ANSTATEU ll lllllllllllllllllllllllll 300901 9328 m:ll2ll This is to certify that the thesis entitled A DEVELOPMENTAL STUDY OF THE DISTRIBUTION OF LUTEINIZING HORMONE RELEASING HORMONE (LHRH)-CONTAINING NEURONS WITH- IN THE BRAIN OF A MUSTELID, THE EUROPEAN FERRET presented by Yu Ping Tang has been accepted towards fulfillment of the requirements for MA degree in Psychology Wiw M or professor Date 1/8/91 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution LIBRARY ‘ Michigan State University ‘— PLACE iN RETURN BOX to remove this checkout from your record. TO AVOID FINES Mum on or before date duo. DATE DUE DATE DUE DATE DUE J Tll‘l 1 MSU in An Affirmative Action/Equal Opportunity institution owns-pt A DEVELOPMENTAL STUDY OF THE DISTRIBUTION OF LUTEINIZING HORMONE RELEASING HORMONE (LHRH)-CONTAINING NEURONS WITHIN THE BRAIN OF A MUSTELID, THE EUROPEAN FERRET By Yu Ping Tang A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF ARTS Department of Psychology 1991 ABSTRACT A DEVELOPMENTAL STUDY OF THE DISTRIBUTION OF LUTEINIZING HORMONE RELEASING HORMONE (LHRH)-CONTAINING NEURONS WITHIN THE BRAIN OF A MUSTELID, THE EUROPEAN FERRET By Yu Ping Tang The neuropeptide luteinizing hormone releasing hormone (LHRH) controls gonadotropin secretion from the pituitary gland. Reproductive maturation is characterized by enhanced release of the gonadotropins. This study determined whether a change in the number or morphology of LHRH neurons is correlated with the pubertal rise in gonadotropin release. LHRH-containing neurons were identified immunocytochemically in male ferrets at four ages spanning puberty. Immunopositivc (LHRH-0 cell bodies were diffusely distributed from rostral forebrain through caudal hypothalamus. There were no age-related differences in the total number of LHRH+ neurons or in mean somal area. However, when brain subareas were examined separately, there were significantly fewer LHRH+ somata within the arcuate nucleus in peri- and postpubertal ferrets compared to prepubertal fenets. Thus, an inability to produce LHRH cannot account for the minimal release of gonadotropins prepubertally. Instead, the LHRH neurons in the arcuate nucleus may inhibit the episodic release of LHRH in reproductively immature ferrets. ACKNOWLEDGMENTS I would like to thank my committee members, Dr. Glenn Hatton, Dr. Tony Nunez and Dr. Cheryl Sisk for supporting me throughout this thesis. To Cheryl Sisk. who gave me the chance to understand the neuroscience research, guided me into the neuroendocrine field and taught me everything in all these years. T_hanl;s_! To labmates (Lee Ann Berglund, Isabel Sanchis and Jane Venier) and friends, thank you for always being there when I needed you. To my little boy, Mang-Git, thank you for bringing happiness to the family. To Wai Man, your understanding and encouragement help me through all the frustrations, thanks. To Mom and Dad, words cannot express my appreciation for all you have given me. iii TABLE OF CONTENTS LIST OF TABLES LIST OF FIGURES LIST OF ABBREVIATIONS INTRODUCTION METHODS Animals and housing Experimental design Experiment 1 Experiment 2 Tissue preparation LHRH immunocytochemistry Immunostaining controls Data analysis RESULTS Experiment 1 Experiment 2 DISCUSSION REFERENCES iv PAGE vi vii 10 ll 20 31 38 LIST OF TABLES TABLE 1. PAIRED TESTICULAR WEIGHT OF MALE FERRETS DURING PUBERTY TABLE 2. NEURAL STRUCTURES CONT AH‘HNG LHRH+ CELL BODIES TABLE 3. QUANTIFICATION OF LHRH+ CELL BODIES IN MALE FERRET S DURING PUBERTY TABLE 4. QUANTIFICATION OF LHRH+ CELL BODIES WITHIN SEVEN SUBAREAS OF THE BRAIN TABLE 5. SOMAL AREA OF LHRH+ NEURONS IN MALE FERRETS DURING PUBERTY TABLE 6. QUANTIFICATION OF LHRH+ CELL BODIES IN CONTROL AND COLCHICINE-TREATED MALE FERRETS TABLE 7. QUANTIFICATION OF LHRH+ CELL BODIES WITHIN ARCUATE NUCLEUS IN MALE FERRETS DURING PUBERTY PAGE 12 14 23 26 27 28 30 LIST OF FIGURES PAGE Figure 1. Coronal sections of male fenet brain illustrating the presence of 13 LHRH-immunopositive cell bodies in (A) diagonal band of Broca; (B) medial basal hypothalamus; and (C) arcuate nucleus/median eminence. Magnification bar = 100 um. (ARC-arcuate nucleus; 3V-third ventricle). Figure 2. Line drawings of coronal sections of a representative ferret brain 15 (25 wk old) through forebrain and diencephalon. Dots depict location of LHRH+ cell bodies. Each drawing shows a composite of the locations of cell bodies in 3-5 sections anterior and posterior to the section shown. All cell bodies identified in this individual are represented. (AC-anterior commissure; AMY -amygdala; CA-caudate nucleus; LOT-lateral olfactory tract; LS-lateral septum; ME-median eminence; OC-optic chiasm; OT-optic tract; POA-preoptic area; VMH-ventromedial hypothalamus; 3V-third ventricle). Figure 3. Coronal sections of male ferret brain illustrating the distribution 19 of LHRH+ fibers (arrows) in (A) organum vasculosum of the lamina tenninalis; (B) amygdala; and (C) median eminence. Magnification bar = 100 um. (3V-third ventricle). ' Figure 4. Line drawing of a midsagittal section of male ferret brain. 21 Photomicrographs illustrate the distribution of LHRH+ fibers in dark field. Magnification bar = 100 um. (AC-anterior commissure; ARC-arcuate nucleus; CC-corpus callosum; OC-optic chiasm; VMH-ventromedial hypothalamus; 3V-third ventricle). Figure 5. Histograms of the number of LHRH+ cell bodies found in each 24 40 um coronal section examined in brains of representative individual ferrets from the different age groups. Each section is 80 pm from the next since only every other section was reacted. The scale on the x axis gives distance (tun) rostral and caudal to an arbitrary zero point defined as the section which first contained the suprachiasmatic nucleus (SCN). The approximate locations of preoptic area (FDA) and arcuate nucleus (ARC) are noted. LIST OF ABBREVIATIONS . AC - anterior commissure AMY - amygdala AN OVA - analysis of variance ARC - arcuate nucleus BNST - bed nucleus of stria terminalis CA - caudate nucleus CC - corpus callosum DBB - diagonal band of Broca FSH - follicle stimulating hormone GnRH - gonadotropin releasing hormone HIPP - hippocampus LH - luteinizing hormone LHRH - luteinizing hormone releasing hormone LHRH+ - LHRH irnmunopositive LOT - lateral olfactory tract LS - lateral septum LV - lateral ventricle MBH - mediobasal hypothalamus ME - median eminence MM - mammillary bodies LIST OF ABBREVIATIONS (Cont’d) 0C - optic chiasm OT - optic tract OVLT - organum vasculosum of the lamina terminalis PBS - phosphate buffered saline PBS-TX - phosphate buffered saline with Triton X-100 POA - preoptic area PVN - paraventricular nucleus SCN - suprachiasmatic nucleus VMH - ventromedial hypothalamus 3V - third ventricle INTRODUCTION Luteinizing hormone releasing hormone (LHRH) is a neuropeptide present in high concentrations within the hypothalamus. The amino acid sequence of this decapeptide, originally determined from porcine hypothalarnic extracts, is Glu-His-Trp- Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2 (Matsuo, et al., 1971). LHRH is cleaved from a larger 92 amino acid pre-prohormone, which, in addition to LHRH, also yields a 56 amino acid peptide known as LHRH-associated peptide (Adelman, et al., 1986; Seeburg & Adelman, 1984). The best known function of LHRH is stimulation of the synthesis and release of luteinizing hormone (LH) and follicle stimulating hormone (FSH) from the anterior pituitary gland. Thus, LHRH is also known as gonadotropin