Presynaptic GABAB autoreceptor modulation of P/Q-type calcium channels and GABA release in rat suprachiasmatic nucleus neurons

GABA is the primary transmitter released by neurons of the suprachiasmatic nucleus (SCN), the circadian clock in the brain. Whereas GABAB receptor agonists exert a significant effect on circadian rhythms, the underlying mechanism by which GABAB receptors act in the SCN has remained a mystery. We found no GABAB receptor-mediated effect on slow potassium conductance, membrane potential, or input resistance in SCN neurons in vitro using whole-cell patch-clamp recording. In contrast, the GABAB receptor agonist baclofen (1-100 microM) exerted a large and dose-dependent inhibition (up to 100%) of evoked IPSCs. Baclofen reduced the frequency of spontaneous IPSCs but showed little effect on the frequency or amplitude of miniature IPSCs in the presence of tetrodotoxin. The activation of GABAB receptors did not modulate postsynaptic GABAA receptor responses. The depression of GABA release by GABAB autoreceptors appeared to be mediated primarily through a modulation of presynaptic calcium channels. The baclofen inhibition of both calcium currents and evoked IPSCs was greatly reduced (up to 100%) by the P/Q-type calcium channel blocker agatoxin IVB, suggesting that P/Q-type calcium channels are the major targets involved in the modulation of GABA release. To a lesser degree, N-type calcium channels were also involved. The inhibition of GABA release by baclofen was abolished by a pretreatment with pertussis toxin (PTX), whereas the inhibition of whole-cell calcium currents by baclofen was only partially depressed by PTX, suggesting that G-protein mechanisms involved in GABAB receptor modulation at the soma and axon terminal may not be identical. We conclude that GABAB receptor activation exerts a strong presynaptic inhibition of GABA release in SCN neurons, primarily by modulating P/Q-type calcium channels at axon terminals.     

A comparative study of synchronised and conventional culture methods on the micronucleus dose-response curve

The involvement of GABAA and GABAB receptors in neural mechanisms responsible for the production of theta rhythms in hippocampal formation (HPC) slices is addressed in the present study. In a number of papers published in the last decade, we have demonstrated that theta-like activity can be successfully recorded in the limbic cortex maintained in vitro when the cholinergic agonists, acetylcholine, carbachol or muscarine, were added to the bath. Recently, we have also shown a strong GABAA modulation of the cholinergic-induced in vitro theta-like activity. This study presents a report of the first demonstration of in vitro theta-like field responses induced a consequence of simultaneously inhibiting hippocampal GABAA and GABAB receptors. HPC slices (350 microns) were maintained in a gas-liquid interface chamber (35 degrees C). Theta-like activity was induced in the presence of bath perfusion of bicuculline (GABAA antagonist) and 2-hydroxysaclophen (GABAB antagonist). This in vitro induced field response was antagonized both by muscimol (GABAA agonist) and baclophen (GABAB agonist). In addition, the experiments presented here revealed that bicuculline/2-hydroxysaclophen-induced in vitro theta-like activity also had a strong cholinergic M1 involvement: it was abolished by hemicholinium-3 (choline transport blocker) and pirenzepine (specific antagonist of M1 receptor), but not by gallamine (specific antagonist of M2 receptor). The results of the present study provided further evidence for a strong GABAergic/cholinergic interaction in the neural mechanism responsible for production of theta-like activity in the hippocampal formation slices.     

GABAB receptor activation causes a depression of low- and high-voltage-activated Ca2+ currents, postinhibitory rebound, and postspike afterhyperpolarization in lamprey neurons

