Fast inhibitory postsynaptic potentials and responses to inhibitory amino acids of sympathetic preganglionic neurons in the adult cat.

Intracellular recordings were obtained from sympathetic preganglionic neurons (SPNs) of the intermediolateral nucleus (IML) in slices of upper thoracic spinal cord of the anesthetized cat. A total of 44 neurons was studied. Single shock stimulation of an area of white matter dorsolateral to the IML, close to the recording electrode (< 0.5 mm), evoked fast IPSPs with rise time of 3.8 ms and 1/2 decay time of 14.7 ms (n = 12). In 17 other cells only fast EPSPs were recorded but, after suppression of the EPSPs by the excitatory amino acid receptor antagonists CNQX (20 microM) and APV (100-250 microM), fast IPSPs were unmasked. The IPSP reversed polarity at -63 mV (-67 mV in the presence of CNQX and APV). The reversal potential shifted to a less negative value when the extracellular chloride concentration was reduced. The IPSP was reversibly abolished by the GABAA receptor antagonist bicuculline in 32% of the cells, by the glycine receptor antagonist strychnine in 47% of the cells and by the combination of the two in 21% of the cells. The IPSP was abolished by TTX (0.5 microM), had constant latency and showed no failures during high frequency stimulation. The IPSP presumably resulted from the excitation of inhibitory axons and/or inhibitory neuron somata with monosynaptic connections to the SPN. Glycine and GABA (1-3 mM) produced hyperpolarization associated with decreased membrane resistance. Sixty-nine percent of cells responded to both agonists, 19% to glycine only and 12% to GABA only. The GABAB agonist baclofen (5 microM) had no effect.(ABSTRACT TRUNCATED AT 250 WORDS)     

Actions of (-)-baclofen on rat dorsal horn neurons.

The actions of a gamma-aminobutyric acid B (GABAB) agonist, (-)-baclofen, on the electrophysiological properties of neurons and synaptic transmission in the spinal dorsal horn (laminae I-IV) were examined by using intracellular recordings in spinal cord slice from young rats. In addition, the effects of baclofen on the dorsal root stimulation-evoked outflow of glutamate and aspartate from the spinal dorsal horn were examined by using high performance liquid chromatography (HPLC) with flourimetric detection. Superfusion of baclofen (5 nM to 10 microM) hyperpolarized, in a stereoselective and bicuculline-insensitive manner, the majority (86%) of tested neurons. The hyperpolarization was associated with a decrease in membrane resistance and persisted in a nominally zero-Ca2+, 10 mM Mg(2+)- or a TTX-containing solution. Our findings indicate that the hyperpolarizing effect of baclofen is probably due to an increase in conductance to potassium ions. Baclofen decreased the direct excitability of dorsal horn neurons, enhanced accommodation of spike discharge, and reduced the duration of Ca(2+)-dependent action potentials. Baclofen depressed, or blocked, excitatory postsynaptic potentials evoked by electrical stimulation of the dorsal roots. Spontaneously occurring synaptic potentials were also reversibly depressed by baclofen. Whereas baclofen did not produce any consistent change in the rate of the basal outflow of glutamate and aspartate, the stimulation-evoked release of the amino acids was blocked. The present results suggest that baclofen, by activating GABAB receptors, may modulate spinal afferent processing in the superficial dorsal horn by at least two mechanisms: (1) baclofen depresses excitatory synaptic transmission primarily by a presynaptic mechanism involving a decrease in the release of excitatory amino acids, and (2) at higher concentrations, the hyperpolarization and increased membrane conductance may contribute to the depressant effect of baclofen on excitatory synaptic transmission in the rat spinal dorsal horn.     

Presynaptic inhibition by muscimol through GABAB receptors

The gamma-aminobutyric acid type A (GABAA) receptor agonist muscimol is widely used as a tool for reducing neuronal activities particularly in experiments in vivo. At the synapse formed by the calyx of Held in the rat brainstem slice, the GABAA receptor agonist muscimol (> 10 microM) attenuated the amplitude of excitatory post synaptic currents (EPSCs) accompanied by an increase in the coefficient of variation of EPSCs, suggesting its presynaptic inhibitory effect. This muscimol effect was not affected by bicuculline but occluded the presynaptic inhibitory effect of the GABAB receptor agonist baclofen and was abolished by the type B GABA (GABAB) receptor-specific antagonist (+)-5, 5-dimethyl-2-morpholineacetic acid (SCH 50911; 20 microM). We conclude that muscimol activates presynaptic GABAB receptors thereby attenuating synaptic transmission.     

