Expression of Rab3D N135I inhibits regulated secretion of ACTH in AtT-20 cells.

Rab proteins are small molecular weight GTPases that control vesicular traffic in eucaryotic cells. A subset of Rab proteins, the Rab3 proteins are thought to play an important role in regulated exocytosis of vesicles. In transfected AtT-20 cells expressing wild-type Rab3D, we find that a fraction of the protein is associated with dense core granules. In the same cells, expression of a mutated isoform of Rab3D, Rab3D N135I, inhibits positioning of dense core granules near the plasma membrane, blocks regulated secretion of mature ACTH, and impairs association of Rab3A to membranes. Expression of Rab3D N135I does not change the levels of ACTH precursor or the efficiency with which the precursor is processed into ACTH hormone and packaged into dense core granules. We also find that cells expressing mutated Rab3D differentiate to the same extent as untransfected AtT-20 cells. We conclude that expression of Rab3D N135I specifically impairs late membrane trafficking events necessary for ACTH hormone secretion.

Sexual dimorphism in the synaptic input to gonadotropin releasing hormone neurons.

GnRH neurons form the final common pathway regulating the secretion of gonadotropins from the anterior pituitary. Since the patterns of gonadotropin release display profound sexual dimorphism among mammals including the rodent, we undertook an ultrastructural analysis to determine whether these neurosecretory cells were differentially innervated between the sexes. As a further exploration of the organization of the neurocircuitry integrating GnRH neurons with the central nervous system, we also determined the degree to which GnRH cells and their processes were innervated by terminals containing either the endogenous opiate, beta-endorphin (BE) or GnRH itself. Sections from the diagonal band of Broca and the preoptic area of adult male and diestrus II female rats were immunocytochemically processed for dual localization of GnRH and BE. GnRH neurons cut through the plane of the nucleus were identified in 1 micron sections. Serial ultrathin sections were made and analyzed for 1) total synaptic input to both cell bodies and dendrites; 2) BE input; and 3) input arising from GnRH itself. We report that GnRH neuronal cell bodies in females received approximately twice the number of synapses as did those of males. The input to the GnRH dendrites, when measured as percent of plasma membrane in synaptic contact, also showed a profound sexual dimorphism with the female having a larger proportion of the dendrite in synaptic apposition. BE innervation contributed to this dimorphism at the level of both the cell body and dendrite. In contrast, the distribution and number of GnRH terminals did not differ between the sexes. In both they were confined to the dendritic arbor. We hypothesize that the capacity of the female rodent GnRH system to show neurogenic derived alterations in GnRH output not seen in the male may be due in part to these anatomical differences.

Novel associations among gonadotropin-releasing hormone neurons.

GnRH is secreted in bursts into the hypophyseal portal vasculature by a small dispersed population of neurons. The means by which the activity of these intrinsically pulsatile cells is coordinated are unknown. This study was initiated as a continuation of our examination of the synaptic input to these cells and their anatomical relationships. Brain tissue from female rhesus monkeys and male and female rats was prepared for the immunocytochemical demonstration of GnRH. At the light microscopic level, GnRH neurons were occasionally found to be in close apposition. Such pairs (or small groups) were randomly distributed throughout the population of GnRH neurons from the diagonal band of Broca through the anterior hypothalamic area in rats and monkeys and in the medial basal hypothalamus in monkeys. The percentage of neurons found in such associations was small (2-7% in rats and 3-15% in monkeys) and was independent of the hormonal condition of the animal. GnRH neurons, either singly or in pairs, were serially sectioned for electron microscopic examination. The sparsity of synaptic input to the cell body that we had reported earlier on the basis of random sampling was confirmed. No soma had more than a dozen synapses, but none totally lacked innervation. The most significant result of serial reconstruction was the discovery of intercellular bridges or passageways between contiguous pairs of GnRH neurons. These were formed by the fusion of processes extending from the two cells or by fusion and opening of passageways in the membranes along regions of contiguity between the two cells. They were found in four of seven pairs of neurons examined in the rat and in four of eight pairs in the monkey. This syncytial arrangement along with GnRH-GnRH synaptic interactions could contribute to the coordination of dispersed influences on these neurons and the propagation of coordinated pulsatile release of GnRH.

