József Kiss

Immunoreactive corticotropin-releasing hormone in the hypothalamoinfundibular tract.

Ovine corticotropin-releasing hormone (CRF)-like immunoreactivity has been examined in the rat hypothalamus by light microscopy. Immunoreactivity was found in nerve fibers of the median eminence, mainly in the external zone around the portal vessels. In rats pretreated with colchicine or with hypothalamic knife cuts, small to moderate sized cells with two (bipolar) or rarely more (multipolar) dendrites, showing CRF-like immunoreactivity were present in the anterior and medial parvocellular subdivisions of the paraventricular nucleus. Scattered CRF-like immunopositive cells were found in the periventricular and medial preoptic nuclei. CRF-like immunoreactivity was clearly enhanced in the median eminence and paraventricular nucleus 8-10 days after bilateral adrenalectomy. A variety of hypothalamic transections had to be performed to determine reliably the topography of CRF-like nerve fibers projecting to the stalk-median eminence. Axons left the paraventricular nucleus in a lateral direction, turned ventrally in the lateral hypothalamus then medially as they approached the base of the hypothalamus above and behind the optic chiasm (lateral retrochiasmatic area). Fibers reached the median eminence by traveling caudally and medially from the rostral half of the lateral retrochiasmatic area. Scattered fibers were present in the retroinfundibular (posterior) portion of the median eminence. No immunoreactive fibers remained in the stalk-median eminence 1 or 4 weeks after transection of that loop-like pathway of CRF-containing fibers in the lateral retrochiasmatic area.

Glucocorticoid Implants around the Hypothalamic Paraventricular Nucleus Prevent the Increase of Corticotropin-Releasing Factor and Arginine Vasopressin Immunostaining Induced by Adrenalectomy

The site of inhibitory action of glucocorticoids on the hypothalamic corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) was studied using a combination of glucocorticoid implantation and immunohistochemistry. Adrenalectomy increased the number and the staining intensity of the neurons containing CRF-like immunoreactivity in the anterior and medial parvicellular subdivisions of the paraventricular nucleus (PVN) and induced the appearance of AVP-like immunoreactivity in the same cell population. These effects of adrenalectomy were inhibited only by those dexamethasone implants which were placed close to the PVN. Unilateral implantation of dexamethasone into the PVN inhibited the adrenalectomy-induced changes in CRF and AVP immunostaining only on the implanted side. Dexamethasone implants placed into the hippocampus decreased the effect of adrenalectomy in the PVN while similar implants into the amygdala and cerebral cortex were ineffective. These results suggest that the primary site of glucocorticoid feedback inhibition on the hypothalamic secretagogues of adrenocorticotropin is the PVN.

Localization of glutamatergic/aspartatergic neurons projecting to the hypothalamic paraventricular nucleus studied by retrograde transport of [3H]D-aspartate autoradiography.

Morphological and functional data indicate that glutamatergic innervation of the hypothalamic paraventricular nucleus plays an important role in the control of this prominent cell group. Sources of this neural input are unknown. The present investigations were aimed at studying this question. The retrograde tracer [3H]D-aspartate, which is selectively taken up by the terminals of neurons that use glutamate or aspartate as a neurotransmitter, and is retrogradely transported to their perikarya, was injected into the paraventricular nucleus. The brain was examined for labelled neurons visualized by autoradiography. Labelled neurons were detected in the paraventricular nucleus itself, in several hypothalamic areas including medial and lateral preoptic area, suprachiasmatic nucleus, anterior hypothalamic area, ventromedial nucleus, dorsomedial nucleus, lateral hypothalamic area, posterior part of arcuate nucleus, ventral premammillary nucleus and supramammillary nucleus. Outside the hypothalamus labelled neurons were found in the thalamic paraventricular nucleus and in certain telencephalic regions including lateral septum, bed nucleus of the stria terminalis and amygdala. All of them are known to project to the hypothalamic paraventricular nucleus. We failed to detect labelled neurons in the lower brainstem. From these findings we conclude that firstly, there are glutamatergic/aspartatergic interneurons in the paraventricular nucleus; secondly, all intrahypothalamic and telencephalic, but not lower brainstem afferents to this nucleus contain glutamatergic/aspartatergic fibres; and thirdly, the glutamatergic/aspartatergic innervation of this heterogeneous cell group is extremely complex.

Cellular architecture of the nucleus reuniens thalami and its putative aspartatergic/glutamatergic projection to the hippocampus and medial septum in the rat

Little is known about the neurochemical features of the nucleus reuniens thalami (RE). In the present study, immunocytochemical experiments were performed to characterize the expression pattern of certain neurochemical markers, e.g. the calcium‐binding proteins calbindin and calretinin and several neuropeptides. Colocalization studies revealed that half of the calbindin‐positive cells express calretinin, and numerous calretinin‐immunoreactive neurons contain calbindin. In contrast, immunolabelling for neuropeptides did not reveal cell bodies in the RE. The RE establishes widespread connections with several limbic structures. To correlate these projection patterns with the neurochemical characteristics of RE neurons, the retrograde tracer [3H]d‐aspartate, which is selectively taken up by high affinity uptake sites that use glutamate as neurotransmitter, and the nonselective retrograde tracer wheatgerm agglutinin‐conjugated colloidal gold was injected into the stratum lacunosum moleculare of the hippocampal CA1 subfield and into the medial septum. The results provide direct anatomical demonstration of aspartatergic/glutamatergic projection from the RE to the hippocampus and to the medial septum. Nearly all of the projecting neurons proved to be calbindin‐immunopositive and many of them expressed calretinin. Both retrograde labelling techniques revealed that neurons projecting to the hippocampus were located in clusters in the dorsolateral part of the RE, whereas neurons projecting to the medial septum were mainly distributed in the ventromedial portion of the nucleus, indicating that different cell populations project to these limbic areas. These results suggest that neurons in the RE are heterogeneous and contribute to the excitatory innervation of the septo‐hippocampal system.