G-protein-coupled GABAB receptors inhibit Ca2+ channels and modulate transmitter release in descending turtle spinal cord terminal synapsing motoneurons

Presynaptic gamma-aminobutyric acid type B receptors (GABA(B)Rs) regulate transmitter release at many central synapses by inhibiting Ca(2+) channels. However, the mechanisms by which GABA(B)Rs modulate neurotransmission at descending terminals synapsing on motoneurons in the spinal cord remain unexplored. To address this issue, we characterized the effects of baclofen, an agonist of GABA(B)Rs, on the monosynaptic excitatory postsynaptic potentials (EPSPs) evoked in motoneurons by stimulation of the dorsolateral funiculus (DLF) terminals in a slice preparation from the turtle spinal cord. We found that baclofen depressed neurotransmission in a dose-dependent manner (IC(50) of approximately 2 microM). The membrane time constant of the motoneurons did not change, whereas the amplitude ratio of the evoked EPSPs in response to a paired pulse was altered in the presence of the drug, suggesting a presynaptic mechanism. Likewise, the use of N- and P/Q-type Ca(2+) channel antagonists (omega-conotoxin GVIA and omega-agatoxin IVA, respectively) also depressed EPSPs significantly. Therefore, these channels are likely involved in the Ca(2+) influx that triggers transmitter release from DLF terminals. To determine whether the N and P/Q channels were regulated by GABA(B)R activation, we analyzed the action of the toxins in the presence of baclofen. Interestingly, baclofen occluded omega-conotoxin GVIA action by approximately 50% without affecting omega-agatoxin IVA inhibition, indicating that the N-type channels are the target of GABA(B)Rs. Lastly, the mechanism underlying this effect was further assessed by inhibiting G-proteins with N-ethylmaleimide (NEM). Our data show that EPSP depression caused by baclofen was prevented by NEM, suggesting that GABA(B)Rs inhibit N-type channels via G-protein activation.     

Activation of GABAB receptors causes presynaptic inhibition at synapses between muscle spindle afferents and motoneurons in the spinal cord of bullfrogs

Baclofen, a specific GABAB receptor agonist, was used to study the functional role of activation of GABAB receptors in synaptic transmission between muscle spindle afferents and motoneurons in the isolated spinal cord of bullfrogs. (+/-)-Baclofen (5 microM) reversibly reduced the amplitude of the excitatory postsynaptic potential (EPSP) evoked by simulation of various brachial muscle nerves and recorded extracellularly from the ventral root by 47% without shortening the falling phase of the EPSP. Neither the time course nor the amplitude of the action potentials in the sensory afferents was affected. Thus, baclofen caused synaptic inhibition without reducing either the potential change occurring in the muscle sensory afferents or the motoneuronal membrane resistance. Quantal analysis, performed using a deconvolution technique, of the monosynaptic EPSPs in brachial motoneurons evoked by activity in single triceps muscle spindle afferents showed that transmission at these synapses was quantal, and baclofen lowered the quantal content without altering the quantal size. Furthermore, quantal analysis of the electrical component of these unitary EPSPs showed that it did not fluctuate in amplitude, either in normal saline or with baclofen. The inhibition produced by activation of GABAB receptors is therefore presynaptic but is not likely to be caused by conduction failures in the sensory terminals.     

Effects of baclofen and bicuculline on inhibition in the fascia dentata and hippocampus regio superior

