Evidence for estrogenic regulation of gonadotropin-releasing hormone neurons by glutamatergic neurons in the ewe brain: An immunohistochemical study using an antibody against vesicular glutamate transporter-2

Gonadotropin-releasing hormone (GnRH) secretion is controlled by various factors, including the excitatory neurotransmitter glutamate. Estrogen (E) regulates GnRH secretion by means of E-responsive cells in the brain that relay the feedback effects to the preoptic area (POA). We used an antibody to vesicular glutamate transporter 2 (VGluT2) to label glutamatergic neurons in the areas of the ewe brain that control GnRH secretion. VGluT2-immunoreactive cells were observed in the arcuate nucleus (ARC)/ventromedial hypothalamic nucleus (VMH) complex, POA, bed nucleus of stria terminalis (BnST), and A1 and A2 cell groups in the brainstem. In three ewes, E receptor-alpha was detected in 52-61% of glutamatergic neurons in ARC/VMH, 37-52% of neurons in the POA, and 37-58% of neurons in the BnST. E injection (i.m. or i.v.) increased the percentage of glutamatergic cells that expressed Fos protein in the ARC (P < 0.01 and P < 0.001, respectively). In six ewes, injection of the retrograde tracer Fluoro-Gold into the POA labeled cells in the ARC and 6-29% of these were also VGluT2-immunoreactive. Double-labeling of varicosities in the POA showed colocalization of VGluT2 in 12.5 +/- 3% of dopamine beta-hydroxylase-immunoreactive terminals, indicating that a subset of glutamatergic inputs could arise from brainstem noradrenergic neurons cells. In the POA, 60% of GnRH neurons had close appositions that were VGluT2-immunoreactive. We conclude that E-responsive glutamatergic neurons arising from the brainstem, the BnST, and ARC/VMH provide input to the POA and may be involved in the regulation of GnRH secretion.     

Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat

The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.     

Vesicular glutamate transporter 2 protein and mRNA containing neurons in the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus is the key structure of the control of circadian rhythms and has a rich glutamatergic innervation. Besides the presence of glutamatergic afferents, several findings also suggest the existence of glutamatergic efferents from the suprachiasmatic nucleus to its target neurons in various prominent hypothalamic cell groups. However, there is no direct neuromorphological evidence for the presence of glutamatergic neurons in the suprachiasmatic nucleus. Therefore, the purpose of the present investigations was to try to clarify this question. Immunocytochemistry was used at the light and electron microscopy level to identify vesicular glutamate transporter type 2 (VGluT2) immunopositive (presumed glutamatergic) neurons in the rat suprachiasmatic nucleus. In addition VGluT2 mRNA expression in neurons of the nucleus was also addressed with radioisotopic in situ hybridization. Both at the light and electron microscopy level we detected VGluT2 positive neurons, which did not contain GABA, vasoactive intestinal polypeptide or vasopressin. Further, we demonstrated the expression of VGluT2 mRNA in a few cells within the suprachiasmatic nucleus; these glutamatergic cells were distinct from somatostatin mRNA expressing neurons. As VGluT2 is a selective marker of glutamatergic neuronal elements, the present observations provide direct neuromorphological evidence for the presence of glutamatergic neurons in the cell group.     

Glutamatergic innervation of growth hormone-releasing hormone-containing neurons in the hypothalamic arcuate nucleus and somatostatin-containing neurons in the anterior periventricular nucleus of the rat

Growth hormone-releasing hormone (GHRH) and somatostatin are the two main hypothalamic neurohormones, which stimulate or inhibit directly hypophysial growth hormone (GH) release. Majority of the GHRH neurons projecting to the median eminence is situated in the arcuate nucleus and the somatostatin neurons in the anterior periventricular nucleus. Data suggest that the excitatory amino acid glutamate may play an important role in the control of hypothalamic neuroendocrine neurons and processes including the control of GH. There is a dense plexus of glutamatergic fibres in the hypothalamic arcuate and anterior periventricular nucleus. The aim of the present studies was to examine the relationship of these fibres to the GHRH neurons in the arcuate nucleus and to somatostatin neurons in the anterior periventricular nucleus. Double-labelling immuno-electron microscopy was used. Glutamatergic structures were identified by the presence of vesicular glutamate transporter 2 (VGluT2) (a selective marker of glutamatergic elements) immunoreactivity. A significant number of VGluT2-immunoreactive boutons was observed to make asymmetric type of synapses with GHRH-immunostained nerve cells in the arcuate and with somatostatin neurons in the anterior periventricular nucleus. A subpopulation of somatostatin-immunoreactive neurons displayed also VGluT2 immunoreactivity. Our findings provide direct neuromorphological evidence for the view that the action of glutamate on GH release is exerted, at least partly, directly on GHRH and somatostatin neurons releasing these neurohormones into the hypophysial portal blood.     

