (nAChR) with mecamylamine significantly reduced the light-evoked IJP. Bath application of the purinergic P2Y1 antagonist, MRS 2179, was sufficient to abolish the IJP response, while the muscarinic ACh receptor antagonist, Scopolamine, abolished the EJP response. The data suggest that BLS of ChR2 activates cholinergic EMNs and cholinergic interneurons, and that activation of the cholinergic interneurons activates purinergic only IMNs that supply the smooth muscle, resulting in a predominant purinergic only IJP. Taken together, this work provides evidence for a diverse and more complex enteric neural circuit of GI motility. Future experiments should, however, focus on studying these enteric circuits at the level of the neuron, as these studies can provide a more in-depth analysis of the enteric circuitry.
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Title
CNGB1 mutation in papillon dogs : the identification, characterization and cure
Creator
Winkler, Paige A.
Date
2015
Collection
Electronic Theses & Dissertations
Description
Progressive retinal atrophy (PRA) is an inherited retinal dystrophy that affects over 100 breeds of dogs. It is characterized by a bilateral retinal degeneration commonly resulting in blindness. Affected dogs typically present with loss of dim light vision, attenuation of retinal blood vessels and tapetal hyperreflectivity. The purpose of this study was to identify the underlying cause of PRA in Papillon dogs and to characterize the phenotype at the cellular and molecular levels. I identified...
Show moreProgressive retinal atrophy (PRA) is an inherited retinal dystrophy that affects over 100 breeds of dogs. It is characterized by a bilateral retinal degeneration commonly resulting in blindness. Affected dogs typically present with loss of dim light vision, attenuation of retinal blood vessels and tapetal hyperreflectivity. The purpose of this study was to identify the underlying cause of PRA in Papillon dogs and to characterize the phenotype at the cellular and molecular levels. I identified a mutation in the gene CNGB1 which accounts for 70% of the PRA in the Papillon dogs. The CNGB1 mutation involves a 6 base pair insertion and a 1 base pair deletion resulting in exon skipping and a premature stop codon cause by a frameshift. CNGB1 encodes the β-subunit of a cyclic nucleotide-gated ion (CNG) channel. CNGB1 has multiple splice variants expressed in rod photoreceptors, olfactory sensory neurons and other tissues. CNG channels are directly involved in rod phototransduction and olfactory transduction. The retinal phenotype of the CNGB1 affected dogs was characterized by in vivo and ex vivo analyses. Electroretinograms (ERGs) and behavioral vision testing were conducted to assess retinal function throughout the course of the disease. The CNGB1 affected dogs had decreased and abnormal rod function at the earliest age tested but cone function was preserved until 5.5 years of age. Histological analyses showed that the morphology of rod photoreceptors deteriorate slowly over the first ~1.5 years of life while cone photoreceptor morphology is preserved for longer.Adeno-associated viral (AAV) vector therapy was used to treat five CNGB1 affected dogs with a wild-type copy of canine CNGB1 cDNA. One eye was injected with a low titer (1x1012) of an AAV vector delivering CNGB1 cDNA, six eyes were injected with a higher titer (5x1012) and one eye was injected with a GFP-expressing construct as a vehicle and procedural control. All dogs treated with the AAV vector containing the wild-type copy of CNGB1 showed rescue of rod function that was maintained throughout the time course of the study (9 months).The CNGB1 affected dog olfactory phenotype was investigated using in vivo and ex vivo techniques. The olfactory epithelium and the olfactory bulbs were abnormal in the CNGB1 affected dogs when compared to control dogs. I developed a behavioral test that could assess olfactory function in the CNGB1 dogs. The CNGB1 affected dogs had decreased but not absent olfactory function. The detailed descriptions of the retinal and olfactory phenotypes, in addition to the successful gene replacement therapy trial, in the CNGB1 affected dogs have laid the ground work for future studies including working with clinicians to advance gene replacement therapy trials in human patients with mutations in the CNGB1 gene.
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Title
Investigating the role of ventral tegmental area TORC2 in stress and stress-induced changes in opiate reward
Creator
Kaska, Sophia
Date
2017
Collection
Electronic Theses & Dissertations
Description
"Depression and opiate addiction are two prevalent neuropsychiatric diseases that produce a significant societal burden in terms of health and economic costs. Despite the substantial prevalence of depression and addiction both in the United States and worldwide, our understanding of the underlying neurobiological mechanisms remains incomplete, and elucidation of these neuroadaptations is necessary to develop more effective pharmacological therapies. Depression and addiction are often co...
