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J W Witkin

Publications and source records attributed to J W Witkin.

36 records · Page 2Linked to original sources
PubMed1990

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)

Animals↗
PubMed1990

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.

Aging↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

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.

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PubMed1989

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.

Animals↗
PubMed1989

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.

Animals↗
PubMed1989

Morphology of luteinizing hormone-releasing hormone neurons as a function of age and hormonal condition in the male rat.

Previous work in many laboratories has shown that luteinizing hormone-releasing hormone (LHRH) neurons display a range of shapes from smooth-contoured to extremely irregular or 'thorny'. To determine if these forms reflect or can be modified by the gonadal steroid milieu, the morphology of LHRH neurons was examined in male rats from each of four experimental groups: adult (2-3 months); aged (23 months) and adult castrate (2-3 months, castrated for 4 weeks) with or without testosterone replacement. The region studied extended from the level of the medial septum and diagonal band of Broca posteriorly through the lateral preoptic and anterior hypothalamic areas, throughout which region the majority of LHRH neurons reside. Neurons were divided into groups based on the degree of irregularity of their profiles ('thorniness'). The total numbers of neurons did not differ among the experimental groups and smooth-countoured neurons predominated. However, there were fewer thorny LHRH neurons in the castrate animals than in the other experimental groups. LHRH neurons in the young adult and aged intact rats and testosterone-replaced animals did not differ in their morphology. These results suggest that LHRH neurons in the male rat tend to lose somatic or dendritic spines under conditions in which gonadal steroids are dramatically reduced. It also appears that the levels to which testosterone is reduced in the aged rat are not associated with a decrease in the incidence of spine-like projections on LHRH neurons.

Aging↗
PubMed1987

Immunocytochemical demonstration of luteinizing hormone-releasing hormone in optic nerve and nasal region of fetal rhesus macaque.

The brain and nasal region of a fetal (124 days) female rhesus macaque (Macaca mulatta) were fixed in situ, decalcified, sectioned and treated for the immunocytochemical demonstration of LHRH. Immunoreactive neurons and fibers were found in expected sites and also in sites not reported to date in primates: there was an abundant distribution along the pathway of the nervus terminalis and clusters of neurons anterior and ventral to the olfactory bulbs and scattered in the nasal septum. Fibers extended into the epithelium lining the nasal septum. Most unexpectedly, LHRH fibers and a few neurons were observed within the optic nerve.

Animals↗
PubMed1987

Localization of a peptide sequence contained in the precursor to gonadotropin releasing hormone (GnRH).

The precursor protein that contains the sequence for the neurohormone gonadotropin releasing hormone (GnRH) also contains an additional fragment (amino acids 14-26, designated pHGnRH14-26) that can release luteinizing hormone (LH) and follicle stimulating hormone (FSH) in vitro. An immunocytochemical study was carried out to determine if this sequence could be found in its processed form in hypothalamic nerve terminals. In rats, ewes and rhesus monkeys pHGnRH14-26 was demonstrated in both neuronal cell bodies and in axon terminals. In mice, immunoreactivity was present in terminals only. No reaction was found in the hamster hypothalamus. Double label immunocytochemical studies for GnRH and pHGnRH14-26 showed that both sequences could be found in the rat septal-preoptic-hypothalamic continuum in some but not all neuronal cell bodies. Control experiments strongly suggest that the 14-26 immunoreactivity represents the fragment after it has been cleaved from the precursor protein and that this peptide could be available for release into the hypophysial portal system.

Amino Acid Sequence↗
PubMed1987

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.

Aging↗
PubMed1987

Nervus terminalis, olfactory nerve, and optic nerve representation of luteinizing hormone-releasing hormone in primates.

The luteinizing hormone-releasing hormone (LHRH) system was examined immunocytochemically in olfactory bulbs of adult monkeys, including two New World species (squirrel monkey, Saimiri sciureus and owl monkey, Aotus trivirgatus) and one Old World species (cynomolgus macaque, Macaca fasciculata), and in the brain and nasal region of a fetal rhesus macaque Macaca mulatta. LHRH neurons and fibers were found sparsely distributed in the olfactory bulbs in all adult monkeys. There was more LHRH in the accessory olfactory bulb (which is absent in Old World monkeys). In the fetal macaque there was a rich distribution of LHRH neurons and fibers along the pathway of the nervus terminalis, anterior and ventral to the olfactory bulb, and in the nasal septum, with fibers branching into the olfactory epithelium. In addition, there were LHRH neurons and fibers in the optic nerve.

Animals↗
PubMed1985

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.

Animals↗
PubMed1983

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.

Animals↗
PubMed1982

The luteinizing hormone-releasing hormone (LHRH) systems in the rat brain.

Immunocytochemical procedures on thick, unembedded sections were used to visualize the neurons and their processes that contain LHRH-immunoreactive material in the rat central nervous system (CNS). In animals pretreated with colchicine (75 micrograms, intraventricularly), cell bodies could be observed as far anterior as the olfactory bulb and posterior to the retrochiasmatic area of the basal hypothalamus. Several new observations for the rat were made in this study, including LHRH neurons in the accessory olfactory bulb and other olfactory-related structures, and in the anterior hippocampus and the induseum griseum. As in studies from other laboratories, we observed many LHRH cells in the periventricular medial preoptic area, diagonal band of Broca and septal nuclei, and fewer positive cells in the anterior hypothalamic area and the region of the supraoptic commissure. The LHRH fibers from all of these cells are widely dispersed in the CNS. In addition to the dense innervation of the median eminence, positive fibers are found innervating other circumventricular organs, coursing close to the ependymal wall of the ventricular system or in close association with cerebral arteries and areas of the pia mater and subarachnoid space. LHRH fibers may also innervate neurons in several regions of the CNS. A novel projection of LHRH fibers for the rat was found originating from supracallosal neurons and coursing through both cingulate and neocortex. The possible distribution of efferents from each LHRH cell group is discussed.

Animals↗
PubMedDate not supplied

Synaptology of luteinizing hormone-releasing hormone neurons in rat preoptic area.

The ultrastructural appearance of luteinizing hormone-releasing hormone (LHRH) immunoreactive elements was studied in the medial preoptic area (MPOA) of adult male Fischer 344 rats. The purpose of the study was to determine the distribution and morphology of innervation of the LHRH neuron. Although not numerous, both axo-somatic and axo-dendritic synapses were present and generally of the asymmetric (Gray's II) category. Analyses of 56 profiles of 11 separate perikarya revealed only 7 axo-somatic terminals. The synaptic input to LHRH dendrites was a fraction of that to non-identified dendrites in the same electron micrographic fields; 0.4% of LHRH dendritic membrane was in synaptic contact compared to 6.6% of nonidentified dendritic membrane. In addition to receiving an input, LHRH processes were also seen to make synapses onto non-immunoreactive elements. Close examination of this material for evidence of contact between LHRH elements revealed two clear examples of synaptic interaction and several instances of close association in which no other elements intervened.

Animals↗
PubMedDate not supplied

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.

Aging↗
PubMedDate not supplied

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.

Aging↗