Location of glutamatergic/aspartatergic neurons projecting to the hypothalamic ventromedial nucleus studied by autoradiography of retrogradely transported [³H]D-aspartate.

The hypothalamic ventromedial nucleus is a prominent cell group, which is involved in the control of feeding, sexual behavior and cardiovascular function as well as having other functions. The nucleus receives inputs from various forebrain structures and has a dense glutamatergic innervation. The aim of the present investigations was to reveal the location of glutamatergic neurons in the telencephalon and diencephalon projecting to this hypothalamic cell group. [(3)H]d-aspartate retrograde autoradiography was used injecting the tracer into the ventromedial nucleus. We detected radiolabeled neurons in telencephalic structures including the lateral septum, bed nucleus of the stria terminalis and the amygdala, and in various diencephalic regions, such as the medial preoptic area, hypothalamic paraventricular nucleus, periventricular nucleus, anterior hypothalamic area, ventral premamillary nucleus, thalamic paraventricular and parataenial nuclei and in the hypothalamic ventromedial nucleus itself. Our observations are the first data on the location of glutamatergic neurons terminating in the hypothalamic ventromedial nucleus. The findings indicate that glutamatergic innervation of the ventromedial nucleus is very complex.

Semicarbazide-sensitive amine oxidase: current status and perspectives.

Semicarbazide-sensitive amine-oxidase (SSAO) is present in various human tissues and in plasma. Oxidative deamination of short-chain aliphatic amines is catalyzed by this enzyme to afford the corresponding aldehydes, ammonia and hydrogen peroxide. Methylamine and aminoacetone have been recognized to be physiological substrates for SSAO. There are several pathological states where increased serum SSAO activity have been found, such as diabetes mellitus, congestive heart failure, multiple types of cerebral infarction, uraemia, and hepatic cirrhosis. The role of SSAO in pathophysiology of diabetes has been most extensively investigated. The elevated formation of the potentially cytotoxic products of the enzyme may contribute to the endothelial injury of blood vessels, resulting in the early development of severe atherosclerosis; it may also contribute to the pathogenesis of diabetic angiopathy. It is now suggested that SSAO inhibitors may prevent the development of atherosclerosis and diabetic complications as well. Inhibitors can be conveniently subdivided into the main groups of hydrazine derivatives, arylalkylamines, propenyl- and propargylamines, oxazolidinones, and haloalkylamines. Of them, aryl(alkyl)hydrazines, and 3-halo-2-phenylallylamines are generally very strong SSAO inhibitors. Most of these inhibitors of SSAO have been originally developed for other purposes, or they are simple chemical reagents with highly reactive structural element(s); these compounds have not been able to fulfil all criteria of high potency, selectivity, and acceptable toxicity. New potent compounds with selectivity and low toxicity are needed, which may prove useful tools for understanding the roles and function of SSAO, or they may even be valuable substances for treatment of various diseases.

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.

Location of putative glutamatergic neurons projecting to the medial preoptic area of the rat hypothalamus.

The medial preoptic area is a key structure in the neural control of reproduction. Considerable evidence has accumulated indicating that glutamatergic innervation of the area plays an important role in this control. Sources of the glutamatergic input are unknown. Present investigations were aimed at studying this question. [3H]D-aspartate, which is selectively taken up by high-affinity uptake sites at presynaptic endings that use glutamate or aspartate as a transmitter, and is transported back to the cell body, was injected into the medial preoptic area. The neurons retrogradely labelled with [3H]D-aspartate were detected autoradiographically. Labelled cells were found in several telencephalic and diencephalic structures, but not in the brainstem. Within the telencephalon, labelled neurons were detected in the lateral septum, bed nucleus of the stria terminalis and amygdala. Diencephalic structures included the medial preoptic area itself, hypothalamic paraventricular, suprachiasmatic, ventromedial, arcuate, ventral premammillary, supramammillary and thalamic paraventricular nuclei. All of them are known to project to this area. The findings provide the first neuromorphological data on the location of putative glutamatergic neurons projecting to the medial preoptic area. Furthermore, they indicate that local putative glutamatergic neurons as well as several telencephalic and diencephalic structures contribute to the glutamatergic innervation of the area.

Identification of neurones of the brain and spinal cord involved in the innervation of the ductus deferens using the viral tracing method.