1. Activation of gamma-aminobutyric acid-B (GABAB) receptors during N-methyl-D-aspartate (NMDA)-induced fictive locomotor activity in the lamprey spinal cord reduces the burst frequency and changes the intersegmental coordination. Presynaptic inhibition of both the excitatory and inhibitory synaptic transmission from spinal premotor interneurons occurs through GABAB receptor activation. To further analyze the cellular mechanisms underlying the GABABergic modulation of the locomotor network, the present study investigates somatodendritic effects of GABAB receptor activation on interneurons and motoneurons in the lamprey spinal cord in vitro using single-electrode current- and voltage-clamp techniques. 2. High- (HVA) and low- (LVA) voltage-activated calcium currents were studied with single-electrode voltage clamp when Na+ and K+ currents were blocked--using tetrodotoxin, tetraethylammonium (TEA), and CsCl electrodes--after substituting Ca2+ with Ba2+. Cobalt-sensitive inward barium currents, activated at -50 mV, became larger when the holding potential was set to a more hyperpolarized level, thus suggesting the existence of an LVA calcium current. The presence of cobalt-sensitive inward barium currents activated at -30 and -10 mV suggests the existence of an HVA calcium current. GABAB receptor activation (baclofen) reduced the peak amplitude of both the LVA and HVA Ca2+ component. 3. The late phase of the afterhyperpolarization (AHP), which follows the action potential, was reduced in amplitude by cobalt, thus lending further support to the notion that the Ca2+ influx, and the subsequent activation of Ca(2+)-dependent K+ channels (KCa2+), constitutes the major part of the AHP generation. Application of the GABAB agonist baclofen also reduced the peak amplitude of the AHP in interneurons and motoneurons, and this reduction was counteracted by the GABAB antagonist 2(OH)saclofen. Baclofen reduced the duration of action potentials broadened by TEA, thus suggesting that the Ca2+ inflow was reduced. Intracellular injection of the GTP analogue GTP gamma S also reduced the duration of the action potential and the peak amplitude of the AHP in TEA, thus supporting the notion that a GTP-binding protein (G-protein)-mediated GABAB receptor activation reduced the calcium inflow, leading to less activation of KCa channels and, consequently, to a smaller peak amplitude of the AHP. 4. Baclofen suppressed the subthreshold depolarization induced by a depolarizing current pulse injection without affecting either the spike threshold or the resting membrane conductance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Astigmatism, accommodation, the oblique effect and meridional amblyopia

Agonist binding to GABAB receptors modulates the activity of the guanine nucleotide binding proteins, Go and Gi. These G proteins are ADP-ribosylated by pertussis toxin and this prevents them from coupling to the GABAB receptor resulting in a reduction in high-affinity GABAB binding. GTP, which binds to a different site on the G protein alpha subunit, also reduces the affinity of the receptor for the G protein, and this can be used as a "marker" for G protein-GABAB receptor linkage. We have examined GABAB binding site distribution in rat brain after unilateral intrahippocampal pertussis toxin injection in vivo, and after incubating brain slices in pertussis toxin in vitro, using the technique of receptor autoradiography. The effect of pertussis toxin was compared with that of GTP gamma S on GABAB binding. Intrahippocampal pertussis toxin administration reduced GABAB but not GABAA receptor binding and the effects appeared to be limited by pertussis toxin diffusion. More widespread reductions in GABAB binding were seen after incubation of brain slices in vitro but the extent varied in different brain regions. No reduction was detected in the corpus striatum. GABAB binding was also reduced in membranes prepared from cerebral cortex, hippocampus and cerebellum but there was no significant reduction in the corpus striatum after pertussis toxin treatment. GTP gamma S reduced GABAB binding to a similar extent in all areas studied irrespective of their sensitivity to pertussis toxin suggesting that while GABAB binding sites are linked to G proteins throughout the rat brain, those in the corpus striatum may be predominantly pertussis toxin insensitive.     

The pharmacology of adenylyl cyclase modulation by GABAB receptors in rat brain slices