Effects of gonadal steroids on the ultrastructure of GnRH neurons in the rhesus monkey: synaptic input and glial apposition.

The secretion of the gonadotropins is modulated by the gonadal steroids, but the means by which these effects are mediated are not well understood. The present anatomical study was undertaken to investigate the possibility that the GnRH system responds to alterations in the gonadal steroid environment with reversible changes in synaptic input and glial wrapping such as have been observed in other neuroendocrine systems. The ultrastructure of GnRH neurons was studied in the preoptic area and medial basal hypothalamus of rhesus monkeys in various steroid conditions including five intact cycling, four long-term ovariectomized animals, two long-term ovariectomized animals with steroid replacement (LtOVX+), and two animals replaced with steroid at the time of ovariectomy (StOVX+). Electron micrographic montages of GnRH neuronal profiles were analyzed using computerized morphometrics, and the percentages of the length of perikaryal membrane immediately apposed by glial processes and that with postsynaptic modification were calculated. Ovariectomy resulted in a significant increase in the apposition of glial processes to GnRH perikaryal membranes and a significant decrease in their innervation in both brain regions. There was also a higher incidence of GnRH neurons with immunostaining confined to secretory granules and a decrease in the volume of nucleoli, both of which could be interpreted as indications that GnRH peptide synthesis was reduced in ovariectomized animals. After an ovarian steroid replacement regimen which mimicked two menstrual cycles, the innervation of GnRH neurons was increased and the glial ensheathment was partially reduced. This was true for both the LtOVX+ and StOVX+ steroid-replacement groups. GnRH neurons in the medial basal hypothalamus received more synaptic input than did those in the preoptic area, regardless of the steroid condition of the animal. The degree of glial ensheathment of GnRH neurons in the preoptic area became significantly greater than that in the medial basal hypothalamus after ovariectomy. These observations suggest there may be differences in the role of GnRH neurons in these two brain regions. These immunocytochemical ultrastructural studies provide strong evidence that alterations in the gonadal steroid milieu can produce morphological changes in the GnRH neuron and its immediate environment in the primate.

Embryonic development of the gonadotropin-releasing hormone (GnRH) system in the chick: a spatio-temporal analysis of GnRH neuronal generation, site of origin, and migration.

We present a quantitative immunocytochemical study of GnRH migration by developmental stage. GnRH peptide was detected in cells of the olfactory epithelium at stage 19. Migration was initiated a few hours later at stage 20. Of interest is the observation that GnRH neurons paused at the central nervous system border for 3 days, entering the brain at stage 29. The major expansions of the GnRH population occurred at two points; stages 26 and 42. In one animal a third population expansion occurred after hatching, with the number of GnRH cells reaching 6600. To determine the site of origin of GnRH cells, embryos were exposed to tritiated thymidine and killed 5 h later. Most GnRH cells incorporated label in the olfactory epithelium; however, some autoradiographically labeled GnRH cells, possessing a neuronal morphology, were found in the olfactory nerve and the forebrain, suggesting that some GnRH neurons divide as they migrate. A cumulative labeling method employing tritiated thymidine was used to examine the birth date of GnRH neurons. Postmitotic GnRH cells were first detected at stages 19-21. At stage 24, a peak in GnRH neurogenesis preceded the increase in GnRH neurons expressing their peptide at stage 26. After stage 24, there was a gradual addition of postmitotic cells to the population through stage 35. A pulse-chase paradigm indicated that birth date did not influence the final GnRH cell distribution. Injections at stage 29, when 10% of the GnRH neurons are born, generated double labeled cells in all locations where placode-derived GnRH neurons reside.

FOS expression in the gonadotropin-releasing hormone (GnRH) neuron does not increase during the ovarian steroid-induced GnRH surge in the rhesus monkey.

