Two high affinity binding sites for pituitary adenylate cyclase-activating polypeptide have different tissue distributions.

Two bioactive products of pituitary adenylate cyclase-activating polypeptide (PACAP) prohormone have been isolated from ovine hypothalamus: PACAP38 with 38 residues and PACAP27 corresponding to the N-terminal 27 residues of PACAP38. Immunocytochemical and RIA results support the existence of PACAP in the rat brain, posterior pituitary, and various peripheral tissues. Furthermore, high affinity PACAP-binding sites have been detected in the rat brain, anterior pituitary, and cultured astrocytes which differ from those in lung, liver, and cultured mouse splenocytes. In the present study additional rat tissues were examined to elucidate the location and characteristics of PACAP-binding sites using [125I] PACAP27 with conventional methods of receptor autoradiography and RRA. Binding specificity was established by displacement with unlabeled PACAP27 or a related peptide, vasoactive intestinal polypeptide (VIP). PACAP27-binding sites were localized autoradiographically in the testis, epididymis, adrenal gland, lung, liver, prostate gland, and seminal vesicle; binding sites were not detected in the heart, kidney, or thymus. In the testis and epididymis, a PACAP27-binding site was localized on germinal cells and in the adrenal gland on medullary chromaffin cells. Excess VIP did not displace PACAP27 binding localized in these three tissues. A site with a greater affinity for PACAP27 than for VIP was detected in adrenal gland and epididymis, characteristic of a site recognized previously in hypothalamus, anterior pituitary, and cultured astrocytes. The PACAP-specific site was more abundant in these tissues than a second site to which PACAP27 and VIP bound with similar affinities. Accordingly, the first site has been named type I. In lung, liver, prostate, and seminal vesicle, VIP displaced PACAP27 binding localized autoradiographically. Lung and liver contained an abundant site to which PACAP27 and VIP bound with similar affinities. This binding site, measured previously in lung, liver, and cultured splenocytes, may be shared by PACAP and VIP and has been named type II. Taken together, these data support the existence of two high affinity binding sites for PACAP with different tissue distribution.

Does alpha-melanocyte-stimulating hormone from the pars intermedia regulate suckling-induced prolactin release? Supportive evidence from morphological and functional studies.

Recent evidence suggests that substances derived from the hypophyseal intermediate lobe (IL) play a crucial role in the regulation of suckling-induced PRL secretion. The purpose of the present study was to explore this possibility further by determining whether the suckling stimulus acutely increases the secretory activity of the IL and whether alpha MSH, a major secretory product of the IL, plays a specific role in suckling-induced PRL release. Light microscopic morphometric analysis of serial pituitary sections obtained from lactating rats revealed that as little as 1 min of suckling caused a significant increase in the proportion of the IL that was in secretory configuration (11.8 +/- 0.7% vs. 6.7 +/- 0.5%; 1-min suckled vs. nonsuckled control; mean +/- SE). Moreover, the fraction of the IL in secretory configuration continued to increase after 5 and 10 min of nursing (to 16.0 +/- 0.8% at 5 min and 18.2 +/- 0.7% at 10 min). In contrast, serum PRL was not significantly elevated above the control level after 1 min of suckling (18.1 +/- 13.5 vs. 9.9 +/- 6.5 ng/ml, 1-min suckled vs. control). In fact, a significant rise in PRL levels (to 314.4 +/- 19.4 ng/ml) could be detected only after 10 min of nursing. Thus, secretion by the IL in response to suckling preceded the release of adenohypophyseal PRL, suggesting that a secretory product(s) from the pars intermedia is involved in the modulation of nursing-induced PRL release. Having established a sequential temporal relationship between these two phenomena, we next investigated whether alpha MSH was the IL factor involved in the regulation of suckling-induced PRL secretion. To this end, lactating rats were injected either with antiserum to alpha MSH or preimmune serum and then allowed to nurse their pups. Serial blood samples were taken from the mothers 15, 30, 60, and 90 min after the litters were returned, and serum PRL was measured by RIA. We found that the suckling-induced rise in serum PRL was severely attenuated in animals that received anti-alpha MSH serum. This suppression was most evident at 15 min (70.1 +/- 13.4 vs. 323.5 +/- 127.0 ng/ml, antibody treated vs. preimmune serum control) and persisted throughout the entire 90-min test period. When taken together, our results suggest that suckling-induced PRL secretion is mediated at least in part by alpha MSH released from the hypophyseal IL.