GABAB receptor activation inhibits forskolin-stimulated adenylyl cyclase activity but augments noradrenaline-stimulated adenylyl cyclase activity. The present study investigated the pharmacology of these two GABAB receptor mediated responses. In a cross-chopped rat cortical slice preparation, it was confirmed that (-)baclofen inhibited forskolin-stimulated adenylyl cyclase activity and augmented noradrenaline-stimulated adenylyl cyclase activity. The potency of five further agonists was investigated (SKF97541, CGP47656, CGP44533, 3-APA and CGP44532). Of these agonists two compounds were significantly more potent as inhibitors of forskolin-stimulated adenylyl cyclase than as augmenters of noradrenaline-stimulated adenylyl cyclase activity, these were (-)baclofen (pEC50 = 6.07 +/- 0.29 and 5.04 +/- 0.17, respectively (p < 0.05)), and CGP47656 (pEC50 = 6.44 +/- 0.05 and 4.48 +/- 0.26, respectively (p < 0.05)). It is possible to explain this difference in potency by proposing that these compounds have low intrinsic efficacy, and the augmentation of noradrenaline-stimulated adenylyl cyclase has a low receptor reserve. In addition six antagonists (CGP49311A, CGP46381, CGP45024, CGP45397, CGP36742) were also tested for their ability to antagonize 10 microM (-)baclofen in these two assays. These antagonists ranged in potency as inhibitors of forskolin-stimulated adenylyl cyclase activity from CGP49311A (pEC50 = 5.45 +/- 0.30) to CGP36742 (pEC50 = 3.87 +/- 0.16). Each antagonist had similar potency in the two assays, suggesting that these two responses are mediated by pharmacologically similar receptors.     

GABAB receptor-mediated inhibition of GABAA receptor calcium elevations in developing hypothalamic neurons

In the CNS, gamma-aminobutyric acid (GABA) affects neuronal activity through both the ligand-gated GABAA receptor channel and the G protein-coupled GABAB receptor. In the mature nervous system, both receptor subtypes decrease neural excitability, whereas in most neurons during development, the GABAA receptor increases neural excitability and raises cytosolic Ca2+ levels. We used Ca2+ digital imaging to test the hypothesis that GABAA receptor-mediated Ca2+ rises were regulated by GABAB receptor activation. In young, embryonic day 18, hypothalamic neurons cultured for 5 +/- 2 days in vitro, we found that cytosolic Ca2+ rises triggered by synaptically activated GABAA receptors were dramatically depressed (>80%) in a dose-dependent manner by application of the GABAB receptor agonist baclofen (100 nM-100 microM). Coadministration of the GABAB receptor antagonist 2-hydroxy-saclofen or CGP 35348 reduced the inhibitory action of baclofen. Administration of the GABAB antagonist alone elicited a reproducible Ca2+ rise in >25% of all synaptically active neurons, suggesting that synaptic GABA release exerts a tonic inhibitory tone on GABAA receptor-mediated Ca2+ rises via GABAB receptor activation. In the presence of tetrodotoxin the GABAA receptor agonist muscimol elicited robust postsynaptic Ca2+ rises that were depressed by baclofen coadministration. Baclofen-mediated depression of muscimol-evoked Ca2+ rises were observed in both the cell bodies and neurites of hypothalamic neurons taken at embryonic day 15 and cultured for three days, suggesting that GABAB receptors are functionally active at an early stage of neuronal development. Ca2+ rises elicited by electrically induced synaptic release of GABA were largely inhibited (>86%) by baclofen. These results indicate that GABAB receptor activation depresses GABAA receptor-mediated Ca2+ rises by both reducing the synaptic release of GABA and decreasing the postsynaptic Ca2+ responsiveness. Collectively, these data suggest that GABAB receptors play an important inhibitory role regulating Ca2+ rises elicited by GABAA receptor activation. Changes in cytosolic Ca2+ during early neural development would, in turn, profoundly affect a wide array of physiological processes, such as gene expression, neurite outgrowth, transmitter release, and synaptogenesis.     

An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells

It has been established that GABAA and GABAB receptors can exist separately and/or co-exist in the membrane of dorsal root ganglion neurons. In our previous investigation it has been shown that co-existence of these two kinds of receptors is about 80% of the neurons examined (20/25). The present study was aimed to explore whether the activation of these two kinds of receptors could interact with each other using intracellular and whole-cell patch-clamp recordings. Baclofen, a specific GABAB receptor agonist, was found to exert negative modulatory effects on the responses mediated by GABAA receptor. In experiments with intracellular recording, GABA (0.3-1000 microM)- and muscimol (100-1000 microM)-induced depolarization was attenuated markedly and reversibly by preapplication of baclofen (100 microM) (15/21 and 17/21, respectively). In whole-cell patch-clamp recordings GABA (100 microM) and two specific GABAA receptor agonists, muscimol (10 microM) and isoguvacine (50 microM), activated currents were inhibited markedly by preapplication of baclofen 30 s or more and the inhibition was concentration dependent (1-100 microM baclofen) and reversible. The possible mechanisms underlying the inhibition by baclofen of the responses mediated by GABAA receptor and the physiological significance implicated are discussed.     

