GABAA receptors on rat cerebellar granule cells are potently activated by muscimol but only slightly modulated by the benzodiazepine agonist flunitrazepam

Summary GABA_A receptors present on rat cerebellar granule cells in culture were studied by the whole cell patch clamp technique. Muscimol appeared to be more potent than GABA itself in activating Cl^– currents. A benzodiazepine, flunitrazepam, only slightly (10%) potentiated the GABA action.These results support the previous suggestion that GABA_A receptors containing the subunit, such as those in the cerebellum granule cells, are potently activated by muscimol. The present results also bear out the concept that GABA action on receptors containing the subunit is not potentiated by benzodiazepines.     

GABA A -receptors: Structural requirements and sites of gene expression in mammalian brain

GABA_A-receptors, the major synaptic targets for the neutotransmitter GABA, are gated chloride channels. By their allosteric drug-induced modulation they serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. Despite their functional prominence, the structural requirements of fully functional GABA_A-receptors are still elusive. Expression of cDNAs coding for the ₁- and ₁-subunits of rat brain yielded GABA-gated chloride channels which were modulated by barbiturates but displayed only agonistic responses to ligands of the benzodiazepine receptor. GABA_A-receptors with fully functional benzodiazepine receptor sites were formed when the ₁- and ₁-subunits were coexpressed with the ₂-subunit of rat brain. These receptors, however, failed to show cooperativity of GABA in gating the channel. In order to determine the subunit repertoire available for receptor assembly in different neuronal populations in vivo, the sites of subunit gene expression were (₁, ₂, ₃, ₅, ₆, ₁, ₂, ₃, ₂) mapped by in situ hybridization histochemistry in brain sections. The mRNAs of the ₁-, ₁- and ₂-subunits were co-localized e.g. in mitral cells of olfactory bulb, pyramidal cells of hippocampus as well as granule cells of dentate gyrus and cerebellum. The lack of colocalization in various other brain areas points to an extensive receptor heterogeneity. The presence of multiple GABA_A-receptors in brain may contribute to synaptic plasticity, differential responsiveness of neurons to GABA and to variations in drug profiles.Special issue dedicated to Dr. Erminio Costa     

GABAA Receptors of Cerebellar Granule Cells in Culture: Interaction with Benzodiazepines

GABA_A receptor mediated inhibition plays an important role in modulating the input/output dynamics of cerebellum. A characteristic of cerebellar GABA_A receptors is the presence in cerebellar granule cells of subunits such as α6 and δ which give insensitivity to classical benzodiazepines. In fact, cerebellar GABA_A receptors have generally been considered a poor model for testing drugs which potentially are active at the benzodiazepine site. In this overview we show how rat cerebellar granule cells in culture may be a useful model for studying new benzodiazepine site agonists. This is based on the pharmacological separation of diazepam-sensitive α1 β2/3 γ2 receptors from those which are diazepam-insensitive and contain the α6 subunit. This is achieved by utilizing furosemide/Zn²⁺ which block α6 containing and incomplete receptors.     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Effect of flumazenil on GABAA receptors in isolated rat hippocampal neurons

Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABA_A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA_A response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABA_A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA_A receptors.     

Methylmercury modulates GABAA receptor complex differentially in rat cortical and cerebellar membranes in vitro

Effects of methylmercury (MetHg) on the specific [³H]flunitrazepam binding were studied in rat cortical and cerebellar P₂-fractions in vitro. MetHg did not affect significantly the specific [³H]flunitrazepam binding in unwashed P₂-fraction but increased it marginally (by 16%) at 100 M in washed P₂-fraction, in both brain regions.Muscimol (3 M), a GABA_A agonist, stimulated the [³H]flunitrazepam binding by 30% to 50% depending on the brain region. In washed cerebellar membranes the enhancing response of muscimol was 10 to 14% lower after preincubation of the tissue with MetHg but in cerebral cortex MetHg did not modulate the muscimol response at all. The results indicate that Met-Hg may have region specific effects on GABA_A receptors in vitro and the effect may depend on the occupational state of the GABA binding domain of the receptor complex.     

