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The cellular localization of binding sites for the vasoactive peptides 125I-neuropeptide Y (NPY) and 3H-bradykinin (BK) was studied in explant cultures of rat cerebellum, brain stem and spinal cord by means of autoradiography. The majority of astrocytes in these cultures expressed binding of 125I-NPY and 3H-BK over their cell bodies and processes. Simultaneous staining of the cultures with anti-glial fibrillary acidic protein (GFAP) has shown that the labelled cells were GFAP-positive and could therefore be identified as astrocytes. In addition to glial cells, a great number of neurones also revealed binding sites for the neuropeptides. Our autoradiographic findings together with recent electrophysiological studies provide good evidence for the existence of NPY- and BK-receptors on astrocytes.

Electrophysiological studies have been made of the actions of the vasoactive peptides atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), neuropeptide Y (NPY) and bradykinin (BK) on astrocytes in explant cultures of rat brain stem and spinal cord. Addition of ANP, BNP and NPY to the bathing solution at concentrations of 10(-8) and 10(-7) M caused depolarizations of the majority of astrocytes studied, some cells were hyperpolarized and on a small number of cells the peptides had no action. BK (10(-8) M) only induced depolarizations of almost all astrocytes studied. Our electrophysiological data provide strong evidence for the presence of receptors for these vasoactive peptides on astrocytes. When ANP, BNP, NPY and BK were tested on the same cell, all peptides were effective, suggesting a coexistence of peptidergic receptors on astrocytes.

The cellular localization of binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide was studied in explant cultures of rat spinal cord, brain stem and cerebellum by means of autoradiography. In brain stem cultures, especially in the nucleus of the solitary tract, a great number of neurons revealed binding sites for both peptides. In spinal cord cultures, many neurons of various sizes were labelled by [125I]arginine vasopressin, whereas only a small number of cells showed binding sites for [125I]atrial natriuretic peptide. Neurons in cerebellar cultures revealed little or no binding for the peptides. In addition to neurons, binding sites for [125I]arginine vasopressin and [125I]atrial natriuretic peptide were also observed on glial cells. Simultaneous staining of the cultures with glial fibrillary acidic protein has shown that the labelled cells were glial fibrillary acidic protein-positive and could therefore be identified as astrocytes. Labelling of the cells by [125I]arginine vasopressin and [125I]atrial natriuretic peptide was more intense in spinal cord and brain stem cultures than in cultures of cerebellum, providing evidence for a heterogeneity of astrocytes in different regions of the central nervous system. Binding of both [125I]arginine vasopressin and [125I]atrial natriuretic peptide to neurons and astrocytes could be competed by the unlabelled peptides, suggesting specific binding of the radioligands. Our autoradiographic studies provide good evidence that in addition to neurons, astrocytes also express receptors for arginine vasopressin and atrial natriuretic peptide.

By means of intracellular recordings we have studied the actions of the vasoactive peptides endothelin-1 and -3 (ET-1, ET-3) and arginine vasopressin (AVP) on the membrane potential of astrocytes in explant cultures of rat spinal cord and brainstem. Addition of ET-1, ET-3 or AVP to the bathing solution at concentrations of 10(-8)-10(-6) M depolarized the membrane of the majority of glial cells studied. The AVP antagonist d(CH2)5(1), Tyr(Me)2, Arg8-vasopressin reversibly blocked the depolarizations by AVP. Our electrophysiological findings together with autoradiographic binding studies strongly suggest the existence of ET and AVP receptors on astrocytes.

The cellular localization of binding sites for 125I-endothelin-1 and -3 (ET-1, ET-3) was studied in explant cultures of rat cerebellum, brain stem and spinal cord by means of autoradiography. The majority of astrocytes in these cultures expressed binding sites for both ET-1 and ET-3. There was a difference in the intensity of labelling between glial cells in the same culture. Some cells revealed intense radioactivity whereas neighbouring astrocytes were only slightly labelled. Besides glial cells, cerebellar neurones presumably Purkinje cells and granule cells as well as medium-sized and large neurones in brain stem and spinal cord cultures showed binding sites for both peptides. Our results provide strong evidence for the existence of ET receptors on astrocytes.

A comparison has been made of the localization and ontogenetic development of acetylcholinesterase (AChE) and succinic dehydrogenase (SDH) in sections of fetal human apinal cord and brain stem using histochemical methods. Spinal cord sections of young fetuses (8-10 weeks in utero) showed low to moderate enzymatic activity whereas sections of older fetuses (12-18 weeks in utero) revealed high concentrations of AChE and SDH in the ventral and laeral horns. A similar increase in enzymatic activity during development was also observed in brain stem sections where AChE and SDH were mainly confined to cranial nerve nuclei. A comparison of our histochemical findings with investigations on the development of motor activity in the spinal cord suggests that there is a correlation between the increase in AChE activity and functional development.

Binding of the delta sleep-inducing peptide (DSIP) was studied in cultures of rat brainstem by means of autoradiography. Binding sites for [3H]DSIP were observed on small, medium-sized and large brainstem neurons but not on glial cells. Addition of unlabeled DSIP inhibited or markedly reduced binding of [3H]DSIP. It is suggested that brainstem neurons might possess receptors for this sleep-inducing peptide.

By means of light-microscopic autoradiography, binding sites for the delta sleep-inducing peptide (3H-DSIP) were observed on the soma and processes of cultured brain stem neurones but not on glial cells. Unlabelled DSIP at high concentrations inhibited binding of 3H-DSIP, indicating specific binding of this peptide. Our data suggest that brain stem neurones might possess receptors for the sleep-inducing peptide.