GABAergic effects on nucleus tractus solitarius neurons receiving gastric vagal inputs

Single units in the region of the medial nucleus tractus solitarius (NTS), responding to electrical stimulation of gastric vagal fibers, were recorded in an in vitro neonatal rat brainstem-gastric preparation. gamma-Aminobutyric acid (GABA) subreceptor agonists and antagonists were applied to the gastric and brainstem compartments of the bath chamber to evaluate the peripheral gastric and central brainstem GABAergic effects on NTS neuronal activity. The gastric effects of the GABAA receptor agonist muscimol and GABAB receptor agonist baclofen were evaluated on 55 tonic units that received the gastric vagal inputs. For approximately 58% (32 of 55) and 38% (21 of 55) of the units observed, muscimol (30 microM; IC50 = 2.0 microM) and baclofen (30 microM; IC50 = 1.5 microM) in the gastric compartment induced a concentration-dependent inhibition of 36.2 +/- 3.1% (mean +/- S.E.) and 31.0 +/- 2.9% of the control level of the NTS neuronal activity, respectively. The brainstem effects of muscimol and baclofen were tested on 51 units. For approximately 90% (46 of 51) and 78% (40 of 51) of the units tested, muscimol (30 microM; IC50 = 1.3 microM) and baclofen (30 microM; IC50 = 1.1 microM) in the brainstem compartment produced a concentration-dependent inhibition of 54.1 +/- 3.4% and 48.9 +/- 3. 5% of the control level, respectively. The remaining NTS units were not affected by these two GABA agonists. Bicuculline (10 microM) and saclofen (10 microM), the GABAA and GABAB subreceptor antagonists, competitively antagonized the gastric and brainstem effects by muscimol and baclofen, respectively. Our results demonstrated that both GABAA and GABAB receptors in the stomach and brainstem play an important role in activity modulation of the medial NTS neurons receiving gastric vagal inputs in neonatal rats.     

Seizure sensitivity and GABAergic modulation of ethanol sensitivity in selectively bred FAST and SLOW mouse lines

FAST and SLOW selected mouse lines were bred for differences in locomotor response to low-dose ethanol. FAST mice exhibit an extreme stimulant response and SLOW mice exhibit locomotor depression at the same ethanol dose. We tested the hypothesis that gamma-aminobutyric acid (GABA) systems modulate ethanol's stimulant effects by examining convulsant responses to GABAA receptor ligands, and by assessing the effects of GABAA and GABAB ligands on locomotor activity in the presence and absence of EtOH. FAST mice were more sensitive to the convulsant effects of GABAA drugs, and to one of two non-GABAergic drugs also tested. FAST and SLOW mice differed in locomotor responses to two benzodiazepines, but not to other GABAA receptor ligands. Ethanol's stimulant effects were not selectively altered by bicuculline or picrotoxin. The selected lines differed in sensitivity to the locomotor depressant effects of the GABAB agonist, baclofen. Ethanol-stimulated activity of FAST mice was inhibited by baclofen, and this effect was reversed by administration of the GABAB antagonist, CGP-35348. These GABAB receptor mediated effects were replicated in DBA/2J inbred mice that exhibit extreme sensitivity to ethanol's stimulant effects. In summary, we found moderate to strong evidence that some sites on the GABAA receptor complex were altered as a consequence of selection of FAST and SLOW mice, but found little support for GABAA mediation of EtOH-stimulated activity. In contrast, we found moderate evidence for differential alteration of GABAB receptor function; however, GABAB receptor involvement in ethanol-stimulated activity was strongly supported by results in the selected lines and an inbred strain.     

