Central-type benzodiazepines and the octadecaneuropeptide modulate the effects of GABA on the release of alpha-melanocyte-stimulating hormone from frog neurointermediate lobe in vitro

The involvement of the GABA-benzodiazepine receptor complex in the regulation of melanotropin secretion has been investigated using perfused frog neurointermediate lobes. The GABAA agonist 3-amino-1 propane sulfonic acid mimicked the biphasic effect of GABA on alpha-melanocyte-stimulating hormone secretion: a brief stimulation followed by an inhibition of melanotropin secretion. The GABAA antagonist SR 95531 (10(-4) M) inhibited both stimulation and inhibition of alpha-melanocyte-stimulating hormone release induced by GABA (10(-4) M). Since the inhibitory effect of baclofen (10(-4) M) was partially antagonized by SR 95531 (10(-4) M), it appears that the GABAergic control of alpha-melanocyte-stimulating hormone release is mainly achieved through activation of GABAA receptors. GABA-induced stimulation of alpha-melanocyte-stimulating hormone release was inhibited by tetrodotoxin (10(-5) M), an Na+ -channel blocker, or nifedipine (10(-5) M), a voltage-dependent Ca2+ -channel blocker, suggesting that Na+ and Ca2+ ions are involved in the stimulatory phase of GABA action. Only central-type benzodiazepine binding site agonists such as clonazepam (10(-4) M) modified alpha-melanocyte-stimulating hormone release. In fact, clonazepam (10(-7) to 10(-5) M) led to a dose-dependent potentiation of both GABA-induced stimulation and inhibition of alpha-melanocyte-stimulating hormone release. This potentiating effect was antagonized by the GABAA antagonist SR 95531 (10(-4) M) or by the central-type benzodiazepine binding site antagonist flumazenil (10(-4) M), whereas picrotoxin (10(-4) M) abolished only the stimulatory phase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reduction of inhibition by a benzodiazepine antagonist, Ro15-1788, in the rat hippocampal slice

The effects of extracellular applications of benzodiazepine agonists and the benzodiazepine antagonist, Ro15-1788, were investigated on pyramidal neurons in the CA1 region of rat hippocampal slices. The benzodiazepine agonists, chlordiazepoxide and diazepam, enhanced gamma-aminobutyrate synaptic inhibition, as tested by extracellular recordings during a paired-pulse inhibition paradigm. In contrast, Ro15-1788 (0.1-1 microM) depressed paired-pulse inhibition in a dose-dependent manner that suggested agonist activity at higher (10-100 microM) concentrations. Intracellular recordings from CA1 neurons showed that Ro15-1788 reduced both orthodromically and antidromically evoked inhibitory postsynaptic potentials. The reduction of the inhibitory postsynaptic potential probably resulted from a postsynaptic effect on the conductance mechanism of the inhibitory postsynaptic potential, since there were no changes in resting input resistance, the inhibitory postsynaptic reversal potential or the frequency of spontaneous inhibitory postsynaptic potentials. These data suggest that in the hippocampal slice preparation either (1) an endogenous benzodiazepine agonist exists that can be displaced by Ro15-1788 or (2) Ro15-1788 has inverse agonist activity.     

Ro 15-1788 is a potent antagonist of benzodiazepines in the olfactory cortex slice

The olfactory cortex slice from the guinea pig has been used to test the benzodiazepine antagonist, Ro 15-1788. Bath application of muscimol has a GABA-mimetic effect on the resting input conductance of these neurones. Benzodiazepines increase the potency of muscimol and increase the duration of postsynaptic inhibitory conductance. To measure the effect of muscimol, the input conductance was measured either directly using intracellular microelectrodes or by measuring its effect on the amplitude of the evoked compound potentials recorded from the slice surface after stimulating the lateral olfactory tract. The potentiation of postsynaptic inhibition produced by benzodiazepines was measured indirectly by their effect on the amplitude of the second of two evoked compound potentials. All of the potentiating effects of diazepam, clonazepam, flurazepam and chlordiazepoxide were blocked by Ro 15-1788 (0.01-10 mumol/l). Ro 15-1788 up to a concentration of 10 mumol/l had no effect on any of the synaptic or electrical responses when applied alone. General anaesthetics which also potentiate inhibition were unaffected by Ro 15-1788. It is concluded that Ro 15-1788 is a highly potent and specific benzodiazepine antagonist in this preparation.     