Light and electron microscopic demonstration of mGluR5 metabotropic glutamate receptor immunoreactive neuronal elements in the rat cerebellar cortex.

The cellular and subcellular localization of the mGluR5 metabotropic glutamate receptor subtype was studied in the rat cerebellar cortex, by using the preembedding immunoperoxidase and immunogold techniques. Light microscopic observations revealed an abundant, intense labeling of neurons in the granular layer as well as in the molecular layer. Lugaro and Golgi cells exhibited an intense mGluR5 immunoreactivity, while only a fraction of the neurons in the molecular layer were found to be mGluR5 immunopositive. In addition to a dense plexus of immunoreactive dendrites in the molecular layer of the cerebellar cortex, the mGluR5 immunopositive Golgi cell dendrites resembling axons at the light microscopic level were also labeled in the granular layer. At the ultrastructural level, mGluR5 immunoreactivity was present in neuronal elements postsynaptic to axon terminals of different morphology. By using a pre-embedding immunogold method, it was found that mGluR5 immunoreactivity is accumulated at the plasma membranes extrasynaptically as well as at the periphery of the postsynaptic specializations, mainly of the parallel fiber synaptic contacts. These findings provide morphological evidence that mGluR5 is expressed by a population of neurons in the cerebellar cortex and can synaptically be activated via the parallel fiber system.

Immunohistochemical study on the distribution of neuropeptides within the pontine tegmentum--particularly the parabrachial nuclei and the locus coeruleus of the human brain.

The topographical distribution of neuropeptide-containing cell bodies, fibers and terminals was studied in human parabrachial nuclei and the pontine tegmentum with immunohistochemical stainings. Brains of seven adult human subjects of 35-72 years were fixed within 2 h post mortem. Serial sections were immunostained by antisera of 14 different neuropeptides--oxytocin, vasopressin, thyrotropin-releasing hormone, angiotensin II, calcitonin gene-related peptide, beta-endorphin, dynorphin A, dynorphin B, leucine-enkephalin, alpha-melanocyte stimulating hormone, substance P, neuropeptide Y, cholecystokinin and galanin--alternately. All of these peptides were found to be present in nerve fibers and terminals, but only two, angiotensin II and dynorphin B, in cell bodies of the parabrachial nuclei. Calcitonin gene-related peptide-, neuropeptide Y-, cholecystokinin- and galanin-immunoreactive cells were present in other areas of the pontine tegmentum, like the motor trigeminal nucleus, locus coeruleus, periventricular gray matter but not in the parabrachial nuclei. Peptidergic fibers were distributed unevenly throughout the pontine tegmentum having unique, individual distribution patterns. In the parabrachial nuclei, substance P, neuropeptide Y, cholecystokinin and galanin showed the highest density of immunoreactive neuronal networks. Moderate to low concentrations of immunoreactive processes were detected by calcitonin gene-related peptide, alpha-melanocyte stimulating hormone, dynorphin B, thyrotropin releasing hormone, leucine-enkephalin, dynorphin A, angiotensin II, beta-endorphin, vasopressin and oxytocin antisera, respectively. Other pontine tegmental areas, like the locus coeruleus, dorsal tegmental, pontine raphe and motor trigeminal nuclei as well as the central gray of the tegmental region exhibited a varying assortment of neuropeptides with distinct, individual localization patterns. Their detailed topographical distributions are mapped and given in coronal sections.

Different populations of vasoactive intestinal polypeptide-immunoreactive interneurons are specialized to control pyramidal cells or interneurons in the hippocampus.

The postsynaptic targets of three vasoactive intestinal polypeptide-containing GABAergic interneuron types were examined in the rat hippocampus. Two of them showed remarkable target selectivity for other GABAergic neurons, while the third contacted the somata and proximal dendrites of pyramidal cells. Vasoactive intestinal polypeptide-positive interneurons innervating the stratum oriens/alveus border in the CA1 region were shown to establish multiple contacts with horizontal GABAergic interneurons immunoreactive for type 1 metabotropic glutamate receptor. Similarly, identified axons of vasoactive intestinal polypeptide-positive interneurons projecting to stratum radiatum were found to establish symmetrical synapses largely on GABAergic dendrites. The majority of these postsynaptic GABAergic neurons were shown to contain calbindin or vasoactive intestinal polypeptide. In contrast to the first two vasoactive intestinal polypeptide-containing cell populations, vasoactive intestinal polypeptide-positive interneurons arborizing in stratum pyramidale formed baskets around pyramidal cells. These results revealed a new element in cortical microcircuits, interneurons which are specialized to innervate other GABAergic interneurons. The role of this new component may be the synchronization of dendritic inhibition, or an input-specific disinhibition of pyramidal cells in various dendritic domains. In contrast, vasoactive intestinal polypeptide-containing basket cells are likely to be involved in perisomatic inhibition of pyramidal neurons, and represents a new basket cell type different from that containing parvalbumin.