Using the viral transneuronal tracing technique cell groups of the spinal cord and brain transsynaptically connected with the ductus deferens were identified. Neurotropic (pseudorabies) virus was injected into the muscular coat of the ductus deferens and after survival times of 3, 4 and 5 days the spinal cord and brain were processed immunocytochemically. Virus-labelled neurones could be detected in the preganglionic sympathetic neurones and the dorsal commissural nucleus (upper lumbar segments) and in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei including the gigantocellular and paragigantocellular nucleus, the lateral reticular nucleus, the nucleus of the solitary tract, the caudal raphe nuclei, the A1/C1, A2, A5 and A7 noradrenergic cell groups and the locus coeruleus. In the hypothalamus significant numbers of virus-infected neurones could be detected in the paraventricular nucleus. In most cases moderate numbers of virus-labelled cells were present in the lateral hypothalamic area, in the retrochiasmatic area, in the periventricular region and in the median preoptic area. Double-labelling immunofluorescence detection of virus-infected neurones and thyrosine hydroxylase (TH) showed colocalization of virus protein and TH in portion of neurones of the A1/C1, A2, A5 and A7 noradrenergic cell groups, in the locus coeruleus and in the hypothalamic paraventricular nucleus. The present results provide the first morphological data on the multisynaptic circuit of neurones innervating the ductus deferens.

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.

Brain structures mediating the suckling stimulus-induced release of prolactin.

Suckling-induced prolactin release is a widely studied neuroendocrine reflex, comprising a neural afferent and a humoral efferent component. The information on the brain structures involved in this reflex is fairly limited. The present studies focused on this question. The following hypothalamic interventions were made in lactating rats and the dams were tested for the suckling-induced prolactin response: (i) unilateral or (ii) bilateral frontal cuts at the level of the anterior and posterior hypothalamus; (iii) administration of 5,7-dihydroxytryptamine or (iv) 6-hydroxydopamine into the hypothalamic paraventricular nucleus (PVN) to destroy serotonergic and catecholaminergic innervation of the cell group, respectively; (v) lesion of the medial subdivision of the PVN; and (vi) horizontal knife cuts below the PVN. Bilateral posterior and bilateral or unilateral anterior frontal cuts caused blockade of the suckling-induced release of prolactin. Likewise, most dams receiving 5,7-dihydroxytryptamine in the PVN did not respond to the suckling stimulus. Immunocytochemistry revealed that, in those rats which did not show a rise in plasma prolactin, there were almost no serotonergic fibres and terminals in the PVN, while in dams which exhibited a response, numerous serotonergic elements were evident. 6-Hydroxydopamine treatment did not cause significant alteration in the prolactin response. Lesion of the medial, largely parvocellular subdivision of the PVN, or horizontal knife cuts below this cell group, blocked the hormone response. The findings demonstrate for the first time that: (i) interruption of the connections between the brain stem and the hypothalamus interferes with the prolactin response to the suckling stimulus; (ii) serotonergic fibres terminating in the hypothalamic PVN are involved in the mediation of the suckling stimulus; and (iii) within the PVN, neurones in the medial, largely parvocellular subdivision of the cell group take part in the transfer of the neural signal, eventually inducing prolactin release.

Transneuronal labelling of nerve cells in the CNS of female rat from the mammary gland by viral tracing technique.

Using the viral transneuronal tracing technique, the cell groups in the CNS transneuronally connected with the female mammary gland were detected. Lactating and non-lactating female rats were infected with pseudorabies virus injected into the mammary gland. The other group of animals was subjected to virus injection into the skin of the back. Four days after virus injection, infected neurons detected by immunocytochemistry, were present in the dorsal root ganglia ipsilateral to inoculation and in the intermediolateral cell column of the spinal cord. In addition, a few labelled cells could be detected in the dorsal horn and in the central autonomic nucleus (lamina X) of the spinal cord. At this survival time several brain stem nuclei including the A5 noradrenergic cell group, the caudal raphe nuclei (raphe obscurus, raphe pallidus, raphe magnus), the A1/C1 noradrenergic and adrenergic cell group, the nucleus of the solitary tract, the area postrema, the gigantocellular reticular nucleus, and the locus coeruleus contained virus-infected neurons. In some animals, additional cell groups, among others the periaqueductal gray and the red nucleus displayed labelling. In the diencephalon, a significant number of virus-infected neurons could be detected in the hypothalamic paraventricular nucleus. In most cases, virus-labelled neurons were present also in the lateral hypothalamus, in the retrochiasmatic area, and in the anterior hypothalamus. In the telencephalon, in some animals a few virus-infected neurons could be found in the preoptic area, in the bed nucleus of the stria terminalis, in the central amygdala, and in the somatosensory cortex. At the longer (5 days) survival time each cell group mentioned displayed immunopositive neurons, and the number of infected cells increased. The pattern of labelling was similar in animals subjected to virus inoculation into the mammary gland and into the skin. The distribution and density of labelling was similar in lactating and non-lactating rats. The present findings provide the first morphological data on the localization of CNS structures connected with the preganglionic neurons of the sympathetic motor system innervating the mammary gland. It may be assumed that the structures found virus-infected belong to the neuronal circuitry involved in the control of the sympathetic motor innervation of the mammary gland.