Contrasting regulation by GABA of the displacement of benzodiazepine antagonist binding by benzodiazepine agonists and purines

Gaba increases the potency of the benzodiazepines chlordiazepoxide, clonazepam, diazepam, nitrazepam and oxazepam, and the triazolopyridazine CL 218,872 in displacing specific binding of the benzodiazepine antagonist [3H]Ro 15-1788. In contrast, the potencies of the purines 1-methyl- and 1-ethylisoguanosine for benzodiazepine antagonist binding sites were decreased by GABA, while the potencies of inosine, hypoxanthine, 6-dimethylaminopurine, and the non-benzodiazepine anxiolytic, zopiclone, were unaltered by GABA. The results suggest that the purines and 'classical' benzodiazepine agonists may bind to different conformations or populations of receptors.     

Inhibition of adenosine accumulation by a CNS benzodiazepine antagonist (Ro 15-1788) and a peripheral benzodiazepine receptor ligand (Ro 05-4864)

Although benzodiazepines can inhibit adenosine uptake into central neurones, this effect is not antagonized by behaviourally effective 'benzodiazepine antagonists' such as Ro 15-1788. We now report that Ro 15-1788 and the 'peripheral' benzodiazepine ligand Ro 05-4864 themselves inhibit adenosine accumulation by rat brain synaptosomes. The inhibition of adenosine accumulation may thus underlie those behavioural effects of benzodiazepines which are mimicked but not antagonized by Ro 15-1788.     

Benzodiazepine Ro 15-1788: electrophysiological evidence for partial agonist activity

The effects of diazepam and Ro 15-1788 were assessed upon responses of mouse spinal cord (SC) neurons in cell culture to the amino acid neurotransmitters 4-aminobutyric acid (GABA) and S-glutamic acid. Diazepam (100 nM) enhanced GABA responses by 65 +/- 3% (113 cells), while Ro 15-1788 (100 nM) failed to alter GABA responses but reduced their enhancement by diazepam. Higher Ro 15-1788 concentrations (1 microM or 10 microM) enhanced GABA responses to a moderate extent, while blocking further enhancement of GABA by diazepam. Neither diazepam nor Ro 15-1788 affected glutamate responses or resting membrane potential or conductance of spinal cord neurons. These results provide electrophysiological support for partial agonist, rather than pure antagonist, activity of Ro 15-1788.     

Functional characterization of GABA(A) receptors in neonatal hypothalamic brain slice

The hypothalamus influences a number of autonomic functions. The activity of hypothalamic neurons is modulated in part by release of the inhibitory neurotransmitter GABA onto these neurons. GABA(A) receptors are formed from a number of distinct subunits, designated alpha, beta, gamma, delta, epsilon, and theta, many of which have multiple isoforms. Little data exist, however, on the functional characteristics of the GABA(A) receptors present on hypothalamic neurons. To gain insight into which GABA(A) receptor subunits are functionally expressed in the hypothalamus, we used an array of pharmacologic assessments. Whole cell recordings were made from thin hypothalamic slices obtained from 1- to 14-day-old rats. GABA(A) receptor-mediated currents were detected in all neurons tested and had an average EC(50) of 20 +/- 1.6 microM. Hypothalamic GABA(A) receptors were modulated by diazepam (EC(50) = 0.060 microM), zolpidem (EC(50) = 0.19 microM), loreclezole (EC(50) = 4.4 microM), methyl-6,7-dimethoxy-4-ethyl-beta-carboline (EC(50) = 7.7 microM), and 5alpha-pregnan-3alpha-hydroxy-20-one (3alpha-OH-DHP). Conversely, these receptors were inhibited by Zn(2+) (IC(50) = 70.5 microM), dehydroepiandrosterone sulfate (IC(50) = 16.7 microM), and picrotoxin (IC(50) = 2.6 microM). The alpha4/6-selective antagonist furosemide (10-1,000 microM) was ineffective in all hypothalamic neurons tested. The results of our pharmacological analysis suggest that hypothalamic neurons express functional GABA(A) receptor subtypes that incorporate alpha1 and/or alpha2 subunits, beta2 and/or beta3 subunits, and the gamma2 subunit. Our results suggest receptors expressing alpha3-alpha6, beta1, gamma1, and delta, if present, represent a minor component of functional hypothalamic GABA(A) receptors.     