The effects of microiontophoretically applied baclofen, bicuculline and phaclofen were studied on evoked field responses, paired-pulse (PP) plasticity and single-unit activity of dentate granule cells (DGCs) and CA1 pyramidal cells (PCs) in anesthetized rats. The GABAB agonist, baclofen, increased population spike (PS) amplitudes in the dentate evoked by perforant path stimulation but decreased PS amplitudes in CA1 evoked by Schaffer collateral stimulation, whereas the GABAA antagonist, bicuculline, increased PS amplitudes in both regions. Neither baclofen nor bicuculline had significant effects on dendritically recorded population excitatory postsynaptic potentials (EPSPs) in the dentate or CA1 evoked by stimulation of their respective afferents. Control PP curves in the dentate revealed a triphasic response of inhibition/potentiation/inhibition, whereas control PP curves in CA1 manifested a biphasic response of inhibition/potentiation of test/conditioned PS amplitudes. Baclofen and bicuculline reversed the early and late phases of PP inhibition in the dentate and the early phase of PP inhibition in CA1. The GABAB antagonist, phaclofen, selectively reversed the effects of baclofen on PP inhibition in both the dentate and CA1. Whereas baclofen had no effect, bicuculline incre sed and phaclofen decreased DGC single-unit spontaneous firing rate, while baclofen decreased and bicuculline and phaclofen increased PC firing rate. These results support and extend studies suggesting that GABAergic feedback inhibition of DGCs and PCs is mediated by postsynaptic GABAA receptors and feedback inhibition of PCs is mediated by postsynaptic GABAB receptors. Our results also provide significant new evidence suggesting that postsynaptic inhibition in the dentate is not regulated by GABAB receptors and that feedback and feedforward inhibition of DGCs and PCs is regulated by presynaptic GABAB receptors located on GABAergic interneurons.     

Functional GABAB receptors are present in guinea pig nodose ganglion cell bodies but not in peripheral mechanosensitive endings

The effects of the GABAB-selective agonist baclofen were studied on guinea pig nodose ganglion neurones using grease gap and intracellular recording techniques, and on peripheral mechanosensitive endings in the guinea pig oesophagus and stomach with extracellular recordings. GABA dose-dependently reduced the amplitude of the compound action potential of C-type neurones (C spikes, EC50 = 30.9 microM), which was prevented by the GABAA antagonist bicuculline (10 microM). The GABAB agonist baclofen (1-300 microM) did not produce any significant effect on the amplitude of C spikes. In microelectrode studies, baclofen (100 microM) evoked hyperpolarisation (by 2.53 +/- 0.51 mV, n = 6, N = 5) in a subset of nodose neurones (6 out of 26, N = 18). In seven out of eight neurones (N = 8) with a slow after-hyperpolarisation following action potentials, baclofen significantly inhibited its amplitude by 19 +/- 4% (n = 7, p < 0.05). GABA (100 microM) evoked a depolarisation of 9.3 +/- 2.4 mV (10 nodose neurones, N = 9, p < 0.05) associated with a decrease in input impedance of 49 +/- 12% (N = 4, p < 0.05). Baclofen (100-200 microM) did not affect either spontaneous or stretch-evoked firing of distension-sensitive vagal mechanoreceptors of the guinea pig oesophagus and stomach but did inhibit mechanoreceptors in the ferret oesophagus. Antibodies to GABAB receptor 1a splice variants labelled most of the neurones and numerous fibres in the guinea pig nodose ganglion while antibodies to GABAB receptor 1b splice variants stained only nerve cell bodies. There were numerous nerve fibres showing GABAB receptor 1a- and 1b-like immunoreactivity in the myenteric plexus in the guinea pig oesophagus and stomach but not in anterogradely labelled extrinsic vagal nerve fibres. The result indicates that most guinea pig C-type nodose ganglion neurones have GABAB receptors on their cell bodies but their density on distension-sensitive peripheral endings is too low to allow modulation of mechanotransduction. There is a significant species-dependent difference in the expression of GABAB receptors on peripheral vagal mechanosensitive endings.     

Local GABAergic modulation of acetylcholine release from the cortex of freely moving rats

Cortical perfusion with GABA agonists and antagonists modulates the spontaneous release of cortical acetylcholine and GABA in freely moving rats. Twenty-four hours after implantation of a dialysis fibre, cerebral cortex spontaneously released acetylcholine (3.8 +/- 0.2 pmol/10 min) and GABA (6.6 +/- 0.4 pmol/10 min) at a stable rate. Local administration of GABA (1 or 5 mM) or the GABAA agonist muscimol (25 or 50 microM) had no effect on the spontaneous release of acetylcholine. However, bicuculline (1-25 microM), a GABAA antagonist, added to the dialysis perfusate, elicited a concentration-dependent increase of acetylcholine release to approximately double that of control. This effect of bicuculline (25 microM) was completely prevented by coperfusion with muscimol (50 microM). Local administration of the GABAB receptor agonist baclofen (10 or 50 microM) elicited a concentration-dependent increase in spontaneous acetylcholine release with a maximal increase of about 60%. Intracortical administration of baclofen also decreased the spontaneous release of GABA. The GABAB receptor antagonist CGP 35348 (1 mM), administered alone for 20 min through the dialysis fibre, was without effect on spontaneous acetylcholine release; however, it completely blocked both the baclofen-induced increase in acetylcholine release and the decrease in GABA release. These results suggest that cortically released GABA exerts a tonic influence on cholinergic activity.     