Topography of the Somatostatin-Immunoreactive Fibers to the Stalk-Median Eminence of the Rat

The course of the hypothalamo-infundibular somatostatin containing pathway was visualized by immunocytochemistry (the unlabeled antibody peroxidase-antiperoxidase complex method) combined with various knife cuts in the hypothalamus. Somatostatin immunoreactive fibers left the perikarya in the anterior hypothalamic-preoptic periventricular cell mass in a lateral direction. After a loop-like path through the lateral hypothalamus, fibers enter the medial basal hypothalamus mainly in the lateral retrochiasmatic area near the ventral surface and project to the ipsilateral half of the median eminence and the pituitary stalk. Somatostatin immunoreactivity almost completely disappeared from the stalk-median eminence 3-7 days following a complete anterolateral transection while transections dorsal or posterior to the lateral retrochiasmatic area failed to influence it. 7 weeks after anterolateral deafferentation an increased density of somatostatin containing terminals and immunopositive perikarya were seen in the arcuate and ventromedial nuclei but these neurones failed to reinnervate the stalk-median eminence. Thus there appears to be a neuron system containing somatostatin-like immunoreactivity within the medial basal hypothalamus, cell bodies being most abundant in the posterior divisions of the ventromedial and arcuate nuclei. However, most if not all of the somatostatin immunoreactivity in the stalk median eminence originates from outside of the medial-basal hypothalamus. Further, we have demonstrated the importance of the careful histological evaluation of each individual operation if functional studies are to be carried out after hypothalamic deafferentation.

Immunoreactivity to vasoactive intestinal polypeptide (VIP) and thyreotropin-releasing hormone (TRH) in hypothalamic neurons of the domesticated pigeon (Columba livia). Alterations following lactation and exposure to cold.

SummaryThe distribution of VIP- and TRH-immunoreactivity in neurons and processes within the hypothalamus of the pigeon was investigated with light-microscopic immunocytochemical techniques. Most of the VIP-containing neurons are concentrated in the middle and caudal parts of the hypothalamus, with the greatest concentration of perikarya occurring in the medial and lateral part of the ventromedial hypothalamic nucleus and the infundibular nucleus. These cells give rise to axons that seem to extend into the median eminence. An extensive network of VIP-immunoreactive fibers and varicosities occupy the external layer of the median eminence. The majority of TRH-containing neurons is found in the anterior hypothalamus with the greatest concentration of cells in the magnocellular preoptic, medial preoptic, suprachiasmatic and paraventricular nuclei. TRH-immunoreactive fibers and varicosities form a dense arborization in the external layer of the median eminence. Lactation seems to induce substantial changes in VIP as well as in TRH-immunostaining in the median eminence and other hypothalamic regions as compared to control, sexually active animals. Furthermore, TRH-immunoreactivity decreased in the median eminence following 60-min exposure to cold. These results suggest that VIP- and TRH-containing pathways in the pigeon hypothalamus are involved in the mediation of neuroendocrine responses.

Entorhinal cortical innervation of parvalbumin‐containing neurons (basket and chandelier cells) in the rat ammon's horn

Physiological data suggest that in the CA1–CA3 hippocampal areas of rats, entorhinal cortical efferents directly influence the activity of interneurons, in addition to pyramidal cells. To verify this hypothesis, the following experiments were performed: 1) light microscopic double‐immunostaining for parvalbumin and the anterograde tracer Phaseolus vulgaris‐leucoagglutinin injected into the entorhinal cortex; 2) light and electron microscopic analysis of cleaved spectrin‐immunostained (i.e., degenerating axons and boutons) hippocampal sections following entorhinal cortex lesion; and 3) an electron microscopic study of parvalbumin‐immunostained hippocampal sections after entorhinal cortex lesion. The results demonstrate that in the stratum lacunosum‐moleculare of the CA1 and CA3 regions, entorhinal cortical axons form asymmetric synaptic contacts on parvalbumin‐containing dendritic shafts. In the stratum lacunosum‐moleculare, parvalbumin‐immunoreactive dendrites represent processes of GABAergic, inhibitory basket and chandelier cells; these interneurons innervate the perisomatic area and axon initial segments of pyramidal cells, respectively. A feed‐forward activation of these neurons by the entorhinal input may explain the strong, short‐latency inhibition of pyramidal cells.

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

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 β‐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 β‐endorphin‐positive neurons. VGluT2‐positive perikarya were also observed, part of them also contained β‐endorphin. Nerve terminals containing both VGluT2 and β‐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.

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.

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 γ‐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.