The purpose of this study was to investigate the expression of the immediate early gene, c-fos, in GnRH neurons in female rhesus monkeys as a function of generation of the LH surge. Adult monkeys were either intact (n = 6) or ovariectomized (n = 10). Intact animals received estradiol benzoate (EB; 330 micrograms in oil, sc; n = 5) or oil (n = 1). Ovariectomized animals received either EB (n = 5) or EB, followed by progesterone (P; 2.5 ml in oil, im; n = 4), or oil (n = 1). Animals were killed from 31-75 h after EB treatment. Blood samples were collected to document LH release in response to steroid treatment. A surge of LH was initiated in most animals that received EB alone or EB plus P about 30 h after steroid treatment. Animals were perfused with 4% paraformaldehyde, and brain blocks encompassing the region known to contain the majority of GnRH neurons (septum through the medial basal hypothalamus) were cut on the vibratome. Sites of FOS and GnRH immunoreactivities were demonstrated using double labels with a variety of chromogens. Regardless of the time in the surge, there were very few GnRH neurons with FOS immunoreactivity in their nuclei (0-9%). FOS-positive nuclei were seen in many other neurons in various brain regions, including the suprachiasmatic and supraoptic nuclei. There were no differences in FOS expression in GnRH neurons in intact and ovariectomized animals or in steroid- or oil-treated animals. These results suggest that FOS activation in GnRH neurons is not associated with the initiation of the secretory GnRH stimulus to the LH surge in the rhesus monkey. If confirmed, these data suggest that the GnRH nerve terminal may be the primary site for the control of the GnRH surge.

Some hypothalamic hamartomas contain transforming growth factor alpha, a puberty-inducing growth factor, but not luteinizing hormone-releasing hormone neurons.

Activation of LH-releasing hormone (LHRH) secretion, essential for the initiation of puberty, is brought about by the interaction of neurotransmitters and astroglia-derived substances. One of these substances, transforming growth factor alpha (TGFalpha), has been implicated as a facilitatory component of the glia-to-neuron signaling process controlling the onset of female puberty in rodents and nonhuman primates. Hypothalamic hamartomas (HH) are tumors frequently associated with precocious puberty in humans. The detection of LHRH-containing neurons in some hamartomas has led to the concept that hamartomas advance puberty because they contain an ectopic LHRH pulse generator. Examination of two HH associated with female sexual precocity revealed that neither tumor had LHRH neurons, but both contained astroglial cells expressing TGFalpha and its receptor. Thus, some HH may induce precocious puberty, not by secreting LHRH, but via the production of trophic factors--such as TGFalpha--able to activate the normal LHRH neuronal network in the patient's hypothalamus.

Increased synaptic input to gonadotropin-releasing hormone neurons in aged, virgin, male Sprague-Dawley rats.

Using double-label ultrastructural immunocytochemistry, we found the synaptic input to gonadotropin-releasing hormone (GnRH) neurons in the preoptic area of aged (20 months old), virgin, male Sprague-Dawley rats to be denser than that in young adults (3 months old). These results confirmed earlier observations on F-344 virgin male rats. The aging F-344 rat, however, is prone to testicular tumor and so it was essential to see if the phenomenon was reproducible in another rat strain. In the first study, a portion of the increase in synaptic density was due to an increase in the proportion of synapses containing pleiomorphic vesicles, frequently associated with the neurotransmitter GABA. We tested the possibility directly using a double-label protocol for GnRH and glutamic acid decarboxylase (GAD). However, in the present study the density of input by GABA did not change with age. This inhibitory amino acid represented about 10% of the total innervation in young animals; but, in aged animals, because the total synaptic input was greater, GABA represented only about 4% of the innervation. Synaptic vesicles within GAD-immunoreactive terminals were uniformly clear and spherical, suggesting that pleiomorphic vesicle shape is not an appropriate criterion for GABAergic innervation.