Show more"Depression and opiate addiction are two prevalent neuropsychiatric diseases that produce a significant societal burden in terms of health and economic costs. Despite the substantial prevalence of depression and addiction both in the United States and worldwide, our understanding of the underlying neurobiological mechanisms remains incomplete, and elucidation of these neuroadaptations is necessary to develop more effective pharmacological therapies. Depression and addiction are often co-morbid, suggesting that similar neuroadaptations may underlie both diseases. The ventral tegmental area (VTA) is an important brain region in the reward circuit with a well-established role in the effects of drugs of abuse and development of addiction. Interestingly, increasing evidence suggests that dysfunction of the reward pathway may also contribute to depression. Similar biochemical changes, specifically the phosphorylation of AKT, a serine/threonine kinase, at S473, occurs in the VTA in response to chronic social defeat stress (CSDS), a pre-clinical model of depression, and chronic morphine treatment. Importantly, manipulation of AKT S473 phosphorylation alters both depressive- and addictive-like behaviors. Thus, regulation of AKT S473 phosphorylation may be a critical determinant in the development of depression and addiction. The protein complex responsible for phosphorylation of AKT S473 is mammalian (or mechanistic) Target of Rapamycin Complex 2 (TORC2). Previous work has demonstrated a critical role for VTA TORC2 in mediating morphine reward, but its role in stress had not been examined. Thus, this dissertation investigated whether VTA TORC2 signaling mediated susceptibility to CSDS. Furthermore, it has been established that CSDS alters the rewarding effects of drugs of abuse, including morphine. Given that both CSDS and chronic morphine similarly decrease AKT S473 phosphorylation, these studies also determined whether TORC2 signaling was necessary for stress-induced changes in morphine reward. Finally, one mechanism by which TORC2 signaling is thought to mediate long-term adaptations underlying behavioral changes is the alteration of VTA DA neuronal morphology. Therefore, these studies also sought to determine whether TORC2 promotes cytoskeletal remodeling in the VTA via regulation of Rac1 signaling. Through viral and genetic manipulation of Rictor, an essential protein for TORC2 activity, this dissertation investigated the central hypothesis that alteration of TORC2 signaling in the VTA contributes to changes in stress-induced morphine reward and CSDS susceptibility through modulation of specific downstream signaling molecules such as Rac1. Overall, the results of these studies reveal novel information and significance regarding the physiological role of VTA TORC2. While decreased TORC2 signaling in the VTA, or dopamine neurons, is not sufficient to increase susceptibility to CSDS or stress-induced drug reward, we identified a novel role for VTA TORC2 signaling in general consummatory behavior. Moreover, the data from our studies suggest that catecholaminergic TORC2 signaling might regulate behavior in a sex-specific matter, presenting novel opportunities for future studies. Finally, these data indicate that VTA TORC2 does not alter Rac1-PAK-Cofilin signaling and thus, further studies are needed to elucidate the mechanism by which TORC2 facilitates morphine-induced changes in VTA DA morphology."--Pages ii-iii.
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Title
Neuro- and gliogenesis in the pubertal rat brain : implications for female reproduction
Creator
Mohr, Margaret Ann
Date
2015
Collection
Electronic Theses & Dissertations
Description
"The goal of the experiments conducted in this dissertation was to test the hypothesis that cells added to the AVPV [anteroventral periventricular nucleus] during puberty are functionally incorporated into the neural circuitry involved in female-specific regulation of the hypothalamic-pituitary-gonadal (HPG) axis that controls the LH [leuteinizing hormone] surge and ovarian cyclicity."--from the abstract.
Title
Impaired function of prejunctional adenosine receptors on perivascular sympathetic nerves in salt-sensitive hypertension
Creator
Sangsiri, Sutheera
Date
2013
Collection
Electronic Theses & Dissertations
Description
Hypertension is a major public problem that will affect more than 1.6 billion people worldwide by 2025. Essential hypertension is associated with sympathetic nerve hyperactivity and 50% of the patients are salt sensitive. Deoxycorticosterone acetate (DOCA)-salt rat model is a low rennin form of hypertension with transient salt and water retention and is a model of salt-sensitive human hypertension. Norepinephrine (NE) and ATP are the major cotransmitters relased from perivascular sympathetic...
