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The cellular localization of binding sites for [125I]galanin was studied in explant cultures of rat neocortex, cerebellum, locus coeruleus and spinal cord by means by of autoradiography. Binding sites for the peptide were observed on a great number of astrocytes in all CNS regions studied. In addition to astrocytes, many neurones were intensely labelled by [125I]galanin. Binding of [125I]galanin (10(-8) M) to both astrocytes and neurones was markedly reduced or inhibited by the unlabelled peptide at high concentration (10(-6) M), suggesting 'specific' binding of the radioligand. Evidence for the colocalization of galanin and cholinergic receptors on astrocytes was provided by combined autoradiographic and immunohistochemical studies. Many astrocytes were labelled by [125I]galanin and immunostained with antibodies to either muscarinic or nicotinic receptors. Electrophysiological studies revealed that addition of galanin (10(-9) to 10(-7) M) to the bathing fluid caused a dose-dependent hyperpolarization of the majority of astrocytes studied. When galanin (10(-8) M) and the cholinergic agonists muscarine and nicotine (10(-6) M) were tested on the same astrocyte, all three compounds induced a hyperpolarization, suggesting a colocalization of functional galanin and cholinergic receptors on the glial membrane.

Electrophysiological studies have shown that somatostatin (SOM; 10(-8) and 10(-7) M) causes a hyperpolarization of the majority of astrocytes in explant cultures of rat spinal cord and cortex. When SOM and the cholinergic agonists muscarine and nicotine (10(-6) M) were tested on the same cell, all three compounds produced hyperpolarizations, suggesting a colocalization of functional cholinergic and SOM receptors on the glial membrane. Combined immunohistochemical and autoradiographic binding studies demonstrating that almost all astrocytes which were immunostained by the monoclonal muscarinic or nicotinic antibodies were also intensely labelled by 125I-SOM, provide further evidence for the coexistence of cholinergic and SOM receptors on astrocytes.

The action of cholecystokinin (CCK) and bombesin (Bom) was studied on the membrane potential of astrocytes in explant cultures of rat cortex, cerebellum, brain stem and spinal cord. Both peptides (10(-8) and 10(-7) M) caused a hyperpolarization of most astrocytes studied. The hyperpolarization by CCK was markedly reduced or blocked by the CCKB-antagonist L-365,260 whereas addition of the Bom-antagonist [D-Phe12,Leu14]-Bom antagonized the effects of Bom, suggesting a specific action of these peptides. When CCK and Bom were tested on the same cell, both peptides were effective, indicating a colocalization of receptors for CCK and Bom on the glial membrane. Our electrophysiological investigations provide strong evidence for the existence of functional CCK and Bom receptors on astrocytes.

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.

The action of gamma-aminobutyric acid A (GABAA) and B (GABAB)-agonists has been studied on the membrane potential of astrocytes in explant cultures of rat spinal cord by means of intracellular microelectrode recordings. The GABAB-agonists (-)-baclofen and CGP 27 492 (3-aminopropyl phosphonous acid; 10(-6) to 10(-4) M) caused a hyperpolarization of the majority of astrocytes studied. On approximately 25% of the cells, the compounds had no effect. The hyperpolarization by baclofen (10(-4) M) was reversibly antagonized by the GABAB-antagonist 5-hydroxysaclofen (10(-4) M). GABA and the GABAA-agonist muscimol (10(-4) and 10(-3) M) depolarized approximately two thirds of the glial cells tested, whereas the remaining third remained unaffected. The GABAA-antagonist bicuculline (10(-4) and 10(-3) M) only reduced the depolarization by GABA (10(-4) M) but did not completely block it. On half of the cells tested, the depolarization by GABA was not affected by bicuculline, suggesting that the glial GABAA-receptor is different from the neuronal GABAA-receptor. Our electrophysiological investigations together with recent autoradiographic binding studies strongly suggest the existence of GABAB-receptors on astrocytes whereas there is less evidence for GABAA-sites on these cells.