Activation of GABAA receptors causes presynaptic and postsynaptic inhibition at synapses between muscle spindle afferents and motoneurons in the spinal cord of bullfrogs

The functional role of GABAA receptors in inhibition of synaptic transmission between muscle spindle afferents and spinal motoneurons was studied in the isolated spinal cord of bullfrogs. Extracellular recording from the ventral root showed that activation of GABAA receptors by muscimol (primarily a GABAA receptor agonist) at 50 microM produced a 38% reduction in the amplitude of the excitatory postsynaptic potential (EPSP) evoked by stimulation of triceps muscle sensory afferents and a 66% reduction in the EPSP half-width, suggesting a large increase in the conductance of the motoneuronal membrane. Quantal analysis of unitary triceps EPSPs recorded intracellularly from motoneurons showed that muscimol reduced the quantal content of release (presynaptic inhibition). In addition, muscimol decreased the quantal size (postsynaptic inhibition) when the postsynaptic conductance change was large. Because the effects of muscimol were completely and reversibly blocked by 100 microM bicuculline (a specific GABAA receptor antagonist), both the pre- and the postsynaptic inhibition caused by muscimol were due to activation of GABAA receptors. Activation of GABAA receptors thus causes both pre- and postsynaptic inhibition of synaptic transmission between muscle afferents and spinal cord motoneurons in the frog.     

An intracellular study of the synaptic input to sympathetic preganglionic neurones of the third thoracic segment of the cat

In chloralose anaesthetized, paralyzed and artificially ventilated cats intracellular recordings were obtained from sympathetic preganglionic neurones (SPN) of the third thoracic segment of the spinal cord identified by antidromic stimulation of the white ramus T3. The synaptic input to SPNs was assessed, in cats with intact neuraxis or spinalized at C3, by electrical stimulation of segmental afferent fibres in intercostal nerves and white rami of adjacent thoracic segments and by stimulation of the ipsi- and contralateral dorsolateral funiculus and of the dorsal root entry zone of the cervical spinal cord. In both preparations SPNs showed on-going synaptic activity which predominantly consisted of excitatory post-synaptic potentials (EPSPs). Inhibitory post-synaptic potentials (IPSPs) were rarely observed. EPSPs were single step (5 mV) or, less frequently, large (up to 20 mV) summation EPSPs. The proportion of SPNs showing very low levels of on-going activity was markedly higher in spinal than in intact cats. Stimulation of somatic and sympathetic afferent fibres evoked early EPSPs (amplitude 3 mV, latency 5-22.3 ms), and late, summation EPSPs (amplitude up to 20 mV, latency 27-55 ms). Early and late EPSPs were evoked in nearly all SPNs in which this synaptic input was tested in the intact preparation (from 79-93% of the SPNs). In spinal cats, early EPSPs were evoked in 88% of the SPNs, whereas late EPSPs were recorded only in half of the neurones. No evidence for a monosynaptic pathway from these segmental afferent fibres to SPNs was obtained. In both intact and spinal cats, stimulation of the dorsolateral funiculus evoked early and late EPSPs in SPNs. Late EPSPs were recorded in 70% and 37% of the SPNs in intact and spinal cats, respectively. Early EPSPs, however, were evoked in all neurones. The early EPSPs evoked by stimulation of the dorsolateral funiculus had several components which are suggested to arise from stimulation of descending excitatory pathways with different conduction velocities. The following conduction velocities were calculated in intact (spinal) cats: 9.5-25 m/s (7.8-13.2 m/s), 5.7-9.5 m/s (5.5-7.8 m/s), 3.8-5.7 m/s (3.2-5.5 m/s), and 2.6-3.8 m/s (2.1-3.2 m/s). EPSPs of these various groups were elicited in a varying percentage in SPNs. EPSPs of the most rapidly conducting pathway were subthreshold for the generation of action potentials; some EPSPs of this group had a constant latency suggesting a monosynaptic pathway to SPNs. Stimulation of the dorsal root entry zone at the cervical level yielded essentially the same results as stimulation of the dorsolateral funiculus.(ABSTRACT TRUNCATED AT 400 WORDS)     