Altered balance of γ‐aminobutyric acidergic and glutamatergic afferent inputs in rostral ventrolateral medulla‐projecting neurons in the paraventricular nucleus of the hypothalamus of renovascular hypertensive rats

An imbalance of excitatory and inhibitory functions has been shown to contribute to numerous pathological disorders. Accumulating evidence supports the idea that a change in hypothalamic γ‐aminobutyric acid (GABA)‐ergic inhibitory and glutamatergic excitatory synaptic functions contributes to exacerbated neurohumoral drive in prevalent cardiovascular disorders, including hypertension. However, the precise underlying mechanisms and neuronal substrates are still not fully elucidated. In the present study, we combined quantitative immunohistochemistry with neuronal tract tracing to determine whether plastic remodeling of afferent GABAergic and glutamatergic inputs into identified RVLM‐projecting neurons of the hypothalamic paraventricular nucleus (PVN‐RVLM) contributes to an imbalanced excitatory/inhibitory function in renovascular hypertensive rats (RVH). Our results indicate that both GABAergic and glutamatergic innervation densities increased in oxytocin‐positive, PVN‐RVLM (OT‐PVN‐RVLM) neurons in RVH rats. Despite this concomitant increase, time‐dependent and compartment‐specific differences in the reorganization of these inputs resulted in an altered balance of excitatory/inhibitory inputs in somatic and dendritic compartments. A net predominance of excitatory over inhibitory inputs was found in OT‐PVN‐RVLM proximal dendrites. Our results indicate that, along with previously described changes in neurotransmitter release probability and postsynaptic receptor function, remodeling of GABAergic and glutamatergic afferent inputs contributes as an underlying mechanism to the altered excitatory/inhibitory balance in the PVN of hypertensive rats. J. Comp. Neurol. 518:567–585, 2010. © 2009 Wiley‐Liss, Inc.     

Synaptic contacts of vesicular glutamate transporter 2 fibres on chemically identified neurons of the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore, the purpose of the present study was to examine the relationship between the afferent glutamatergic axon terminals and the vasoactive intestinal polypeptide (VIP)-, arginine-vasopressin (AVP)- and gamma-aminobutyric acid (GABA)-positive neurons of the SCN. Glutamatergic elements were revealed via immunocytochemical double-labelling for vesicular glutamate transporter type 1 (VGluT1) and type 2 (VGluT2), and brain sections were imaged via confocal laser-scanning microscopy and electron microscopy. Numerous VGluT2-immunoreactive axons were observed to be in synaptic contact with VIP- and GABA-positive neurons, and only a few synapses were detected between VGluT2 boutons and AVP neurons. VGluT1 axon terminals exhibiting very moderate distribution in this cell group were observed to be in synaptic contact with chemically unidentified neurons. The findings provide the first morphological data on the termination of presumed glutamatergic fibres on chemically identified neurons of the rat SCN, and indicate that all three prominent cell types of the cell group receive glutamatergic afferents.     

Glutamatergic afferent projections to the dorsal raphe nucleus of the rat

Based on WGA-apo-HRP-gold (WG) retrograde tracing, the present study revealed that different subdivisions of the dorsal raphe (DR) such as dorsomedial, ventromedial, lateral wing, and caudal regions receive unique, topographically organized afferent inputs, that are more restricted than previously reported. Phaseolus vulgaris leucoagglutinin anterograde tracing studies confirmed that the medial prefrontal cortex provides the major afferent input to each subdivision of the DR. Double-labeling studies combining WG tracing and glutamate immunostaining indicated that the medial prefrontal cortex, various hypothalamic nuclei including perifornical, lateral, and arcuate nuclei, and several medullary regions such as lateral and medial parabrachial nuclei, and laterodorsal tegmental nucleus provide the major glutamatergic input to each subregion of the DR. It should be noted that the degree of glutamatergic input from these afferent sites was specific for each DR subdivision. The present findings indicated that dorsomedial, ventromedial, lateral wing, and caudal subdivisions of the DR receive excitatory inputs from both cortical and subcortical sites which might be involved in regulation or modulation of a broad range of systems, including sensory and motor functions, arousal and sleep-wake cycle, biorhythmic, cognitive, and affective behaviors.     