Innervation of different peptide-containing neurons in the hippocampus by GABAergic septal afferents.

The termination pattern of septohippocampal axons visualized by anterograde transport of Phaseolus vulgaris leucoagglutinin was studied in the hippocampal formation in the rat, with special reference to the innervation of neurons immunoreactive for the neuroactive peptides cholecystokinin, somatostatin or vasoactive intestinal polypeptide. The type I, GABAergic, septohippocampal afferents were shown to terminate on neurons immunoreactive for each of the three peptides. The cholecystokinin-like immunoreactive neurons in all regions, and the somatostatin-immunoreactive cells in stratum oriens of CA1 region were the most preferred targets. Cholecystokinin-immunoreactive cells, especially those in the granule cell layer of the dentate gyrus, were often seen to be contacted by type II (presumed cholinergic) axons as well. The somatostatin-immunoreactive cells in the hilus were also innervated by type I septohippocampal axons, although less frequently than those in stratum oriens of the CA1 subfield. Each type of peptidergic neuron received multiple symmetrical synaptic input from the Phaseolus vulgaris leucoagglutinin-labelled septal afferents, as confirmed by correlated electron microscopy. The majority of these neuropeptide-containing cells are known to be GABAergic, and to have distinct input and output relationships. Thus, the present results demonstrate that the GABAergic septohippocampal pathway can control a wide range of putative inhibitory circuits, and thereby influence the pattern of electrical activity in the hippocampal formation.

The use of gold-substituted silver-intensified diaminobenzidine (DAB) and non-intensified DAB for simultaneous electron microscopic immunoperoxidase labeling of tyrosine hydroxylase and glutamic acid decarboxylase immunoreactivity in the rat medial preoptic area.

An improved gold-substituted silver intensification procedure for the peroxidase-diaminobenzidine (DAB) reaction product was developed. The method was applied in the rat medial preoptic area to label tyrosine hydroxylase (TH)-immunoreactive profiles. Following the gold toning, the same sections were immunostained for glutamic acid decarboxylase (GAD) immunoreactivity with non-intensified peroxidase-DAB. Single DAB-labeled GAD axons were found in symmetric synaptic connection with unlabeled dendrites as well as with gold-toned immunoperoxidase-containing TH neurons.

New aspects of the neuroendocrine role of PACAP.

The presence of PACAP was revealed in the anterior pituitary with RIA, HPLC, and with the demonstration of its mRNA. The level of PACAP mRNA in the anterior pituitary is the highest during the proestrous LH surge. In our immunohistochemical studies we were able to demonstrate PACAP immunoreactive cells in the anterior pituitary. The shape and the distribution of PACAP immunoreactive cells were very similar to that of the gonadotropes; however, the number of PACAP cells was less than that of LH cells. Additionally, another PACAP-positive cell population with small diameter appeared in the proestrous stage, during pregnancy and lactation. Double labeling revealed that the major part of large PACAP cells exhibited LH immunoreactivity and those with a small diameter contained PRL. It is not clear whether the pituitary- or the hypothalamic-born PACAP, or both, influence pituitary LH and PRL secretion. I.c.v. administration of PACAP just prior to the critical period in the proestrous stage inhibited the expected ovulation and blocked the proestrus LH and PRL surge, although i.v. administration of PACAP had no effect. PACAP antiserum did not interfere with ovulation when i.c.v. or i.v. injection was used. Our results support the view that PACAP has a role in the control of LH and PRL secretion during the estrous cycle, pregnancy, and lactation. The inhibitory effect of PACAP on ovulation is mediated through the hypothalamus.

Metabotropic glutamate receptor in GHRH and beta-endorphin neurones of the hypothalamic arcuate nucleus.