Tyr-MIF-1 binding in brain is not altered by ligands selective for the GABAA/benzodiazepine receptor

Binding of benzodiazepines to the benzodiazepine gamma-aminobutyric acid (GABA) receptor-chloride channel complex has been shown to be altered by Tyr-MIF-1 (Tyr-Pro-Leu-Gly-NH2). This raised the possibility of allosteric binding interactions between Tyr-MIF-1 sites and the GABAA receptor complex. We tested this possibility in rat brain by examining the binding of Tyr-MIF-1 to brain membranes in the presence of clonazepam, GABA, a combination of clonazepam and GABA, RO15788, or picrotoxinin. None of the tested substances affected Tyr-MIF-1 binding. We also tested mouse cortex for changes in Tyr-MIF-1 binding in the presence of ligands that bind to the GABA/benzodiazepine/chloride channel complex. Clonazepam, flunitrazepam, RO15788, and picrotoxinin at concentrations ranging from 10(-13) to 10(-5) M, each in the absence or presence of GABA at concentrations ranging from 10(-9) to 10(-5) M, each in the absence or presence of GABA at concentrations ranging from 10(-9) to 10(-6) M, did not significantly alter the binding of Tyr-MIF-1. The results indicate that simple bidirectional allosteric interactions between Tyr-MIF-1 binding sites and benzodiazepine, GABA or chloride channel binding sites are not likely to be the mechanism by which Tyr-MIF-1 affects binding at this complex.     

EDPC: a novel high affinity ligand for the benzodiazepine site on rat GABA(A) receptors.

Rat recombinant alpha1beta2gamma2 gamma-aminobutyric acid type A (GABAA) receptors were functionally expressed in Xenopus laevis oocytes and analyzed for the action of EDPC (Ethyl 3-(1,3-dithian-2-yl)-1H-pyrrolo[2,3-c]pyridine-5-carboxylate) using electrophysiological techniques. EDPC inhibited GABA currents at low concentrations (IC50 approximately/= 2 nM). The inhibition by 100 nM EDPC could be reversed by 1 microM of the benzodiazepine antagonistflumazenil (Ro 15-1788), indicating a negative allosteric modulation via the benzodiazepine binding site. In line with this conclusion are radioactive ligand binding studies. EDPC inhibited the binding of 2 nM [3H]flunitrazepam to membranes from the cerebellum or the cortex with IC50 values of about 8 and 25 nM, respectively.     