Vesicular glutamate (VGlut), GABA (VGAT), and acetylcholine (VACht) transporters in basal forebrain axon terminals innervating the lateral hypothalamus

The basal forebrain (BF) is known to play important roles in cortical activation and sleep, which are likely mediated by chemically differentiated cell groups including cholinergic, gamma-aminobutyric acid (GABA)ergic and other unidentified neurons. One important target of these cells is the lateral hypothalamus (LH), which is critical for arousal and the maintenance of wakefulness. To determine whether chemically specific BF neurons provide an innervation to the LH, we employed anterograde transport of 10,000 MW biotinylated dextran amine (BDA) together with immunohistochemical staining of the vesicular transporter proteins (VTPs) for glutamate (VGluT1, -2, and -3), GABA (VGAT), or acetylcholine (ACh, VAChT). In addition, we applied triple staining for the postsynaptic proteins (PSPs), PSD-95 with VGluT or Gephyrin (Geph) with VGAT, to examine whether the BDA-labeled varicosities may form excitatory or inhibitory synapses in the LH. Axons originating from BDA-labeled neurons in the magnocellular preoptic nucleus (MCPO) and substantia innominata (SI) descended within the medial forebrain bundle and extended collateral varicose fibers to contact LH neurons. In the LH, the BDA-labeled varicosities were immunopositive (+) for VAChT ( approximately 10%), VGluT2 ( approximately 25%), or VGAT ( approximately 50%), revealing an important influence of newly identified glutamatergic together with GABAergic BF inputs. Moreover, in confocal microscopy, VGluT2+ and VGAT+ terminals were apposed to PSD-95+ and Geph+ profiles respectively, indicating that they formed synaptic contacts with LH neurons. The important inputs from glutamatergic and GABAergic BF cells could thus regulate LH neurons in an opposing manner to stimulate vs. suppress cortical activation and behavioral arousal reciprocally.     

Evidence for vesicular glutamate transporter synapses onto gonadotropin-releasing hormone and other neurons in the rat medial preoptic area

The medial preoptic area is a key structure in the control of reproduction. Several data suggest that excitatory amino acids are involved in the regulation of this function and the major site of this action is the medial preoptic region. Data concerning the neuromorphology of the glutamatergic innervation of the medial preoptic area are fragmentary. The present investigations were focused on: (i) the morphology of the vesicular glutamate transporter 1 (VGluT1)- and vesicular glutamate transporter 2 (VGluT2)-immunoreactive nerve terminals, which are considered to be specific to presumed glutamatergic neuronal elements, in the medial preoptic area of rat; and (ii) the relationship between these glutamate transporter-positive endings and the gonadotropin-releasing hormone (GnRH) neurons in the region. Single- and double-label immunocytochemistry was used at the light and electron microscopic level. There was a weak to moderate density of VGluT1- and a moderate to intense density of VGluT2-immunoreactive elements in the medial preoptic area. Electron microscopy revealed that both VGluT1- and VGluT2-immunoreactive boutons made asymmetric type synaptic contacts with unlabelled neurons. VGluT2-labelled, but not VGluT1-labelled, axon terminals established asymmetric synaptic contacts on GnRH-immunostained neurons, mainly on their dendrites. The present findings are the first electron microscopic examinations on the glutamatergic innervation of the rat medial preoptic area. They provide direct neuromorphological evidence for the existence of direct glutamatergic innervation of GnRH and other neurons in the rat medial preoptic area.     