Growth hormone-releasing hormone (GHRH) and beta-endorphin are mainly synthesized in neurones of the hypothalamic arcuate nucleus. Arcuate neurones also contain both ionotropic and metabotropic glutamate receptors. The aim of present study was to investigate whether glutamate receptors are present in GHRH and beta-endorphin containing nerve cells of this hypothalamic area. Using double-label immunocytochemistry as well as the mirror technique, we found that almost all GHRH and beta-endorphin immunoreactive arcuate neurones contain the metabotropic glutamate receptor la. The observations provide morphological evidence for the view that glutamate, which appears to be a major excitatory neurotransmitter in the hypothalamus, may directly stimulate GHRH and beta-endorphin neurones of the medial hypothalamus.

Immunohistochemical visualization of a metabotropic glutamate receptor.

The immunocytochemical localization of the recently cloned metabotropic glutamate receptor 1 alpha (mGluR1 alpha) was demonstrated with a C-terminus specific antibody in rat cerebellar cortex. This antibody detects a 138-140 kDa major, and a 46 kDa minor band in membrane preparations of rat cortex and cerebellum. mGluR1 alpha immunoreactivity (mGRi) was present in Purkinje and basket cells. Purkinje cell dendritic spines and their postsynaptic membranes showed selective labelling. Presynaptic membranes, parallel fibres and glial processes were devoid of mGRi. It is suggested that the selective postsynaptic localization of this receptor at the dendritic spines of Purkinje cells serves as the morphological basis for long term depression processes in the molecular layer of the cerebellar cortex.

Cellular and subcellular localization of the mGluR5a metabotropic glutamate receptor in rat spinal cord.

The cellular and subcellular distribution of the mGluR5a metabotropic glutamate receptor was studied in the spinal cord of the rat using an antibody raised against a mGluR5a-specific carboxy-terminal peptide. Strong mGluR5a-immunoreactivity (mGluR5a-ir) was found in the laminae I-II of the dorsal horn, which gradually decreased towards the deeper layers. At the electron microscopical level, mGluR5a-ir was present exclusively in neuronal somata and dendrites. Immunometal labelling revealed that mGluR5a-ir is concentrated at the periphery of postsynaptic densities of asymmetrical synapses or localized extrasynaptically at dendritic and somatic membranes. The mGluR5a-immunoreactive dendritic profiles were often targeted by synaptic boutons with the morphological characteristics of C-fibre terminals. These observations provide evidence for mGluR5a being involved in the nociceptive transmission at the dorsal horn.

Cortical cholinergic decline parallels the progression of Borna virus encephalitis.

Borna disease virus (BDV)-induced meningoencephalitis is associated with the dysfunction of the cholinergic system. Temporal development of this cholinergic decline during pre-encephalitic and encephalitic stages of BDV infection remains however elusive. Changes in choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) activities were therefore determined in the cerebral cortex, hippocampus, striatum, amygdala and cholinergic basal forebrain nuclei (ChBFN) of rats infected with BDV. Immunocytochemistry for ChAT and vesicular acetylcholine transporter (VAChT) was employed to identify morphological consequences of BDV infection on cholinergic neurons. Whereas both ChAT and AChE activities changed only slightly under pre-encephalitic conditions, the encephalitic stage was characterized by a significant decrease of ChAT activity in the cerebral cortex, horizontal diagonal band of Broca (hDBB), hippocampus and amygdala concomitant with a marked reduction of AChE activity in the cerebral cortex, hDBB and hippocampus. The striatum and medial septum remained unaffected. ChAT and VAChT immunocytochemistry revealed prominent axonal degeneration in affected cortical and limbic projection areas of ChBFN. In summary, our data indicate progressive deterioration of forebrain cholinergic systems that parallels the progression of BDV encephalitis.

VIP fibers in rat optic chiasm and optic nerve arising from the hypothalamus.

This is the first report showing VIP fibers in the optic chiasm and the optic nerves of intact rats. These fibers form a fan-shaped dorso-medial bundle in the optic nerves. After colchicine injection into the vitreous body VIP fibers could be followed farther in the optic nerve toward the eye when compared to intact rats. After removal of eyes (enucleation) the VIP fiber-bundle became more prominent and VIP immunoreactive perikarya appeared in the supraoptic and para ventricular nuclei. When five-nine months after the enucleation Phaseolus vulgaris leucoagglutinin was administered to the paraventricular or supraoptic area, the anterogradely transported tracer was demonstrated in the optic nerve. These observations suggest the existence of a hypothalamic projection to the eye, which is, at least in part, VIP immunoreactive.

