Expression of Vesicular Glutamate Transporter 2 (vGluT2) on Large Dense-Core Vesicles within GnRH Neuroterminals of Aging Female Rats

The pulsatile release of GnRH is crucial for normal reproductive physiology across the life cycle, a process that is regulated by hypothalamic neurotransmitters. GnRH terminals co-express the vesicular glutamate transporter 2 (vGluT2) as a marker of a glutamatergic phenotype. The current study sought to elucidate the relationship between glutamate and GnRH nerve terminals in the median eminence—the site of GnRH release into the portal capillary vasculature. We also determined whether this co-expression may change during reproductive senescence, and if steroid hormones, which affect responsiveness of GnRH neurons to glutamate, may alter the co-expression pattern. Female Sprague-Dawley rats were ovariectomized at young adult, middle-aged and old ages (~4, 11, and 22 months, respectively) and treated four weeks later with sequential vehicle + vehicle (VEH + VEH), estradiol + vehicle (E₂ + VEH), or estradiol + progesterone (E₂+P₄). Rats were perfused 24 hours after the second hormone treatment. Confocal microscopy was used to determine colocalization of GnRH and vGluT2 immunofluorescence in the median eminence. Post-embedding immunogold labeling of GnRH and vGluT2, and a serial electron microscopy (EM) technique were used to determine the cellular interaction between GnRH terminals and glutamate signaling. Confocal analysis showed that GnRH and vGluT2 immunofluorescent puncta were extensively colocalized in the median eminence and that their density declined with age but was unaffected by short-term hormone treatment. EM results showed that vGluT2 immunoreactivity was extensively associated with large dense-core vesicles, suggesting a unique glutamatergic signaling pathway in GnRH terminals. Our results provide novel subcellular information about the intimate relationship between GnRH terminals and glutamate in the median eminence.     

A novel pathway regulates thyroid hormone availability in rat and human hypothalamic neurosecretory neurons

Hypothalamic neurosecretory systems are fundamental regulatory circuits influenced by thyroid hormone. Monocarboxylate-transporter-8 (MCT8)-mediated uptake of thyroid hormone followed by type 3 deiodinase (D3)-catalyzed inactivation represent limiting regulatory factors of neuronal T3 availability. In the present study we addressed the localization and subcellular distribution of D3 and MCT8 in neurosecretory neurons and addressed D3 function in their axons. Intense D3-immunoreactivity was observed in axon varicosities in the external zone of the rat median eminence and the neurohaemal zone of the human infundibulum containing axon terminals of hypophysiotropic parvocellular neurons. Immuno-electronmicroscopy localized D3 to dense-core vesicles in hypophysiotropic axon varicosities. N-STORM-superresolution-microscopy detected the active center containing C-terminus of D3 at the outer surface of these organelles. Double-labeling immunofluorescent confocal microscopy revealed that D3 is present in the majority of GnRH, CRH and GHRH axons but only in a minority of TRH axons, while absent from somatostatin-containing neurons. Bimolecular-Fluorescence-Complementation identified D3 homodimers, a prerequisite for D3 activity, in processes of GT1-7 cells. Furthermore, T3-inducible D3 catalytic activity was detected in the rat median eminence. Triple-labeling immunofluorescence and immuno-electronmicroscopy revealed the presence of MCT8 on the surface of the vast majority of all types of hypophysiotropic terminals. The presence of MCT8 was also demonstrated on the axon terminals in the neurohaemal zone of the human infundibulum. The unexpected role of hypophysiotropic axons in fine-tuned regulation of T3 availability in these cells via MCT8-mediated transport and D3-catalyzed inactivation may represent a novel regulatory core mechanism for metabolism, growth, stress and reproduction in rodents and humans.     