3-Aminopropanephosphinic acid is a potent agonist at peripheral and central presynaptic GABAB receptors

The actions of the GABA analog 3-aminopropanephosphinic acid (3-APA) were studied in the guinea-pig isolated ileal preparation and at synapses between cultured rat hippocampal neurons. Like the GABAB receptor agonist, baclofen, 3-APA inhibited the electrically evoked ileal twitch. The EC50 for 3-APA was 0.8 microM; the EC50 for baclofen was 9 microM. In addition, the depressant responses to 3-APA and baclofen were blocked by the GABAB receptor antagonists phaclofen, saclofen, 2-hydroxy-saclofen and delta-aminovaleric acid. 3-APA also mimicked the presynaptic action of baclofen at GABAergic synapses between embryonic rat hippocampal neurons in culture. 3-APA reduced the amplitude of inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) by greater than 50% at a concentration of 1 microM, while baclofen reduced synaptic transmission to a similar degree at 10 microM. 3-APA did not alter membrane conductance, nor did the drug alter postsynaptic responses to GABA. These data show that 3-APA is a potent agonist at presynaptic GABAB receptors in the periphery and on GABAergic neurons from the central nervous system. The activity of 3-APA at central postsynaptic GABAB receptors remains to be studied.     

gamma-Aminobutyric acid, through GABAA receptors, inhibits the potassium-stimulated release of calcitonin gene-related peptide- but not that of substance P-like material from rat spinal cord slices.

Superfusion of slices of the dorsal zone of the lumbar enlargement with an artificial cerebrospinal fluid was used to investigate the possible modulation by GABA receptor ligands of the in vitro release of calcitonin gene-related peptide- and substance P-like materials (CGRPLM and SPLM) from the rat spinal cord. Whereas the spontaneous outflow of both peptides remained unaffected, the K+ (30 mM)-evoked overflow of CGRPLM could be partially inhibited (approx. -30%) by GABA (1 microM-0.1 mM) and muscimol (10 microM-0.1 mM) but not by baclofen (1-10 microM). Bicuculline methiodide (1 microM) completely prevented the inhibition by GABA (1 microM) and muscimol (10 microM) as expected from an action through GABAA receptors. By contrast, the K(+)-evoked SPLM overflow was altered neither by GABA nor muscimol and baclofen. These data further support that GABA exerts a presynaptic inhibitory control of (CGRP-containing) primary afferent fibres within the rat dorsal horn.     

Interaction between GABAergic neurotransmission and rat hypothalamic corticotropin-releasing hormone secretion in vitro

Corticotropin-releasing hormone (CRH) has been considered a major coordinator of the overall physical and behavioral response to stress. Moreover, prolonged hypersecretion of CRH has been implicated in the pathogenesis of disorders characterized by anxiety and/or depression. Drugs acting through the gamma-aminobutyric acid/benzodiazepine (GABA/BZD) receptor system have anxiolytic and/or antidepressant properties whereas benzodiazepine inverse agonists cause anxiety and stimulate the pituitary-adrenal axis in vivo. To examine the involvement of the GABA/BZD system in the regulation of hypothalamic CRH secretion, we studied the effects of various agonists and antagonists of GABAA and GABAB receptors using a sensitive rat hypothalamic organ culture with radioimmunoassayable CRH (IR-rCRH) as endpoint. The GABAA and GABAB receptor agonist GABA inhibited serotonin (5-HT)-induced IR-rCRH secretion from 10(-9) to 10(-6) M, but failed to do so at 10(-5) M. The GABAA receptor agonist muscimol was a weak inhibitor of 5-HT-induced IR-rCRH secretion, being effective only at the concentration of 10(-6) M. In contrast, the specific GABAB receptor agonist baclofen was able to inhibit 5-HT-induced IR-rCRH secretion from 10(-7) to 10(-5) M. The rank of potency was thus, GABA much greater than baclofen greater than muscimol. Bicuculline, a GABAA receptor antagonist, partially reversed the inhibitory effects of GABA. Diazepam, a classic benzodiazepine which interacts with the benzodiazepine-site of the GABAA receptor complex, inhibited 5-HT-induced IR-rCRH secretion from 3.3 X 10(-9) to 10(-5) M, an effect that could be reversed by the BZD inactive ligand Ro15-1788.(ABSTRACT TRUNCATED AT 250 WORDS)     