Possible recognition of the GnRH receptor by an antiserum against a peptide encoded by nucleotide sequence complementary to mRNA of a GnRH precursor peptide.

Two peptides, rHRnG and hproHRnG, which were encoded by the nucleotide sequences complementary to mRNA of rat hypothalamic gonadotropin-releasing hormone (GnRH) and human placental proGnRH(-3-13), respectively, were synthesized. A remarkable hydropathic anti-complementarity was observed in the N-terminal region between hproHRnG and human proGnRH(-3-13). Neither hproHRnG nor rHRnG bound GnRH in ELISA unless extremely high concentrations of peptides were used. 125I-GnRH failed to bind with either rHRnG or hproHRnG previously coated polypropylene tubes. Antisera against these peptides were generated in rabbits. All the rabbits produced antibodies with high titer as tested by ELISA. One rabbit immunized with hproHRnG showed markedly reduced serum testosterone levels as compared with those of other rabbits. Intravenous administration of 1 ml serum from this rabbit, antiserum R281, into ovariectomized rats significantly decreased plasma LH. Using antiserum R281, about 10% of female rat pituitary cells were stained by immunohistochemistry. The staining was specific to hproHRnG since it was abolished by preabsorption of the antiserum with hproHRnG, but not with rHRnG, GnRH, LH nor any other peptide tested. This particular antiserum may have recognized the GnRH receptor, and thereby interfered with the action of endogenous GnRH. These results appear to be in agreement with the view that there is a structural similarity between the receptor for a peptide and the so-called complementary peptide.

Neuropeptide Y innervation of ACTH-immunoreactive neurons in the arcuate nucleus of rats: a correlated light and electron microscopic double immunolabeling study.

A fairly high number of neuropeptide Y (NPY) and adrenocorticotropic hormone (ACTH) immunoreactive (ir) neuronal perikarya and dense networks of NPY-ir fibers are present in the hypothalamic arcuate nucleus of rats. Light and electron microscopic double immunolabeling techniques were used to demonstrate morphological connections between NPY-ir nerve fibers and ACTH-ir neurons here. Silver-gold intensified diaminobenzidine (DAB) labeling of perikaryal-dendritic immunoreactivity followed by a second, non-intensified DAB chromogen labeling of immunoreactive nerve terminals was used and recommended in the above sequence as a method of choice for the detection of synaptic contacts with double-labeling technique. By this way, NPY-immunoreactivity was localized in axons and axonal terminals forming a variety of conventional synaptic contacts with ACTH-ir neuronal perikarya and dendrites in the arcuate nucleus.

Enkephalin and serotonin innervation of somatostatin-immunoreactive medullary reticular formation neurones.

Somatostatin cells in the rat medullary reticular formation (Md) have been studied using peroxidase-antiperoxidase immunocytochemistry. Large fusiform and multipolar somatostatin immunoreactive cells were observed in the ventral subnucleus (MdV) running in a band close to the border with the dorsal subnucleus (MdD). In the same region somatostatin-, serotonin- and enkephalin-immunoreactive fibres occur and double staining revealed that these all contact the somatostatin-immunoreactive cells, with enkephalin making a particularly dense innervation.

Immunocytochemical mapping of NPY and VIP neuronal elements in the cat subcortical visual nuclei, with special reference to the pretectum and accessory optic system.

The aim of this study was to describe the distribution patterns of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP)-immunoreactive (ir) neuronal elements in subcortical visual centers of the cat. Numerous NPY-ir neurons were present in the feline nucleus of the optic tract and in the anterior pretectal nucleus. Only a few NPY-ir neurons were found in the posterior, medial and olivary pretectal nuclei and in the accessory optic nuclei. Diffuse and heavily beaded NPY-ir fiber plexuses were observed throughout the superior colliculus, pretectum, and accessory optic system. Extensively arborising NPY-ir fibers were present in the mesencephalon and ventral lateral geniculate nucleus, while the dorsal visual thalamic nuclei contained only a few NPY-ir fibers. VIP-ir cells were present mainly in the accessory optic nuclei, and they were absent in the dorsal visual thalamus. Both NPY- and VIP-ir neurons were multipolar and fusiform in shape in the regions studied. Enucleation did not alter the appearance of NPY- and VIP-containing neuronal elements in the superior colliculus and pretectum while in the thalamus a subset of NPY-ir fiber population disappeared, indicating their retinal origin. Although there is a partial overlap in the topographical localization of the NPY- and VIP-ergic neurons in the pretectum, the colocalization of the two peptides could not be demonstrated. The present observations demonstrate the existence of two different and separate peptidergic (NPY and VIP) neuronal populations in the pretectum.