Show moreHypertension is a major public problem that will affect more than 1.6 billion people worldwide by 2025. Essential hypertension is associated with sympathetic nerve hyperactivity and 50% of the patients are salt sensitive. Deoxycorticosterone acetate (DOCA)-salt rat model is a low rennin form of hypertension with transient salt and water retention and is a model of salt-sensitive human hypertension. Norepinephrine (NE) and ATP are the major cotransmitters relased from perivascular sympathetic nerve varicosities supplying arteries and veins. NE and ATP regulate pheripheral arterial resistance and venous capacitance. Release of NE and ATP is precisely regulated by prejnctional autoreceptors. NE acts on the α2-adrenergic receptors (α2AR) to inhibit NE and ATP release and the α2AR function is impaired in DOCA-salt rats. ATP is hydrolyzed to adenosine which activates prejunctional adenosine A1 receptors (A1Rs) and A2ARs to modulate NE and ATP release. This work determined role of prejunctional A1Rs and A2ARs in adrenergic transmission. I also tested the hypothesis that prejunctional A1R and A2AR function is impaired in sympathetic nerves supplying mesenteric arteries and veins of DOCA-salt rats. Electricall-evoked NE release and constriction of blood vessel were measured in vitro by using amperometry and video microscopy. Drug effects on the kinetics of NE oxidtion currents were measured. My results show that prejunctional A1Rs couple to inhibition of NE release and A1R function is impaired in periarterial nerves of DOCA-salt rats. I also showed that prejunctional A2ARs couple to stimulation of adenosine transporter in perivenous sympathetic nerves and this receptor function is also compromised in DOCA-salt hypertension. These data suggests that the perivascular sympathetic nerve varicosities are an important target for the pathophysiology of salt sensitive hypertension. Furthermore, sympathetic autoreceptor dysfunction is not specific to α2-adrenergic receptors, but there is a more general disruption of prejunctional mechanisms controlling sympathetic neurotransmitter release. Finally, the data illustrate differential autoreceptor dysfunction in arteries and veins of DOCA-salt hypertension.
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Title
Mechanisms of GNAO1-associated neurological disorders
Creator
Feng, Huijie
Date
2019
Collection
Electronic Theses & Dissertations
Description
Tremendous advances in the genetics of neurodevelopmental disorders have markedly improved the understanding of disease mechanisms. This project will focus on understanding the mechanisms of GNAO1 encephalopathies, a devastating but complex disorder, which exhibit multiple neurological symptoms. These include developmental delay and variable components of early onset epilepsy and/or hyperkinetic movement disorders (MDs). These symptoms are associated with mutations in the GNAO1 gene, which...
Show moreTremendous advances in the genetics of neurodevelopmental disorders have markedly improved the understanding of disease mechanisms. This project will focus on understanding the mechanisms of GNAO1 encephalopathies, a devastating but complex disorder, which exhibit multiple neurological symptoms. These include developmental delay and variable components of early onset epilepsy and/or hyperkinetic movement disorders (MDs). These symptoms are associated with mutations in the GNAO1 gene, which encodes the Gαo protein. GNAO1 mutation-associated neurological disorders include neurodevelopmental delay with involuntary movements (NEDIM, OMIM#617493) and early infantile epileptic encephalopathy (EIEE17, OMIM#615473). The number of identified patients and mutant alleles for EIEE17 or NEDIM is increasing rapidly. Gαo is the most abundant membrane protein in the mammalian central nervous system. It couples to multiple G protein-coupled receptors (GPCRs) including GABAB, α2 adrenergic, D2 dopamine, and adenosine A1 receptors; all are associated with both MDs and epilepsy. In addition, GPCRs are readily targetable by agonists and antagonists. This provides the possibility of treating GNAO1-associated neurological disorders. This project revealed a fundamental mechanistic distinction among these GNAO1 mutations: Loss-of-function (LOF) GNAO1 alleles are associated with epilepsy, while gain-of-function (GOF) GNAO1 alleles are associated primarily with MDs. However, this simple model is insufficient to explain all clinical observations. To explore this correlation, we have created mouse models carrying two of the most common human GNAO1 mutant alleles (G203R and R209H). They largely share the human pathophysiology; the G203R mouse model exhibits both MD and enhanced seizure propensity, while the R209H mutant results in MD alone, as seen in children with those mutations. Using these models, we can further explore mechanisms that lead to distinct patterns in human GNAO1 encephalopathies. To explore electrophysiological alterations in the Gnao1 G203R mutant mouse model, I performed patch clamp studies on cerebellar Purkinje cells. The results show a decreased frequency of both miniature inhibitory postsynaptic currents (mIPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs), suggesting a reduced presynaptic GABA release.This study provides a molecular and physiological understanding of different GNAO1 alleles in vitro, and identifies key candidate alleles for further analysis in in vivo mouse models and in human GNAO1-associated neurological disorders. Furthermore, our study may serve as a prototype for other correlations between reported monogenic mutations and human neurological disorders.