Immunohistochemical localization of GABAA receptors in the scotopic pathway of the cat retina

The distribution of GABA_A receptors in the inner plexiform layer of cat retina was studied using monoclonal antibodies against the 2/3 subunits. A dense band of receptor labeling was found in the inner region of the inner plexiform layer where the rod bipolar axons terminate. Three forms of evidence indicate that the GABA_A receptor labeling is on the indoleamine-accumulating, GABAergic amacrine cell that is synaptically interconnected with the rod bipolar cell terminal. (1) Electron microscopy showed that the anti-GABA_A receptor antibody (62-3G1) labeled profiles that were postsynaptic to rod bipolar axons and made reciprocal synapses. (2) Indoleamine uptake (and the subsequent autofluorescence) combined with GABA_A receptor immunohistochemistry showed co-localization of the two markers in half of the receptor-positive amacrine cells. (3) Double labeling demonstrated that half of the receptor-positive somata also contained GABA. These results indicate that a GABAergic amacrine cell interconnected with the rod bipolar cell, most likely the so-called A17 amacrine cell, itself bears GABA_A receptors.     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

Changes of [3H]Muscimol Binding and GABAA Receptor β2-Subunit mRNA Level by Tolerance to and Withdrawal from Pentobarbital in Rats

Effects of continuous pentobarbital administration on binding characteristics of [³H]muscimol were examined by autoradiography, and levels of GABA_A receptor 2-subunit mRNA were investigated by in situ hybridization histochemistry in the rat brain. In order to eliminate the induction of hepatic metabolism by systemic administration of pentobarbital, an i.c.v. infusion model of tolerance to and withdrawal from pentobarbital was used. An experimental model of barbiturate tolerance and withdrawal was developed using i.c.v. infusion of pentobarbital (300 g/10 l/hr for 7 days) by osmotic minipumps and abrupt withdrawal from pentobarbital. The levels of [³H]muscimol binding were elevated in cingulate of frontal cortex (46%) and granule layer of cerebellum (32%) of rats 24-hr after withdrawal from pentobarbital, while it was only elevated in cingulate (58%) of tolerant rats. The GABA_A receptor 2-subunit mRNA was increased in the withdrawal rats only: in the cortex (9–14%), hippocampus (15–21%), inferior colliculus (21%), and granule layer of cerebellum (24%). These results show the involvement of GABA_A receptor and its 2-subunit up-regulations in pentobarbital withdrawal rats, and suggest that the levels of [³H]muscimol binding and GABA_A receptor 2-subunit mRNA are altered in a region-specific manner during pentobarbital withdrawal.     

Modulation of the expression of GABAA receptors in rat cerebellar granule cells by protein tyrosine kinases and protein kinase C

The expression of GABA_A receptors in rat cerebellar granules in culture has been studied by β_2/3 subunit immunocytochemistry and fluorescence confocal microscopy. These cells show labeling all over the cell bodies' plasma membrane and dendrites. Treatment with the protein tyrosine kinase (PTK) inhibitor genistein results in a decrease of the labeling associated with the β_2/3 subunit in both cell bodies and dendrites. No effect was found with an inactive genistein analogue, daidzein. A similar effect was found with a protein kinase C (PKC) activator, phorbol myristate acetate (PMA). The effects of genistein and PMA are additive.The interpretation of the results is that PTK inhibition blocks exocytotic deposit of newly synthesized GABA_A receptors onto the neuronal plasma membrane. On the other hand, PKC activation speeds up endocytotic removal of GABA_A receptors.     

Possible Involvement of GABAA and GABAB Receptors in the Inhibitory Action of Lindane on Transmitter Release from Cerebellar Granule Neurons

Cerebellar granule cells in culture express receptors for GABA belonging to the GABA_A and GABA_B classes. In order to characterize the ability of the insecticide lindane to interact with these receptors cells were grown in either plain culture media or media containing 150 M THIP as this is known to influence the properties of both GABA_A and GABA_B receptors. It was found that lindane regardless of the culture condition inhibited evoked (40 mM K⁺) release of neurotransmitter ([³H]D-aspartate as label for glutamate). In naive cells both GABA_A and GABA_B receptor active drugs prevented the inhibitory action of lindane but in THIP treated cultures none of the GABA_A and GABA_B receptor active drugs had any effect on the inhibitory action of lindane. This lack of effect was not due to inability of baclofen itself to inhibit transmitter release. It is concluded that lindane dependent on the state of the GABA_A and GABA_B receptors is able to indirectly interfere with both GABA_A and GABA_B receptors. In case of the latter receptors it was shown using [³H]baclofen to label the receptors that lindane could not displace the ligand confirming that lindane is likely to exert its action at a site different from the agonist binding site.     