releasing hormone (GnRH). Since LHRH fibers are found in other brain structures in addition to the median eminence, where LHRH is released into the pituitary portal system, LHRH probably also functions as a neurotransmitter or neuromodulator. LHRH can alter electrophysiological activity of neurons in the hypothalamus (Dudley & Moss, 1987; Dyer & Dyball, 1974; Moss, 1977; Renaud, et al., 1975; Rothfeld, et al., 1985) and hippocampus (Palovcik & Phillips, 1986). Depending on the cell, LHRH can elicit either excitatory or inhibitory responses. Although a precise role for LHRH within the mammalian central nervous system has not been established, it may be involved in facilitation of lordosis in female rats and of ejaculation in male rats (Boyd & Moore, 1985; Dudley & Moss, 1987; Kow & Pfaff, 1988; Moss, 1977 & 1979; Moss & McCann, 1975; Myer & Baum, 1980; Ryan & Frankel, 1978; Schiess, et al., 2 1987). The behavioral effects of LHRH are independent of its effects on LH secretion (Kastin, et al., 1980; Moss, 1977; Moss & McCann, 1975). The neuroanatomical distribution of LHRH has been studied immunocytochemically in numerous mammalian species, including marsupials, rodents, carnivores, ungulates, and primates. In all species, LHRH-immunopositive (LHRH+) cell bodies are diffusely distributed along a rostral—caudal continuum extending from the olfactory bulb, diagonal band of Broca (DBB), medial septum, and preoptic area (POA) through caudal hypothalamus (Anthony, et al., 1984; Boissin-Agasse, et al., 1988; Caldani, et al., 1988; Glass, 1986; Goldsmith & Song, 1987; Jennes & Stumpf. 1980; Kineman, et al., 1988; King & Anthony, 1984; King, et al., 1982; Lehman, et al., 1986; Leshin, et al., 1988; Merchenthaler, et al., 1984; Polkowska, et al., 1980; Ronnekleiv et al., 1987; Schwanzel-Fukuda, et al., 1987, 1988; Shivers, et al., 1983a; Silverman, et al., 1982; Takahashi, et al., 1988; Witkin, 1986, 1987; Witkin, et al., 1982; Wray & Hoffman, 1986a & b; Zheng, et al., 1988; reviewed in Silverman, 1988). Species differences in this distribution are typically only in the relative proportion of cell bodies anterior to hypothalamus, and not in the specific structures in which the neurons are found. For example, in rats, more than half of the LHRH+ neurons reside rostrally in the septum, POA and anterior hypothalamus (e.g., Wray & Hoffman, 1986a). In contrast, in primates, ferrets, and bats, the majority of LHRH+ neurons are present more caudally in basal retrochiasrnatic hypothalamus, extending as far posteriorly as the median eminence (ME) (Goldsmith & Song, 1987; King & Anthony, 1984; Tang & Sisk, 1988). One exception to this generalization is that LIRH+ neurons are not found within the arcuate nucleus of rodents, but are present in relatively high numbers (compared to other structures) in the arcuate nucleus of primates and camivores (King and Anthony, 1984; Silverman, et al., 1982; Sisk, ct al., 1988). LHRH+ axons are abundant in the ME and the organum vasculosum of 3 the lamina terminalis (OVLT). Sparser projections are found in the medial mammillary nuclei, the epithalamus, the amygdala (AMY), midbrain central grey, and hippocampus (HIPP) (Anthony, et al., 1984; Boissin-Agasse, et al., 1988; Caldani, et al., 1988; Glass, 1986; Goldsmith & Song, 1987; Jennes & Stumpf, 1980; Kineman, et al., 1988; King and Anthony, 1984; King, et al., 1982; Lehman, et al., 1986; Leshin, et al., 1988; Merchenthaler, et al., 1984; Polkowska, et al., 1980; Ronnekleiv et al., 1987; Schwanzel-Fukuda, et al., 1987, 1988; Shivers, et al., 1983a; Silverman, et al., 1982; Takahashi, et al., 1988; Witkin, 1986, 1987; Witkin, et al., 1982; Wray & Hoffman 1986a & b; Zheng, et al., 1988; reviewed in Silverman, 1988). LHRH neurons are usually round or fusiforrn and bipolar or multipolar. Somal area ranges from 100—300 urn2 (Cameron, et al., 1985). In certain species, e.g., the rat, sheep, and monkey, some LHRH+ cell bodies and dendrites have either thin protuberances (pedunculated spines) or knob-er protuberances (sessile spines). These types of cells have been termed "spiny" or "thomy" neurons and can be morphologically distinguished from "smooth" neurons, which are characterized by the absence of such protuberances (Lehman, et al., 1986; Witkin, 1986; Wray & Hoffman, 1986a & b). In other species, such as the ferret, pig, and golden hamster, spiny LHRH neurons have not been identified (Jennes & Stumpf, 1980; Kineman, et al., 1988; King & Anthony, 1984; Sisk, et al., 1988). Ultrastructurally, LHRH+ cell bodies are characterized by a large round or ovoid nucleus, and the presence of Golgi complexes, ribosomes and rough or smooth endoplasmic reticulum (Kozlowski, et al., 1980). Both smooth and spiny LHRH neurons have the same organelles in similar number and distribution. Initially, Spiny LHRH neurons were reported to have more synaptic contacts compared to smooth LHRH neurons (Jennes, et al., 1985). However, using a systematic morphometn‘c method, Witkin and Demasio (1990) more recently found no difference in the number 4 of synapses between smooth and thomy LHRH neurons. LHRH+ neurons are innervated by catecholaminergic, serotoninergic and opiatergic neurons (Chen, et al., 1989; Kiss & Halasz, 1985; Leranth, et al., 1988; Meister, et al., 1988), all of which may play an important role in the regulation of activity of LHRH neurons (Gallo, 1980; Kalra, 1985; Kalra, et al., 1988; McCann, 1980; Negro-Vilar, et al., 1986; Ramirez, et al., 1984; Wuttke, et al., 1982). LHRH+ cell bodies and dendrites are also innervated by LHRH+ axons (Chen, et al., 1989). Research in our laboratory is focused on central nervous system mechanisms of puberty onset. One endocrine hallmark of puberty in mammals is an increase in the frequency of release of LH from the anterior pituitary gland occasioned by an increase in the episodic release of LHRH into the median eminence (for reviews, see Foster, 1988; Ojeda & Urbanski, 1988; Plant, 1988). The mechanisms underlying the pubertal increase in episodic LHRH secretion are not fully understood. Changes in both LHRH+ cell morphology and number have been correlated with pubertal development. LHRH+ neurons in the adult monkey are significantly larger in total cross-sectional area and in cyt0p1asmic cross-sectional area than in the juvenile monkey (Cameron, et al., 1985). While the total number of LHRH+ cell bodies remains constant throughout pubertal development in the rat, the proportion of cell bodies classified as spiny increases (Wray & Hoffman, 1986a & b). Phot0period— induced delay of puberty in Djungarian hamsters results in a smaller number of immunocytochemically identified LHRH cell bodies compared to that observed in normally maturing controls (Yellon, et al., 1987). These anatomical findings have all been interpreted as evidence for activation of the LHRH neuronal system during reproductive maturation. Experiments in our laboratory have employed the male European ferret (Mustcla putorius furo) as an animal model for the study of central mechanisms of puberty 5 onset. As in other male mammals, LH pulse frequency increases during puberty in the ferret (Sisk, 1987). Since pituitary responsiveness to LHRH is not diminished in prepubertal ferrets compared to postpubertal fenets (Berglund & Sisk, 1990), the infrequent secretion of LH in prepubertal ferrets is the result of insufficient LHRH release from the hypothalamus. The pubertal increase in LH pulse frequency in the ferret is due