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Title
The role of Nrf2 in the activation of primary CD4 T cells from mice and humans
Creator
Turley, Alexandra Elizabeth
Date
2018
Collection
Electronic Theses & Dissertations
Description
Nuclear factor erythroid 2-related factor 2 (Nrf2) is a cytoprotective transcription factor activated by cellular stressors such as reactive oxygen species, electrophilic xenobiotics, and other forms of oxidative stress that regulates the transcription of a number of antioxidant, detoxification, and cytoprotective genes. Commonly used Nrf2 activators include 1[2-Cyano-3,12-di oxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) and the food additive tert-butylhydroquinone (tBHQ). Nrf2 was...
Show moreNuclear factor erythroid 2-related factor 2 (Nrf2) is a cytoprotective transcription factor activated by cellular stressors such as reactive oxygen species, electrophilic xenobiotics, and other forms of oxidative stress that regulates the transcription of a number of antioxidant, detoxification, and cytoprotective genes. Commonly used Nrf2 activators include 1[2-Cyano-3,12-di oxooleana-1,9(11)-dien-28-oyl]imidazole (CDDO-Im) and the food additive tert-butylhydroquinone (tBHQ). Nrf2 was initially characterized as a cytoprotective transcription factor, and has since been shown to have additional effects, including a role in the immune system. Nrf2-null mice are more sensitive to a number of inflammatory stimuli, and cell-type specific effects of Nrf2 have been observed, mainly in macrophages and dendritic cells. However, the role of Nrf2 in T cell function has not been well studied. Prior studies from our lab have shown that activation of Nrf2 skews murine CD4 T cell differentiation towards a Th2 (allergy-like) phenotype. However, the role of Nrf2 in T cell activation, which sets up T cells for differentiation, and in human T cells is unknown. The purpose of the present studies is to determine the role of Nrf2 in primary CD4 T cell activation to fill this gap. To do this, the Nrf2 activators tBHQ and CDDO-Im were used to determine effects on T cell activation in murine or human primary CD4 T cells. In mice, tBHQ inhibited production of the cytokines interleukin-2 (IL-2), interferon gamma (IFNγ), and granulocyte-macrophage colony stimulating factor (GM-CSF) in both wild-type and Nrf2-null splenocytes, and inhibited tumor necrosis factor alpha (TNFα) only in wild-type splenocytes. CDDO-Im inhibited IFNγ and TNFα production, and increased IL-2 production, in a largely Nrf2-dependent manner, and increased GM-CSF independently of Nrf2. Both tBHQ and CDDO-Im had little effect on expression of the cell surface proteins CD25 and CD69. Both tBHQ and CDDO-Im increased nuclear translocation of the transcription factor c-Jun in wild-type but not Nrf2-null splenocytes, and inhibited p65 NF-κB DNA binding in wild-type splenocytes. Overall, the data suggest that Nrf2 activation has differential effects on murine T cell activation. Nrf2 activation inhibits IFNγ and TNFα secretion and p65 NF-κB DNA binding, and increases production of IL-2 and translocation of c-Jun, with little effect on CD25 and CD69 expression. In primary human CD4 T cells, tBHQ inhibits events of T cell activation, including production of IL-2 and IFNγ, expression of CD25 and CD69, and p65 NF-κB DNA binding. CDDO-Im also inhibits IL-2 and IFNγ production, and CD25 and CD69 expression in primary human CD4 T cells. However, the role of Nrf2 in mediating these effects was undetermined, so a primary human CD4 T cell Nrf2 knockdown model was developed. Nucleofection with siRNA directed against Nrf2 knocked down Nrf2 protein expression by ~90% 12 h after transfection. Suppression of IL-2, IFNγ, TNFα, and GM-CSF production by tBHQ occurred in both the scrambled control and Nrf2-deficient CD4 T cells, as did inhibition of CD25 and CD69 expression. RNA-seq analysis was performed and identified a number of differentially expressed genes between the scrambled control and Nrf2-deficient CD4 T cells, as well as genes differentially expressed with tBHQ treatment, indicating potential areas in which Nrf2 may play a role in primary human CD4 T cell activation.