Inhibition of type A GABA receptors by L-type calcium channel blockers

Modulation of type A GABA receptors (GABAA) by L-type Ca++ channel blockers was investigated. The dihydropyridines nifedipine and nitrendipine, and the phenylalkylamine verapamil inhibited recombinant rat alpha1beta2gamma2 receptors recorded from human embryonic kidney (HEK) 293 cells; nifedipine at low concentrations also elicited modest stimulatory effects on GABA-gated current. The IC50 for GABA current inhibition was lowest for nitrendipine (17.3 +/- 1.3 microM), so subsequent studies were focused on further exploring its mechanism and possible site of action. When co-applied with GABA, nitrendipine had minimal effects on initial current amplitude, but significantly enhanced current decay rate. Nitrendipine-mediated inhibition was subunit-selective, as its IC50 was 10-fold lower in alpha1beta2 receptors. Nitrendipine's effect in recombinant human alpha1beta2gamma2 receptors was similar (IC50=23.0 +/- 1.3 microM) to that observed in rat receptors of the same configuration, indicating the site of action is conserved in the two species. The inhibitory effects were dependent on channel gating, were independent of transmembrane voltage, and were also observed in GABAA receptors recorded from hypothalamic brain slices. The pharmacologic mechanism of inhibition by nitrendipine was non-competitive, indicating it does not act at the GABA binding site. Nitrendipine block was retained in the presence of the benzodiazepine antagonist flumazenil, indicating it does not interact at the benzodiazepine site. The actions of nitrendipine were not affected by a mutation (beta2T246F) that confers resistance to the channel blocker picrotoxin, and they were not altered in the presence of the picrotoxin site antagonist alpha-isopropyl-alpha-methyl-gamma-butyrolactone, demonstrating nitrendipine does not act at the picrotoxin site of the GABAA receptor. Possible interaction of nitrendipine with the Zn++ site was also eliminated, as mutation of beta2 H267 to A, which confers resistance to Zn++, had no effect on nitrendipine-mediated inhibition. Our data suggest some of the central effects of dihydropyridines may be due to actions at GABAA receptors. Moreover, the effects may be mediated through interaction with a novel modulatory site on the GABAA receptor.     

Aging does not alter the sensitivity of benzodiazepine receptors to GABA modulation

The effects of GABA on benzodiazepine receptor binding in cerebral cortical, hippocampal, and cerebellar membranes from 2-3 months old and 28-32 months old rats were studied. GABA modulation of agonist, antagonist, and inverse agonist binding to the receptor was examined using the displacement of 3H-Ro15-1788 by diazepam, Ro15-1788, and beta-carboline-3-carboxylate methyl ester, respectively, in the absence and presence of 100 microM GABA and 150 mM sodium chloride. GABA modulation was alike in old and young rats, with respect to the particular ligand and brain region. The results support the hypothesis that, in the brain regions studied, the allosteric modulation of benzodiazepine receptor binding by GABA remains intact as a function of aging.     

Selective blockade of benzodiazepine receptors by Ro 15-1788 prevents foot shock-induced decrease of low affinity gamma-aminobutyric acid receptors

The cerebral cortex of unstressed rats has a higher density of low affinity gamma-aminobutyric acid (GABA) receptors than that of stressed animals. Stress (handling or foot shock) produces a sudden decrease in the total number of low-affinity GABA receptors in the cerebral cortex of unstressed rats but leaves unchanged the density of GABA receptors in the cortex of stressed animals. The in vivo administration of Ro 15-1788 (30 mg/kg per os), a specific benzodiazepine receptor antagonist, completely prevents the effect of footshock on the low-affinity GABA receptors. The results suggest that (a) benzodiazepine recognition sites are involved in the action of stress on GABA receptors, and (b) stress may release an endogenous ligand for the benzodiazepine recognition site.     

Multiple effects of drugs acting on benzodiazepine receptors

The actions on [³H]noradrenaline release of chlordiazepoxide and FG 7142 were investigated in rat hippocampal synaptosomes. The release evoked by GABA, an action mediated by GABA_A receptors, was enhanced by chlordiazepoxide and depressed by FG 7142. K⁺-evoked release, however, was depressed by both chlordiazepoxide and FG 7142 and occurred in the absence of GABA. The actions on both GABA-evoked and K⁺-evoked release were blocked by Ro 15-1788. The results suggest that the distinction between agonist and inverse agonist applies to the GABA-dependent but not to the GABA-independent action of benzodiazepines.     

Modulation of decay kinetics and frequency of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents in hippocampal neurons.

