Inhibitions mediated by glycine and GABAA receptors shape the discharge pattern of bulbar respiratory neurons

Experiments were performed to identify the glycinergic or GABAergic nature, and the timing of discharge, of the neurons which produce chloride-dependent inhibitions on other bulbar respiratory neurons (RNs) during their silent and active phases. RNs recorded extracellularly in pentobarbital-anesthetized or decerebrate cats, were subjected to iontophoretic applications of glutamate, of the glycine antagonist strychnine, and of the GABA_A receptor antagonist bicuculline. Both antagonists induced discharge or increased discharge frequency in restricted parts of the respiratory cycle without affecting the discharge frequency in other parts of the cycle. Strychnine most often elicited activity in late-inspiration and early-expiration, but also in early inspiration and in late expiration. Bicuculline was most often effective throughout the entire discharge period of each neuron with no effect during the silent period, although it also acted selectively during late-inspiration in inspiratory neurons, an effect attributed to GABA_A receptor blockade. The convergence of glycinergic afferent inputs during late inspiration and early expiration suggests that glycinergic neurons may play an important role in the inspiratory to expiratory phase transition.     

Modulation of acetylcholine release via GABAA and GABAB receptors in rat striatum

In order to investigate whether changes in acetylcholine (ACh) release induced by GABA receptors are due to a direct or indirect effect on cholinergic neurons in the striatum, GABA_A and GABA_B receptor bindings were assayed in the striatum microinjected with ethylcholine mustard aziridinium ion (AF64A), a cholinergic neurotoxin. Intra-striatal injection of a selective concentration of AF64A (10 nmol) reduced GABA_A receptor binding without significantly altering GABA_B receptor binding. Treatment with a higher, less selective concentration of AF64A (20 nmol) reduced all markers examined. These results suggest that GABA_A, but not GABA_B receptors, are located on cholinergic neurons in the striatum, and that GABA can directly modulate ACh release through stimulation of GABA_A receptors. Findings further suggest that GABA can also indirectly modulate ACh release through stimulation of GABA_B receptors located on non-cholinergic neuronal elements in the striatum.     

Peptide heterogeneity of GABAA/benzodiazepine receptors in bovine cerebral cortex and cerebellum

The GABA_A/benzodiazepine receptor complex has been purified from both bovine cerebral cortex and cerebellum by immunoaffinity chromatography on immobilized monoclonal antibody 62-3G1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified receptor from either cerebral cortex or cerebellum revealed 3 main bands corresponding to 51 000, 55 000 and 57 000M_r silver-stained peptides In addition, a minor band corresponding to a 53 000M_r peptide was also found. The difference between the two receptor preparations were: (1) that the main silver-stained 55 000M_r subunit was present in a relative smaller quantity in cerebellum than in cerebral cortex, and (2 when the membrane-bound receptor was photoaffinity-labeled with [³H]flunitrazepam and subsequently immunoaffinity-purified, two photolabeled peptide bands of 51 000 and 57 000M_r were found in cerebral cortex while only the 51 000M_r photolabeled peptide was detected cerebellum following one-dimension sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Peptide maps of the 57 000M_r [³H]flunitrazepam photoaffinity-labeled peptide indicated that it was composed of two closely migrating photolabeled peptides of 55 000M_r and 57 000M_r 0899 Peptide mapping and deglycosylation experiments using the [³H]flunitrazepam photolabeled receptor suggested that the photolabeled peptides commonly present in cerebellum and cerebral cortex are qualitatively similar if not identical. The results suggest that there are subunits of some type(s) of GABA_AR/BZDR complex(es) which are more abundant in cerebral cortex than in cerebellum. Photoaffinity labeling with [³H]muscimol showed similar photolabeled peptides in both cerebral cortex and cerebellum: two main peptides of 54 000 and 57 000M_r wer photolabeled with [³H]muscimol to a similar extent in both receptor preparations. Following deglycosylation, the mobility shifts of the peptides that were photolabeled with [³H]flunitrazepam or [³H]muscimol were different, suggesting that the co-migrating 54 000 – 57 000M_r peptides that have high affinity binding sites for [³H]flunitrazepam or [³H]muscimol are different receptor subunits.     