Immunocytochemical study of the GABAergic innervation of the mouse pituitary by use of antibodies against gamma-aminobutyric acid (GABA)

Summary The GABAergic innervation of the mouse pituitary, including the median eminence, was studied at light microscopic and ultrastructural levels by use of a pre-embedding immunocytochemical technique with antibodies directed against GABA. In the median eminence, a high density of GABA-immunoreactive fibers was found in the external layer where the GABAergic varicosities were frequently observed surrounding the blood vessels of the primary capillary plexus. In the internal and subependymal layers, only few fibers were immunoreactive. The intense labeling of the external layer was observed in the entire rostro-caudal extent of the median eminence. In the pituitary proper, a dense network of GABA-immunoreactive fibers was revealed throughout the neural and intermediate lobes, entering via the hypophyseal stalk. The anterior and tuberal lobes were devoid of any immunoreactivity. The GABA-immunoreactive terminals were characterized in the median eminence, and in the intermediate and posterior lobes at the electron-microscopic level. They contained small clear vesicles, occasionally associated with dense-core vesicles or neurosecretory granules. In the intermediate lobe they were seen to be in contact with the glandular cells. In the posterior lobe and in the median eminence, GABA-immunoreactive terminals were frequently located in the vicinity of blood vessels. These results further support the concept of a role of GABA in the regulation of hypophyseal functions, via the portal blood for the anterior lobe, directly on the cells in the intermediate lobe, and via axo-axonic mechanisms in the median eminence and posterior lobe.     

Co-transport of sex hormone-binding globulin/SHBG with oxytocin in transport vesicles of the hypothalamo-hypophyseal system.

The intrinsic expression of sex hormone binding globulin (SHBG) in magnocellular hypothalamic neurons, in part co-localized with either vasopressin or oxytocin, was recently described. This study is focused on the ultrastructural localization of SHBG in the hypothalamo-neurohypophyseal pathway in rats. Immunostaining for SHBG in the hypothalamic perikarya was increased by colchicine treatment, indicating that the steroid-binding globulin is subject to rapid axoplasmic transport along with the classical posterior lobe peptides. With immunoelectron-microscopic double labeling, we found co-localization of oxytocin and sex hormone binding globulin in a portion of the large dense-core vesicles in paraventricular and supraoptic perikarya and in axonal varicosities in the median eminence and in the posterior lobe. Our observations show that SHBG is processed, transported and stored along with oxytocin suggesting that SHBG is released from nerve terminals in the posterior lobe, the median eminence and possibly the brain similarly to and in conjunction with oxytocin.     

Fine structure of the median eminence and the pars nervosa of the bullfrog,Rana catesbeiana

Summary The hypothalamic neurosecretory system of the bullfrog, Rana catesbeiana, was studied with light- and electron microscopy. The median eminence is roughly divided into two portions. The upper portion mostly consists of ependymal cells, glial cells and preoptico-hypophysial nerve tract, whereas in the lower portion, neurosecretory axons, glial cells, processes of glial and ependymal cells, and fine blood vessels of the hypothalamic portal vein are located. A part of the neurosecretory axons of the preoptico-hypophysial tract proceeds to the lower portion of the median eminence. These axons are arranged perpendicularly to the capillaries of the hypothalamic portal vein. The glial cells are densely located in the area of the median eminence where neurosecretory material is abundant. The neurosecretory material in the neurosecretory cells, their axons, the median eminence and the pars nervosa of the bullfrog shows a positive reaction to PAS treatment.The neurohemal area of the median eminence is occupied by many neurosecretory and non-neurosecretory axons, containing neurosecretory granules and/or synaptic vesicles. The axonal portions with the synaptic vesicles which are considered to be the nerve endings abut on the capillaries of the portal system. The size of synaptic vesicles in the axon terminals containing few neurosecretory granules is larger than those in the endings with many neurosecretory granules. Infrequently glial and ependymal processes are interposed between the nerve endings and the capillary wall.In the hilar region of the infundibulum, synapses are frequently observed between the thin fibers with or without neurosecretory granules and dendrites of non-neurosecretory neurons. The probable functions of these synapses are briefly discussed on the basis of our findings. Both in the hilar region of the infundibulum and in the pars nervosa, electron-dense neurosecretory granules of two different sizes were observed. The median eminence contains only one type of granules.The fine structure of the pars nervosa shows similar structures to those of the median eminence. Both in the median eminence and the pars nervosa, the fenestrated endothelium of the capillaries was frequently observed. The thick perivascular connective tissue space containing fibroblasts and collagen fibrils was observed both in the median eminence and the pars nervosa. Vesicles in the cytoplasm of the endothelial cells which appear to take a part in the transendothelial transport were observed.This investigation was supported in part by United States Public Health Service Research Grant, No. A-3678, to Hideshi Kobayashi from the National Institute of Arthritis and Metabolic Diseases and partly by a grant for Fundamental Scientific Research from the Ministry of Education of Japan. The authors wish to express their thanks to Prof. K. Takewaki for his kind encouragement.     