Luteinizing hormone releasing hormone (LHRH) neurons in aging female rhesus macaques.

Luteinizing hormone releasing hormone (LHRH) elements were demonstrated immunocytochemically in coronal sections of a portion of the left hypothalamus in twenty female rhesus macaques ranging in age from just over two years to over 21 years. This time period includes the juvenile preovulatory phase. The region studied included the retrochiasmatic region as far dorsally as the superior extent of the optic tract and as far posteriorly as the mammillary body. There was a range in total LHRH neurons per animal from 55 to 470. No trends in cell number as a function of age was observed. The range in cell number among the youngest animals (two year olds) was about the same as that in adult (6-15 year olds) and older (20+ year olds) animals. These results suggest that there is no age-related decline in the potential for manufacture of LHRH, at least into the pre-menopausal period, in the female rhesus macaque.

Luteinizing hormone-releasing hormone (LHRH) in rat olfactory systems.

The luteinizing hormone-releasing hormone (LHRH) systems of rat olfactory bulbs and nasal areas were studied in neonatal and adult rats. Animals were perfused with Zamboni's fixative and olfactory bulbs with nasal olfactory areas intact were removed, postfixed, and decalcified. LHRH was immunohistochemically demonstrated in unembedded frozen or vibratome sections. Luteinizing hormone-releasing hormone immunoreactive elements were found along the course of the nervus terminalis (NT) and within both the main and accessory olfactory bulbs (MOB and AOB, respectively). Both LHRH neurons and fibers were present in the AOB, but only fibers were detected in the MOB. The fibers of the AOB were not confined to any particular lamina while fibers in the MOB were found mainly in the external plexiform layer. LHRH fibers were found in the mucosa of the olfactory epithelium of the vomeronasal organ in both neonatal and adult rats. The NT probably serves as a source of LHRH fibers for both the AOB and the MOB and for fibers observed in the olfactory epithelium of the vomeronasal organ. Other likely sources of LHRH fibers in the olfactory bulb are discussed.

beta-Endorphin and gonadotropin-releasing hormone synaptic input to gonadotropin-releasing hormone neurosecretory cells in the male rat.

Physiological and pharmacological evidence has suggested that both endogenous opiates and gonadotropin-releasing hormone (GnRH) itself can act centrally to exert a tonic inhibition on gonadotropin secretion via an inhibition of the neurosecretion of GnRH. To determine if the effects of these two peptides might be mediated via a direct synaptic input to the GnRH neuron, we undertook a double label ultrastructural study. We were able to localize in the same tissue section beta-endorphin and GnRH. Analysis of serial sections through GnRH perikarya and dendrites in the male rat diagonal band/preoptic area revealed that almost 10% of the synapses impinging on the GnRH neuron contained beta-endorphin; an additional 10% of the terminals contained GnRH. These data provide anatomical evidence in support of both a direct modulation of GnRH release by opiates and of the presence of an ultrashort feedback loop.

Aging changes in synaptology of luteinizing hormone-releasing hormone neurons in male rat preoptic area.