Explant cultures of rat spinal cord, brain stem and cerebellum were used to visualize GABAA-receptors by means of immunohistochemistry. For these studies we have incubated the cultures with the monoclonal antibody bd 17 against the beta-subunit of the GABAA/benzodiazepine/chloride channel complex. In spinal cord cultures, many interneurones were immunoreactive whereas only a small number of large neurones, probably motoneurones was specifically stained. In brain stem cultures, groups of large and medium-sized neurones showed immunoreactivity. In cultures of cerebellum, a great number of neurones was specifically stained. Granule cells showed the strongest immunoreactivity whereas other neurones, presumably Purkinje cells and interneurones, were only moderately stained. The immunoreactivity was mainly confined to the cell bodies of the neurones while their processes were only weakly or not stained. In contrast to neurones, no immunoreactivity could be detected 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.

Substance P caused a depolarization of cultured spinal neurones which was often accompanied by a decrease in membrane conductance. The peptide usually had no effect on the membrane potential and resistance of glial cells. In contrast to the depolarization by glutamate, which is associated with an influx of Na+ and an efflux of K+, the action of substance P may be due to a decrease of the K+-conductance.

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.

The actions of adenosine, R-N6-phenylisopropyladenosine (R-PIA) and N-(ethyl)carboxamidoadenosine (NECA) were tested on the membrane potential of astrocytes of cultured rat spinal cord and cerebellum. All 3 compounds hyperpolarized the majority of astrocytes studied. A considerable number of cells did, however, not respond to adenosine and its analogues, suggesting that only a certain type of astrocyte possesses adenosine receptors. The hyperpolarizations by adenosine, R-PIA and NECA were reversibly blocked by their antagonist 8-phenyltheophylline indicating that these compounds activate specific adenosine receptors. In agreement with biochemical and autoradiographic binding studies, our electrophysiological data strongly suggest the existence of adenosine receptors on astrocytes.

The cellular localization of binding sites for [125I] vasoactive intestinal polypeptide and [3H]angiotensin II was studied in explant cultures of rat spinal cord, brain stem, cerebellum and cortex by means of autoradiography. In spinal cord cultures, interneurons of the dorsal horn and motoneurons of the ventral horn were labelled by [125I]vasoactive intestinal polypeptide and [3H]angiotensin II. In many brain stem cultures, groups of large neurons revealed intense binding of both peptides. In contrast, only few medium-sized cerebellar neurons, probably interneurons, showed binding sites for [125I]vasoactive intestinal polypeptide and [3H]angiotensin II. Furthermore, the intensity of labelling of cerebellar neurons was usually weaker than that of neurons of the brain stem and spinal cord. Many neurons in cultures of neocortex were also labelled by [125I]vasoactive intestinal polypeptide, whereas little binding was found with [3H]angiotensin II. In addition to neurons, binding sites for both peptides were also observed on astrocytes. Labelling of these cells was more intense in spinal cord and brain stem cultures than in cultures of cerebellum and cortex, suggesting that only a certain type or a certain population of astrocytes possesses receptors for vasoactive intestinal polypeptide and angiotensin II, or that glial cells in different parts of the CNS have different physiological and pharmacological properties.

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.

By means of light microscopic autoradiography, binding sites for the gamma-aminobutyric acid (GABA) analogues, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol ([7-3H]THIP), [3H]isoguvacine and [3H]baclofen were found on many cultured cerebellar and spinal neurons of fetal and newborn rats. The number of neurons labelled by [3H]THIP was considerably smaller than that by the other two radioligands. Unlabelled THIP, GABA and bicuculline methiodide inhibited binding of [3H]THIP and [3H]isoguvacine, whereas binding of [3H]baclofen was inhibited by unlabelled GABA and baclofen but not by bicuculline methiodide. Our results indicate that cultured cerebellar and spinal neurons possess both GABAA and GABAB binding sites and that [3H]THIP possibly binds to a subclass of GABAA 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.

