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The cellular localization of binding sites for [125I]1-tyramine somatostatin ([125I]SS) was studied in explant cultures of rat CNS by autoradiography. In cultures from cortex, brain stem and spinal cord many neurones revealed binding sites for the peptide whereas in cerebellar cultures only little binding of [125I]SS was observed. In addition to neurones, astrocytes were also labelled by the peptide. By combined immunohistochemical and autoradiographic techniques, it was demonstrated that the majority of neurones which expressed binding sites for [125I]SS were also immunostained by the monoclonal cholinergic muscarinic or nicotinic receptor antibodies (M 35 and W 6, respectively), providing evidence for a colocalization of cholinergic and somatostatin receptors on the neuronal membrane.

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.

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.

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.

Using autoradiographic techniques, studies on the cellular localization of binding sites for second messengers in explant cultures of rat cerebellum have been performed. Binding of [3H]inositoltrisphosphate ([3H]IP3), [3H]phorbol dibutyrate ([3H]PDBu) occurred to Purkinje cells whereas binding sites for [3H]forskolin were mainly confined to granule cells and outgrowing nerve fibres. A great number of astrocytes was also labelled by all three radio-ligands. Binding of [3H]IP3, [3H]PDBu and [3H]forskolin was displaced by unlabelled compounds, suggesting specific binding. Our observations provide strong evidence that in addition to neurones, astrocytes also express binding sites for second messengers.

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.

The time course of the depolarization of cultured astrocytes and neurones by glutamate and aspartate corresponds well with the increase of the extracellular K+ concentration ([K +]0) measured with an ion-sensitive microelectrode placed in the close vicinity of the cells tested. It is concluded that the depolarization of glial cells is caused by an efflux of K+ from neighbouring neurones during their excitation by the amino acids.

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.

Electrophysiological studies have been made of the actions of vasoactive intestinal peptide (VIP) and angiotensin II (Ang II) on astrocytes in explant cultures of rat brain stem and spinal cord. Addition of VIP to the bathing solution at concentrations of 10(-8) to 10(-6) M caused hyperpolarizations. Ang II at the same concentrations depolarized the majority of astrocytes tested. At 10(-9) M, both peptides had no action or only small effects. In approximately one third of the cells, the depolarizations by Ang II were accompanied by rhythmic oscillations. The Ang II antagonist saralasin reversibly blocked the depolarizations by Ang II. These findings, together with autoradiographic binding studies from our laboratory strongly suggest the existence of VIP and Ang II receptors on astrocytes.

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.

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.

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.

Intracellular recordings were made from glial cells of human and rat spinal cord grown in tissue culture. The membrane potentials of the glial cells were usually higher than those recorded from neurones. Addition of the putative neurotransmitters glycine, GABA, glutamate and aspartate to the bathing fluid in a concentration of 10(-4) M had no effect on the membrane potential of most glial cells tested.

4-Aminopyridine (4-AP) which selectively blocks K+-conductance of excitable membranes, reversibly abolished the depolarization by gamma-aminobutyric acid (GABA) of cultured satellite glial (SG) cells, but did not or only slightly affect the action of GABA on dorsal root ganglion (DRG) neurons. It is therefore suggested that the depolarization of glial cells by GABA is an indirect effect due to the accumulation of K+ which is released from adjacent neurons during their depolarization by the amino acid. Tetraethylammonium (TEA) had no effect on the GABA depolarization at a concentration of 15 mM but produced a slight reduction at higher concentrations (60--70 mM).

gamma-Hydroxybutyrate (GHB) and GABA caused a hyperpolarization of cultured spinal, brainstem and cerebellar neurones. When KCl electrodes were used, the hyperpolarizations were reversed to depolarizations, suggesting that the actions of GHB and GABA are associated with an increase in conductance for chloride ions. Both the hyperpolarizations and depolarizations by these compounds were accompanied by an increase in membrane conductance and were reversibly blocked by the GABA antagonist bicuculline. It is suggested that GHB might either exert its effects by mimicking the action of GABA, or act as neurotransmitter or neuromodulator in the mammalian CNS.

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.