This study was undertaken to examine some aspects of the anatomical substrate for reproductive senescence. Immunocytochemically identified luteinizing hormone-releasing hormone neurons and their processes in the male rat brain preoptic area were compared in young adult (2-4 months), middle-aged (12-14 months) and old (20-23 months) animals. At the light microscopic level there were no age-dependent differences in total numbers or sizes of LHRH neurons nor in their distribution in the brain. Examination of these neurons at the electron microscopic level did reveal significant differences in certain organelles and in the degree and kind of synaptic input. Random sections of middle-aged luteinizing hormone-releasing hormone neurons more frequently passed through the nucleolus and the incidence of nematosomes was higher than in luteinizing hormone-releasing hormone neurons from the young and old animals. Quantitative measures of synaptic input to luteinizing hormone-releasing hormone soma and dendrites as well as to unidentified neurons in the same thin section were made. These are reported as percent of membrane that showed synaptic structure. Dendrites of both luteinizing hormone-releasing hormone and nonidentified neurons were more densely innervated than perikarya. The density of synaptic input to luteinizing hormone-releasing hormone neurons was significantly greater than that to nonidentified neurons in young and middle-aged animals, but was equal to that of nonidentified neurons by old age. Age-related changes were noted in synaptic organization with the most significant change being an increased input to luteinizing hormone-releasing hormone perikarya. Indeed, synaptic input to luteinizing hormone-releasing hormone perikaryal membrane was increased three-fold by middle age and ten-fold by old age. Density of synaptic input to luteinizing hormone-releasing hormone dendritic membrane did not change with age. There were no aging changes in percentage of membrane with synaptic structure in nonidentified elements. Synapses were also classified on the basis of their synaptic vesicle content. There were proportionately more synaptic boutons containing round clear than pleomorphic vesicles in the young sample. The proportion of synapses with pleomorphic vesicles increased with age onto both luteinizing hormone-releasing hormone perikarya and their dendrites. The proportion of boutons containing some electron dense-core vesicles along with clear vesicles decreased with age onto both luteinizing hormone-releasing hormone and nonidentified neurons and their processes.

Synaptology of luteinizing hormone-releasing hormone neurons in the preoptic area of the male rat: effects of gonadectomy.

Ultrastructural analysis of the synaptic input to luteinizing hormone-releasing hormone neurons has previously shown that in male rats these cells acquire an increased density of innervation with increasing age [Witkin J. W. (1987) Neuroscience 22, 1003-1013]. To determine if this aging phenomenon might be due to changes in the steroid environment, we examined luteinizing hormone-releasing hormone neurons, using methods identical to the earlier study, in sham operated and 1 day and 4 week castrated male rats. The density of synaptic input to luteinizing hormone-releasing hormone neurons did not vary among the three experimental groups. Furthermore, there were no differences in the relative numbers of various morphological categories of synaptic boutons (characterized by vesicle type: clear vs dense and round vs pleomorphic) among the groups. Long term castration did result in a slight decrease in immunocytochemically detectable luteinizing hormone-releasing hormone neuron numbers. These results suggest that gonadal steroid deprivation does not alter the total density or morphological characteristics of synaptic input to the luteinizing hormone-releasing hormone neuron in the male rat.

Access of luteinizing hormone-releasing hormone neurons to the vasculature in the rat.

Luteinizing hormone-releasing hormone is secreted into the hypophysial portal vasculature through which it controls the release of the gonadotropins. The peptide also acts as a neurotransmitter in various loci within the central nervous system. It is not known whether these roles are performed by separate populations of luteinizing hormone-releasing hormone neurons. Some recent tracing experiments suggest that this is the case (Silverman et al., J. Neurosci. 7, 2312, 1987; Jennes and Stumpf, Neuroscience 18, 403, 1986). One aspect of this question was addressed in the current study by intraperitoneal introduction of Fluoro-Gold (a retrograde tracer) into male and female rats under various age and hormonal conditions. Brain sections from the anterior olfactory nuclei to the median eminence were treated for the immunocytochemical demonstration of luteinizing hormone-releasing hormone. In all cases, regardless of the age, sex or hormonal condition of the animal, the Fluoro-Gold tracer was found in more than 90% of the luteinizing hormone-releasing hormone neurons. We conclude that virtually all luteinizing hormone-releasing hormone neurons in the rat secrete outside the blood-brain barrier, including those which project to sites within the central nervous system.

Comparison of ultrastructural characteristics of gonadotropin-releasing hormone neurons in prepubertal and adult male rats.