Glutamatergic innervation of neuropeptide Y and pro-opiomelanocortin-containing neurons in the hypothalamic arcuate nucleus of the rat

Abstract The hypothalamic arcuate nucleus contains a number of neurochemically different cell populations, among others neuropeptide Y (NPY)- and pro-opiomelanocortin (POMC)-derived peptide-expressing neurons; both are involved in the regulation of feeding and energy homeostasis, NPY neurons also in the release of hypophysiotropic hormones, sexual behaviour and thermogenesis. Recent observations indicate that there is a dense plexus of glutamatergic fibres in the arcuate nucleus. The aim of the present studies was to examine the relationship of these fibres to the NPY and POMC neurons in the arcuate nucleus. Double-label immunoelectron microscopy was used. Glutamatergic elements were identified by the presence of vesicular glutamate transporter 1 (VGluT1) or 2 (VGluT2) (selective markers of glutamatergic elements) immunoreactivity. A significant number of VGluT2-immunoreactive terminals was observed to make asymmetric type of synapses with NPY and with beta-endorphin (a marker of POMC neurons)-immunostained nerve cells of the arcuate nucleus. About 15% of VGluT2 synapsing terminals established asymmetric synapses with NPY-positive cells and more than 40% of VGlut2-positive terminals formed synapse on beta-endorphin-positive neurons. VGluT2-positive perikarya were also observed, part of them also contained beta-endorphin. Nerve terminals containing both VGluT2 and beta-endorphin were demonstrated in the cell group. Only very few VGluT1 fibres were detected. Our observations provide the first direct neuromorphological evidence for the existence of glutamatergic innervation of NPY and POMC neurons of the arcuate nucleus.     

Glutamatergic innervation of corticotropin-releasing hormone- and thyrotropin-releasing hormone-synthesizing neurons in the hypothalamic paraventricular nucleus of the rat

Glutamate plays a role in the central regulation of the hypothalamic-pituitary-adrenal (HPA) and thyroid (HPT) axes. Until the recent discovery of vesicular glutamate transporters (VGLUT1-3), there was no specific tool for the examination of the putative morphological relationship between the glutamatergic and the hypophysiotropic systems. Using antisera against VGLUT2, corticotropin-releasing hormone (CRH), and prothyrotropin-releasing hormone (proTRH) (178-199), we performed double-labeling immunocytochemistry at light and electron microscopic levels in order to study the glutamatergic innervation of the CRH- and TRH-synthesizing neurons in the hypothalamic paraventricular nucleus (PVN). Fine VGLUT2-immunoreactive (IR) axons very densely innervated the parvocellular subdivisions of the PVN. VGLUT2-IR axons established juxtapositions with all parvocellular CRH- and TRH-synthesizing neurons. The innervation was similarly intense in all parvocellular subdivisions of the PVN. At ultrastructural level, VGLUT2-IR terminals frequently established synapses with perikarya and dendrites of the CRH- and proTRH-IR neurons. These findings demonstrate that glutamatergic neurons directly innervate hypophysiotropic CRH and TRH neurons in the PVN and, therefore, support the hypothesis that the glutamate-induced activation of the HPA and HPT axes may be accomplished by a direct action of glutamate on hypophysiotropic CRH and TRH systems.     

Glutamatergic pathways from the Kölliker-Fuse nucleus to the phrenic nucleus in the rat

After ipsilateral injections of biotinylated dextran amine (BDA) into the K?lliker-Fuse (KF) nucleus and cholera toxin B subunit (CTb) into the ventral horn in C4 to C5 segments of the spinal cord, an overlapping distribution of BDA-labeled axon terminals and CTb-labeled neurons was found in the rostral ventral respiratory group (rVRG) region ipsilateral to the injection sites. After ipsilateral injections of BDA into the KF and Fluoro-Gold (FG) into the ventral horn in C4 to C5 segments of the spinal cord, BDA-labeled axons were found to make asymmetrical synapses with the somata and dendrites of FG-labeled neurons within the neuropil of the rVRG region. Using retrograde tracing combined with immunohistochemistry for phosphate-activated glutaminase (PAG), we observed that as many as 72% of the rVRG neurons projecting to the PhN were immunoreactive for PAG and that approximately 62% and 75% of the KF neurons projecting respectively to the rVRG region and PhN contain PAG immunoreactivity. Using anterograde tracing combined with immunohistochemistry for vesicular glutamate transporter 2 (VGluT2), we further demonstrated that the KF axon terminals in the rVRG and PhN regions as well as the rVRG axon terminals in the PhN region contain VGluT2 immunoreactivity. The present results suggest that the glutamatergic pathways from the KF to the PhN directly and indirectly via the rVRG region may exist and underlie the inspiratory responses that are elicited by activation of the KF neurons.     