solely to a developmental decline in hypothalamic responsiveness to steroid negative feedback; in the absence of gonadal steroids, the pattem of episodic LH secretion in prepubertal fenets is comparable to that of gonadectomized adult ferrets (Ryan, et al., 1988; Sisk, 1987). Gonadal steroids must either directly or indirectly inhibit LHRH synthesis and/or release into the median eminence, and a change in the interaction between steroid target neurons and LHRH neurons must be critical for the onset of puberty in the ferret. One way in which a change in response to the inhibitory effects of steroid might be reflected at the cellular level is by a change in the ability of neurons to synthesize LHRH; that is, the number of neurons capable of producing LHRH may increase during puberty as the ability of testosterone to exert negative feedback decreases. A LHRH has been immunocytochemically localized in the adult ferret (Boissin- Agasse, et al., 1988; King & Anthony, 1984; Sisk, et al., 1988), however, a quantitative analysis of the distribution of these neurons in this species has not been performed. In addition, it is not known whether the number or morphology of LHRH-producing cells changes during pubertal development in this species. LHRH+ cells are reported to be more numerous in adult male ferrets during the breeding season than during the non-breeding season, although exact numbers in each reproductive state were not provided (Boissin-Agasse, et al., 1988). The purpose of this experiment was to determine whether there is a change in the number, anatomical distribution, or morphological features of LHRH+ neurons that is correlated with 6 activation of the hypothalamic-pituitary-gonadal axis at the time of puberty in male ferrets. METHODS Ani n in Weanling (8 wk old) male fenets were purchased from Marshall Farms (North Rose, NY) and housed in stainless steel cages (51 x 60 x '38 cm) with Purina Cat Chow or Purina Ferret Chow (Ralston Purina, St. Louis, MO) and water available at all times. The light-dark cycle in the colony room consisted of 8 hr light and 16 hr dark per day (LD 8:16; lights on 0900 h), and room temperature was maintained at 23 3; 1° C. In our colony, ferrets continuously maintained from weaning on 8 hr light/day (short days) undergo puberty beginning at approximately 18-20 wk of age. Adulthood is achieved by 25-28 weeks of age, as indicated by large descended testes, frequent secretion of LH pulses, and the presence of mature spermatozoa within the testes (Sisk, 1990). Exmrimentfl design Exmriment 1 Ten-wk-old (n=3), lS-wk-old (n=3), 20—wk-old (n=3) and 25-wk-old (n=3) ferrets were deeply anesthetized with an overdose of Equithesin (2.5 ml/kg ip) and perfused intracardially with 350 ml 0.87% heparinized saline (sodium heparin; Elkins-Sinn, Inc. Cherry Hill, NJ; 10000 units/100 m1 saline), followed by 350 ml of Zamboni’s fixative (1.8% parafonnaldehyde/picric acid in 0.1 M phOSphate buffered saline (PBS)). The brains were removed, blocked at extreme rostral forebrain and at midbrain and stored in 20% sucrose in Zamboni’s fixative for 2-3 days. Just after anesthetization and prior to perfusion, the testes were removed from each ferret, weighed, and homogenized in 0.15 M NaCl and 0.25 mM thimerosal containing 0.05% Triton X-100. Samples of the testicular homogenates were microscopically 8 examined for the presence of mature spermatozoa. Exmriment 2 Eight ferrets, four 10- and four 25-wk-old, were used to determine whether treatment with a blocker of axonal transport, colchicine, wOuld enable visualization of a larger number of LHRH+ cell bodies. Two animals from each age group received an intracerebro-ventricular injection (third ventricle) of 250 ttg colchieine in 10 ul of artificial CSF under Equithesin anesthesia (2.5 milkg ip). Stereotaxic coordinates were: 9.1 mm anterior to ear bar zero, on the midline, and 7.0 mm ventral from dura. Fenets were anesthetized and perfused with saline and Zamboni’s fix 24 hr later, and the brains were cryoprotected as in Experiment 1. Tissue pmparation Coronal sections (40 um) were cut on a freezing microtome and every other section (every fourth section for Experiment 2) was saved and processed for immunocytochemical identification of LHRH neurons. One brain obtained from a 25 wk old ferret (in Experiment 1) was sectioned in the sagittal plane for better visualization of descending LHRH projections. A LHRH immungcmxghemistry Sections were washed 3x in 0.1 M PBS with 0.2% Triton X-100 (PBS-TX) and then incubated in 0.3% H202 in methanol for 30 min to remove remaining aldehydes and reduce endogenous peroxidase activity. Sections were then incubated sequentially in normal goat serum (Vectastain ABC Kit, Vector Laboratories, Burlingame, CA; 30 minutes), rabbit anti-LHRH LR-l (obtained from Dr. Robert Benoit, The Montreal General Hospital Research Institute) at a dilution of 1:10,000 in PBS-TX (16-24 hr), secondary antibody (goat anti-rabbit immunoglobulins; Vectastain ABC Kit; 2 hr), avidin—biotin-HRP complex (Vectastain ABC Kit; 1 hr) and diaminobenzidene-glucose oxidase (Sigma, St. Louis, MO; about 30 minutes, or until reaction product appeared). 9 Sections were washed 3x in PBS-TX in between each incubation. All incubations were at room temperature, except for that with primary antibody, which was at 4°C. The optimal dilution of primary antibody was determined in pilot experiments in which a range of dilutions (1:5,000-1:25,000) of primary antibody was tested. The reagents from the Vectastain ABC Kit were prepared according to manufacturer’s instructions. After the chromogen reaction, sections were washed 5x in PBS-TX, mounted onto gelatinized slides, dried, counterstained with cresyl violet or thionin, and coverslipped. Immungstaining controls The antibody LR-l used in this experiment is directed against amino acid residues 2-4 and 7-10 of the decapeptide; it does not cross-react with thyrotropic releasing hormone, substance P, neurotensin, arginine vasopressin, somatostatin, met- enkephalin or leu-enkephalin (Benoit, et al., 1987). Pre-immune serum did not bind to LHRH in a radioirnmunoassay (Benoit, personal communication, 1990). Specificity of immunostaining was verified in two ways. First, some sections were incubated in primary antiserum which had been preabsorbed with synthetic LHRH (Sigma, St. Louis, MO; 100 ug/ml) for 24 hours at 4°C. Other sections were processed in the absence of primary antiserum. No immunocytochemical staining of cell bodies and fibers was observed after either control procedure. Da a a1 i All sections were examined microscopically under brightfield illumination at 100- 400x. A neuron was identified as LHRH+ if the brown immunocytochemical reaction product was present within the cytoplasm. Line tracings of each section were made and the locations of LHRH+ cell bodies and fibers were marked on the drawings. In addition to counting the total number of LHRH+ neurons, the number of LHRH+ somata within certain specific areas was determined. These areas were 10 DBB, bed nucleus of stria terminalis (BNST), POA, mediobasal hypothalamus (MBH), AMY, arcuate nucleus (ARC), and ME. The somal area of all LHRH+ cells found in every fourth section from each individual in Experiment 1 was calculated using an Olympus Cue 2 Image Analysis System. The effect of age on testicular weight, on total LHRH+ cell number within the brain and subareas, and on somal area in ferrets from Experiment 1 was assessed by