Gonadotropin-releasing hormone neurons from prepubertal (29-day-old) and adult (three-month-old) male rats were demonstrated immunocytochemically using the LR1 antibody, and prepared for electron microscopic examination. Gonadotropin-releasing hormone neurons were equally immunoreactive in the two age groups, but there were heavy deposits of reaction product in the outer nuclear envelope of these neurons in prepubertal animals. Point count stereology on electron micrographic montages of gonadotropin-releasing hormone neurons at x25,000 was used to compare the relative proportion of cytoplasm containing various subcellular organelles. More of the cytoplasm was occupied by Golgi apparatus and secretory vesicles in the prepubertal animals. The representation of mitochondria was equal in the two age groups, while there were more lysosomes in the gonadotropin-releasing hormone neurons from adult animals. The density of synaptic input to the neurons was estimated using quantitative morphometrics on electron micrographs of three levels of section through the neuron, magnified x25,000. The percentage of the perikaryal membrane with synaptic contacts was greater in the gonadotropin-releasing hormone neurons from adults. Most strikingly, there were gonadotropin-releasing hormone terminals on gonadotropin-releasing hormone soma of these neurons in prepubertal animals, but not in the adults. The highly immunoreactive outer nuclear envelope and relative larger representation of Golgi and secretory vesicles in gonadotropin-releasing hormone neurons in prepubertal animals suggest that these cells are actively synthesizing peptides, including gonadotropin-releasing hormone. The large representation of Golgi apparatus may also reflect the active biosynthesis of membrane in association with the elaboration of neuronal processes.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural differences between smooth and thorny gonadotropin-releasing hormone neurons.

It has been suggested that gonadotropin-releasing hormone neurons with irregular contours receive more synaptic input than do those with smooth contours. To test this hypothesis, a morphometric analysis of gonadotropin-releasing hormone neurons of differing shapes was made. Male Sprague-Dawley rats were perfused and 40-microns tissue sections from the preoptic area were treated for the demonstration of gonadotropin-releasing hormone immunoreactivity, using the avidin-biotin horseradish peroxidase method with 3,3'-diaminobenzidine as the chromogen. Gonadotropin-releasing hormone neurons with either a smooth or a highly irregular outline, sampled at three depths ultrastructurally, were compared for density of synaptic innervation and for relative content of various subcellular organelles. Point counting stereology comparisons of five gonadotropin-releasing hormone neurons of the two shapes in four rats revealed that smooth neurons contained less cytoplasm than "thorny" neurons, but the size of their nuclei was the same. There were more and larger nucleoli in smooth cells. Thorny neurons contained more Golgi apparatus and more mitochondria, but the amount of rough endoplasmic reticulum was the same in neurons of the two morphological types. There was no difference in the incidence of synapses or in the density of synaptic input as measured per unit of neuronal membrane in smooth and thorny gonadotropin-releasing hormone neurons. These results suggest that smooth-contoured gonadotropin-releasing hormone neurons may be more actively transcribing a message while thorny neurons are more actively engaged in peptide processing and packaging.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructural changes in gonadotropin-releasing hormone neurons as a function of age and ovariectomy in rats.

In this study we examined the effects of aging on various aspects of the ultrastructure of gonadotropin-releasing hormone neurons in female rats, including the density of synaptic input and the volume fraction of various subcellular organelles. In addition, we explored the possibility that removal of estrogen might provide a protective effect on the aging of the gonadotropin-releasing hormone neuron as exposure to gonadal steroids alters the time course of reproductive aging. Our experimental groups included four- and 18-20-month-old virgin female rats divided as follows: young intact, young short-term ovariectomized, old intact, old short-term ovariectomized and old long-term ovariectomized. Brain tissue was processed for immunocytochemical detection of gonadotropin-releasing hormone neurons and selected cells from the preoptic area were chosen for electron microscopic examination. The percentage of plasma membrane containing synaptic modification was quantified using a morphometrics program, and the volume fraction of lysosomes/lipofuscin, rough endoplasmic reticulum and Golgi apparatus were estimated using point count stereology. Whereas we had previously found a significant increase in the density of synaptic input to gonadotropin-releasing hormone neurons in aged virgin male rats, the density of synaptic input to gonadotropin-releasing hormone cells in the virgin female was not affected by age. The volume fraction of lysosomes/lipofuscin was increased in all age groups. Aging produced a dramatic decrease in the volume fraction of rough endoplasmic reticulum as well as a decrease in Golgi, suggesting a general decrease in biosynthetic activity of the cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptic interactions of luteinizing hormone-releasing hormone (LHRH) neurons in the guinea pig preoptic area.