Fos expression in forebrain afferents to the hypothalamic paraventricular nucleus following swim stress

The paraventricular nucleus of the hypothalamus (PVN) serves as the origin of the final common pathway in the secretion of glucocorticoid hormones in response to stress. Various stress-related inputs converge upon the cells of the medial parvocellular division of the PVN. These neurons, which synthesize and release corticotropin-releasing hormone, arginine vasopressin, and other secretagogues, are responsible for a cascade of events which culminates in the adrenocorticotropin-induced release of corticosteroids from the adrenal cortex. Previous data have suggested complex afferent regulation of PVN neurons, although the neuronal pathways by which the effects of stress are mediated remain to be fully disclosed. The present experiment sought to identify forebrain areas potentially involved in afferent regulation of the PVN in response to an acute stressor. Discrete injections of the retrograde tracer Fluoro-gold were delivered to the PVN, and rats were subsequently subjected to an acute swim stress. Brains were processed immunocytochemically for the simultaneous detection of the tracer and Fos, the protein product of the immediate early gene c-fos, utilized as a marker for neuronal activation. The majority of Fluoro-gold/Fos labeled neurons were detected in the parastrial nucleus, the medial preoptic area, the anterior hypothalamic area, the dorsomedial hypothalamic nucleus and adjacent posterior hypothalamic area, and, to a lesser extent, the supramammillary nucleus. These findings are discussed in relation to neural pathways mediating activation and inhibition of the hypothalamic-pituitary-adrenocortical axis. © 1996 Wiley-Liss, Inc.     

Chronic stress‐induced neurotransmitter plasticity in the PVN

Chronic stress precipitates pronounced enhancement of central stress excitability, marked by sensitization of hypothalamic‐pituitary‐adrenocortical (HPA) axis responses and increased adrenocorticotropic hormone (ACTH) secretagogue biosynthesis in the paraventricular nucleus of the hypothalamus (PVN). Chronic stress‐induced enhancement of HPA axis excitability predicts increased excitatory and/or decreased inhibitory innervation of the parvocellular PVN. We tested this hypothesis by evaluating chronic variable stress (CVS)‐induced changes in total (synaptophysin), glutamatergic (VGluT2), GABAergic (GAD65), and noradrenergic (DBH) terminal immunoreactivity on PVN parvocellular neurons using immunofluorescence confocal microscopy. CVS increased the total PVN bouton immunoreactivity as well as the number of glutamatergic and noradrenergic immunoreactive boutons in apposition to both the corticotropin‐releasing hormone (CRH)‐immunoreactive cell bodies and dendrites within the parvocellular PVN. However, the number of GABAergic‐immunoreactive boutons in the PVN was unchanged. CVS did not alter CRH median eminence immunoreactivity, indicating that CVS does not enhance CRH storage within the median eminence. Taken together, the data are consistent with a role for both glutamate and norepinephrine in chronic stress enhancement of HPA axis excitability. These changes could lead to an enhanced capacity for excitation in these neurons, contributing to chronic stress‐induced hyperreactivity of stress effector systems in the brain. J. Comp. Neurol. 517:156–165, 2009. © 2009 Wiley‐Liss, Inc.     

Postnatal changes of vesicular glutamate transporter (VGluT)1 and VGluT2 immunoreactivities and their colocalization in the mouse forebrain

Vesicular glutamate transporter 1 (VGluT1) and VGluT2 accumulate neurotransmitter glutamate into synaptic vesicles at presynaptic terminals, and their antibodies are thus considered to be a good marker for glutamatergic axon terminals. In the present study, we investigated the postnatal development and maturation of glutamatergic neuronal systems by single- and double-immunolabelings for VGluT1 and VGluT2 in mouse forebrain including the telencephalon and diencephalon. VGluT2 immunoreactivity was widely distributed in the forebrain, particularly in the diencephalon, from postnatal day 0 (P0) to adulthood, suggesting relatively early maturation of VGluT2-loaded glutamatergic axons. In contrast, VGluT1 immunoreactivity was intense only in the limbic regions at P0, and drastically increased in the other telencephalic and diencephalic regions during three postnatal weeks. Interestingly, VGluT1 immunoreactivity was frequently colocalized with VGluT2 immunoreactivity at single axon terminal-like profiles in layer IV of the primary somatosensory area from P5 to P10 and in the ventral posteromedial thalamic nucleus from P0 to P14. This was in sharp contrast to the finding that almost no colocalization was found in glomeruli of the olfactory bulb, patchy regions of the caudate-putamen, and the ventral posterolateral thalamic nucleus, where moderate to intense immunoreactivities for VGluT1 and VGluT2 were intermingled with each other in neuropil during postnatal development. The present results indicate that VGluT2-loaded glutamatergic axons maturate earlier than VGluT1-laden axons in the mouse telencephalic and diencephalic regions, and suggest that VGluT1 plays a transient developmental role in some glutamatergic systems that mainly use VGluT2 in the adulthood.     