Baclofen: Effects on evoked field potentials and amino acid neurotransmitter release in the rat olfactory cortex slice

A study has been made of the in vitro effects of (+/-)- and (-)-baclofen on the evoked field potentials and release of endogenous amino acid neurotransmitter candidates (aspartate, glutamate, GABA and possibly taurine) which accompany electrical stimulation of the excitatory input to the olfactory cortex slice, the lateral olfactory tract. Baclofen appears to reduce the excitatory input to the GABA-utilizing inhibitory interneurones; this action was manifest as a drug-induced abolition of the field potential known as the P-wave (IC50 for (-)-baclofen, 1.7 +/- 0.4 microM) together with a simultaneous reduction in the synaptically evoked release of aspartase and glutamate from the cut surface of slices. Both these actions of baclofen exhibited concentration dependence and stereospecificity and were not antagonized by picrotoxin (25 microM) thereby suggesting that they are directly related. The consequence of this action of baclofen was the abolition of GABA-mediated presynaptic and postsynaptic inhibition together with their respective field potential correlates, the late N- and I-waves. (+/-)-Baclofen (5 and 25 microM) also inhibited the potassium-evoked release of aspartate and glutamate from small cubes of tissue but, except at a high concentration (1 mM), had no effect on GABA release. Baclofen (up to 1 mM) did not affect transmission either at the lateral olfactory tract-superficial pyramidal cell synapse, a site where aspartate is the likely neurotransmitter, or at the superficial pyramidal cell collateral-deep pyramidal cell excitatory synapse. It is proposed that: (i) the actions of baclofen on the olfactory cortex are the result of inhibition of aspartate and glutamate release, probably from deep pyramidal cell collaterals; and (ii) not all neurones utilizing excitatory amino acids as their neurotransmitters are subject to the inhibitory action of baclofen.     

GABAA receptor mediated elevation of Ca2+ and modulation of gonadotrophin-releasing hormone action in alphaT3-1 gonadotropes

gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter in the CNS, mediating fast inhibitory synaptic transmission, by activating GABAA receptors. However, these GABA-gated Cl- channels can also be excitatory, causing depolarization, and increasing Ca2+ entry via voltage-operated Ca2+ channels (VOCCs). Evidence exists for excitatory ionotropic GABA receptors in anterior pituitary cells, including gonadotropes, but these have not been directly characterized and their pharmacology remains controversial. Here we have measured the cytosolic Ca2+ concentration ([Ca2+]i) in alphaT3-1 gonadotropes, to test for expression of excitatory GABA receptors. The GABAA agonists, GABA and muscimol, both caused rapid, robust and dose-dependent increases in [Ca2+]i (EC50 values 2.7 and 1 microM), whereas the GABAB agonist, baclofen, did not. The GABAA antagonist, bicuculline, inhibited muscimol's effect, whereas the GABAB antagonist, phaclofen, did not. The neuroactive steroid 5alpha-pregnan-3alpha-ol-11,20-dione (an allosteric activator of GABAA receptors) increased [Ca2+]i, and this effect, like that of muscimol, was inhibited by picrotoxin. The muscimol effect on [Ca2+]i was blocked by the VOCC antagonist, nifedipine, or by Ca2+-free medium. When cells were pretreated with muscimol this increased the spike phase of the [Ca2+]i response to subsequent stimulation with gonadotropin-releasing hormone (GnRH). Similar amplification was seen in muscimol-pretreated cells stimulated with GnRH in Ca2+-free medium, but not when cells were pretreated with muscimol in Ca2+-free medium. The amplification was not, however, GnRH receptor-specific, because the spike response to ionomycin was also increased by muscimol pretreatment. These data provide the first direct evidence for expression of excitatory GABAA receptors, and the first demonstration of acute steroid effects, on GnRH-responsive pituitary cells. They also reveal a novel mechanism by which GABAA activation modulates GnRH action, raising the possibility that this may also influence gonadotrophin secretion from non-immortalized gonadotropes.     