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Title
Characterization and disruption of sea lamprey sex pheromone communication
Creator
Scott, Anne M. (Anne Marie) (Graduate of Michigan State University)
Date
2018
Collection
Electronic Theses & Dissertations
Description
Modulation of the sea lamprey (Petromyzon marinus) pheromone communication system may offer additional effective and environmentally benign approaches to manage populations in the Laurentian Great Lakes where they are invasive predators of large fishes. Previous studies showed that pheromones are indispensable cues that mediate sea lamprey migration and reproduction. Mature male sea lampreys release a multi-component sex pheromone that contains 3-keto petromyzonol sulfate (3kPZS) as a main...
Show moreModulation of the sea lamprey (Petromyzon marinus) pheromone communication system may offer additional effective and environmentally benign approaches to manage populations in the Laurentian Great Lakes where they are invasive predators of large fishes. Previous studies showed that pheromones are indispensable cues that mediate sea lamprey migration and reproduction. Mature male sea lampreys release a multi-component sex pheromone that contains 3-keto petromyzonol sulfate (3kPZS) as a main component. 3kPZS is a potent odorant that induces upstream movement of ovulated females to the spawning grounds with nesting mature males. While the function and potential management implications of 3kPZS are well-studied, the identity of other putative sex pheromone components and the utility of pheromone antagonists that disrupt the sea lamprey pheromone communication system remain largely unknown. In this dissertation, I characterize novel pheromone components released by mature male sea lampreys and identify antagonists that disrupt the olfactory and behavioral responses to the sea lamprey sex pheromone components. In Chapter 1, I provide evidence that spermine is a sex pheromone released by mature male sea lampreys through the emission of milt (fish semen). Spermine is detected by two trace amine-associated receptors expressed in the olfactory epithelium, induces olfactory responses at a sub-femtomolar concentration, and attracts ovulated females but not mature males. These results reveal a new source and chemical template of sea lamprey pheromones in addition to identifying a secondary benefit of male gamete release; that is to recruit additional mates in sea lampreys. In Chapter 2, I provide evidence that PZS treatment interferes with the responses to 3kPZS. Electro-olfactogram recordings from sea lampreys indicate PZS reduces the olfactory response to 3kPZS in a concentration-dependent manner. Behavioral results from maze assays and field experiments in-stream indicate the PZS disrupts 3kPZS-mediated behavioral responses in ovulated females as well. In Chapter 3, I provide evidence that petromyzonol-3,7,12,24-tetrasulfate (3sPZS), which was identified as a candidate 3kPZS antagonist through virtual screening in a previous study, selectively reduces the 3kPZS olfactory response. 3sPZS also reduces the behavioral preference of ovulated females for 3kPZS in the maze and reduces upstream movement, entry, and retention in artificial nest baited with 3kPZS in a natural spawning environment. The collective results of electrophysiology through field trials in a natural spawning environment provide a proof of concept that pheromone antagonists may be useful to disrupt vertebrate pheromone communication. In Chapter 4, I describe the methodology of bioassay-guided fractionation as an effective and interdisciplinary approach to isolate and characterize the structure, olfactory potency, and behavioral response of putative pheromones of sea lampreys. In Appendices A−C, I report the structures, olfactory potencies, and female behavioral preferences of eight novel bile salts or bile alcohols released from mature male sea lampreys. The results further elucidate the identity and bioactivity of the multi-component sex pheromone released by mature male sea lampreys. In addition, the identified naturally occurring and synthetic pheromone antagonists may offer new control tactics to manipulate behavioral responses or disrupt mating of sea lampreys. Taken together, the results on sea lamprey pheromones and pheromone antagonists can help guide an effective, integrated sea lamprey control program in the Laurentian Great Lakes.