Nervous tissue cultures have proved to be an excellent tool for investigating the cellular localization of the uptake of transmitter substances using autoradiography. The uptake of [3H]GABA and [3H]beta-alanine was studied in cultures of cerebellum, brain stem, spinal cord and dorsal root ganglia (DRG) of the rat. In cultures of brain stem and spinal cord, [3H]GABA was taken up by a great number of neurones and by almost all glial cells, suggesting that glial elements might also be involved in the inactivation of this amino acid. In cerebellar cultures, [3H]GABA was accumulated by many interneurones, by Purkinje cells and by a great number of glial cells. The finding that cultured Purkinje cells accumulated [3H]GABA contrasts with observations made in the cerebellum in vivo or in slices where no labelling of Purkinje cells could be demonstrated. Since GABA is taken up to a great extent by Bergman glia which tightly surround Purkinje cells in vivo, it has been suggested that the barrier formed by these glial cells prevents the uptake of the amino acid into Purkinje cells. In cultured cerebellum this glial barrier might be disrupted or absent and therefore Purkinje cells are able to accumulate [3H]GABA. A similar glial barrier preventing the uptake of [3H]GABA might also exist in DRG, where neurones that were completely surrounded by satellite glial cells did not take up the amino acid, whereas isolated neurones that were deprived of their glial envelopments became intensely labelled. After incubation with [3H]beta-alanine, it was observed that only glial cells were labelled in cerebellar and DRG cultures, whereas in spinal cord and brain stem both neurones and glial cells took up the amino acid providing further evidence that beta-alanine might act as a transmitter substance in these two regions.

The cellular localization of the uptake of [3H]adenosine and of binding of labelled adenosine analogues was studied in explant cultures of rat cerebellum and spinal cord by means of autoradiography. [3H]Adenosine was taken up by many neurons and astrocytes in both cerebellar and spinal cord cultures. The uptake of adenosine was inhibited in the absence of sodium or at 0 degrees C, suggesting an active transport mechanism. In both types of cultures, a great number of neurons showed binding sites for the A1-receptor agonist [3H]R-N6-phenylisopropyladenosine and for the mixed A1/A2-agonist [3H]N(ethyl)carboxamidoadenosine. Binding sites for both radioligands were also found on astrocytes, suggesting that these cells have receptors for the purinergic neurotransmitter adenosine. This suggestion is further supported by recent electrophysiological studies from our laboratory demonstrating that adenosine and its analogues produce hyperpolarizations of astrocytes which are blocked by the adenosine antagonist theophylline.

Autoradiographic investigations on the uptake of L-glutamate and L-aspartate have shown that the amino acids were taken up by neurones as well as by glial cells of cultured human and rat spinal cord. The activity of glutamate and aspartate varied considerably between individual neurones, whereas glial cells showed a more even distribution of the labelled amino acids. Our results suggest that both neurones and glial cells are involved in the uptake of amino acid transmitters.

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.

By means of light microscopic autoradiography, binding sites for the GABA catabolite [3H]gamma-hydroxybutyrate (GHB) were observed on cultured cerebellar and spinal neurones but not on glial cells. [3H]GHB was bound to similar types of neurones as [3H]GABA. However, the number of neurones labelled by [3H]GHB was considerably smaller than that by [3H]GABA, and the intensity of labelling by [3H]GHB was usually weaker. It is suggested that GHB might interact with neurotransmission mediated by GABA or play a role as neuromodulator or neurotransmitter in the mammalian central nervous system.

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.

The cellular localization of binding sites for [3H]dopamine, and dopamine-antagonists (D1 and D2) was studied in organotypic cultures of rat striatum and spinal cord by means of autoradiography. In both types of cultures, many astrocytes were labelled by [3H]dopamine, the D1-antagonist [3H]cis-flupenthixol and the D2-antagonists [3H]domperidone and [3H]spiperone (10(-9) to 10(-8) M). Addition of unlabelled dopamine and antagonists at high concentrations (10(-6) to 10(-4) M) inhibited or markedly reduced binding of the radioligands indicating 'specific' binding of the compounds. Our autoradiographic studies are consistent with biochemical investigations by other authors, suggesting that astrocytes possess receptors for dopamine.

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]