The effects of orchidectomy and replacement therapy on the ultrastructure and gonadotropin-releasing hormone content of the median eminence of the rat

The effects of 2 weeks of orchidectomy and replacement therapy with testosterone upon the content and distribution of gonadotropin-releasing hormone (GnRH) in the median eminence were determined by means of radioimmunoassay and electron microscopy. Photographic montages were prepared from electron micrographs of the lateral median eminence at the point of deepest invagination of the tuberoinfundibular sulcus. Morphometric analysis of photographs of tissues immunohistochemically stained for GnRH was performed to determine changes in the volume density of GnRH-containing axon profiles following the experimental treatments. A decrease in GnRH content after orchidectomy was observed both by morphometric analysis of axon volume density and radioimmunoassay of total GnRH content. Testosterone treatment of orchidectomized animals prevented the postorchidectomy loss of GnRH. Morphometric analysis of conventional electron micrographs revealed an increase in the number of axons containing no dense-core vesicles following orchidectomy, but no decrease in volume density of the neuropil. The results indicate that the change in volume density of immunostained axons was related to the loss of immunostainable dense-core vesicles and not to a change in the size or number of axons. The area corresponding to the location of the highest concentration of GnRH-containing axons was observed to be largely avascular and separated from the vessels of the tuberoinfundibular sulcus by a "border zone" composed of glial foot processes. The unique morphology of the GnRH area has suggested the name "compact zone" to distinguish it from the palisade zone with which it is continuous medially. GnRH axons in this region are probably part of a tract extending farther caudally rather than a terminal field.     

Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion

Neurotransmitters and hormones are released from neurosecretory cells by exocytosis (fusion) of synaptic vesicles, large dense-core vesicles and other types of vesicles or granules. The exocytosis is terminated and followed by endocytosis (retrieval). More than fifty years of research have established full-collapse fusion and clathrin-mediated endocytosis as essential modes of exo-endocytosis. Kiss-and-run and vesicle reuse represent alternative modes, but their prevalence and importance have yet to be elucidated, especially in neurons of the mammalian CNS. Here we examine various modes of exo-endocytosis across a wide range of neurosecretory systems. Full-collapse fusion and kiss-and-run coexist in many systems and play active roles in exocytotic events. In small nerve terminals of CNS, kiss-and-run has an additional role of enabling nerve terminals to conserve scarce vesicular resources and respond to high-frequency inputs. Full-collapse fusion and kiss-and-run will each contribute to maintaining cellular communication over a wide range of frequencies.     

A comparative study of the ultrastructure of the median eminence,infundibular stem and neural lobe of the hypophysis of the rat

Summary The ultrastructure of the infundibulum has been studied and compared with that of neural lobe in normal rats. The neurohemal areas of the median eminence are similar to those of the stem but differ from those of neural lobe. The infundibular axons which end around the primary capillaries of the portal system are of a significantly finer caliber. Secondly they contain a different vesicle population. They lack the large (1500 Å–2100 Å) neurosecretory vesicles so abundant in neural lobe axon terminals but contain a smaller (less than 1000 Å) type of vesicle with an osmiophilic center. These dense-core vesicles are consistently present in the many infundibular levels examined, although they are not as numerous as the neurosecretory ones of neural lobe. They are outnumbered by vesicles of the synaptic type, whereas in neural lobe the neurosecretory ones predominate. Another difference involves the electron lucent, neurosecretory vesicle. These are abundant in neural lobe axons, but comparable aggregations of them have not been seen in infundibular axon endings of the neurohemal areas. In contrast, the internal zone of median eminence and the interior of the stem display, in addition to the fine axons, many large fibers which by size and content match the ones of neural lobe. However, careful study indicates that these are axis cylinders and not axon endings.These observations lead to the conclusion that the small calibered axons which terminate around the infundibular capillaries of the portal system constitute a separate group, and are clearly distinguishable at the ultrastructural level from the large supraoptico-neurohypophyseal axons. The latter normally traverse the infundibulum but terminate in neural lobe.This investigation was supported by U.S.P.H.S. Research Grant 5 RO 1 NB 02321-05, National Institute of Neurological Diseases and Blindness. — The author is particularly indebted to Mrs. Nora Tong for her excellent technical assistance throughout the course of this study.     

