Joan W. Witkin

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.

Gonadotropin-releasing hormone neurons in the rhesus macaque are not immunoreactive for the estrogen receptor.

The issue of whether gonadotropin-releasing hormone (GnRH) neurons in the primate contain the estrogen receptor was examined by immunocytochemistry using prepubertal and adult (intact and ovariectomized) female rhesus macaques. No GnRH neurons were found to contain nuclei that were immunoreactive for the estrogen receptor. These results confirm in primates what has been reported in other species and leave open the question of how the effects of gonadal steroids on GnRH neurons are mediated.

Role of the Cysteine-rich Domain of the t-SNARE Component, SYNDET, in Membrane Binding and Subcellular Localization

Wild-type syndet is efficiently recruited at the plasma membrane in transfected AtT-20 cells. A deletion at the cysteine-rich domain abolishes palmitoylation, membrane binding, and plasma membrane distribution of syndet. Syndet, SNAP-25A, and SNAP-25B share four cysteine residues, of which three, Cys2, Cys4, and Cys5, are absolutely conserved in all three homologs. Mutations at any pair of cysteines within cysteines 2, 4, and 5 shift syndet from the cell surface into the cytoplasm. Thus, at least two cysteines within the conserved triplet are necessary for plasma membrane localization. Syndet C1S/C3S, with substitutions at the pair Cys1 and Cys3, distributes to the plasma membrane, a Golgi-like compartment, and the cytosol. We conclude that Cys1 and Cys3 are not absolutely necessary for membrane binding or plasma membrane localization. Our results show that the cysteine-rich domain of syndet plays a major role in its subcellular distribution.

SNAP‐25 and Synaptotagmin 1 Function in Ca2+‐Dependent Reversible Docking of Granules to the Plasma Membrane

In neuroendocrine cells, Ca2+ triggers fusion of granules with the plasma membrane and functions at earlier steps by increasing the size of the readily releasable pool of vesicles. The effect of Ca2+ at early steps of secretion may be due to the recruitment at the plasma membrane of granules localized in the cytoplasm. To study the mechanism of granule docking, a new in vitro assay is designed using membrane fractions from mouse pituitary AtT‐20 cells. By using this assay, it is found that granule docking to the plasma membrane is controlled by Ca2+ concentrations in the micromolar range, is reversible and requires intact SNAP‐25, but not VAMP‐2. In the docking assay, addition of Ca2+ induces the formation of a SNAP‐25‐Synaptotagmin 1 complex. The cytosolic domain C2AB of Synaptotagmin 1 and anti‐Synaptotagmin 1 antibodies block granule docking. These results show that Ca2+ modulates dynamic docking of granules to the plasma membrane and that this process is due to a Ca2+‐dependent interaction between SNAP‐25 and Synaptotagmin 1.

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.

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.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 ...

Synchronized neuronal networks: The GnRH system

The anatomical substrate for coordinated release from the dispersed gonadotropin‐releasing hormone (GnRH) neuronal population remains obscure. There is physiological evidence that the GnRH hormone itself has a role in tonic inhibition or modulation of GnRH function. This has led to the hypothesis that there is an ultrashort negative feedback mechanism subserved by axon collaterals acting back on the GnRH neurons. Recent ultrastructural studies have revealed GnRH synapses on GnRH neurons and their processes. Furthermore, there are alterations in the frequency of these synapses with the age and hormonal condition of the animal. Another candidate for coordination of neuronal activity for which there is some evidence in the magnocellular system, is the gap junction. Recently, physiological and anatomical evidence for gap junctional modifications among an immortalized GnRH‐secreting cell line (GT1) has been reported. However, at present there is no immunocytochemical or ultrastructural evidence for gap junctions between GnRH neurons. A third and highly unorthodox anatomical relationship between (among) these cells has been suggested by serial ultrastructural reconstructions of pairs of GnRH neurons in close association. In some regions, GnRH neuronal processes can be seen to extend from each member of a pair of GnRH neurons. These meet and merge, forming an intercellular bridge. This phenomenon has been observed in several pairs of GnRH neurons in rat and monkey. The important caveat in making these observations is that techniques employed to demonstrate sites of antigenicity can severely compromise the ultrastructural integrity of membrane components. For this reason, further verification of the existence of intercellular bridges is being pursued. Should their existence be confirmed, they would be prime candidates for the coordination of secretory events among the scattered GnRH neuronal population. Microsc. Res. Tech. 44:11–18, 1999.

Glial Ensheathment of GnRH Neurons in Pubertal Female Rhesus Macaques

During the period of development, prior to full sexual maturity, gonadotropin hormone‐releasing hormone (GnRH) neurons are fully capable of synthesizing and processing the GnRH decapeptide. Nonetheless, the secretion of the hormone is not adequate to stimulate adult patterns of gonadotropin release. The present study was undertaken to examine ultrastructural characteristics of the GnRH neuron and its relationship to its environment in early‐ midpubertal female rhesus monkey. These neurons bore all the ultrastructural immunocytochemical characteristics of those in mature animals, but quantitative morphometrics revealed that they were extensively apposed by glial processes. Such ensheathment was described earlier in ovariectomized adult animals and was found to be reversible by administration of gonadal steroids. The density of synaptic input to GnRH neurons in the pubertal animals did not differ significantly from that of adult intact or ovariectomized animals from a previous study. Chemical identification will be required to determine whether there are age or hormonal differences in the innervation of these neurons.

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.

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.