Inhibitory postsynaptic currents mediated by spontaneous activation of GABAA receptors were studied using whole-cell voltage-clamp recordings in granule cells of the adult rat (postnatal day 60+) dentate gyrus in 400-microns-thick coronal half-brain slices maintained at 34-35 degrees C. The average amplitude of spontaneous inhibitory postsynaptic currents remained constant during a given recording period (i.e. no rundown was noted). The spontaneous currents had an average conductance between 200-400 pS, were mediated by Cl- flux through GABAA receptor/channels since they reversed at the Cl- equilibrium potential and were blocked by bicuculline or picrotoxin. Their mono-exponential decay time-constants (range: 4.2-7.2 ms) were prolonged by midazolam and pentobarbital in a dose-dependent manner. The effect of midazolam was reversed by the benzodiazepine receptor antagonist flumazenil (RO 15-1788) which, by itself, had no effect on the decay time-constant. The decay time-constant was also dependent on membrane voltage and on temperature. A 132-mV change in membrane potential produced an e-fold prolongation of the decay while the Q10 (between 22-37 degrees C) of the decay rate was 2.1. Within a given neuron, the frequency of spontaneous GABAergic events was remarkably constant over long time-periods, though the mean frequency among different cells showed large variability. Spontaneous miniature inhibitory postsynaptic currents also persisted under experimental conditions such as the presence of extracellular tetrodotoxin (1 microM), Cd2+ (200 microM) or lowered extracellular Ca2+/elevated Mg2+, which effectively abolished all stimulus-evoked GABAergic neurotransmission. The frequency of tetrodotoxin-resistant miniature events was increased by elevating extracellular K+ concentration and was diminished by the GABAB receptor agonist (-)baclofen only at a dose (50 microM) which was an order of magnitude larger than that required to depress stimulus-evoked responses. These findings are consistent with different mechanisms being responsible for the spontaneous and stimulus-evoked release of GABA from interneuron terminals and also identify pre- and postsynaptic modulatory factors of the endogenous, action-potential-independent, GABAergic neurotransmission as being important determinants of the excitability level of mammalian CNS neurons.     

Labelling of high (D-2 receptor) and low affinity sites by [3H]domperidone in homogenates of the corpus striatum of the rat

The effects of several purines and the purine uptake inhibitor, dipyridamole, on the binding, to rat brain membranes, of 4 benzodiazepines with different pharmacological specificities were studied. While all purines tested displaced the binding of [3H](+)-3-methyl-clonazepam and [3H]Ro15-1788, selective agonist and antagonist ligands respectively for 'central' benzodiazepine receptors, purines had little or no affinity for [3H]Ro5-4864 'peripheral'-type binding sites in brain, heart or kidney. These results suggest that purines interact with a pharmacologically relevant class of central benzodiazepine 'receptors', and not with central and peripheral 'acceptor' sites labelled by the benzodiazepine Ro5-4864.     

Role of fructose 2,6-bisphosphate in the regulation of glycolysis in various types of cultivated brain cell

Low doses of Ro 15-1788 and CGS 8216 were without effect on variable-interval self-stimulation, but completely abolished the enhancement of responding produced by chlordiazepoxide (5 mg/kg). Higher doses of Ro 15-1788, unlike other benzodiazepine receptor antagonists, produced an increase in response rates similar to that found after chlordiazepoxide. This result is consistent with its suggested action as a partial agonist. The combination of a high (benzodiazepine-like) dose of Ro 15-1788 with chlordiazepoxide produced a depression of responding similar to that seen with high doses of benzodiazepines. High doses of CGS 8216 produced a depression of self-stimulation, which was not reversed by chlordiazepoxide (5 mg/kg). Thus, the present procedure is able to distinguish contrasting behavioural effects of benzodiazepine antagonists.     

GABAA receptor-mediated tonic currents in substantia gelatinosa neurons of rat spinal trigeminal nucleus pars caudalis

In the present study, we describe GABAA receptor-mediated tonic inhibitory currents in the substantia gelatinosa (SG) region of rat spinal trigeminal nucleus pars caudalis (Vc). The GABA(A) receptor-mediated tonic currents were identified by bath-application of the GABAA receptor antagonists, picrotoxin (1mM), SR95531 (100microM) and bicuculline (100microM). All three antagonists completely blocked outward spontaneous (phasic) inhibitory postsynaptic currents, but only picrotoxin and bicuculline induced a significant (>5pA) inward shift of holding currents at a holding potential (Vh) of 0mV in 60-70% of SG neurons, revealing the existence of tonic outward currents. The tonic currents were resistant to further the blockades of glycine receptors or those in addition to glutamate receptors and voltage-dependent sodium channels. An acute bath-application of THDOC (0.1microM), the stress-related neurosteroid, did enhance tonic currents, but only in a small population of SG neurons. In addition, slices incubated with THDOC for 30min increased the probability of neurons with significant tonic currents. The GABAergic tonic inhibition demonstrated in this study may play a significant role in the sensory processing system of the Vc.     