one way Analysis of Variance (ANOVA). Two way ANOVA was used to assess the effects of colchicine and age on total LHRH+ cell body number in ferrets from Experiment 2. Scheffe F-test was used for post hoc comparisons. RESULTS Extrema Paired testicular weights of ferrets from the different age groups are shown in Table 1. ANOVA indicated a significant main effect of age (F(3,10) = 113.42, p < 0.0001). Post hoc comparisons revealed no differences among mean weights of the 10-, 15-, and 20-wk-old groups. There was a dramatic increase in gonadal size by 25 wk of age which resulted in significant differences between mean testicular weight of this group and that of the other three groups (all p < 0.05). Mature spermatozoa were found in testicular homogenates from the 25-wk-old group only. In all age groups, LHRH+ cell bodies were diffusely distributed throughout the telencephalon and diencephalon in structures ranging as far anteriorly as medial and lateral septum, DBB, and POA to structures extending as far caudally as the ARC, ME and mammillary bodies (MM). Most hypothalamic LHRH+ somata were located near the base of the brain. In the more rostral areas, such as the vertical limb of DBB and POA, most LHRH+ cell bodies were found near the midline and scattered dorsally to ventrally. Photomicrographs of representative LHRH+ cell bodies are shown in Figure 1. All structures in which one or more LHRH+ cell bodies were found are listed in Table 2. Figure 2 contains line drawings of coronal sections through a representative brain with locations of LHRH+ cell bodies indicated. The most dense projection of LHRH+ fibers was found in the ME (Figure 3C). Sparser projections were seen in the OVLT and AMY (Figures 3A & 3B). Scattered fibers were observed in the olfactory tubercle, DBB, BNST, POA, MBH, HIPP and MM. Figure 4 shows LHRH+ fibers in the midsagittal plane. The mean total number of LHRH+ cell bodies found in sections from each age group is shown in Table 3. There was no significant main effect of age on the total 11 12 TABLE 1. PAIRED TESTICULAR WEIGHT OF MALE FERRETS DURING PUBERTY Age n Testis weight (mg) 10 wk 3 221.97 :1: 13.69* 15 wk 3 289.20 :1: 1423* 20 wk 3 586.70 :1: 192.08* 25 wk 2 4243.60 1: 378.90 Values are MEAN :i: SEM * p < 0.05 compared to 25-wk—old 13 Figure 1. Coronal sections of male ferret brain illustrating the presence of LHRH- immunopositive cell bodies in (A) diagonal band of Broca; (B) mediobasal hypothalamus; and (C) arcuate nucleus/median eminence. Magnification bar = 100 um. (ARC-arcuate nucleus, 3V-third ventricle). 14 TABLE 2. NEURAL STRUCTURES CONTAINING LHRH+ CELL BODIES Anterior hypothalamic area Arcuate nucleus Bed nucleus of the stria terminals Cingulate gyrus Corpus callosum Cortical and medial nuclei of the amygdala Diagonal band of Broca Dorsal hypothalamic area Fomix Hippocarnpus Lateral and medial septum Lateral hypothalamus Lateral olfactory tract Median eminence Neocortex Nucleus accumbens Olfactory tubercle Optic chiasm Optic tract Premammillary area Preoptic area Pyriform cortex Retrochiasmatic mediobasal hypothalamus 15 Figure 2. Line drawings of coronal sections of a representative ferret brain (25 wk old) through forebrain and diencephalon. Dots depict location of LHRH+ cell bodies. Each drawing shows a composite of the locations of cell bodies in 3-5 sections anterior and posterior to the section shown. All cell bodies identified in this individual are represented. (AC-anterior commissure; AMY -amygdala; CA-caudate nucleus; LOT-lateral olfactory tract; LS-lateral septum; ME-median eminence; OC-optic chiasm; OT-optic tract; POA-preoptic area; VMH- ventromedial hypothalamus; 3V-third ventricle). Figure 3. Coronal sections of male fenet brain illustrating the distribution of LHRH+ fibers (arrows) in (A) organum vasculosum of the lamina terminalis; (B) amygdala; and (C) median eminence. Magnification bar = 100 um. (3V-third ventricle). 20 number of LHRH+ cell bodies (p > 0.05). Approximately 25-35% of the LHRH+ somata were located rostral to the optic chiasm and 65-75% were caudal to the chiasm in all age groups (Table 3; Figure 5). The number of LHRH+ cell bodies within the DBB, BNST, POA, AMY, MBH, ARC and ME were counted and all areas except ME were analyzed by one way ANOVA. ME could not be analyzed because this structure had torn away from some of the brains. There was no significant age related difference in LHRH+ cell body number in any of the structures examined (Table 4; p > 0.05). However, there was a tendency toward a decrease in the number of cell bodies in the ARC (p < 0.09) as age and reproductive maturity progressed. The small number of ferrets in each group may have prevented detection of a statistically significant difference in this analysis. Mean somal area of the sample of analyzed LHRH+ cells from each age group is shown in Table 5. There were no statistical differences in somal areas at the different ages. Exmriment 2 The dose of colehicine administered was effective in blocking axonal transport as evidenced by the significantly larger mean somal area of LHRH+ neurons in colehicine-treated ferrets compared to control ferrets (F(1,4) = 29.91, p < 0.01). The number of LHRH+ cell bodies found in the brains of colehicine-treated and control ferrets at 10 and 25 wk of age is shown in Table 6. Two way ANOVA revealed that there was neither an effect of colehicine treatment nor an effect of age on the total number of visualized cell bodies. Because the results from Experiment 1 indicated a tendency toward a decreased number of LHRH+ neurons in ARC of older ferrets, data were combined from the two control ferrets at each age from Experiment 2 with data from Experiment 1 for a statistical analysis. Since every other section was reacted in Experiment 1, but every 21 Figure 4. Line drawing of a midsagittal section of male fenet brain. Photomicrographs illustrate the distribution of LHRH+ fibers in dark field Magnification bar = 100 tun. (AC- anterior commissure; ARC-arcuate nucleus; CC-corpus callosum; OC-optic chiasm; 3V-third ventricle). 23 TABLE 3. QUANTIFICATION OF LHRH+ CELL BODIES IN MALE FERRETS DURING PUBERTY Age 11 Total # cells # caudal to optic % caudal to chiasm optic chiasm 10wk 3 358i38 257:1:9 73:1:6% 15 wk 3 359 i 55 263 :t: 33 74 :i: 4 % 20wk 3 258:1:44 180i30 70:i:2% 25 wk 2 305 :1: 47 195 :i:14 64 i15 % Values are MEAN :1: SEM 24 Figure 5. Histograms of the number of LHRH+ cell bodies found in each 40 um coronal section examined in brains of representative individual fenets from the different age groups. Each section is 80 run from the next since only every other section was reacted. The scale on the x axis gives distance (tun) rostral and caudal to an arbitrary zero point defined as the section which first contained the suprachiasmatic nucleus (SCN). The approximate locations of preoptic area (POA) and arcuate nucleus (ARC) are noted. NUMBER OF LHRH IMMUNOPOSITIVE CELL BODIES 201 153 10 wk 15 wk 20 wk 25 wk -2400 -1600 -800 0 SCN 25 800 1600 2400 ARC 3200 4000 um 26 3 (n=2), b (n=1) TABLE 4. QUANTIFICATION OF LHRH+ CELL BODIES WITHIN SEVEN SUBAREAS OF THE BRAIN Number of LHRH+ Cell Bodies Age DBB BNST POA AMY MBH ARC ME 10 wk 23.33 3.33 45.67 10.67 60.00 101.00 26.00 $12.14 $1.20 $12.03 $1.85 $12.53 $14.87 $8.02 15 wk 15.33 3.33 60.67 18.00 50.33 67.00 26.00 $1.45 $1.20 $12.99 $1.73 $2.85 $10.58 $8.00a 20 wk 7.33 1.67 44.00 13.00 46.00 59.67 20.33 $3.48 $0.33 $4.73 $1.53 $18.19 $12.41 $7.62 25 wk 18.00 5.00 