Intracellular retention of recombinant GABAB receptors

gamma-Aminobutyric acid type B (GABAB) receptors mediate the transmission of slow and prolonged inhibitory signals in the central nervous system. Two splice variants of GABAB receptors, GABABR1a and GABABR1b, were recently cloned from a mouse cortical and cerebellar cDNA library. As predicted, these receptors belong to the G protein-coupled receptor superfamily. We have used epitope-tagged versions of GABABR1a receptors to study the cellular distribution of these proteins in a variety of non-neuronal and neuronal cell types. Here we report that recombinant GABAB receptors fail to reach the cell surface when expressed in heterologous systems and are retained in the endoplasmic reticulum when introduced into COS cells. In addition, we prove that recombinant GABAB receptors are excluded from the cell surface when overexpressed in ganglion neurons and we further demonstrate that they fail to activate in superior cervical ganglion neurons. Together our observations suggest that recombinant GABAB receptors require additional information for functional targeting to the plasma membrane.     

G-Protein-coupled modulation of presynaptic calcium currents and transmitter release by a GABAB receptor

Presynaptic GABAB receptors play a regulatory role in central synaptic transmission. To elucidate their underlying mechanism of action, we have made whole-cell recordings of calcium and potassium currents from a giant presynaptic terminal, the calyx of Held, and EPSCs from its postsynaptic target in the medial nucleus of the trapezoid body of rat brainstem slices. The GABAB receptor agonist baclofen suppressed EPSCs and presynaptic calcium currents but had no effect on voltage-dependent potassium currents. The calcium current-EPSC relationship measured during baclofen application was similar to that observed on reducing [Ca2+]o, suggesting that the presynaptic inhibition generated by baclofen is caused largely by the suppression of presynaptic calcium influx. Presynaptic loading of the GDP analog guanosine-5'-O-(2-thiodiphosphate) (GDPbetaS) abolished the effect of baclofen on both presynaptic calcium currents and EPSCs. The nonhydrolyzable GTP analog guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) suppressed presynaptic calcium currents and occluded the effect of baclofen on presynaptic calcium currents and EPSCs. Photoactivation of GTPgammaS induced an inward rectifying potassium current at the calyx of Held, whereas baclofen had no such effect. We conclude that presynaptic GABAB receptors suppress transmitter release through G-protein-coupled inhibition of calcium currents.     

The gamma-aminobutyric acid uptake inhibitor NO-711 potentiates 3-aminopropylphosphinic acid-induced actions in rat neocortical slices

In rat neocortical slices maintained in Mg2+-free Krebs medium, the GABAB receptor agonists baclofen and 3-aminopropylphosphinic acid dose-dependently reduced the frequency of spontaneous discharges, 3-aminopropylphosphinic acid being 10 times less potent than baclofen. These were sensitive to the antagonist CGP 52432 (3-[[3,4-dichloro-phenyl)methyl]-amino]propyl](-P-diethoxymethyl)- phosphinic acid) (1, 5 and 10 microM). The GABA uptake inhibitor NO-711 (1-(2-(((diphenylmethylene)amino)oxy)ethyl)-1,2,5,6-tetrahydro-3-+ ++pyridinecarboxylic acid) (5 and 10 microM) produced 2.9 and 9 fold increases in the potency of 3-aminopropylphosphinic acid without affecting baclofen-induced responses. In this study, the low potency of 3-aminopropylphosphinic acid when compared to baclofen, may be attributed to its uptake by NO-711-sensitive GABA transporters.     

Role of GABAB receptors in the control of hypoglossal motoneurons in vivo

There is little information on GABAB receptor-mediated effects on orofacial motoneurons. We recorded the inspiratory activity from both hypoglossal (XII) nerves in urethane-anesthetized, paralyzed, vagotomized and artificially ventilated rats. A GABAB receptor agonist, baclofen, or antagonist, CGP-35348, was microinjected into one XII nucleus. Baclofen rapidly reduced the XII nerve activity in a dose-dependent manner by over 50%. The antagonist caused a delayed suppression of activity by 40%. We conclude that: (1) GABAB receptors within the XII nucleus may suppress the activity of inspiratory XII motoneurons, but they are not tonically active under the conditions of our experiment; (2) there is a net endogenous excitatory effect in XII motoneurons that is mediated by GABAB receptors located in the reticular formation surrounding the XII nucleus.     