Musciomol and flunitrazepam binding sites in a subcellular fraction enriched in rat cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of GABA_A and of benzodiazepine binding sites between membranes derived from this fraction (fraction G) and from a total cerebellar homogenate (fraction T) was studied. The benzodiazepine and GABA binding sites were measured by the binding of agonists [³H]flunitrazepam and [³H]muscimol, respectively. The results indicate that both binding sites are present, but only slightly enriched, in the glomerular synapses. We found a muscimol/flunitrazepam binding site ratio of two, which is consistent with the enrichement of muscimol binding sites in the granular layer shown by both autoradiographic with radioactive glutamatergic ligands and in situ hybridization experiments respectively.     

Clozapine's Antipsychotic Effects do not Depend on Blockade of 5-HT3 Receptors

Sixteen known 5-HT₃ receptor blockers, including clozapine, fully or partially reverse the inhibitory effect of 1 M GABA on [³⁵S]TBPS binding, indicating that they are also GABA_A antagonists, some of them selective for subsets of GABA_A receptors. The 5-HT₃ receptor blocker, ondansetron, has been reported to produce some antipsychotic and anxiolytic effects. However, no antipsychotic effects have been reported for a large number of highly potent 5-HT₃ receptor blockers. Like clozapine, ondansetron partially reverses the inhibitory effect of GABA on [³⁵S]TBPS binding. Additivity experiments suggest that ten 5-HT₃ receptor blockers tested at low concentrations preferentially block subtypes of GABA_A receptors that are among those blocked by clozapine. Wiley and Porter (29) reported that MDL-72222, the most potent GABA_A antagonist decribed here, partially generalizes (71%) with clozapine in rats trained to discriminate an interoceptive clozapine stimulus, but only at a dose that severly decreases responding. Tropisetron (ICS-205,930) exhibits both GABA-positive and GABA-negative effects. R-(+)-zacopride is 6-fold more potent than S-(–)-zacopride as a GABA_A antagonist. We conclude that the observed antipsychotic and, possibly, anxiolytic effects of some 5-HT₃ receptor blockers are due to selective antagonism of certain GABA_A receptors, and not to blockade of 5-HT₃ receptors. We speculate that the anxiolytic and sedative effects of clozapine and several other antipsychotic drugs may be due to selective blockade of 122 GABA_A receptors which are preferentially located on certain types of GABAergic interneurons (probably parvalbumin positive). Blockade of these receptors will increase the inhibitory output of these interneurons. So far, no highly potent GABA_A antagonists with clozapine-like selectivity have been identified. Such compounds may exhibit improved clozapine-like antipsychotic activity.     

Calcium accumulation in neurites and cell bodies of rat cerebellar granule cells in culture: effects on GABAA receptor function

Summary. Accumulation of calcium in rat cerebellar granule cells in culture was studied by two photon laser scanning microscopy. Depolarizations by high extracellular potassium induced short-lived increases in calcium in both cell bodies and neurites. However, although the increase in neurites subsided completely after the initial peak, in cell bodies there was a persistent plateau until the high potassium stimulus was removed. On the contrary, the calcium signal due to NMDA receptors activation was persistent in both cell bodies and neurites and remained until the agonist was present.The nature of these calcium signals provides an interpretation key for the effects of NMDA receptors activation on GABA_A receptors. In particular, the persistent calcium increase in neurites may explain the decrease in GABA activated chloride currents which are related to activation of dendritic/synaptic GABA_A receptors.     

[3H]muscimol and [3H]flunitrazepam binding sites in the developing cerebellum of mice treated with methylazoxymethanol at different postnatal ages

Two models of perturbed cerebellar ontogenesis were obtained by a single administration of methylazoxymethanol (MAM), a potent antimitotic agent, to mouse pups either on the day of birth (MAM₀ mice) or at postnatal day 5 (MAM₅ mice). The alterations of the cerebellar GABAergic system were studied by measuring glutamic acid decarboxylase activity, [³H]muscimol binding sites, which are known to be concentrated in the GABA_A receptors in the internal granular layer, and [³H]flunitrazepam binding sites, which are more abundant in the molecular layer. The primary target of the antimitotic agent are the precursors of the glutamatergic and GABAceptive granule cells. In both models GABAergic structures, as revealed by GAD activity measurements, appear to be relatively spared, and recovery of granule cell numbers occurs during development in MAM₅ mice. In MAM treated mice the number of [³H]muscimol binding sites (on a per cerebellum basis) decrease as the number of granule cells decrease, although some recovery occurred in MAM₅ mice, but not in MAM₀ mice. In MAM₅ mice, [³H]flunitrazepam binding sites (on a per cerebellum basis) were relatively unaffected, while they were decreased significantly, but to a lesser extent than [³H]muscimol binding sites, in MAM₀ animals. The more significant reduction of granule cell numbers and the cytoarchitectural disruption resultant from the more precocious application of the antimitotic appear responsible for the significant alteration and lack of recovery in MAM₀ mice.     