The role of pre-synaptic GABA and benzodiazepine receptors in the control of noradrenaline release in rat hippocampus

Noradrenaline release from synaptosomes of rat hippocampus is modulated by both GABA and benzodiazepines. GABAA and GABAB receptors are present on these nerve terminals, and agonists at these receptors enhance spontaneous release and depress K+ -evoked release, respectively. Chlordiazepoxide has a dual effect: it enhances the action of GABA at GABAA receptors and depresses K+ -evoked release of [3H]noradrenaline in the absence of GABA. The mechanism of these actions and their in vivo role are discussed.     

5-HT1B receptor-mediated presynaptic inhibition of GABA release in the suprachiasmatic nucleus

The suprachiasmatic nucleus (SCN) receives a dense serotonergic innervation that modulates photic input to the SCN via serotonin 1B (5-HT1B) presynaptic receptors on retinal glutamatergic terminals. However, the majority of 5-HT1B binding sites in the SCN are located on nonretinal terminals and most axonal terminals in the SCN are GABAergic. We therefore tested the hypothesis that 5-HT1B receptors might also be located on SCN GABAergic terminals by examining the effects of the highly selective 5-HT1B receptor agonist CP-93,129 on SCN miniature inhibitory postsynaptic currents (mIPSCs). Whole cell patch-clamp recordings of mIPSCs were obtained from rat and mouse SCN neurons in hypothalamic slices. Using CsCl-containing microelectrodes with QX314, we isolated mPSCs that were sensitive to the GABAA receptor antagonist, bicuculline. Bath application of CP-93,129 (1 microM) decreased the frequency of mIPSCs by an average of 22% (n = 7) in rat SCN neurons and by an average of 30% (n = 8) in mouse SCN neurons with no clear effect on mIPSC amplitude. In mice lacking functional 5-HT1B receptors, CP-93,129 (1 microM) had no clear effect on the frequency or the amplitude of mIPSCs recorded in any of the cells tested (n = 4). The decrease in the frequency of mIPSCs of SCN neurons produced by the selective 5-HT1B receptor agonist CP-93,129 is consistent with the interpretation that 5-HT1B receptors are located on GABA terminals in the SCN and that 5-HT inhibits GABA release via a 5-HT1B presynaptic receptor-mediated mechanism.     

GABAA and GABAB receptors on porcine pars intermedia cells in primary culture: functional role in modulating peptide release

A primary culture of porcine pars intermedia cells with particularly high yields has been developed. The cells, grown in monolayers, secrete the pro-opiomelanocortin-derived peptide alpha-melanocyte-stimulating hormone over several weeks. The patch-clamp technique has been used to demonstrate the presence of gamma-aminobutyrateA (GABAA) receptors on the cells. GABA or the selective GABAA receptor agonist isoguvacine produced a depolarizing increase in chloride conductance that desensitized rapidly. The response was antagonized by bicuculline and by the aminopyridazine derivative of GABA (SR 95103), a novel GABAA receptor antagonist. The effects of specific agonists for each receptor were tested on peptide release from cells maintained in a perfusion system. Isoguvacine (10 microM) potentiated Ba2+-evoked release of alpha-melanocyte-stimulating hormone, whereas (-)-baclofen (50 microM) decreased both basal and stimulated hormone release. This negative effect on peptide secretion was reproduced when GABA (50 microM) was perfused in the presence of bicuculline (10 microM) to block GABAA receptor activation. The possible mechanisms underlying these GABAA and GABAB effects on stimulus-secretion coupling in this neuroendocrine model are discussed.