Possible origin of glutamatergic projections to the midbrain periaqueductal gray and deep layer of the superior colliculus of the rat

The possible origin of glutamatergic input to the rodent periaqueductal gray (PAG) was analyzed utilizing a combined retrograde transport-immunocytochemical technique. Injections of wheat germ agglutinin-horseradish peroxidase were made into the PAG of 12 adult rats and into the deep layer of the superior colliculus in 2 rats. The brain tissue was first reacted histochemically to demonstrate the retrograde tracer and subsequently processed with immunohistochemical techniques using a recently developed monoclonal glutamate antibody. Following PAG injections, several brain areas were found to contain double-labeled neurons. The greatest number of double-labeled glutamate-like immunoreactive neurons were observed in the zona incerta, spinal trigeminal nucleus, cuneiform nucleus, cingulate cortex, cerebellar interpositus nucleus, deep mesencephalic nucleus and the PAG itself. Double-labeled neurons were also observed in several other nuclei including the pretectal nuclei, the frontal and occipital cortex, several reticular nuclei, the dorsomedial hypothalamic nucleus, and the substantia nigra. Many of the same nuclei contained double-labeled neurons following collicular injections, but in addition, double-stained cells were found in the primary visual cortex, lateral dorsal and lateral posterior thalamic nuclei, nucleus of the posterior commissure, ventral lateral geniculate nucleus, dorsal column nuclei and several additional pretectal nuclei. The results of this double-labeling study raise the possibility that these nuclei may provide glutamatergic inputs to the midbrain PAG and/or superior colliculus. These putative glutamatergic afferent projections may ultimately influence the PAG's role in several important functions including antinociception, defensive mechanisms or vocalization and may also play a role in the superior collicular involvement in defensive mechanisms, in visuo-motor integration in the orienting reflex and in facilitating shifts in gaze.     

Forebrain afferents to the cat posterior hypothalamus: a double labeling study

Using a double immunostaining technique with cholera toxin (CT) as a retrograde tracer, we examined the cells of origin and the histochemical nature of afferents to the cat posterior hypothalamus. After injection in the tuberomamillary nucleus, a number of CT-labeled cells were observed in: medial preoptic area, nuclei of the septum and the stria terminalis, amygdaloid complex, anterior hypothalamic, ventromedial hypothalamic and premamillary nuclei. CT injections in the lateral hypothalamic area gave an additional heavy labeling of neurons in: lateral preoptic area, nuclei of the diagonal band of Broca, substantia innominata, and nucleus accumbens. The posterior hypothalamus receives: 1) cholinergic inputs from the septum, the lateral preoptic area and the nuclei of the diagonal band of Broca; 2) dopaminergic afferents from A11, A13, and A14 groups; 3) histaminergic afferents from the posterior hypothalamus; and 4) peptidergic afferents such as methionin-enkephalin, galanin and neurotensin, substance P and corticotropin-releasing factor from the medial preoptic area, the nucleus of the stria terminalis and/or the posterior hypothalamic structures.     