Differential intracellular regulation of cortical GABAA and spinal glycine receptors in cultured neurons

Using patch-clamp techniques we studied several aspects of intracellular GABA_A and glycine Cl⁻ current regulation in cortical and spinal cord neurons, respectively. Activation of PKA with a permeable analog of cyclic AMP (cAMP) produced a potentiation of the Cl⁻ current activated with glycine, but not of the current induced with GABA. The inactive analog was without effect. Activation of PKC with 1 μM PMA reduced the amplitude of the GABA_A and glycine currents. Internal application of 1 mM cGMP, on the other hand, had no effect on the amplitude of either current. The amplitude of these inhibitory currents changed slightly during 20 min of patch-clamp recording. Internal perfusion of the neurons with 1 μM okadaic acid, a phospatase inhibitor, induced potentiation in both currents. The amplitude of GABA_A and glycine currents recorded with 1 mM internal CaCl₂ and 10 mM EGTA (10 nM free Ca²⁺) decayed by less than 30% of control. Increasing the CaCl₂ concentration to 10 mM (34 μM free Ca²⁺) induced a transient potentiation of the GABA_A current. A strong depression of current amplitude was found with longer times of dialysis. The glycine current, on the contrary, was unchanged by increasing the intracellular Ca²⁺ concentration. Activation of G proteins with internal FAl⁻₄ induced an inhibition of the GABA_A current, but potentiated the amplitude of the strychnine-sensitive Cl⁻ current. These results indicate that GABA_A and glycine receptors are differentially regulated by activation of protein kinases, G proteins and Ca²⁺. This conclusion supports the existence of selectivity in the intracellular regulation of these two receptor types.     

Blockade of GABAA receptors in the medial ventral pallidum elicits feeding in satiated rats

γ-aminobutyric acid (GABA)-containing fibers from the nucleus accumbens shell (AcbSh) terminate in the medial ventral pallidum (VPm) and neurons in the VPm project to the lateral hypothalamus (LH). Therefore, the VPm is anatomically interposed between the AcbSh and LH, two functionally related brain regions that mediate food intake. The present study demonstrates that blockade of GABA_A receptors in the VPm by local administration of bicuculline greatly increases food intake in satiated rats. The data suggest that an AcbSh-VPm-LH circuit may be involved in the control of feeding behavior.     

The GABAA/benzodiazepine receptor complex in rat brain neuronal cultures. Characterization by immunoprecipitation

The expression of the GABA_A/benzodiazepine receptor (GABAR/BZDR) complex in primary neuronal cultures from rat brain embryos has been investigated. The GABAR/BZDR complex was photoaffinity labeled with [³H]flunitrazepam [³H]FNZ and immunoprecipitated with subunit specific antibodies. These were the mAb 62-3G1 which is specific for the 57-kDa GABA binding subunit, and the rabbit antiserum A which recognizes the 51-kDa [³H]FNZ binding subunit. The results indicate that the cultured neurons express 5 different peptides of 51, 53, 54, 57 and 59 kDa that can be photoaffinity labeled with [³H]FNZ and that all of them are physically coupled to the GABA_A receptor. Most of the [³H]FNZ photolabeled peptides have similar mobilities to those found in the brain of the newborn rat. Nevertheless, some of the quantitative changes in the photolabeled peptides observed during the normal development of the rat brain were not observed or occurred at much slower pace in the cultured neurons.     

OFF pathway is preferentially suppressed by the activation of GABAA receptors in carp retina

This study examines the effect of γ-aminobutyric acid (GABA) on the ON and OFF pathways in isolated, superfused carp retina. In most (76%) of amacrine cells bath-applied GABA preferentially suppressed the OFF response. The effect of GABA was blocked by bicuculline. Baclofen did not cause a similar effect. Furthermore, GABA produced a substantial suppression of the OFF bipolar cell response. We conclude that the preferential suppression of the OFF pathway may be due to a presynaptic inhibition mediated via GABA_A receptors at the terminals of OFF bipolar cells.     