5-hydroxytryptamine1B receptors block the GABAB synaptic potential in rat dopamine neurons.

Intracellular recordings were made from presumed dopamine-containing neurons in slices cut from the midbrain of the rat. Focal electrical stimulation produced a hyperpolarizing synaptic potential that was reduced by 75-95% by the GABAB-receptor antagonist 2-hydroxysaclofen (300 microM). 5-HT (3-100 microM) reduced the amplitude of the GABAB synaptic potential by 20-74%, with a 50% reduction at 10 microM, but did not reduce the amplitude of synaptic potentials mediated by GABAA receptors. 5-HT acted presynaptically because hyperpolarizations produced by exogenously administered GABA (1 mM) in picrotoxin (100 microM) were not affected by 5-HT (30 microM). (+/-)-Cyanopindolol (100 nM), a 5-HT1B antagonist, blocked the effect of 5-HT (10 microM); spiperone (1 microM), which is an antagonist at 5-HT1A and 5-HT2 receptors, had no effect. The amplitude of the GABAB synaptic potential was reduced by the 5-HT1B receptor agonists 1-[3-(trifluoromethyl)-phenyl]-piperazine (300 nM) and 7-trifluoromethyl-4-(4-methyl-1-piperazinyl)-pyrrolo[1,2-a]quinoxaline (1 microM), but not by the 5-HT1A agonist N,N-dipropyl-5-carboxamidotryptamine (1 microM) or the 5-HT2 agonist (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-amino-propane (10 microM). We conclude that 5-HT activates presynaptic 5-HT1B receptors that inhibit the release of GABA onto GABAB but not GABAA receptors.     

Reduction of GABAB inhibitory postsynaptic potentials by serotonin via pre- and postsynaptic mechanisms in CA3 pyramidal cells of rat hippocampus in vitro.

The action of serotonin (5-HT) on GABAergic synaptic transmission was investigated with intracellular recordings in CA3 pyramidal cells of rat hippocampal slices. Local application of 5-HT (500 microM) hyperpolarized CA3 pyramidal cells, decreased cellular input resistance, and reduced slow afterhyperpolarizations. Serotonin attenuated the late (GABAB) component of polysynaptic inhibitory postsynaptic potentials (IPSPs; 47% of control) without affecting the early (GABAA) component. During bath application of the excitatory amino acid antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and 2-amino-5-phosphonovalerate (AP-5) (40 microM), 5-HT similarly decreased the amplitude of the late (GABAB) component (17% of control) of monosynaptic IPSPs but did not affect the early (GABAA) component. The mean reversal potentials of poly- and monosynaptic IPSPs were unaffected by 5-HT. The conductance increases associated with the late component of poly- and monosynaptic IPSPs were reduced by 5-HT. Hyperpolarizing responses evoked in CA3 pyramidal cells by somatic applications of gamma-aminobutyric acid (GABA) were unaffected by 5-HT. During bath application of bicuculline (20-50 microM), hyperpolarizing responses elicited by dendritic GABA application were reduced by 5-HT (71% of control). The effect of 5-HT on these direct GABAB hyperpolarizations (29% decrease in response) does not appear sufficient to fully account for the effect of 5-HT on late GABAB IPSPs (53-83% decrease in response). Therefore, 5-HT may reduce GABAB IPSPs in CA3 pyramidal cells 1) by a postsynaptic action on pyramidal cells and 2) by a selective presynaptic action on GABAergic interneurons mediating the GABAB IPSP. This presynaptic action of 5-HT does not appear to involve excitatory afferents onto inhibitory interneurons.     

Pharmacological characterization of GABAB-mediated responses in the CA1 region of the rat hippocampal slice.