Localization of putative glutamatergic/aspartatergic neurons projecting to the supraoptic nucleus area of the rat hypothalamus

Oxytocin and vasopressin neurosecretory neurons of the supraoptic nucleus receive a rich glutamatergic innervation. The nerve cells of this prominent structure express various ionotropic and metabotropic glutamate receptor subtypes and there is converging evidence that glutamate acts as an excitatory transmitter in the control of release of oxytocin and vasopressin synthesized in this cell group. The location of the glutamatergic neurons projecting to this hypothalamic region is unknown. The aim of the present investigation was to study this question. [(3)H]D-aspartate, which is selectively taken up by high-affinity uptake sites at presynaptic endings that use glutamate as a transmitter, and is transported back to the cell body, was injected into the supraoptic nucleus area. The neurons retrogradely labelled with [(3)H]D-aspartate were detected autoradiographically. Labelled nerve cells were found in several diencephalic and telencephalic structures, but not in the brainstem. Diencephalic cell groups included the supraoptic nucleus itself, its perinuclear area, hypothalamic paraventricular, suprachiasmatic, ventromedial, dorsomedial, ventral premammillary, supramammillary and thalamic paraventricular nuclei. Within the telencephalon, labelled neurons were detected in the septum, amygdala, bed nucleus of the stria terminalis and preoptic area. The findings provide neuromorphological data on the location of putative glutamatergic neurons projecting to the supraoptic nucleus and its perinuclear area. Furthermore, they indicate that local putative glutamatergic neurons as well as several diencephalic and telencephalic structures contribute to the glutamatergic innervation of the cell group and thus are involved in the control of oxytocin and vasopressin release by neurosecretory neurons of the nucleus.     

Hypothalamic and brainstem sources of pituitary adenylate cyclase-activating polypeptide nerve fibers innervating the hypothalamic paraventricular nucleus in the rat

The hypothalamic paraventricular nucleus (PVN) coordinates major neuroendocrine and behavioral mechanisms, particularly responses to homeostatic challenges. Parvocellular and magnocellular PVN neurons are richly innervated by pituitary adenylate cyclase-activating polypeptide (PACAP) axons. Our recent functional observations have also suggested that PACAP may be an excitatory neuropeptide at the level of the PVN. Nevertheless, the exact localization of PACAP-producing neurons that project to the PVN is not understood. The present study examined the specific contribution of various brain areas sending PACAP innervation to the rat PVN by using iontophoretic microinjections of the retrograde neuroanatomical tracer cholera toxin B subunit (CTb). Retrograde transport was evaluated from hypothalamic and brainstem sections by using multiple labeling immunofluorescence for CTb and PACAP. PACAP-containing cell groups were found to be retrogradely labeled from the PVN in the median preoptic nucleus; preoptic and lateral hypothalamic areas; arcuate, dorsomedial, ventromedial, and supramammillary nuclei; ventrolateral midbrain periaqueductal gray; rostral and midlevel ventrolateral medulla, including the C1 catecholamine cell group; nucleus of the solitary tract; and dorsal motor nucleus of vagus. Minor PACAP projections with scattered double-labeled neurons originated from the parabrachial nucleus, pericoeruleus area, and caudal regions of the nucleus of the solitary tract and ventrolateral medulla. These observations indicate a multisite origin of PACAP innervation to the PVN and provide a strong chemical neuroanatomical foundation for interaction between PACAP and its potential target neurons in the PVN, such as parvocellular CRH neurons, controlling physiologic responses to stressful challenges and other neuroendocrine or preautonomic PVN neurons.     

Brainstem origins of glutamatergic innervation of the rat hypothalamic paraventricular nucleus

Multiple lines of evidence document a role for glutamatergic input to the hypothalamic paraventricular nucleus (PVH) in stress-induced activation of the hypothalamic-pituitary-adrenocortical (HPA) axis. However, the neuroanatomical origins of the glutamatergic input have yet to be definitively determined. We have previously shown that vesicular glutamate transporter 2 (VGLUT2) is the predominant VGLUT isoform expressed in the basal forebrain and brainstem, including PVH-projecting regions, and that the PVH is preferentially innervated by VGLUT2-immunoreactive terminals/boutons. The present study employed a dual-labeling approach, combining immunolabeling for a retrograde tract tracer, Fluoro-Gold (FG), with in situ hybridization for VGLUT2 mRNA, to map the brainstem and caudal forebrain distribution of glutamatergic PVH-projecting neurons. The present report presents evidence for substantial dual labeling in the periaqueductal gray, caudal portions of the zona incerta and subparafascicular nucleus, and the lateral parabrachial nucleus. The current data also suggest that relatively few PVH-projecting neurons in ascending raphe nuclei, nucleus of the solitary tract, or ventrolateral medulla are VGLUT2 positive. The data reveal multiple brainstem origins of glutamatergic input to PVH that are positioned to play a role in transducing a diverse range of stressful stimuli.