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Title
The function of sympathetic innervation in the spleen and the role of endogenous CB1/CB2 receptor signaling
Creator
Simkins, Tyrell Jonathan
Date
2014
Collection
Electronic Theses & Dissertations
Description
The spleen is a multifunction organ that sits at a unique intersection between the circulatory, immune, and neurologic systems. The work in this dissertation endeavored to shed light on the interaction of the sympathetic nervous system in the spleen with these other vital biologic systems. In addition, the role of signaling by the cannabinoid receptors 1 and 2 was explored as it relates the function of splenic sympathetic innervation. Specifically, it was found that splenic noradrenergic...
Show moreThe spleen is a multifunction organ that sits at a unique intersection between the circulatory, immune, and neurologic systems. The work in this dissertation endeavored to shed light on the interaction of the sympathetic nervous system in the spleen with these other vital biologic systems. In addition, the role of signaling by the cannabinoid receptors 1 and 2 was explored as it relates the function of splenic sympathetic innervation. Specifically, it was found that splenic noradrenergic neurons do not play a role in T cell independent humoral immunity, and that both norepinephrine and adenosine mediate spleen contraction. It was discovered that splenic sympathetic noradrenergic neurons are likely not regulated by CB1 and that cannabinoid-mediated immunosuppression of humoral immunity is likely due solely to CB2 on immune cells. It was also found that CB1/CB2 play a permissive role in maintaining the relationship between NE release from splenic sympathetic neurons and spleen contraction. These findings add to the knowledge base regarding both the spleen and extra-CNS cannabinoid effects and can be built upon for a more complete understanding of these systems.
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Title
Effects of methylmercury on cerebellar granule cells of the tottering mouse
Creator
Marrero-Rosado, Brenda Marie
Date
2013
Collection
Electronic Theses & Dissertations
Description
Methylmercury (MeHg) is an organic highly toxic form of mercury and a persistent environmental neurotoxicant. The most common manner in which humans are exposed to MeHg is by consumption of contaminated fish. Studies from cases of chronic and acute human poisonings have revealed that MeHg causes a massive loss of cerebellar granule cells (CGCs), causing the characteristic dysarthria and ataxic signs. Previous research has found that acute treatment of rat CGCs with MeHg in vitro<...
Show moreMethylmercury (MeHg) is an organic highly toxic form of mercury and a persistent environmental neurotoxicant. The most common manner in which humans are exposed to MeHg is by consumption of contaminated fish. Studies from cases of chronic and acute human poisonings have revealed that MeHg causes a massive loss of cerebellar granule cells (CGCs), causing the characteristic dysarthria and ataxic signs. Previous research has found that acute treatment of rat CGCs with MeHg in vitro causes a time- and concentration-dependent increase in intracellular Ca2+ ([Ca2+]i) that is sufficient to cause CGC death. Voltage-gated Ca2+ channels (VGCCs) are believed to play a role in the mechanism of MeHg-induced cytotoxicity by possibly facilitating access for the metal to intracellular targets. Many subtypes of VGCCs are expressed in CGCs, each characterized by different pharmacological and kinetic properties. The endeavor of the present studies was to investigate the effects of MeHg on CGCs of a mouse model of human Cav2.1 (P/Q-type) channelopathy. The tottering (tg) mouse is the result of a non-lethal deleterious point mutation in the á1A pore-forming subunit of the P/Q-type Ca2+ channel. This VGCC subtype plays a crucial role in the process of neurotransmitter release in mature CGCs of humans and animal models. Data will show that, in low-K+ conditions (closely mimicking their mature state) CGCs isolated from mice homozygous (tg/tg) and heterozygous (+/tg) for the tg mutation present a delay in the onset of MeHg-induced [Ca2+]i increase in vitro. On the other hand, when they are acutely exposed to MeHg under depolarizing environments (mimicking their state in early development) tg/tg CGCs show increased susceptibility to cytotoxicity. Cerebellar organotypic slices from postnatal day (PND) 23-25 (after onset of ataxia) mice were also used to study the response of CGCs to MeHg taking into account the entire local cerebellar circuitry. Interestingly, +/tg cerebellar organotypic slices showed a higher percentage of CGC death than WT and tg/tg. The effect of MeHg on +/tg CGCs was partially eliminated by pretreating the slices with ù-conotoxin GVIA, an N-type VGCC antagonist. This suggests an important role of N-type VGCCs in the greater susceptibility of mature +/tg CGCs to MeHg. Of great importance is that tg-like mutations have been linked to human disorders including episodic ataxia type 2, familial hemiplegic migraine and spinocerebellar ataxia type 6. This work presents evidence of a genotype-environment interaction that could potentially identify human populations with higher risk for the neurotoxic effects of MeHg.