45.50 16.00 65.00 49.00 16.00 $4.00 $2.00 $8.50 $9.00 $17.00 $5.00 $0.00b Values are MEAN $ SEM 27 TABLE 5. SOMAL AREA OF LHRH+ NEURONS IN MALE FERRETS DURING PUBERTY Age 11 # cells measured Somal area ( tun2 ) 10 wk 3 100.00 $ 8.08 189.71 $ 1.05 15 wk 3 87.33 $ 14.17 234.55 $ 16.05 20 wk 3 56.33 $ 11.72 232.54 $ 18.98 25 wk 2 53.50 $ 2.50 208.55 $ 27.84 Values are MEAN :1: SEM 28 TABLE 6. QUANTIFICATION OF LHRH+ CELL BODIES IN CONTROL AND COLCHICINE-TREATED MALE FERRETS Age Treatment II Total # cells Control 2 199 $ 20 10 wk Colchicine 2 196 $ 20 Control 2 124 $ 16 25 wk Colchicine 2 163 $ 30 Values are MEAN $ SEM 29 fourth section reacted in Experiment 2, every other section in Experiment 1 was eliminated from analysis in order to have approximately the same number sections from animals in both experiments. Using these combined data, total and ARC LHRH+ cell body number were analyzed by one way ANOVA. There were again no differences in total cell body number. However, there was a significant main effect of age on LHRH+ cell body number in ARC (F(3,14) = 8.53, p < 0.005). Post hoc comparisons indicated significantly smaller numbers of LHRH+ cell bodies in 20- and 25-wk-old ferrets (F = 5.39 & 6.46 respectively, p < 0.05) compared to 10-wk-old ferrets (Table 7). TABLE 7. QUANTIFICATION OF LHRH+ CELL BODIES WITHIN ARCUATE NUCLEUS IN MALE FERRETS DURING PUBERTY Age Number of LHRH+ cell bodies 10 wk 58.00 $ 5.81 15 wk 36.67 $ 5.21 20 wk 27.67 $ 3.84* 25 wk 27.50 $ 456* Values are MEAN $ SEM Data from Experiments 1 and 2 are combined. * p < 0.05 compared to 10-wk-old DISCUSSION These experiments demonstrate that the total number of neuronal cell bodies that are immunopositive for LHRH does not increase with reproductive maturation in the male fenet. This finding indicates that the central nervous system is capable of LHRH synthesis and storage throughout pubertal development, and does not support the hypothesis that an inability to synthesize LHRH is the limiting factor in reproductively immature animals. This conclusion is strengthened by three additional findings. First, colehicine treatment did not reveal additional pepulations of LHRH+ cell bodies in prepubertal or postpubertal fenets. Thus, the absence of a pubertal increase in LHRH+ cells cannot be due to rapid transport of LHRH from the cell bodies in adults that results in somal peptide content below the level of immunocytochemical detectability. Second, even when discrete areas of the brain were examined individually, no evidence was found for a developmental increase in cell number within subpopulations of LHRH neurons. Third, an increase in somal area, which would be indicative of increased peptide synthesis, was not observed. Taken together, these data suggest that it is unlikely that the increase in frequency of release of LH during puberty in the male ferret is the result of an increase in the number of LHRH-producing neurons, or of enhanced synthesis of peptide within LHRH+ cells. This study is in general agreement with studies in other species which also show that LHRH+ number does not increase during puberty (Cameron, et al., 1985; Jennes & Stumpf, 1980; Takahashi, et al., 1988; Wray & Hoffman, 1986a,b). More recently, Wiemann, et a1. (1989) found no difference in LHRH mRNA between prepubertal and adult male rats in four brain regions, including the medial septum, the vertical limb of the DBB, the medial POA, and the lateral POA. These data provide strong evidence 31 32 that LHRH neurons in prepubertal and postpubertal animals are equally capable of LHRH biosynthesis. LHRH neurons originate from the olfactory placode during embryologic development, and then migrate across the nasal septum and enter the forebrain with the nervus terminalis. Later in embryological development, but still before birth. LHRH neurons eventually reach the septal-preoptic area and hypothalamus (Schwanzel-Fukuda & Pfaff, 1989; Wray, et al., 1989). Once the full complement of LHRH neurons is bom, no further increase in the number of immunocytochemically identified LHRH cell bodies has been found. Taken together, the data from experiments in which immunopositive LHRH neurons have been examined across various stages of reproductive development suggest that these peptidergic neurons are present and in place prenatally and do not increase in number thereafter. Furthermore, they appear to make and store peptide throughout the life of the animal, even during times when the reproductive system is quiescent. There is some indirect evidence for an increase in LHRH synthesis during puberty in some species. Cameron, et al. (1985) reported that the total cross-sectional area of LHRH+ perikarya and cytoplasm in the adult male monkey is larger than that in the juvenile male monkey. Takahashi, et a1. (1988) found an increase in LHRH content in the mid-hypothalamic area of male rats during puberty. While these studies are consistent with enhanced synthesis of LHRH during puberty, Wiemann, et a1. (1989) reported no change in the biosynthetic capacity of LHRH neurons in the rat. This discrepancy could be explained on the basis of differential LHRH release in pre- and post-pubertal states. In pre-pubertal animals, small amounts of LHRH might continuously be released from axon terminals in the ME, while in peri- and post- pubertal animals, LHRH may accumulate in the terminals in between episodes of LHRH release resulting in higher hypothalamic content. We cannot completely mlc 33 out the possibility of increased synthesis of LHRH during puberty in the fenet, since we did not quantify the density of LHRH immunocytochemical staining. The number of LHRH+ neurons within the brains of different species varies somewhat. There are about 1,300 LHRH cells in rat brain (Wray & Hoffman, 1986b), 2,600 in male monkey and 5,600 in female monkey (Goldsmith & Song, 1987). Based on the present experiments, it is estimated that there are approximately 400-800 LHRH+ neurons in the male ferret brain, and that about 70% of these neurons are caudal to the optic chiasm. This distribution is similar to that of LHRH neurons in primates in that a significant proportion of the somata are within retrochiasmatic MBH (Goldsmith & Song, 1987; King & Anthony, 1984). In contrast, the majority of LHRH+ cell bodies in many other species are found in POA around OVLT (Anthony, et al., 1984; Barry, et al., 1974; Caldani, et al., 1988; Glass, 1986; Jennes & Stumpf, 1980; Kineman, et al., 1988; King, et al., 1982; Lehman, et al., 1986; Leshin, et al., 1988; Marshall & Goldsmith, 1980; Merchenthaler, et al., 1984; Paulin, et al., 1977; Silverman, 1976; Silvennan & Krey, 1978; Silverman, et al., 1982; Silverman, et al., 1987; Witkin, 1982; Wray & Hoffman, 11986a). Whether or not the caudal shift in distribution of LHRH neurons in the ferret and primates is of functional significance is not known. Morphological changes in LHRH+ cells have been correlated with endocrine changes during puberty. Yellon, et a1. (1987) reported a decrease in the ratio of bipolar to unipolar LHRH+ somata when puberty was photoperiodically delayed in hamsters compared to post-pubertal male hamsters. In rats, while the absolute number of LHRH+ cell bodies remain the same throughout pubertal development, the proportion of the cell bodies that are spiny increases from 32% to 66%. This finding was interpreted as evidence for increased synaptic input to LHRH neurons during puberty (Wray & Hoffman, 1986b). However, since