Histological characterization of gonadotropin-releasing hormone (GnRH) in the hypothalamus of the South American plains vizcacha (Lagostomus maximus)

In contrast to most mammalian species, females of the South American plains vizcacha, Lagostomus maximus, show an extensive suppression of apoptosis-dependent follicular atresia, continuous folliculogenesis, and massive polyovulation. These unusual reproductive features pinpoint to an eventual peculiar modulation of the hypothalamo-hypophyseal-gonadal axis through its main regulator, the gonadotropin-releasing hormone (GnRH). We explored the hypothalamic histological landscape and cellular and subcellular localization of GnRH in adult non-pregnant L. maximus females. Comparison to brain atlases from mouse, rat, guinea pig and chinchilla enabled us to histologically define and locate the preoptic area (POA), the ventromedial nucleus, the median eminence (ME), and the arcuate nucleus (Arc) of the hypothalamus in vizcacha's brain. Specific immunolocalization of GnRH was detected in soma of neurons at medial POA (MPA), ventrolateral preoptic nucleus, septohypothalamic nucleus (SHy) and Arc, and in beaded fibers of MPA, SHy, ventromedial hypothalamic nucleus, anterior hypothalamic area and ME. Electron microscopy examination revealed GnRH associated to cytoplasmic vesicles of the ME and POA neurons, organized both in core and non-core vesicles within varicosities, and in neurosecretory vesicles within the myelinated axons of the MPA. Besides the peculiar and unusual features of folliculogenesis and ovulation in the vizcacha, these results show that hypothalamus histology and GnRH immune-detection and localization are comparable to those found in other mammals. This fact leads to the possibility that specific regulatory mechanisms should be in action to maintain continuous folliculogenesis and massive polyovulation.     

Dual Somatic Recordings from Gonadotropin-Releasing Hormone (GnRH) Neurons Identified by Green Fluorescent Protein (GFP) in Hypothalamic Slices

Gonadotropin-Releasing Hormone (GnRH) is a small neuropeptide that regulates pituitary release of luteinizing hormone (LH) and follicle-stimulating hormone (FSH). These gonadotropins are essential for the regulation of reproductive function. The GnRH-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release GnRH from their axon terminals into the hypophysiotropic portal system (1). In the portal capillaries, GnRH travels to the anterior pituitary gland to stimulate release of gonadotropins into systemic circulation. GnRH release is not continuous but rather occurs in episodic pulses. It is well established that the intermittent manner of GnRH release is essential for reproduction (2, 3).Coordination of activity of multiple GnRH neurons probably underlies GnRH pulses. Total peptide content in GnRH neurons is approximately 1.0 pg/cell (4), of which 30% likely comprises the releasable pool. Levels of GnRH during a pulse (5, 6), suggest multiple GnRH neurons are probably involved in neurosecretion. Likewise, single unit activity extracted from hypothalamic multi-unit recordings during LH release indicates changes in activity of multiple neurons (7). The electrodes with recorded activity during LH pulses are associated with either GnRH somata or fibers (8). Therefore, at least some of this activity arises from GnRH neurons.The mechanisms that result in synchronized firing in hypothalamic GnRH neurons are unknown. Elucidating the mechanisms that coordinate firing in GnRH neurons is a complex problem. First, the GnRH neurons are relatively few in number. In rodents, there are 800-2500 GnRH neurons. It is not clear that all GnRH neurons are involved in episodic GnRH release. Moreover, GnRH neurons are diffusely distributed (1). This has complicated our understanding of coordination of firing and has made many technical approaches intractable. We have optimized loose cell-attached recordings in current-clamp mode for the direct detection of action potentials and developed a recording approach that allows for simultaneous recordings from pairs of GnRH neurons.     