Evidence for the involvement of nigral GABAA receptors in seizures of adult rats

Several studies have implicated the substantia nigra GABAergic system in the mediation of seizures in adult rats. The present study examines whether the different GABA receptors (GABA_A and GABA_B), are preferentially involved in this GABAergic seizure suppression mechanism. Adult rats were intranigrally infused with muscimol (GABA_A receptor agonist), bicuculline (GABA_A receptor antagonist) or baclofen (GABA₁ receptor agonist) and were exposed to flurothyl seizures Results indicated that while infusions of muscimol had an anticonvulsant effect, infusions of bicuculline had a proconvulsant effect. Baclofen infusions were found to have no effect on seizures. These findings suggest an involvement of the nigral GABA_A receptors in the mediation of seizures in adult rats.     

Cholinergic cell expression in the developing rat medial septal nucleus in vitro is differentially controlled by GABAA and GABAB receptors

The early appearance and relative abundance of GABAergic neurons in basal forebrain cholinergic nuclei like the medial septum suggest that the maturation of the later developing cholinergic neurons in these nuclei may be controlled by GABA. To examine this possibility, the effects of both exogenous GABA and specific GABA receptor agonists, as well as that of endogenous GABA on the phenotypic expression and survival of the cholinergic neurons in primary cultures from the fetal rat medial septum, were studied. Treatment of these cultures for six days with GABA significantly decreased the enzymatic activity of choline acetyltransferase (EC 2.3.1.6) (ChAT) in a dose-dependent manner. This response to exogenous GABA was blocked by bicuculline, mimicked by muscimol and slightly potentiated by saclofen. Consistent with this latter observation, the GABA_B receptor agonist, baclofen, dose-dependently increased septal ChAT activity. However, while the effect of baclofen on cholinergic expression was lost in the absence of glia, the suppressive effects of GABA or muscimol were more marked. Acetylcholinesterase (EC 3.1.1.7) (AChE) expression in mixed neuronal–glial cultures, was, like ChAT activity, increased or decreased in intensity with the inclusion of baclofen or muscimol, respectively. Although the number of AChE positive neurons in muscimol-treated cultures was significantly lower than that in controls, no changes in neither neuronal nor general cell viability were noted. Finally, as GABA_A or GABA_B receptor antagonists bicuculline and picrotoxin or saclofen, when applied alone to mixed cultures, increased or decreased ChAT activity, respectively, it appears that endogenous GABA, tonically released in the developing septum, may, via specific receptor types, differentially control the biochemical maturation of the cholinergic neurons.     

GABA, benzodiazepine and histamine-H1 receptors in the guinea pig cerebellum: effects of kainic acid injections studied by autoradiographic methods

By using kainic acid (KA) to perform chemical lesions in the guinea pig cerebellum, we have caused degeneration of Purkinje cells without affecting cell morphology. Near the injection site we found a large decrease in autoradiographically labeled histamine-H1 and benzodiazepine receptors of the molecular layer while those receptors distant from the injection site were unaffected. GABA receptors in the granule cell layer remained uniformly constant even immediately adjacent to the lesion site. This evidence suggests that histamine-H1 and benzodiazepine receptors are present on neuronal elements (possibly on Purkinje cell dendrites) in the molecular layer of the cerebellum and that GABA receptors area associated with the KA-resistant granule cells.     

Noradrenaline action on cat retinal ganglion cells is mediated by dopamine (D2) receptors

Effects of iontopheretically applied noradrenaline, dopamine and their receptor antagonists on the retinal ganglion cells, were studied in optically intact eyes of barbiturate-anaesthetized cats. Noradrenaline inhibited visually evoked and spontaneous firing of all classes of retinal ganglion cells: the effect being greater on ON-than on OFF-cells and slightly more potent than dopamine on a given cell. All α- and β-adrenoceptor blockers tested tended to change spikes but were generally ineffective in blocking the noradrenaline-induced inhibition, when not affecting spikes. The noradrenaline-induced inhibition was, however, effectively blocked by dopamine D₂-receptor antagonists. The α- and β-adrenoreceptor antagonists applied alone had no effect, suggesting the absence of endogenous noradrenergic antagonism, although α-type adrenergic antagonism was suggestive on a very small number of cells. These results suggest that: (1) noradrenaline action on cat retinal ganglion cells is mediated via dopamine D₂-receptors; (2) noradrenaline is not generally released on them, except there may be physiologically active α-receptors on a few cells; and (3) many of the adrenoreceptor blockers affect membrane properties of the retinal ganglion cells, in a similar manner to local anaesthetics.     