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Title
Serotonergic signaling at 5-HT3 receptors in sertotonin transporter (SERT) knockout (KO) rat, a sex specific animal model of visceral hypersensitivty
Creator
El-Ayache, Nadine Chahine
Date
2017
Collection
Electronic Theses & Dissertations
Description
The irritable bowel syndrome (IBS) is a functional gastrointestinal motor and visceral sensation disorder that is more common in women compared to men. 5-Hydroxytryptamine (5-HT, serotonin) signaling is disrupted in some IBS patients possibly due to polymorphic variations in the gene encoding the serotonin transporter (SERT) which result in increased extracellular 5-HT availability. Female SERT knockout (KO) rats exhibit visceral hypersensitivity to colonic distention that mimics colonic...
Show moreThe irritable bowel syndrome (IBS) is a functional gastrointestinal motor and visceral sensation disorder that is more common in women compared to men. 5-Hydroxytryptamine (5-HT, serotonin) signaling is disrupted in some IBS patients possibly due to polymorphic variations in the gene encoding the serotonin transporter (SERT) which result in increased extracellular 5-HT availability. Female SERT knockout (KO) rats exhibit visceral hypersensitivity to colonic distention that mimics colonic hypersensitivity known to occur in female IBS patients. Studies performed herein focused on understanding the role of 5-HT3 receptor signaling in SERT KO rats. The visceromotor response (VMR) to colorectal distension (CRD) was determined following inhibition of peripheral and central 5-HT3 receptors in SERT KO and WT rats. In female SERT KO rats spinal 5-HT3 receptor inhibition with alosetron caused an increase in VMR to CRD. In Male SERT KO rats activation of spinal 5-HT3 receptors increased VMR to CRD. Depletion of descending serotonergic input from the brainstem reversed the effects of 5-HT 3 receptor inhibition in SERT KO female rats and activation in SERT KO male rats. Based on these studies, I concluded that this sex specific response observed in SERT KO rats is due to differential pattern of 5-HT3 receptor expression in male and female SERT KO rats. The effects of ovarian hormone on VMR to CRD were also investigated in SERT KO and WT female rats. Ovariectomy and estrogen receptor (ER) antagonist studies were performed in SERT KO and WT female rats. VMR to CRD was enhanced in the proestrus phase of the estrous cycle in SERT KO but not WT female rats. Ovariectomy increased discomfort to CRD in SERT KO female rats in a time dependent manner. VMR to CRD increased on day 7 post-ovariectomy and lasted for up to 3 weeks. Intrathecal administration of ICI 182 780 (ER-α and ER-β) antagonist increased VMR to CRD in SERT KO female rats. The G-protein coupled ER (GPR30) antagonist, G15, did not affect VMR to CRD in SERT KO and WT female rats. These studies suggest that in SERT KO female rats classical ERs (ERα and ERβ) play an antinociceptive role in the presence of serotonergic dysfunction.
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Title
Lactobacillus reuteri reduces visceral hypersensitivity and alters gut motility without changing gut serotonin availability in an animal model of the irritable bowel syndrome
Creator
Jakupovic, Jasmina
Date
2013
Collection
Electronic Theses & Dissertations
Description
Irritable bowel syndrome (IBS) is a functional gastrointestinal (GI) tract disorder characterized by recurrent abdominal pain and changes in stool frequency and form, which are often comorbid with anxiety and depression. It affects up to 14% of the middle-aged population, is twice as prevalent in women as men and places a large cost on both the individual and the healthcare system. IBS results from a combination of genetic, psychosocial and physiological factors that make treatment difficult,...