Bicarbonate-Independent Sodium Conductance of Na/HCO3 Cotransporter NBCn1 Decreases NMDA Receptor Function

The sodium bicarbonate cotransporter NBCn1 is an electroneutral transporter with a channel activity that conducts Na⁺ in a HCO₃^–-independent manner. This channel activity was suggested to functionally affect other membrane proteins which permeate Na⁺ influx. We previously reported that NBCn1 is associated with the NMDA receptors (NMDARs) at the molecular and physiological levels. In this study, we examined whether NBCn1 channel activity affects NMDAR currents and whether this effect involves the interaction between the two proteins. NBCn1 and the NMDAR subunits GluN1A/GluN2A were expressed in Xenopus oocytes, and glutamate currents produced by the receptors were measured using two-electrode voltage clamp. In the absence of CO₂/HCO₃^–, NBCn1 channel activity decreased glutamate currents mediated by GluN1A/GluN2A. NBCn1 also decreased the slope of the current–voltage relationships for the glutamate current. Similar effects on the glutamate current were observed with and without PSD95, which can cluster NBCn1 and NMDARs. The channel activity was also observed in the presence of CO₂/HCO₃^–. We conclude that NBCn1 channel activity decreases NMDAR function. Given that NBCn1 knockout mice develop a downregulation of NMDARs, our results are unexpected and suggest that NBCn1 has dual effects on NMDARs. It stabilizes NMDAR expression but decreases receptor function by its Na⁺ channel activity. The dual effects may play an important role in fine-tuning the regulation of NMDARs in the brain.     

Presenilin-1/γ-Secretase Controls Glutamate Release,Tyrosine Phosphorylation,and Surface Expression of N-Methyl-d-aspartate Receptor (NMDAR) Subunit GluN2B

Abnormally high concentrations of extracellular glutamate in the brain may cause neuronal damage via excitotoxicity. Thus, tight regulation of glutamate release is critical to neuronal function and survival. Excitotoxicity is caused mainly by overactivation of the extrasynaptic NMDA receptor (NMDAR) and results in specific cellular changes, including calcium-induced activation of calpain proteases. Here, we report that presenilin-1 (PS1) null mouse cortical neuronal cultures have increased amounts of calpain-dependent spectrin breakdown products (SBDPs) compared with WT cultures. NMDAR antagonists blocked accumulation of SBDPs, suggesting abnormal activation of this receptor in PS1 null cultures. Importantly, an increase in SBDPs was detected in cultures of at least 7 days in vitro but not in younger cultures. Conditioned medium from PS1 null neuronal cultures at 8 days in vitro contained higher levels of glutamate than medium from WT cultures and stimulated production of SBDPs when added to WT cultures. Use of glutamate reuptake inhibitors indicated that accumulation of this neurotransmitter in the media of PS1 null cultures was due to increased rates of release. PS1 null neurons showed decreased cell surface expression and phosphorylation of the GluN2B subunit of NMDAR, indicating decreased amounts of extrasynaptic NMDAR in the absence of PS1. Inhibition of γ-secretase activity in WT neurons caused changes similar to those observed in PS1 null neurons. Together, these data indicate that the PS1/γ-secretase system regulates release of glutamate, tyrosine phosphorylation, and surface expression of GluN2B-containing NMDARs.     