Effect of neonatal treatment with monosodium glutamate on dopaminergic and L-DOPA-ergic neurons of the medial basal hypothalamus and on prolactin and MSH secretion of rats.

The effect of neonatal treatment with monosodium L-glutamate (MSG) on the dopaminergic systems of the medial basal hypothalamus has been investigated using tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) immunocytochemistry. Changes in plasma levels of prolactin (PRL) and alpha-melanocyte-stimulating hormone (MSH) have also been determined in intact and in MSG-treated rats after inhibition of TH by alpha-methyl-p-tyrosine (alpha-MpT) or without inhibition of enzyme activity. Monosodium glutamate resulted in a 40% reduction in the number of TH immunopositive tuberoinfundibular neurons, but no change in the number of AADC-positive tuberoinfundibular nerve cells, indicating that this reduction has occurred mainly in TH-positive but AADC-negative elements, i.e., in L-DOPA-ergic neurons. In contrast, MSG did not cause changes in the number of TH and AADC immunoreactive neurons of the periventriculohypophysial and tuberohypophysial dopaminergic systems, and it did not influence basal plasma PRL levels. alpha-methyl-p-tyrosine has increased plasma PRL concentrations in both control and MSG-treated rats of both sexes, but significantly higher responses were detected in females. None of the treatments had any effect on plasma MSH level. These findings suggest that MSG affects primarily L-DOPA-ergic neurons located in the ventrolateral part of the arcuate nucleus, but not dopaminergic neurons situated in the dorsomedial part of the arcuate nucleus; neither PRL nor MSH secretion is altered by MSG; a significant sex difference exists in the pituitary PRL response to inhibition of TH, and this response is not affected by MSG.

Identification of CNS neurons involved in the innervation of the epididymis: a viral transneuronal tracing study.

Cell groups of the spinal cord and the brain transsynaptically connected with the epididymis (caput, cauda) were identified by means of the viral transneuronal tracing technique. Pseudorabies virus was injected into the caput or the cauda epididymidis, and after survival times 4 and 5 days, the spinal cord and brain were processed immunocytochemically. Virus-labeled neurons could be detected in the preganglionic sympathetic neurons (lower thoracic and upper lumbar segments) and following virus injection into the cauda epididymidis, also in the sacral parasympathetic nucleus (L6-S1). Virus-infected perikarya were present in several brain stem nuclei (lateral reticular nucleus, gigantocellular and paragigantocellular nucleus, A5 noradrenergic cell group, caudal raphe nuclei, locus coeruleus, Barrington's nucleus, nucleus of the solitary tract, periaqueductal gray) and in the diencephalon (hypothalamic paraventricular nucleus, lateral hypothalamus). At the longer survival time, some telencephalic structures also exhibited virus-labeled neurons. The distribution of infected neurons in the brain was similar after virus injection into the caput or cauda epididymidis; however, earlier onset of infection was observed after inoculation into the cauda. The present findings provide the first morphological data on a multisynaptic circuit of neurons innervating the epididymis and presumably involved in the control of epididymal functions. reserved.

Asymmetry of the neuroendocrine system.

There is information on the lateralization of hypothalamic, limbic, and other brain structures involved in the control of the endocrine glands. Sided differences between paired glands, including their peripheral innervation, and relevant clinical observations on asymmetry are also known. Data suggest predominance of the right half of brain structures in controlling gonadal function.

Dual role of glucocorticoids in suckling-induced prolactin secretion.

The exact contribution of corticosteroids to the control of prolactin secretion in lactating rats is poorly understood. Therefore, the present studies were focused on the effect of adrenalectomy and dexamethasone treatment on the suckling-induced prolactin release. Animals were adrenalectomized on the 3rd day of lactation and tested on the 7th day of lactation. In adrenalectomized animals, the suckling stimulus failed to induce the characteristic increase in plasma prolactin levels. Dexamethasone pretreatment (400 microg/kg b.w. s.c. 24, 48, 72 h before testing) of adrenalectomized rats restored this prolactin response. The same treatment with dexamethasone given to control animals attenuated the suckling stimulus induced prolactin response. The present findings indicate that corticosteroids are essential for a basic prolactin response of lactating rats.

Effect of adrenalectomy and dexamethasone treatment on prolactin secretion of lactating rats.