Show moreIrritable bowel syndrome (IBS) is a functional gastrointestinal (GI) tract disorder characterized by recurrent abdominal pain and changes in stool frequency and form, which are often comorbid with anxiety and depression. It affects up to 14% of the middle-aged population, is twice as prevalent in women as men and places a large cost on both the individual and the healthcare system. IBS results from a combination of genetic, psychosocial and physiological factors that make treatment difficult, creating a need for novel therapies that address the whole spectrum of symptoms. Probiotics have recently emerged as a new treatment option for people suffering from gastrointestinal diseases and numerous studies have shown that probiotics can help alleviate symptoms in IBS patients. Currently, very little is known about the probiotic mechanism of action. Alterations in gut serotonin signaling have been implicated in abnormalities of gut motility and visceral sensitivity seen in IBS patients. In the gut, serotonin (5-hydroxytryptamine, 5-HT) is an important paracrine signaling molecule released by enterochromaffin cells in the mucosa. This 5-HT regulates gut motility, secretion and visceral sensation (pain). Probiotics are ingested with food and directly interact with the gut mucosa so we explored the interactions between a common probiotic and the 5-HT system in the gut to help elucidate possible mechanisms of action.In this work, we studied the beneficial effects of a probiotic, Lactobacillus reuteri 6475, in an animal model of IBS: the serotonin transporter knockout (SERT KO) rat. We showed that SERT KO rats recapitulate some of the human symptoms of IBS such as increased visceral hypersensitivity and decreased fecal output. Alterations in gut 5-HT availability did not fully account for the increased visceral hypersensitivity seen in SERT KO rats nor did SERT KO rats exhibit a sub-clinical level of inflammation. L.reuteri treatment decreased visceral hypersensitivity in both male and female SERT KO rats and increased fecal output in male SERT KO rats only, but did not alter gut 5-HT availability. L.reuteri shows promise as a probiotic that may help alleviate symptoms of IBS.
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Title
Mechanisms of methylmercury-induced astrocyte toxicity : a comparative study between cerebellar and cortical astrocytes
Creator
Jaiman-Cruz, Rosa Julia
Date
2017
Collection
Electronic Theses & Dissertations
Description
"Methylmercury (MeHg) can affect both the peripheral and central nervous system; however, granule cells in the cerebellum are preferential targets of MeHg neurotoxicity. The loss of granule cell after MeHg exposure is believed to be the result of unregulated elevations in intracellular calcium concentrations ([Ca2+]i) that lead to glutamate excitotoxicity. Astrocytes are the main cell type responsible for buffering the excess extracellular glutamate levels, preventing the excitotoxicity of...
Show more"Methylmercury (MeHg) can affect both the peripheral and central nervous system; however, granule cells in the cerebellum are preferential targets of MeHg neurotoxicity. The loss of granule cell after MeHg exposure is believed to be the result of unregulated elevations in intracellular calcium concentrations ([Ca2+]i) that lead to glutamate excitotoxicity. Astrocytes are the main cell type responsible for buffering the excess extracellular glutamate levels, preventing the excitotoxicity of neurons. However, MeHg can also affect astrocytes and increase their [Ca2+]i. MeHg-induced cytotoxicity in astrocytes has been studied in the forebrain cortex. However, effects on cerebellar astrocytes are less studied. Because regional differences can occur in astrocytes between the two areas, in this dissertation we compared MeHg toxicity on cerebellar and cortical astrocytes, and aimed to understand different toxicological effects of MeHg on these astrocytes, such as, the increase in extracellular glutamate levels and cytotoxicity. After an acute exposure to MeHg, there was a regional, but not a typological difference in astrocyte viability, in where cerebellar astrocytes were more susceptible to MeHg than cortical astrocytes. One difference between cortical and cerebellar astrocytes that might contribute to the cerebellar astrocytes susceptibility to MeHg was the contribution of calcium (Ca2+) to cell death. It was found that intracellular Ca2+ plays a role in MeHg-induced cortical astrocyte death. However, both intracellular and extracellular Ca2+ contribute to cerebellar astrocyte death. Another factor that contributes to the regional susceptibility of cerebellar astrocytes to MeHg was the Ca2+-dependent vesicular release of glutamate. These releases occurred in cerebellar astrocytes and not in cortical astrocytes, and were not due to the smooth endoplasmic reticulum (SER), but to the interaction of MeHg with the mitochondria, L-type and N-type voltage-gated Ca2+ channels (VGCCs). However, there were no differences in the contribution of these intracellular storages and Ca2+ channels to the reduction of viability. MeHg can also affect the excitatory amino acid transporters (EAATs), inducing an upregulation and dysfunction of these proteins. By studying the effect of MeHg on astrocytes, we found that the Ca2+-dependent vesicular release of glutamate from cerebellar astrocytes and the dysfunction of the EAATs contribute to cerebellar astrocyte susceptibility. The noticeable effects of MeHg exposure in glutamate levels observed on cerebellar astrocytes might contribute to the preferential sensitivity of the granule cells to MeHg by further increasing neuronal excitotoxicity."-Pages ii-iii.
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