Regulation of N-methyl-D-aspartate receptors by calpain in cortical neurons

The N-methyl-D-aspartate (NMDA) receptor is a cation channel highly permeable to calcium and plays critical roles in governing normal and pathologic functions in neurons. Calcium entry through NMDA receptors (NMDARs) can lead to the activation of the Ca2+-dependent protease, calpain. Here we investigated the involvement of calpain in regulation of NMDAR channel function. After prolonged (5-min) treatment with NMDA or glutamate, the whole-cell NMDAR-mediated current was significantly reduced in both acutely dissociated and cultured cortical pyramidal neurons. The down-regulation of NMDAR current was blocked by bath application of selective calpain inhibitors. Intracellular injection of a specific calpain inhibitory peptide also eliminated the down-regulation of NMDAR current induced by prolonged NMDA treatment. In contrast, dynamin inhibitory peptide had no effect on the depression of NMDAR current, suggesting the lack of involvement of dynamin/clathrin-mediated NMDAR internalization in this process. Immunoblotting analysis showed that the NR2A and NR2B subunits of NMDARs were markedly degraded in cultured cortical neurons treated with glutamate, and the degradation of NR2 subunits was prevented by calpain inhibitors. Taken together, our results suggest that prolonged activation of NMDARs in neurons activates calpain, and activated calpain in turn down-regulates the function of NMDARs, which provides a neuroprotective mechanism against NMDAR overstimulation accompanying ischemia and stroke.     

Activation of presynaptic ionotropic glutamate receptors stimulates GABA release from hippocampal and cortical nerve terminals in rats

One of the pathways implicated in a fine-tuning control of neurosecretory process is the activation of presynaptic receptors. The present study was focused on the role of presynaptic glutamate receptor activation in the regulation of inhibitory synaptic transmission in the rat hippocampus and cortex. We aimed to clarify what types of ionotropic glutamate receptors are involved in the modulation of GABA secretion, and what mechanism underlies this modulation. We have revealed that specific agonists of kainate and NMDA receptors, kainate and NMDA, like glutamate, induced the release of [3H]GABA from hippocampal and cortical nerve terminals suggesting the involvement of both types in the regulation of GABAergic transmission. Our results indicate preferential involvement of vesicular, but not cytosolic, pool in response to glutamate receptor activation. This is based on the finding that NO-711 (a specific inhibitor of plasma membrane GABA transporters), fails to attenuate [3H]GABA release. We have concluded that presynaptic glutamate receptor-induced modulation of the strength of synaptic response is due to increasing the release probability of synaptic vesicles.     

D1 receptor mechanisms in the median eminence and their inhibitory regulation of LHRH release

D1 receptor mechanisms in the median eminence have been studied by means of immunocytochemistry using antisera against dopamine and cyclic AMP-regulated phosphoprotein-32 (DARPP-32) and tyrosine hydroxylase (TH) and by autoradiography using the iodinated analogue of the D1 receptor antagonist SCH-23390. The co-distribution of DARPP-32 and TH immunoreactivity (IR) and of DARPP-32 and luteinizing hormone releasing hormone (LHRH) IR was analysed in the median eminence by means of computer-assisted morphometry and microdensitometry. Functional analysis involved studies on the role of D1 receptors in the regulation of LH serum levels in rats treated with nicotine in the absence and presence of the D1 receptor antagonist. LH serum levels were measured by means of radioimmunoassay procedures.The results on the co-distribution of TH and DARPP-32 IR in the median eminence which were obtained both by analysis of adjacent sections and by two-colour immunocytochemistry on the same section, demonstrated a high degree of overlap of TH and DARPP-32 IR nerve terminals and tanycytes within the medial and lateral palisade zone. Furthermore, studies on LHRH and DARPP-32 IR nerve terminals and tanycytes in the median eminence with the same methodologies demonstrated preferential overlaps within the lateral palisade zone. The overlap area was about 50% of the LHRH or DARPP-32 immunoreactive area in this region. Density maps were also obtained on the distribution of LHRH and DARPP-32 immunoreactive profiles at various rostrocaudal levels. Correlation studies demonstrated a significant and positive co-distribution of LHRH and DARPP-32 immunoreactive terminals and tanycytes within the lateral palisade zone and the subependymal layer (when all DARPP-32 positive squares were considered) of the median eminence. Instead within the medial palisade zone a significant negative correlation coefficient was found, when all the LHRH positive squares were considered.In the receptor autoradiographical analysis a weak-to-moderate labelling was obtained of the part outside the mediobasal hypothalamus using the D1 receptor radioligand [¹²⁵I]SCH-23982, while hardly any labelling was found within the median eminence and the arcuate nucleus.SCH-23390 was found to counteract, in a dose-related way, the inhibitory effects of intermittent nicotine treatment on serum LH levels. The D2 receptor antagonist raclopride in a dose of 1 mg/kg did not counteract the inhibitory effects of nicotine on serum LH levels.The present immunocytochemical, autoradiographic and functional studies suggest the existence of a D1 receptor in the median eminence which can be blocked by the D1 receptor antagonist SCH-23390 in behaviourally relevant doses and which is masked under basal conditions in the male rat. It is proposed that one type of median eminence D1 receptor is located on the axon terminals, not linked to DARPP-32, and which may make possible a rapid regulation of hypothalamic hormone release, e.g. LHRH release from the nerve terminals in the lateral palisade zone as indicated in the present morphological and functional experiments. The other type of median eminence D1 receptor may be located on the tanycytes and linked to DARPP-32. It is suggested that this D1 receptor is responsible for a long-term regulation of hypothalamic hormone release inter alia LHRH release from the terminal and preterminal parts of the LHRH axons in the lateral palisade zone and subependymal layer, respectively.     