Changes of [3H]MK-801, [3H]muscimol and [3H]flunitrazepam Binding in Rat Brain by the Prolonged Ventricular Infusion of Transformed Ginsenosides

Ameliorating effects of ginseng were observed on neuronal cell death associated with ischemia or glutamate toxicity. Ginseng saponins are transformed by intestinal microflora and the transformants would be absorbed from intestine. In the present study, we have investigated the effects of transformed ginsenoside Rg3, Rh2 and compound K on the modulation of NMDA receptor and GABA_A receptor binding in rat brain. The NMDA receptor binding was analyzed by quantitative autoradiography using [³H]MK-801 binding, and GABA_A receptor bindings were analyzed by using [³H]muscimol and [³H]flunitrazepam binding in rat brain slices. Ginsenoside Rg3, Rh2 and compound K were infused (10 g/10 l/h) into rat brain lateral ventricle for 7 days, through pre-implanted cannula by osmotic minipumps (Alzet, model 2ML). The levels of [³H]MK-801 binding were highly decreased in almost all regions of frontal cortex and hippocampus by ginsenoside Rh2 and compound K. The levels of [³H]muscimol binding were elevated in part of frontal cortex and granule layer of cerebellum by the treatment of ginsenoside Rh2 and compound K. However, the [³H]flunitrazepam binding was not modulated by any tested ginsenosides. Ginsenoside Rh2 and compound K induced the downregulation of the [³H]MK-801 binding as well as upregulation of the and [³H]muscimol binding in a region-specific manner after prolonged infusion into lateral ventricle. However, ginsenoside Rg3 did not show the significant changes of ligand bindings. In addition, ginsenoside Rh2 decreased the expression of nNOS in the hippocampus although Rg3 decreased the expression in the cortex. These results suggest that biotransformed ginsenoside Rh2 and compound K could play an important role in the biological activities in the central nervous systems and neurodegenerative disease.     

Monoclonal antibody (M2) to glial and neuronal cell surfaces

A monoclonal antibody designated M2 arose from the fusion of mouse myeloma cells with splenocytes from a rat immunized with particulate fraction from early postnatal mouse cerebellum. Expression of M2 antigen was examined by indirect immunofluorescence on frozen sections of developing and adult mouse cerebellum and on monolayer cultures of early postnatal mouse cerebellar cells. In adult cerebellum, M2 staining outlines the cell bodies of granule and Purkinje cells. A weaker, more diffuse staining is seen in the molecular layer and white matter. In sections of newborn cerebellum, M2 antigen is weakly detectable surrounding cells of the external granular layer and Purkinje cells. The expression of M2 antigen increases during development in both cell types, reaching adult levels by postnatal day 14. At all stages of postnatal cerebellar development, granule cells that have completed migration to the internal granule layer are more heavily stained by M2 antibodies than are those before and in process of migration. In monolayer cultures, M2 antigen is detected on the cell surface Of all GFA protein-positive astrocytes and on more immature oligodendrocytes, that express 04 antigen but not 01 antigen. After 3 days in culture, tetanus toxinpositive neurons begin to express M2 antigen. The same delayed expression of M2 antigen on neurons is observed in cultures derived from mice ranging in age from postnatal day 0 to 10.     

GABA-Dopamine Receptor-Receptor Interactions in Neostriatal Membranes of the Rat

Recent evidence has shown in membrane preparations that the binding of one ligand to its receptor is able to modify the binding parameters of a second receptor (receptor-receptor interactions), allowing the modulation of incoming signals onto a neuron. To further understand the -amino-butyric acid (GABA)-dopamine (DA) interactions in the neostriatum we have carried out experiments to explore whether an activation of the GABA_A receptor could affect the binding characteristics of the D₂ DA receptor in membrane preparations of the rat neostriatum. The results show that GABA (30–100 nM) significantly increases the dissociation constant of the high affinity (K_H) D₂ DA binding site (labelled with the selective D₂ DA receptor antagonist [³H]raclopride and that such an effect is fully counteracted by the GABA_A receptor antagonist bicuculline (1 M). It is suggested that such putative GABA_A/D₂ receptor-receptor interactions may take place in the somato-dendritic membrane of the striato-pallidal GABA neurons and that it may modulate the inhibitory effects of DA on these neurons, mediated via D₂ receptors.