spiny LHRH neurons have not 34 been identified in all species examined, including the ferret, increased synaptic input to LHRH neurons may not be a universal mechanism of puberty onset. In addition, Lee and Hoffman (1989) used a diet-restriction paradigm to delay puberty onset in rats, and found that the shift from smooth to spiny LHRH neurons that normally accompanies postnatal maturation still occurred in diet-restricted rats even though they had not gone through ovarian puberty. A quite unexpected finding of the present study was that the number of LHRH+ cell bodies within the ARC was smaller in fenets at the two older ages. This significant reduction in the number of LHRH+ neurons first observed in 20 wk old ferrets correlates well with the pubertal increase in gonadal size, which in the present study was apparent although not yet significant at 20 weeks of age. This suggests that the peripubertal disappearance of LHRH from approximately half of the ARC LHRH+ neurons present prepubertally may be functionally related to the enhanced LHRH/LH release that necessarily precedes gonadal enlargement. LHRH exerts a steroid independent ultrashortloop negative feedback on its own release (Bedran de Castro, et al., 1985; Valenca, et al., 1987; Zanisi, et al., 1987). In fact, a recent study provides evidence that the LHRH pulse generating mechanism is more sensitive to this autofeedback in prepubertal rats than in postpubertal rats (Bourguignon, et al.. 1990). Hypothalamic explants from peri- or postpubertal rats recover from inhibition of LHRH release by exogenous LHRH within 35 minutes, whereas explants from prepubertal rats have a refractory period of over 50 minutes. In addition, ultrastructural studies demonstrate that LHRH-containing nerve terminals in infundibular lip and ME have synaptic contacts with LHRH-immunoreactive dendrites and perikarya (Thind & Goldsmith, 1988). It is proposed then, that in the ferret, the population of ARC LHRH+ neurons is the source of inhibitory autofeedback on LHRH release into the ME, and that a reduction in this inhibitory signal, reflected 35 anatomically by a reduced number of peptide producing neurons, allows an increase in the frequency of release of LHRH pulses at the time of puberty. One prediction of this hypothesis is that selective destruction of LHRH+ neurons in ARC in prepubertal male ferrets should result in an increase in the frequency of LH pulses, and advance puberty. This proposed mechanism may be applicable just to ferrets and primate, since rodents do not have any LHRH-producing neurons in the ARC (Glass, 1986; Merchenthaler, et al., 1984; Witkin, et al., 1982). Of course, non-arcuate LHRH+ neurons may serve an analogous function in rodents although there are no reports of significant reductions in LHRH+ neurons during puberty in other species. Prepubertal fenets are more sensitive to steroid negative feedback compared to postpubertal ferrets. Perhaps the LHRH-producing neurons in ARC receive input from steroid-responsive neurons, or are themselves Steroid target neurons. Although earlier studies have indicated that the vast majority of LHRH+ neurons are not steroid concentrating neurons, only the lack of estrogen receptors in LHRH+ neurons in adult female rodents has been observed (Shivers, et al., 1983b; Watson, et al., 1990). Whether LHRH+ neurons are target neurons for testosterone in prepubertal males has not been investigated. Experiments are in progress in which steroid autoradiography and LHRH immunocytochemistry will be performed on the same tissue sections to determine whether ARC LHRH+ neurons are target cells for testosterone, and if so, whether these are the cells that are selectively lost during puberty. A trend toward a decrease in the number of LHRH+ neurons in ME was also observed in Experiment 1, although the data could not be subjected to statistical analysis. In any case, it would have been difficult to interpret an observation of a decrease in the number of ME LHRH+ cell bodies, because heavy staining of LHRH+ fibers in ME might obscure the presence of cell bodies within the ME, particularly in the adult. Follow-up experiments are planned in which visualization of LHRH+ cell 36 bodies will be maximized and visualization of LHRH+ axons minimized by colehicine treatment of all animals, and by using a lower concentration of first antibody. These modifications in design will help to determine whether there is truly a reduction in LHRH-producing cell bodies in the ME in adulthood in male ferrets. If so, it may be that both ARC and ME LHRH+ neurons participate in the proposed shortloop negative feedback mechanism. Like many seasonally breeding species, fenets display an annual reproductive cycle which is controlled by day length. Boissin-Agasse, et a1. (1988) reported that within the hypothalamus, LHRH+ neurons increased during breeding season compared to the non-breeding season. Fenets in the present study experienced puberty in the absence of a change in daylength, whereas ferrets in the Boissin-Agasse, et a1. (1988) study experienced changing daylength outdoors. This photoperiod difference may account for this discrepancy. On the other hand, an increase in the number of LHRH+ cells may be characteristic of seasonal but not pubertal activation of the reproductive axis. Experiments are in progress in which the number of LHRH+ neurons will be compared during spontaneous puberty in short photoperiod and during puberty induced by a transition from short to long days. In conclusion, the observation of no increase in the number or somal area of LHRH+ neurons during puberty in the fenet supports the hypothesis that the increase in pituitary stimulation by LHRH during puberty is mediated by enhanced episodic release of the neurohonnone rather than by enhanced synthesis. The present studies provide initial evidence that the mechanism underlying the pubertal transition from infrequent to frequent release of LHRH includes a change in autoregulatory mechanisms involving LHRH neurons in the arcuate nucleus. REFERENCES Adelman, J.P., Mason, A.J., Haylick, IS. and Seeburg, RH. (1986) Isolation of the gene and hypothalamic cDNA for the common precursor of gonadotropin- releasing hormone and prolactin release-inhibiting factor in human and rat. P_Lo_c_. Natl, Acad, Sci, USA, Neumhiglggy 83, 179-183. Anthony, E.L.P., King, J. and Stopa, E.G. (1984) Irnmunocytochemical localization of LHRH in the median eminence, infundibular stalk, and neurohypophysis. Cel Tiss. Res, 236, 5-14. Bany, 1., Dubois, MP. and Carette, B. (1974) Immunofluorescence study of the preoptico-infundibular LRF neurosecretory pathway in the normal, castrated or testosterone-treated male guinea pig. Ends; 25, 1416—1423. Bedran de Castro, J.C., Khorram, O. and McCann, SM. (1985) Possible negative ultra- short loop feedback of luteinizing hormone releasing hormone (LHRH) in the ovariectomized rat. Pmc. Soc, Exp, Biol, Med, 122, 132-135. Benoit, R., Ling, N., Brazeau, P., Lavielle, S. and Guillemin, R. (1987) Strategies for antibody production and radioimmunoassays. Neugomethods, Peptides, Ed. Boulton, A.A., Baker, GB. and Pittman, Q]. The Humana Press, Inc., 6, 43-72. Berglund, LA. and Sisk, CL. (1990) Pituitary responsiveness to luteinizing horrnone- releasing hormone in prepubertal and postpubertal male ferrets. Biol. Reprod. 