Inhibition in the human urinary bladder by gamma-amino-butyric acid

OBJECTIVE: To investigate the effects of gamma-amino-butyric acid (GABA) on detrusor activity in man to determine whether it has any inhibitory effect on detrusor contraction. The inhibitory neurotransmitter GABA has been found in mammalian urinary bladders and the effects of GABA on detrusor activity in the rabbit bladder has previously been described [1]. MATERIALS AND METHODS: Human detrusor muscle strips, obtained at cystectomy, were made to contract by electrical stimulation of their autonomic nerves or by the addition of carbachol in a superfusion apparatus. GABA and its analogues were added to the superfusion chamber and any changes in the responses were measured. RESULTS: The electrically evoked nerve-mediated contractions in human bladder muscle were exclusively cholinergic. GABA inhibited nerve-mediated contractions in human detrusor muscle-strips by the activation of the GABAB receptor, since baclofen (a GABAB receptor agonist) produced similar inhibition and muscimol (a GABAA receptor agonist) did not. There was no inhibition of carbachol-mediated contractions by GABA. CONCLUSION: This in vitro study shows that GABA has a peripherally mediated inhibitory effect on excitatory neurotransmission in human detrusor muscle. The site of action is on the post-ganglionic nerves and appears to be mediated via the GABAB receptor.     

GABA and mood disorders: a brief review and hypothesis

Considerable evidence implicates the neurotransmitter gamma-aminobutyric acid (GABA) in the biochemical pathophysiology of mood disorders. Animal models of depression show regional brain GABA deficits and GABA agonists have antidepressant activity in these models. Somatic treatments for depression and mania upregulate the GABAB receptor, similar to the effect of GABA agonists. Clinical data indicate that decreased GABA function accompanies depressed or manic mood states. GABA agonists are effective antidepressant and antimanic agents. Low GABA levels are found in brain, cerebrospinal fluid and plasma of patients with depression and in plasma of patients with mania. Plasma GABA levels, which reflect brain GABA, are not normalized with treatment and clinical remission in depression, suggesting low GABA is not a marker for mood state. Some somatic treatments, including valproic acid and electroconvulsive shock, reduced plasma GABA and response to these correlates with higher levels of baseline plasma GABA. From these data, a GABA hypothesis for mood disorders is formulated. Low GABA function is proposed to be an inherited biological marker of vulnerability for development of mood disorders. Environmental factors, including stress and excessive alcohol use, may increase GABA, causing symptoms of depression or mania. Treatment, or the passage of time, then returns GABA to its presymptomatic baseline as the symptoms remit. This hypothesis, applicable to a subset of mood disordered persons, is testable.     

Neurotransmitter activation of inwardly rectifying potassium current in dissociated hippocampal CA3 neurons: interactions among multiple receptors

We characterized potassium current activated by G-protein-coupled receptors in acutely dissociated hippocampal CA3 neurons. Agonists for serotonin, adenosine, and somatostatin receptors reliably activated a potassium-selective conductance that was inwardly rectifying and that was blocked by 1 mM external Ba2+. The conductance had identical properties to that activated by GABAB receptors in the same cells. In one-half of the CA3 neurons that were tested, the metabotropic glutamate agonist 1S,3R-ACPD also activated inwardly rectifying Ba2+-sensitive potassium current. Activation of the current by serotonin and adenosine agonists occurred with a time constant of 200-700 msec after a lag of 50-100 msec; on removal of agonist the current deactivated with a time constant of 1-2 sec after a lag of 200-400 msec. These kinetics are similar to GABAB-activated current and consistent with a direct action of G-protein on the channels. For somatostatin, both activation and deactivation were approximately fourfold slower, probably limited by agonist binding and unbinding. The half-maximally effective agonist concentrations were approximately 75 nM for somatostatin, approximately 100 nM for serotonin, and approximately 400 nM for 2-chloroadenosine. Dose-response relationships had Hill coefficients of 1.2-1.9, suggesting cooperativity in the receptor-to-channel coupling mechanism. At saturating concentrations of agonists, the combined application of baclofen and either somatostatin, serotonin, or 2-chloroadenosine produced effects that were subadditive and often completely occlusive. However, at subsaturating concentrations the effects of baclofen and 2-chloroadenosine were supra-additive. Thus, low levels of different transmitters can act synergistically in activating inwardly rectifying potassium current.     