Immunocytochemical localization of mGluR1a metabotropic glutamate receptor in inhibitory interneurons of the cerebellar cortex.

Immunocytochemical investigation of metabotrop glutamate receptor la in the cerebellar cortex of rat revealed a presence of this receptor in all cortical inhibitory nerve cells (Purkinje, basket, stellate, Lugaro and Golgi). In addition to the previously described localization of this receptor in Purkinje dendritic spines supplied by parallel and climbing fibres and considered to be responsible for cerebellar long-term depression, we have observed metabotropic glutamate receptor la immunopositivity also in the synapses between parallel fibres and dendrites of basket, stellate and Golgi neurons. The postsynaptic receptor was also present occasionally in inhibitory synapses between stellate cell axons and Purkinje cells as well as between Purkinje axon collaterals and Lugaro cell dendrites. The possibility, that mGluR1a receptors in basket, Golgi and stellate cells might directly or indirectly contribute to cerebellar LTD, is discussed.

Present status of knowledge about the distribution and colocalization of PACAP in the forebrain.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a recently discovered member of the secretion family. 1. PACAP is a well conserved peptide during the phylogenesis. It has two bioactive amidated forms: PACAP38 and PACAP27 with 38 and 27 residues, respectively. 2. PACAP and its receptors are widely distributed in the central and peripheral nervous systems and in non-neural tissues. 3. In the central nervous system PACAP immunoreactive neuronal elements have been observed in the hypothalamus (magno- and parvocellular cell groups), both layers of the median eminence, the septum, the thalamus, the amygdaloid complex, the hippocampus, and various regions of the cortex. 4. In the periphery, PACAP was found in small sensory and parasympathetic neurons. 5. In the hypothalamus PACAP partially colocalizes with oxytocin- and tyrosine hydroxylase-immunoreactivities. In the septum there is no colocalization between the two immunoreactivities, but PACAP- and tyrosine hydroxylase-immunoreactive fibers were often found to establish synaptic contacts with the same, unlabeled dendrite. It was reported that in the periphery, in sensory neurons PACAP colocalized with substance-P and in parasympathetic neurons with acetylcholin. 6. PACAP functions as a neurotransmitter, hypothalamic releasing factor, posterior pituitary hormone, and trophic factor of the nervous tissue. PACAP also participates in neuro-immunoendocrine mechanisms.

Distribution of metabotropic glutamate receptor 1a in the rat hypothalamus: an immunocytochemical study using monoclonal and polyclonal antibody.

L-glutamate appears to be a major excitatory neurotransmitter in the hypothalamus. Its action is mediated via ionotropic and metabotropic glutamate receptors (mGluR). Eight mGluRs have already been cloned. In the present study the hypothalamic distribution of mGluR1a has been investigated by immunocytochemistry using monoclonal antibodies recently produced by some of the present authors (T. J. G., R. K., T. K.). The observations have been compared with findings obtained with a polyclonal antibody. A widespread and heterogeneous distribution of mGluR1a was found with the monoclonal antibodies. Intense immunolabelling of perikarya and dendrites occurred in several hypothalamic cell groups including the suprachiasmatic, anterior periventricular, anterior hypothalamic (posterior part), paraventricular, supraoptic, arcuate, tuberal magnocellular, dorsomedial and mammillary nuclei (particularly in the medial). It was only the ventromedial nucleus in which several perikarya were stained by the polyclonal antibody but appeared to be negative by the monoclonal antibodies. The findings fit extremely well with the data on the hypothalamic distribution of mGluR1 mRNA with the exception of the ventromedial nucleus. It remains to be elucidated whether alternatively spliced variants of mGluR1 (mGluR1b and 1c) are expressed in this nucleus. Further, they confirm the results of former immunohistochemical studies. In addition, they indicate that a significant part of the neuroendocrine region of the hypothalamus (including the paraventricular, supraoptic and arcuate nuclei) also contains mGluR1 suggesting that this receptor may play a role also in neuroendocrine regulation.