43, 335-339. Boissin-Agasse, L., Alonso, G., Roch, G. and Boissin, J. (1988) Peptidergic neurohonnonal systems in the basal hypothalamus of the ferret and the mink: immunocytochemical study of variations during the annual reproductive cycle. Cell Tiss. Res, 251, 153-159. Bourguignon, J.P., Gerard, A. and Franchimont, P. (1990) Maturation of the 37 38 hypothalamic control of pulsatile gonadotropin-releasing hormone secretion at onset of puberty: II. reduced potency of an inhibitory autofeedback. Endo. 127, 2884-2890. Boyd, SK. and Moore, FL. (1985) Luteinizing hormone-releasing hormone facilitates the display of sexual behavior in male voles (Microtus canicaudus). Hormones egg Behavior 12, 252-264. Caldani, M., Batailler, M., Thiery, J.C., Dubois, MP. (1988) LHRH-immunoreactive structures in the sheep brain. Histeehemistg 82, 129-139. Cameron, J.L., McNeil], T.H., Fraser, H.M., Bremner, W.J., Clifton, D.K. and Steiner, RA (1985) The role of endogenous gonadotropin-releasing hormone in the control of luteinizing hormone and testosterone secretion in the juvenile male monkey, Macaca fascicularis. Biel. Repred, 33, 147-156. Chen, W.P., Witkin, J.W. and Silverman, A.-J. (1989) Beta-endorphin and gonadotropin-releasing hormone synaptic input to gonadotropin-releasing hormone neurosecretory cells in the male rat ,1, Cemp. Neerel, 286, 85-95. Dudley, CA. and Moss, R.L. (1987) Effects of a behaviorally active LHRH fragment and septal area stimulation on the activity of mediobasal hypothalamic neurons. Swami. 240247. Dyer, RC. and Dyball, RE]. (1974) Evidence for a direct effect of LRF and TRF on single unit activity in the rostral hypothalamus. NatuLe 252, 486-488. Foster, BL. (1988) Puberty in the female sheep. In The Physielegy of Reproduction, E. Knobil and ID. Neill, eds. New York: Raven Press, p. 1739-1762. Gallo, R.V. (1980) Neuroendocrine regulation of pulsatile luteinizing hormone release in the rat. W30, 122-131. Glass, JD. (1986) Gonadotropin-Ieleasing hormone neuronal system of the white-footed mouse, Peromyscus leucopus. Neemende, 43, 220-229. 39 Goldsmith, RC. and Song, T. (1987) The gonadotropin-releasing hormone containing ventral hypothalamic tract in the fetal rhesus monkey (Macaca mulatta). I, Cemp, Neerel, 251, 130-139. Jennes, L. and Stumpf, W.E. (1980) LHRH-systems in the brain of the golden hamster. Cell Ties, Res. 202, 239-256. Jennes, L., Stumpf, W.E. and Sheedy, ME. (1985) Ultrastructural characterization of gonadotropin-releasing hormone (GnRH)-producing neurons. J. Comp. Neurol, 232, 534-547. Kalra, SP. (1985) Neural circuits involved in the control of LHRH secretion : a model for estrous cycle regulation. ,1. Steroid Bieehem, 23, 733-742. Kalra, S.P., Allen, L.G., Sahu, A., Kalra, RS. and Crowley, W.R. (1988) Gonadal steroids and neuropeptide Y-opioid-LHRH axis: interactions and diversities. i, Stemid Biochem, 39, 185-193. Kastin, A., Coy, D.H., Schally, AV. and Zadina, J.E. (1980) Dissociation of effects of LHRH analogs on pituitary regulation and reproductive behavior. Pharrnacol. Bieehem, Behav. 13, 913-914. Kineman, R.D., Leshin, L.S., Crim, J.W., Rampacek, 0.3., and Kraeling, RR. (1988) Localization of luteinizing hormone-releasing hormone in the forebrain of the pig. Biol, Repred. 32, 665-672. King, J.C., Tobet, S.A., Snavely, FL, and Arimura, A.A. (1982) LHRH immunopositive cells and their projections to the median eminence and organum vasculosum of the lamina terminalis. 1. Cemp. Neurol. 2%, 287-300. King, LC. and Anthony, E.L.P. (1984) LHRH neurons and their projections in humans and other mammals: species comparisons. Peptides_5, 195-207. Kiss, J. and Halasz, B. (1985) Demonstration of serotoninergic axons terminating on luteinizing hormone-releasing hormone neurons in the preoptic area of the rat 40 using a combination of immunocytochemistry and high resolution autoradiography. Neeresei, 14, 69-78. Kow, L.M. and Pfaff, D.W. (1988) Transmitter and peptide actions on hypothalamic neurons in vitro: implications for lordosis. Brain Res, Bull, 20, 857-861. Kozlowski, G., Chu, L., Hostetter, G. and Kerdelhue, B. (1980) Cellular characteristics of irnmunolabeled luteinizing hormone releasing hormone (LHRH) neurons. W. 37-46. Lee, W.S. and Hoffman, GE. (1989) Diet restriction and LHRH neuron maturation. Seeiety for Negroseieng Abstraets, 15, 1082. Lehman, M.N., Robinson, J.E., Karsch, F]. and Silverman, AJ. (1986) Irnmunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) pathways in the sheep brain during anestrus and the mid-luteal phase of the estrous cycle. ,1. Cemp, Neeml, 244, 19-35. Leranth, C., MacLusky, N.J., Shanabrough, M. and Naftolin, F. (1988) Catecholarninergic innervation of luteinizing hormone-releasing hormone and glutamic acid decarboxyiase immunopositive neurons in the rat medial preoptic area. Neumendo, 48, 591-602. Leshin, L.S., Rurld, L.A., Crim, J.W., and Kiser, TE. (1988) Irnmunocytochemical localization of luteinizing hormone-releasing hormone and proopiomelanocortin neurons within the preoptic area and hypothalamus of the bovine brain. Biol, 829521.12. 963-975. Marshall, P. E. and Goldsmith, RC. (1980) Neuroregulatory and neuroendocrine GnRH pathways in the hypothalamus and forebrain of the baboon. Brain Res. 193. 353-372. Matsuo, H., Baba, Y., Nau, R.M.G., Arimura, A., and Schally, A.V. (1971) Structure of the porcine LH and FSH releasing hormone. 1. Proposed amino acid sequence. 41 Bimhem, Biephye, Res. Cemmen, 43, 1334-1339. McCann, SM. (1980) Control of anterior pituitary hormone release by brain peptides. W31, 355-363. Meister, B., Hokfelt, T., Tsuruo, Y., Hemmings, H., Ouimet, C., Greengard, P. and Goldstein, M. (1988) Damp-32, a dopamine- and cyclic AMP-regulated phosphoprotein in tanycytes of the mediobasal hypothalamus: distribution and relation to dopamine and luteinizing hormone-releasing hormone neurons and other glial elements. Neemsei, 222, 607-622. Merchenthaler, 1., Gorcs, T., Setalo, G., Petrusz, P., and Flerko, B. (1984) Gonadotropin-releasing hormone (GnRH) neurons and pathways in the rat brain. Cell Ties, Res, 232, 15-29. Moss, R.L. and McCann, S. M. (1975) Action of luteinizing hormone-releasing factor (LRF) in the initiation of lordosis behavior in the estrone-primed ovariectomized female rat. W, 309-318. Moss, R.L. (1977) Role of hypophysiotropic neurohonnones in mediating neuronal and behavioral events. Fm. Proe, 36. 1978-1983. Moss, R.L. (1979) Actions of hypothalarnic-hypOphysiotrOpic hormones on the brain. Ann. Rev. Physiol. 41. 617-631. Myers, B.M. and Baum, MJ. (1980) Facilitation of copulatory performance in male rats by naloxone: effects of hypophysectomy, 17er-estradiol, and luteinizing hormone releasing hormone. Phennaeol, Bieehem, Behav, 12, 365-370. Negro-Vilar, A., Culler, MD. and Masotto, C. (1986) Peptide-steroid interactions in brain regulation of pulsatile gonadotropin secretion. J. Steroid Biochem. 25, 741- 747. Ojeda, SR. and Urbanski, HF. (1988) Puberty in the rat. In The Physiolegy ef Repmduetion, E. Knobil and JD. Neill, eds. New York: Raven Press, p. 1699- 42 1738. Palovcik, R. A. and Phillips, M.I. (1986) A biphasic excitatory response of hippocampal neurons to gonadotropin-releasing hormone. Neemende. 44 , 137- 141. Paulin, C., Dubois, M.P., Barry, J. and Dubois, RM. (1977) Immunofluorescence study of LH-RH producing cells in the human fetal hypothalamus. Cell Tiss. Res. 182, 341-345. Plant, T.M. (1988) Puberty in primates. In The Physiolegy ef Repmduetien, E. Knobil and JD. Neill, eds. New York: Raven Press, p. 1763-1788. Polkowska, J., Dubois, M.