Adrenalectomy prevents the stress-induced decrease in in vitro [H]Ro15-1788 binding to GABAA benzodiazepine receptors in the mouse

The effect of a single or repeated swim stress on in vivo benzodiazepine receptor binding to various brain regions in adrenalectomized and sham-operated (control) mice was assessed using the benzodiazepine receptor antagonist, [³H]Ro15-1788. In sham-operated mice the binding of [³H]Ro15-1788 to benzodiazepine receptors was reduced in the hippocampus and hypothalamus (single or repeated stress) and cerebral cortex (repeated swim stress) compared to non-stressed mice. In contrast, no alterations in [³H]Ro15-1788 binding were observed in any brain region in adrenalectomized mice after either single or repeated swim stress. These data suggest that an intact hypothalamic-pituitary-adrenal axis is required for the stress-induced decrease in benzodiazepine receptor occupancy measured using the in vivo binding method.     

Multiple neurochemical action of clozapine: A quantitative autoradiographic study of DA 2, opiate and benzodiazepine receptors in the rat brain after long-term treatment

Summary The atypical neuroleptic clozapine has clinical and behavioral properties that differ not only from the typical compounds, but also from atypical ones. It interacts with the dopaminergic systems, but also produces effects on the serotoninergic, GABA-ergic, cholinergic systems. In spite of the amount of papers devoted to its study, the profile of the neurochemical action of this drug is still confuse. In this paper we investigated the DA 2-, opiate- and benzodiazepine-receptor modifications induced by the long term (21 days) treatment with clozapine 20 mg/kg/day in the rat brain. We found a decrease of DA 2 receptor density in the target areas of the mesolimbocortical system (ventral n. caudate-putamen, cerebral cortex except for the anterior cingulate at the most anterior level and the n. accumbens) and a decrease of opiate and benzodiazepine receptors in the cerebral cortex and in the olfactory tubercle. Opiate receptors increase in the patches of the striatum. We also compared these effects with those produced by long-term (21 days), low-dosage (0.5 mg/kg day) haloperidol.     

Immunohistochemical studies on distribution of GABAA receptor complex in the rat brain using antibody against purified GABAA receptor complex

The distribution of the γ-aminobutyric acid (GABA)_A receptor/benzodizepine receptor/CI⁻ channel complex in the rat brain was examined immunohistochemically using the specific antibody against purified GABA_A receptor complex. The immunization of white albino rabbit with purified GABA_A receptor complex resulted in the formation of specific antibody as indicated by the immunoprecipitation test. Immunohistochemical examinations using the antiserum on rat brain slices by the peroxidase-antiperoxidase method revealed the presence of the following immunoreactive sites which coincided with a previous report using antibody againstl-glutamic acid decar☐ylase; ventromedial nucleus of hypothalamus, red nucleus, globus pallidus, zona compacta and zona reticulata of substantia nigra, layers of Purkinje cells and granular cells of cerebellum, layers III–V of cerebral cortex and stratum radiatum of hippocampus. These results strongly suggest that immunohistochemical application of the antibody against the purified GABA_A receptor complex is a useful tool for identifying GABAergic neurons having GABA_A receptor complex-mediated synapses.     

Adenosine by activating A1 receptors prevents GABAA-mediated actions during hypoxia in the rat hippocampus

The relative contribution of adenosine and γ-aminobutyric acid (GABA) for the hypoxia-induced depression of field excitatory postsynaptic potentials in the CA1 area of rat hippocampal slices, was investigated. It is concluded that both adenosine and GABA, by activating A₁ and GABA_A receptors, could be responsible for the inhibition of synaptic transmission during hypoxia, but the action of endogenous GABA becomes evident only when the adenosine A₁ receptor action is precluded.     