Previous studies from many laboratories have failed to demonstrate a significant synaptic input to luteinizing hormone-releasing hormone (LHRH) neurons in the rodent or primate hypothalamus/preoptic area. Having now developed immunocytochemical procedures that result in excellent ultrastructural preservation as well as in retention of antigenicity (Silverman AJ: J Comp Neurol 227:452, 1984), we have reinvestigated the question of the organization of the synaptic arrangements of LHRH neurons in the medial preoptic area of the guinea pig. Afferent inputs to these LHRH neurons include several varieties of axo-somatic and axo-dendritic synapses. Presynaptic terminals contain either round clear vesicles or a mixture of round and flattened vesicles. Most of these terminals, especially when serial sections are examined, contain dense-core granules. Well-defined synaptic clefts are evident and postsynaptic densities can be identified for asymmetrical connections. However, the presence of reaction product in the postsynaptic structure makes it difficult to categorize symmetrical terminals. In addition to these classical inputs, LHRH neurons also enter into complex heterodox synaptic relationships with their neighbors, including somato-dendritic and dendro-dendritic synapses in which the LHRH neuron can be either the pre- or postsynaptic element. These results suggest that complex synaptic relationships might account for the multiple levels of regulation of neurohormone release.

Light and electron microscopic immunocytochemical analysis of antibodies directed against GnRH and its precursor in hypothalamic neurons.

A battery of antibodies directed against different portions of the precursor to gonadotropin-releasing hormone (GnRH), as well as to the mature decapeptide, were characterized immunocytochemically in two ways. Absorption experiments were used to determine the epitope recognized by each antiserum. Electron microscopic immunocytochemistry was then used to define the subcellular organelles that contained reaction product when tissue was incubated with these reagents. These latter observations helped to determine if the antibody recognized the epitope as part of the intact precursor or only after it had been cleaved from parent protein. Our results demonstrate that the GnRH precursor is routed from the rough endoplasmic reticulum through the Golgi apparatus to the secretory vesicles. Furthermore, we show that initial cleavage and processing of the GnRH precursor begin in the cell soma. These antibodies should be useful in the future in determining changes in processing of precursor in animals that differ in endocrine function.

Reproductive history affects the synaptology of the ageing gonadotropin-releasing hormone system in the male rat.

This study is an examination of the density of synaptic input to gonadotropin-releasing hormone (GnRH) neurons in young adult and aged retired breeder male rats. In earlier experiments on aged virgin male rats we observed an increase in synaptic input to this specific neuronal population, ascribable in part to synapses containing flattened vesicles, suggesting GABAergic input. The present study utilized retired breeders in order to dissect the effects of ageing from those associated with reproductive behavioral history. Tissue from the preoptic area was treated for the simultaneous electron microscopic immunocytochemical demonstration of GnRH with tetramethylbenzidine and glutamic acid decarboxylase (the essential enzyme in the production of GABA) using 3,3'-diaminobenzidine. Estimates of the density of synaptic input to the soma of GnRH neurons were made by calculating the percentage of perikaryal membrane with postsynaptic modification. Five GnRH neurons per animal were measured using computerized morpho-metrics and differences in the percent of membrane with synaptic modification between experimental groups were tested using the Mann-Whitney U non-parametric statistic. There was no difference in the total density of synaptic input to GnRH neurons in the young and old animals, or in the proportion of this input that was immunoreactive for glutamic acid decarboxylase. Similar measurements were made on random, non-identified neurons in the same region and a significant decrease with ageing in total synaptic input was found, though the glutamic acid decarboxylase component was unchanged. The present results are in contrast to our earlier findings on virgin males and suggest that reproductive behavioral experience affects the connectivity of GnRH neurons.