-P. and Domanski, E. (1980) Immunocytochemistry of luteinizing hormone releasing hormone (LHRH) in the sheep hypothalamus during various reproductive stages. Cell Tiss. Res, 208, 327-341. Ramirez, V.D., Feder, H.H. and Sawyer, CH. (1984) The role of brain catecholamines in the regulation of LH secretion: 3 critical inquiry. Ed. by L. Martini and WE Ganong. Raven Press, New York. Frontiers in Neuroendocrinologyji, 27-84. Renaud, L.P., Martin, J.B., and Brazeau, P. (1975) Depressant action of TRH, LH-RH and somatostatin on activity of central neurones. NetLILe 255, 233-235. Ronnekleiv, D.K., Adelman, J.P., Weber, E., Herbert, E. and Kelly, MJ. (1987) Irnmunohistochemical demonstration of proGnRH and GnRH in the preoptic-basal hypothalamus of the primate. Neumende, 45, 518-521. Rothfeld, J.M., Carstens, E., and Gross, D.S. (1985) Neuronal responsiveness to gonadotropin-releasing hormone and its correlation with sexual receptivity in the rat. 2912131915. 603-608. Ryan, EL. and Frankel, AL (1978) Studies on the role of the medial preoptic area in sexual behavior and hormonal response to sexual behavior in the mature male laboratory rat. Biel, Reprod. 19, 971-983. 43 Ryan, K.D., Robinson, S.L., Tritt, SH. and Zeleznik, AJ. (1988) Sexual maturation in the female ferret: circumventing the gonadostat. Ende, 122, 1201-1207. Schiess, M., Dudley, CA. and Moss, R.L. (1987) Estrogen priming affects the sensitivity of midbrain central gray neurons to microiontophoretically applied LHRH but not beta-endorphin. W, 24-31. Schwanzel-Fukuda, M., Garcia, M.S., Morlell, 1.1. and Pfaff, D.W. (1987) Distribution of luteinizing hormone-releasing hormone in the nervus terminalis and brain of the mouse detected by immunocytochemistry. 1, Cemp, Neure, 255, 231-244. Schwanzel-Fukuda, M., Fadem, B.H., Garcia, MS, and Pfaff, D.W. (1988) Irnmunocytochemical localization of luteinizing hormone-releasing hormone (LHRH) in the brain and nervus terminalis of the adult and early neonatal gray short-tailed opossum (Monodelphis domestica). 1. Cemp. Neerol. 276, 44-60. Schwanzel-Fukuda, M. and Pfaff, D.W. (1989) Origin of luteinizing hormone-releasing hormone neurons. Nature 338, 161-164. Seeburg, RH. and Adelman, JP. (1984) Characterization of cDNA for precursor of human luteinizing hormone releasing hormone. Nateee 311, 666-668. Shivers, B.D., Harlan, R.E., Monell, J.I., and Pfaff, D.W. (1983a) Immunocytochemical localization of luteinizing hormone-releasing hormone in male and female rat brains. Neemendo. 36, 1-12. Shivers, B.D., Harlan, R.E., Morlell, J.I., and Pfaff, D.W. (1983b) Absence of oestradiol concentration in cell nuclei of LHRH-immunoreactive neurons. Mule 3&1. 345-347. Silverman, A]. (1976) Distribution of luteinizing hormone-releasing hormone (LHRH) in the guinea pig brain. Endo, 99, 30-41. Silverman, AJ. and Krey, LC. (1978) The luteinizing hormone-releasing hormone (LH-RH) neuronal networks of the guinea pig brain. I. Intra- and extra- 44 hypothalamic projections. Bmin Res, 1512, 233-246. Silverman, A.J., Antunes, J.L., Abrams, G.M., Nilaver, G., Thau, R., Robinson, J.A., Ferin, M. and Krey, LC. (1982) The luteinizing hormone-releasing hormone pathways in rhesus (Macaca mulatta) and pi gtailed‘ (Macaca nemestrina) monkeys: new observations on thick, unembedded sections. J. Cemp. Negro]. 2_1_1, 309-317. Silverman, A.J., Jhamandas, J. and Renaud, LP. (1987) Localization of luteinizing hormone-releasing hormone (LHRH) neurons that project to the median eminence. J, Neemseij, 2312-2319. Silverman, A.J. (1988) The gonadotropin-releasing hormone (GnRH) neuronal systems: immunocytochemistry. In The Physielegy 6f Reprmigetien, E. Knobil and JD. Neill eds., New York: Raven Press, p. 1283-1304. Sisk, CL. (1987) Evidence that a decrease in testosterone negative feedback mediates the pubertal increase in luteinizing hormone pulse frequency in male ferrets. Biel, Repm, 31, 73-81. Sisk, C.L., Moss, R.L. and Dudley CA. (1988) Immunocytochemical localization of hypothalamic luteinizing hormone-releasing hormone in male ferrets. Brain Res. Belletin 29, 157-161. Sisk, CL. (1990) Phot0periodic regulation of gonadal growth and pulsatile luteinizing hormone secretion in male ferrets. 1, Big], Rhyth_ms 5, 177-186. Takahashi, S., Ono, R., Nomura, K., and Kawashima, S. (1988) Luteinizing honnone- releasing hormone (LHRH) neurons in the male and female rats at peripubertal period. Anat. Emegol. 178, 475-480. Tang, Y.P. and Sisk, CL. (1988) Distribution of luteinizing hormone releasing hormone (LHRI-D-containing neurons within the brain of a mustelid, the European fenet. Seeieg fer Neeeeseienee Abstgaets 14, 440. 45 Thind, K.K. and Goldsmith, RC. (1988) Infundibular gonadotropin-releasing hormone neurons are inhibited by direct Opioid and autoregulatory synapses in juvenile monkey. Negroenge, 47, 203-216. Valenca, A.A., Johnstone, C.A., Ching, M. and Negro-Vila‘r, A. (1987) Evidence for a negative ultrashort loop feedback mechanism operating on the luteinizing hormone-releasing hormone neuronal system. Enee, 121, 2256-2259. Watson, Jr.R.E., Langub, Jr.M.C., Landis, J.W. (1990) Further evidence that LHRH neurons are not directly estrogen responsive: LHRH and estrogen receptor irnmunoreactivity in the guinea pig brain. Society for Neuroscience Abstracts 16. 1200. Wiemann, J.N., Clifton, D.K. and Steiner, RA. (1989) Pubertal changes in gonadotrOpin-releasing hormone and proopiomelanocortin gene expression in the brain of the male rat. Ende. 124, 1760-1767. Witkin, J.W., Paden, C.M., and Silverman, A.J. (1982) The luteinizing honnonc- releasing hormone (LHRH) systems in the rat brain. Neeroendoerinology 35, 429-438. ' Witkin, J.W. (1986) Luteinizing hormone releasing hormone (LHRH) neurons in aging female rhesus macaques. Neurobiology of Aging :2, 259-263. Witkin, J.W. (1987) Irnmunocytochemical demonstration of luteinizing honnone- releasing hormone in optic nerve and nasal region of fetal rhesus macaque. Neuregienee Legergs :22, 73-77. Witkin, J.W. and Demasio, K. (1990) Ultrastructural differences between smooth and thomy gonadotr0pin-releasing hormone neurons. Neemsei. 34, 777-783. Wray, S. and Hoffman, G. (19863) A developmental study of the quantitative distribution of LHRH neurons within the central nervous system of postnatal male and female rats. 1. mmp, Neuml, 252, 522-531. 46 Wray, S. and Hoffman, G. (1986b) Postnatal morphological changes in rat LHRH neurons correlated with sexual maturation. Neueeeneeerinelegy 43, 93-97. Wray, S., Nieburgs, A. and Elkabes, S. (1989) Spatiotemporal cell expression of luteinizing hormone-releasing hormone in the prenatal mouse: evidence for an embryonic origin in the olfactory placode. Qevelep, Begin Res, 46, 309-318. Wuttke, W., Roosen-Runge, G., Demling, 1., Stock, K.W. and Vijayan, E. (1982) Neuroendocrine control of pulsatile luteinizing hormone release. Brain and Pimitagr Peptides II, Ferring 8mg" 1-10. Yellon, S.M., Hirata, K.K. and Newman, SW. (1987) Effect of short photoperiod during development on the GnRH neuronal system in the Djungarian hamster. Seeiety fer Neereseienee Aestmets 13, 1578. Zanisi, M., Messi, E., Motta, M. and Martin, L. (1987) Ultrashort feedback control of luteinizing hormone-releasing hormone secretion in vitro. Ende, 121, 2199-2204. Zheng, L.M., Caldani, M. and Jourdan, F. (1988) Irnmunocytochemical identification of luteinizing hormone-releasing hormone-positive fibers and terminals in the olfactory system of the rat. Neerosci, 24, 567-578. 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