GABAA-benzodiazepine receptors in the striatum are involved in the sedation produced by a moderate, but not an intoxicating ethanol dose in outbred Wistar rats

The role of the dorsal striatum in mediating the sedation produced by a moderate (0.75 g/kg) and an intoxicating (1.25 g/kg) EtOH dose was investigated in the open field by determining the capacity of direct intrastriatal injections of RY 008, a partial inverse agonist of the benzodiazepine (BDZ) receptor, to antagonize EtOH's effects. SR 95531, the competitive high-affinity GABA_A antagonist was used as a reference compound. Intrastriatal RY 008 (50, 500 ng) and SR 95531 (50 ng) antagonized the sedation produced by the 0.75 g/kg EtOH dose. However, RY 008 did not alter the sedation produced by the 1.25 g/kg dose. RY 008 alone was without effect. RY 008 also failed to negatively modulate GABAergic function at α1β2γ2 or α6β2γ2 receptor subtypes expressed in Xenopus oocytes. Intrastriatal modulation of the moderate EtOH dose was site specific: no antagonism by RY 008 after intraaccumbens infusions was observed. The results suggest that central GABA_A-BDZ receptors in the dorsal striatum play an important role in mediating the sedation produced by a moderate EtOH dose in the open field.     

Cytotoxic action of lindane in neocortical GABAergic neurons is primarily mediated by interaction with flunitrazepam-sensitive GABAA receptors

The cytotoxic action of the γ-isomer of hexachlorocyclohexane (γ-HCH; lindane) was studied in cultured mouse neocortical neurons by measurements of the reduction in mitochondrial function using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test. The cells were exposed to 30-300 μM lindane in the culture medium for different periods of time and lindane cytotoxicity was found to be time- and concentration-dependent. Lindane cytotoxicity could be ameliorated by addition of gamma aminobutyric acid (GABA) in a concentration-dependent manner but this effect of GABA was not blocked by bicuculline or picrotoxinin (PTX). Lindane induced cytotoxicity was also reduced by the GABA_A receptor agonists muscimol and THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). This effect was enhanced by the simultaneous presence of flunitrazepam but only at the highest lindane concentrations studied (200 and 300 μM). Flunitrazepam by itself had no effect on lindane-induced cytotoxicity. The protective effect of GABA plus flunitrazepam was blocked by the benzodiazepine receptor antagonist flumazenil and by the GABA_A antagonist bicuculline, suggesting the involvement of central benzodiazepine receptors allosterically coupled to the GABA recognition site at the GABA_A receptor. When 100 μM PTX was used to suppress the protective effect of GABA and flunitrazepam, a significant effect of PTX was observed only at 300 μM lindane. The GABA_B receptor agonist, baclophen, only marginally reduced the cytotoxic effect induced by the highest lindane concentrations. It is concluded that the cytotoxic action of lindane in neocortical neurons in culture is mediated primarily through an interaction with allosterically coupled GABA-benzodiazepine recognition sites at the GABA_A receptor. J. Neurosci. Res. 52:276–285, 1998. © 1998 Wiley-Liss, Inc.     

The expression of GABAA receptor subunits in the substantia nigra is developmentally regulated and region-specific

The substantia nigra pars reticulata (SNR) controls the spread of seizures. GABA_Aergic drug (agonist or antagonist) infusions into the SNR have age-specific and site-specific effects on flurothyl-induced seizures. Developmental and cell-specific regulation of GABA_A receptor subunit expression may be responsible for these specific effects. To test this hypothesis,in situ hybridization was used to examine regional expression of α1 and γ2L GABA_A receptor subunit mRNAs in the SNR during development. Distinct temporal and spatial patterns of expression were observed. In rats at postnatal days (PN) 21–60, fewer neurons were labeled with probes directed to α1 and γ2L subunits in SNR_anterior compared with SNR_posterior. In addition, neurons in SNR_anterior contained higher amounts of hybridization grains than in SNR_posterior. In PN 15 rats, the labeling of neurons was relatively diffuse throughout the anterior and posterior SNR regions with moderate amounts of hybridization grains for both subunits. The finding of age-related differential distribution of α1 and γ2L subunit mRNAs in the SNR suggests that GABA_A receptor heterogeneity may play a role in the age-specific and site-specific effects of GABA_Aergic agents on seizures in the SNR.