Ultrastructure of the peptidergic and monoaminergic neurons in the hypothalamic neurosecretory system of Anuran Batracians

Summary In the toad Bufo arenarum Hensel the following regions of the hypothalamic — neurohypophyseal system were studied under the electronmicroscope: preoptic and paraventricular nuclei, median eminence and infundibular process of the neurohypophysis.Neuronal perikarya of the preoptic nucleus are loaded with typical neurosecretory granules of peptidergic nature having a mean diameter of 1660 Å. While most neurons of the winter toad are in a storage stage a few show signs of a more active synthetic activity. A distinctive feature of preoptic neurons is the presence of large lipid droplets. The paraventricular nucleus contains small neurons containing granulated vesicles with a mean diameter of 800-1000 Å. In the region extending between these two nuclei and the median eminence axons containing either neurosecretory elementary granules or granulated vesicles are observed.The inner zone of the median eminence is occupied by axons of the preoptic neurohypophyseal tract; two types of axons, according to the size and density of the neurosecretory granules, may be recognized. The outer zone of the median eminence contains mainly axons and nerve terminals containing granulated vesicles of probable monoaminergic nature and only a few with granules of peptidergic type.The neurohypophysis contains two kinds of axons: one with more dense granules of 1800 Å and the other with granules of lesser electron density and 2100 Å. At the ending proper small clear vesicles of synaptic type are found.A progressive increase in volume of the peptidergic granules along the axon is demonstrated. This is of the order of 218% from the preoptic perikarya down to the infundibular process. The physiological significance of the two neurosecretory systems — i.e. the monoaminergic and the peptidergic — and the probable nature of the two types of peptidergic axons is discussed.Supported by grants from the Consejo Nacional de Investigaciones Científicas y Técnicas and by the Air Force Office of Scientific Research (AF-AFOSR 963-67).The authors want to express their gratitude to Mrs. Defilippi-Novoa and Mr. Alberto Sáenz for their skillful assistance.     

Influence of different neural systems on the secretion of sex hormones in potassium deficient mice

The influence of different neural systems that modulate GnRH secretion by hypothalamic neurons was investigated in mice exposed to hypokalemic conditions, in which the pulsatile release of GnRH has been shown to be altered and associated with a significant decrease of plasma sex steroids. Our results demonstrate that the potentiation of the inhibitory pathways mediated by opiates and GABA may be implicated in the decrease of sex hormones secretion produced by hypokalemia since treatment with higher doses of naloxone or flumazenil are required to restore progesterone or testosterone levels in potassium deficient mice. The combination treatment of prazoxin and naloxone suggests that the inhibitory action of opiates take place through its action on noradrenergic neurons. It is also possible that the inhibition of GnRH release could be due to a decrease in the tonic stimulatory action of noradrenergic pathway implicated in the control of GnRH release. Our results also reveal that it is unlikely that the glutamatergic system may play any relevant direct role in the decrease of sex steroid secretion observed in potassium deficient mice. Finally, these results together with the normal pattern of estradiol levels found along the estrus cycle in potassium deficient mice indicate that factors different from estradiol and acting on neural systems implicated in the regulation of GnRH-secreting neurons participate in the generation of the preovulatory surge of GnRH.     

The role of glutamate and nitric oxide in the reproductive neuroendocrine system.