Cultures of rat brain stem and spinal cord were used to visualize binding sites for [3H]glycine, [3H]beta-alanine and their antagonist [3H]strychnine by light microscopic autoradiography. In spinal cord cultures, all radio-ligands were bound mainly to large neurones, probably motoneurones whereas in brain stem cultures, both medium-sized and large neurones were labelled. In contrast, glial cells did not show binding sites for any of the compounds studied, suggesting that glial elements may not possess receptors for glycine and beta-alanine.

By means of lightmicroscopic autoradiography we have studied the cellular localization of binding sites for 3H-serotonin and 3H-ketanserin in organotypic cultures of rat brain stem and spinal cord. In both types of cultures, a relatively great number of neurones revealed binding sites for 3H-serotonin which predominantly labels S1-receptors. 3H-ketanserin, an S2-antagonist was also bound to many neurones although to a lesser extent than 3H-serotonin. Binding sites for both radio-ligands were also observed on astrocytes. These findings together with electrophysiological investigations indicate that astrocytes possess serotonin receptors.

The cellular localization of cholinergic binding sites was studied in organotypic cultures of rat spinal cord and brain stem by means of autoradiography with radiolabelled muscarinic and nicotinic agonists and antagonists. Many astrocytes in both types of cultures showed intense labelling by 3H-acetylcholine in the presence of nicotine and the muscarinic antagonists 3H-pirenzepine, 3H-quinuclidinyl benzilate and 3H-propylbenzilcholine mustard. Incubation of the cultures with 3H-nicotine or 3H-acetylcholine in the presence of atropine also caused labelling of astrocytes. In addition to glial cells, many neurones showed binding of muscarinic and nicotinic agonists and antagonists. From our results it is suggested that astrocytes, besides neurones, possess both muscarinic and nicotinic receptors.

Autoradiographic studies on the uptake of 3H-beta-alanine have shown that, in spinal cord and brain stem cultures, both neurones and glial cells have accumulated the amino acid. In contrast, in cultures of cerebellum and dorsal root ganglia, 3H-beta-alanine was only t aken up by glial elements.

Binding of substance P (SP) was studied in organotypic cultures of rat central nervous system by means of autoradiography. In spinal cord cultures, binding sites for [3H]SP were observed on many interneurons located in the dorsal horn, whereas in the ventral horn, mainly large neurons, probably, motoneurons, were labelled. Almost no binding was detected on neurons of attached dorsal root ganglia. Binding of [3H]SP was also found on a relatively great number of brain stem neurons of various sizes. In contrast, glial cells did not reveal binding sites for [3H]SP. These binding studies together with electrophysiological investigations provide strong evidence for the existence of SP receptors on spinal and brain stem neurons but not on glial cells.

The actions of acetylcholine, muscarine and nicotine on the membrane potential of astrocytes of cultured rat brainstem and spinal cord were examined. All 3 cholinergic agonists produced hyperpolarizations of the majority of astrocytes tested, although a small number of cells was depolarized. The hyperpolarizations induced by acetylcholine and muscarine were blocked by the muscarinic antagonist atropine. The nicotinic antagonist mecamylamine antagonized the effects by nicotine in all cells tested. In contrast, mecamylamine completely blocked the acetylcholine induced hyperpolarizations in approximately half of the astrocytes whereas in the remaining cells the response was only reduced. Our electrophysiological studies are consistent with recent autoradiographic binding studies from our laboratory, suggesting the existence of muscarinic and nicotinic receptors on astrocytes.

By means of intracellular microelectrodes we have studied the action of histamine H1- and H2-agonists and -antagonists on the membrane potential of astrocytes in cultured rat brainstem and spinal cord. Histamine at high concentrations (10(-4) and 10(-5) M) mainly depolarized the glial membrane, whereas at low concentration (10(-6) M) it usually caused hyperpolarizations. The histamine-induced depolarizations were reversibly blocked by the H1-antagonist pyrilamine, whereas the H2-antagonist cimetidine antagonized the hyperpolarizations. The H1-agonist thiazolethylamine mainly produced depolarizations while impromidine, a H2-agonist, predominantly caused hyperpolarizations. Our findings, together with autoradiographic binding studies, provide strong evidence for the existence of histamine H1- and H2-receptors on astrocytes.

Glutamate and the glutamate analogue AMPA ((RS)-alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid) caused depolarizations of cultured rat spinal and brainstem neurones which were reversibly antagonized by the glutamate antagonist glutamic acid diethylester (GDEE) but not by 2-amino-5-phosphonovalerate (APV), an N-methyl-D-aspartate (NMDA) antagonist. In contrast, depolarizations by NMDA were blocked by APV but not by GDEE. These results suggest that the depolarization by AMPA is caused by the activation of glutamate/quisqualate-preferring receptors.

By means of lightmicroscopic autoradiography we have studied the cellular localization of binding sites for H-serotonin and H-ketanserin in organotypic cultures of rat brain stem and spinal cord. In both types of cultures, a relatively great number of neurones revealed binding sites for H-serotonin which predominantly labels S-receptors. H-ketanserin, an S-antagonist was also bound to many neurones although to a lesser extent than H-serotonin. Binding sites for both radio-ligands were also observed on astrocytes. These findings together with electrophysiological investigations indicate that astrocytes possess serotonin receptors. [ABSTRACT FROM AUTHOR]