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Sommario La sostanza nera pars reticulata (SNR) controlla la propagazione delle crisi. Infusioni di GABA agonisti o antagonisti nella SNR hanno effetti specifici in rapporto all’età e alla sede sulle crisi indotte con flurothyl, per la probabile influenza di fattori regolatori dell’espressione di subunità del receptore GABA_A specificamente legati allo sviluppo e al tipo cellulare. L’ipotesi è stata verificata ana-lizzando l’ espressione regionale del mRNA delle subunità recettoriali GABA_A α1 e γ2L durante lo sviluppo. Diverse modalità di espressione temporale e spaziale sono state osservate. Nel ratto ad una età postnatale (PN) di 21–60 giorni, il numero di neuroni marcati con sonde dirette alle subunità α1 e γ2L è risultato inferiore rispetto alla posteriore. Inoltre, i neuroni di SNR anteriore contenevano una quantità più elevata di granuli di ibridizzazione rispetto a quelli di SNR posteriore. In ratti PN 15, invece, la marcatura di neuroni è risultata relativamente diffusa nell’ambito delle regioni anteriori e posteriori di SNR con una quantità moderata di granuli di ibridizzazione per entrambe le subunità. Il rilievo di una distribuzione differenziale età dipendente dell’mRNA delle subunità α1 e γ2L nella SNR suggerisce che un’eterogeneità recettoriale possa giocare uno specifico ruolo nel determinare gli effetti età e sito-specifici sulle crisi di agenti GABAergici iniettati nella SNR.

     

Heterogeneous distribution of benzodiazepine receptors in the human striatum: a quantitative autoradiographic study comparing the pattern of receptor labelling with the distribution of acetylcholinesterase staining

The distribution of benzodiazepine receptors in the human striatum was studied by quantitative autoradiography following in vitro labelling of cryostat sections with [3H]flunitrazepam, and the pattern of receptor-labelling was compared to the distribution of acetylcholinesterase (AChE) staining in adjacent sections. A heterogeneous pattern of benzodiazepine receptors was found in all regions of the striatum. The highest densities of receptors were seen in the ventral striatum (nucleus accumbens and olfactory tubercle), where very dense receptor patches aligned with both AChE-poor and AChE-rich regions. The dorsal striatum (caudate nucleus and putamen) contained lower concentrations of benzodiazepine receptors, but dense receptor patches were still evident (especially in the caudate nucleus) and these aligned with AChE-poor striosomes.     

Anxiogenic-like activity of L-655,708, a selective ligand for the benzodiazepine site of GABAA receptors which contain the alpha-5 subunit, in the elevated plus-maze test

GABA_A receptor is a transmembrane hetero-oligomeric protein which consists of five subunits, the combination of which confers unique pharmacological properties to the receptor. L-655,708 is a new ligand selective for GABA_A receptors containing an alpha-5 subunit. It is a partial inverse agonist that exhibits a 100-fold higher affinity for alpha-5 containing receptors, compared with alpha-1 containing receptors. The aim of this study was to examine the effects of L-655,708 (0.625–5 mg/kg ip) on anxiety tested in the elevated plus-maze in male mice. A number of classical parameters were collected: (a) Open arm duration; (b) Closed arm duration; (c) Central platform duration; (d) Open arm frequency; (e) Closed arm frequency; and (f) Total number of entries in the arms. Likewise, different ethological measures were also obtained (rears, head-dipping [HD], stretched attend posture [SAP] and grooming). Mice treated with L-655,708 showed a marked increase in the frequency of entries and the time spent in closed arms, as well as a reduction in the frequency of entries and the time spent in open arms, as compared with the control group. Unprotected HDs were also significantly decreased after treatment with the drug. Overall, these results indicate that L-655,708 could exhibit an anxiogenic-like profile in the elevated plus-maze test. This ligand is selective for GABA_A receptors containing an alpha-5 subunit, which is mainly expressed over the hippocampal formation, a region which has been involved in the modulation of anxiety.