The preovulatory surge of gonadotropin releasing hormone (GnRH) is essential for mammalian reproduction. Recent work has implicated the neurotransmitters glutamate and nitric oxide as having a key role in this process. Large concentrations of glutamate are found in several hypothalamic nuclei known to be important for GnRH release and glutamate receptors are also located in these key hypothalamic nuclei. Administration of glutamate agonists stimulate GnRH and LH release, while glutamate receptor antagonists attenuate the steroid-induced and preovulatory LH surge. Glutamate has also been implicated in the critical processes of puberty, hormone pulsatility, and sexual behavior. Glutamate is believed to elicit many of these effects by activating the release of the gaseous neurotransmitter, nitric oxide (NO). NO potently stimulates GnRH by activating a heme containing enzyme, guanylate cyclase, which in turn leads to increased production of cGMP and GnRH release. Recent work has focused on identifying anchoring and (or) clustering proteins that target glutamate receptors to the synapse and couple the glutamate-NO neurotransmission system. The present review will discuss these new findings, as well as the role of glutamate and nitric oxide in important mammalian reproductive events, with a focus on the hypothalamic control of preovulatory GnRH release.     

The zinc iodide-osmium tetroxide (ZIO) reaction on nerve endings in the median eminence of the rat under normal and experimental conditions

Summary The reaction of nerve endings in the median eminence of the rat to zinc iodide-osmium tetroxide (ZIO) staining was examined electron microscopically under normal and experimental conditions. The experimental condition of catecholamine exhaustion in the nerve endings was induced by the administration of H44/68 and reserpine. Vesicles in the terminals of catecholaminergic nerves reacted similarly to ZIO staining in both normal and experimental material. The majority of synaptic vesicles in various terminals gave a positive ZIO reaction. The neurosecretory elementary granules, however, failed to react with ZIO. On the other hand, some nerve terminals in the external layer of the median eminence showed a strong positive reaction in the cytoplasmic matrix, in mitochondria as well as in synaptic vesicles. These findings strongly suggest that the ZIO-positive substance in nerve terminals is not the transmitter itself, i.e. the monoamine, but rather represents a range of substances commonly found in various kinds of synaptic vesicles and is probably proteinaceous in nature. A brief discussion is also given on the difference in ZIO reactivity between neurosecretory elementary granules and small vesicles in the hypothalamo-hypophyseal tract.This work was supported in part by a research grant from the Ministry of Education, Japan     

Computational investigation of the changing patterns of subtype specific NMDA receptor activation during physiological glutamatergic neurotransmission

NMDA receptors (NMDARs) are the major mediator of the postsynaptic response during synaptic neurotransmission. The diversity of roles for NMDARs in influencing synaptic plasticity and neuronal survival is often linked to selective activation of multiple NMDAR subtypes (NR1/NR2A-NMDARs, NR1/NR2B-NMDARs, and triheteromeric NR1/NR2A/NR2B-NMDARs). However, the lack of available pharmacological tools to block specific NMDAR populations leads to debates on the potential role for each NMDAR subtype in physiological signaling, including different models of synaptic plasticity. Here, we developed a computational model of glutamatergic signaling at a prototypical dendritic spine to examine the patterns of NMDAR subtype activation at temporal and spatial resolutions that are difficult to obtain experimentally. We demonstrate that NMDAR subtypes have different dynamic ranges of activation, with NR1/NR2A-NMDAR activation sensitive at univesicular glutamate release conditions, and NR2B containing NMDARs contributing at conditions of multivesicular release. We further show that NR1/NR2A-NMDAR signaling dominates in conditions simulating long-term depression (LTD), while the contribution of NR2B containing NMDAR significantly increases for stimulation frequencies that approximate long-term potentiation (LTP). Finally, we show that NR1/NR2A-NMDAR content significantly enhances response magnitude and fidelity at single synapses during chemical LTP and spike timed dependent plasticity induction, pointing out an important developmental switch in synaptic maturation. Together, our model suggests that NMDAR subtypes are differentially activated during different types of physiological glutamatergic signaling, enhancing the ability for individual spines to produce unique responses to these different inputs.