The contribution of corticosteroids to the control of prolactin secretion in lactating rats was investigated. The prolactin response to domperidone (20 microg/kg b.w., i.v.), a dopamine receptor antagonist and to domperidone plus formalin stress was tested in adrenalectomized and/or dexamethasone-treated continuously nursing rats. Animals were adrenalectomized on the 3rd day of lactation and tested on the 7th day of lactation. Dexamethasone was injected s.c. 24 h before testing (400 microg/kg b.w.) and on the day of testing (200 microg/kg b.w.). Domperidone caused a significant rise in plasma prolactin levels. The prolactin response to domperidone was twice as high in solely adrenalectomized dams and in solely dexamethasone-treated rats compared to controls. In adrenalectomized animals treated with dexamethasone, the prolactin response to domperidone was like in controls. Formalin injection to either adrenalectomized plus domperidone-treated animals or to animals injected with dexamethasone plus domperidone, resulted in a statistically significant depletion of plasma prolactin. In controls and in adrenalectomized animals receiving dexamethasone and domperidone, the prolactin response to formalin was very similar, i.e., plasma prolactin levels did not change after the administration of formalin. The present findings suggest that in lactating rats, corticosteroids are involved in the prolactin response to domperidone and to formalin stress.

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.

Central nervous system structures labelled from the testis using the transsynaptic viral tracing technique.

In the present study, the transneuronal transport of neurotrophic virus technique was used to identify cell groups of the spinal cord and the brain that are transsynaptically connected with the testis. Pseudorabies virus was injected into the testis and after survival times of 3-6 days, the spinal cord and brain were processed immunocytochemically using a polyclonal antibody against the virus. Virus-infected perikarya were detected in the preganglionic neurones of the spinal cord (T10-L1, L5-S1) and in certain cell groups and areas of the brain stem, the hypothalamus and the telencephalon. In the brain stem, the cell groups and areas in which labelled neurones were present included, among others, the nucleus of the solitary tract, the caudal raphe nuclei, the locus coeruleus and the periaqueductal grey of the mesencephalon. In the hypothalamus, virus infected perikarya were observed in the paraventricular nucleus and in certain other cell groups. Telencephalic structures containing labelled neurones included the preoptic area, the bed nucleus of the stria terminalis, the central amygdala and the insular cortex. These data identify a multisynaptic circuit of neurones in the spinal cord and in the brain which may be involved in the control of testicular functions.

Central nervous system structures connected with the endocrine glands. findings obtained with the viral transneuronal tracing technique.

This review is a summary of recent neuromorphological observations on the existence of multisynaptic neural pathways between the endocrine glands and the central nervous system (CNS) and its structures involved in this pathway. Introduction of the viral transneuronal tracing technique has made possible investigations of multisynaptic connections. The utility of this approach is based on the ability of the neurotropic virus to invade and replicate in neurons, and then gradually infect synaptically linked second-order, third-order. etc. neurons. Injecting the virus into the endocrine glands, this technique was used to identify cell groups in the spinal cord and in the brain which are connected with the adrenal gland, the gonads and the pancreas. Injection of the virus into these organs resulted in viral labeling of neurons in practically identical structures of the CNS including the intermediolateral cell column of the spinal cord, the vagal nuclei and certain other cell groups in the brain stem. In the hypothalamus the most intensive labeling was in the parvocellular part of the paraventricular nucleus and in the telencephalon labeled nerve cells were detected in the amygdala, the bed nucleus of the stria terminalis and in the preoptic area. It is known that the labeled CNS structures are members of descending pathways arising from the hypothalamic paraventricular nucleus or from other cell groups and terminating on neurons of the vagal nuclei and the intermediolateral cell column of the spinal cord. Experimental data support the view that the CNS structures and pathways connected with the endocrine glands are involved in the neural control of these organs.

CNS structures presumably involved in vagal control of ovarian function.

The contribution of the vagus nerve to viral transneuronal labeling of brain structures from the ovaries demonstrated recently by us was investigated. Unilateral vagotomy was performed prior to ipsilateral intraovarian virus injection. Virus-infected neurons were visualized by immunostaining. In vagotomized rats such neurons were detected only in certain cell groups of the brain (parapyramidal nucleus, A(1), A(5) cell group, caudal raphe nuclei, hypothalamic paraventricular nucleus, lateral hypothalamus). Vagotomy interfered with labeling of several structures that were labeled in controls, including area postrema, nucleus of the solitary tract, dorsal vagal complex, nucleus ambiguus, A(7) cell group, Barrington's nucleus, locus coeruleus, periaqueductal gray, dorsal hypothalamus. Findings provide a morphological basis to study the functional significance of brain structures presumably involved in the control of ovarian function and acting via the vagus or the sympathetic nerves.

Complexity of the neuroendocrine system.

This paper summarizes briefly the numerous connections and mechanisms existing within the neuroendocrine system. In the last decades it became clear that besides the originally postulated basic mechanisms there are several other, such as autocrine and paracrine mechanisms within the glands, bidirectional neural connections between the target endocrine glands and the hypothalamus and lower brainstem, which are of functional significance. Further we learned that the organization of the neural structures involved is much more complicated than originally thought. In addition, it turned out that the neuroendocrine system and the immune system are closely and intimately linked to each other. The available informations indicate clearly that a physiological integration exists between the nervous, the endocrine and immune systems.