It is generally accepted that the bicuculline-resistant responses to GABA are mediated through the activation of GABAB receptors that mediate a slow IPSP. However, a number of reported observations are difficult to reconcile with this model. Specifically, GABAB antagonists only partially block bicuculline-resistant GABA responses, and both 4-aminopyridine (4-AP) and carbachol have been reported to block responses to the selective GABAB agonist baclofen, but not GABA itself. Thus, it has been argued that baclofen and GABA increase potassium conductance through separate receptor mechanisms. This suggestion is not easily reconcilable with the postulated physiological role of GABAB receptors in mediating the slow IPSP. We have addressed these discrepancies by using the new GABAB antagonists 2-hydroxy-saclofen (2-OH-SAC) and CGP 35348 in the presence of the GABA uptake inhibitor SKF 89976A. The weak antagonism of 2-OH-SAC against the bicuculline-resistant GABA response was improved when the GABA uptake was inhibited with SKF 89976A, allowing for the application of lower GABA concentrations. Under these circumstances, 2-OH-SAC and CGP 35348 strongly antagonized GABA and baclofen responses, but did not have any effect on outward currents evoked by 5-HT. The slow IPSP evoked in the presence of glutamate antagonists was reversibly inhibited by CGP 35348 (IC50 = 14 microM), without affecting the fast IPSP. Carbachol (0.3-20 microM) had no effect on outward currents evoked by either baclofen or GABA. 4-AP (5 microM to 1 mM), despite causing a large increase in cell excitability, did not change baclofen responses. Higher concentrations of 4-AP (5 mM) induced inward current, and reduced both baclofen and GABA outward currents to a similar extent.(ABSTRACT TRUNCATED AT 250 WORDS)     

gamma-Aminobutyric acid inhibits the potassium-stimulated release of somatostatin from rat spinal cord slices

Evidence supports the idea that somatostatin (SO) is a neurotransmitter or neuromodulator of primary afferent neurons involved in nociception. Since gamma-aminobutyric acid (GABA), norepinephrine, and morphine alter nociception at the level of the spinal cord, we examined whether these agents could alter the potassium-stimulated release of somatostatin from rat spinal cord slices. Male Sprague-Dawley rats were decapitated and a 2 cm segment of the lumbar spinal cord removed and chopped into 0.5 x 0.5 mm pieces and perfused at 37 degrees C in individual perfusion chambers with a modified Krebs-bicarbonate buffer at a flow rate of 0.5 ml/min. Perfusates were collected at 2 min intervals and assayed for SO using radioimmunoassay. Exposure of spinal cord tissue to 50 mM KCl resulted in a 3-fold increase in release of SO from a basal level of approximately 0.2 to 0.6 pg/mg tissue/min. This evoked release was calcium dependent. Pre-exposure of tissue to GABA at 10(-4) and 10(-5) M significantly inhibited the potassium-stimulated release of SO, but did not alter basal release. The GABA receptor antagonist, bicuculline methiodide, at 10(-5) but not 10(-6) M attenuated the GABA-induced inhibition of somatostatin release. Bicuculline methiodide alone did not significantly alter either basal or stimulated release. Neither baclofen (10(-5) M, 5 x 10(-5) M), norepinephrine (10(-5) M), nor morphine (10(-5) M) had any significant effect on basal or stimulated release of SO from spinal cord tissue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of somatodendritic dopamine release in the ventral tegmental area by opioids and GABA: an in vivo microdialysis study.

Microdialysis of the ventral tegmental area in conscious rats was used to evaluate the influence of opioids and GABA agonists on extracellular levels of GABA and somatodendritically released dopamine. The administration of morphine through the dialysis probe elicited significant, dose-dependent increases in the levels of extracellular dopamine and significantly reduced the extracellular concentration of GABA. In contrast, a dose-dependent decrease in somatodendritic extracellular dopamine was produced following the administration of the GABAB agonist baclofen. The increase in dopamine levels elicited by morphine (100 microM) was completely blocked by either baclofen (100 microM) coadministration or peripheral injection of naloxone (2 mg/kg, i.p.). Application of the GABAA agonist muscimol produced a significant increase in both extracellular levels of dopamine and locomotor activity. The present results, together with other electrophysiological, neurochemical, and behavioral data, support a hypothesis that stimulation of mu-opioid or GABAA receptors inhibits the activity of GABAergic afferents to dopamine neurons, thereby removing tonic inhibitory regulation, whereas stimulation of GABAB receptors directly inhibits dopamine neurons.