GABAergic drugs and sexual motivation, receptivity and exploratory behaviors in the female rat

Female rats were allowed to pace sexual interactions in a bilevel chamber, where a sexually vigorous male was tethered to the bottom level. Exploratory behaviors (sniffing, rearing), locomotor activity (expressed as number of level changes and periods of inactivity) as well as items of sexual motivation (latency to descend to the male's level, approaches towards the male and genital exploration) were recorded. In addition, sexual receptivity was evaluated in a non-paced situation. A test for motor impairment was also performed. The GABA transaminase inhibitor gamma-acetylene GABA reduced exploratory behaviors at doses much lower than those needed to reduce receptivity. The GABA reuptake inhibitor SKF 100330A did not affect any behavior category at doses of 15 and 30 mg/kg, but had a sedative action at 60 mg/kg. This was shown as impaired motor coordination and an almost total absence of activity in the bilevel chamber. Receptivity was not impaired, however. The mixed GABAA/ GABAB agonist progabide reduced exploratory behaviors and receptivity without producing motor impairment at a dose of 400 mg/kg. The GABAA agonist THIP impaired motor coordination and reduced receptivity and exploratory behaviors at a dose of 10 mg/kg. A larger dose, 20 mg/kg, had a strong sedative action. Only a small proportion of the animals descended to the males level. The GABAB agonist baclofen reduced receptivity at a dose that had no effect on motor coordination or exploratory behaviors. None of the drugs had a specific effect on sexual motivation. Whenever behaviors reflecting motivation were reduced, there were also other behavioral effects indicative of sedation. These data show that GABA receptor agonists, particularly the GABAB agonist baclofen, reduce sexual receptivity at doses that have only slight effect on motor functions or exploratory behaviors. In contrast, non-specific enhancement of GABAergic activity by a transaminase or reuptake inhibitor have effects on motor functions and exploratory behaviors at doses much lower than those needed to reduce receptivity.     

Ontogenesis of presynaptic GABAB receptor-mediated inhibition in the CA3 region of the rat hippocampus

gamma-Aminobutyric acid-B(GABAB) receptor-dependent and -independent components of paired-pulse depression (PPD) were investigated in the rat CA3 hippocampal region. Intracellular and whole cell recordings of CA3 pyramidal neurons were performed on hippocampal slices obtained from neonatal (5-7 day old) and adult (27-34 day old) rats. Electrical stimulation in the hilus evoked monosynaptic GABAA postsynaptic currents (eIPSCs) isolated in the presence of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)2-amino-5-phosphovaleric acid (-AP5, 50 microM) with 2(triethylamino)-N-(2,6-dimethylphenyl) acetamine (QX314) filled electrodes. In adult CA3 pyramidal neurons, when a pair of identical stimuli was applied at interstimulus intervals (ISIs) ranging from 50 to 1,500 ms the amplitude of the second eIPSC was depressed when compared with the first eIPSC. This paired-pulse depression (PPD) was partially blocked by P-3-aminoprophyl -P-diethoxymethylphosphoric acid (CGP35348, 0.5 mM), a selective GABAB receptor antagonist. In neonates, PPD was restricted to ISIs shorter than 200 ms and was not affected by CGP35348. The GABAB receptor agonist baclofen reduced the amplitude of eIPSCs in a dose-dependent manner with the same efficiency in both adults and neonates. Increasing the probability of transmitter release with high Ca2+ (4 mM)/low Mg2+ (0.3 mM) external solution revealed PPD in neonatal CA3 pyramidal neurons that was 1) partially prevented by CGP35348, 2) independent of the membrane holding potential of the recorded cell, and 3) not resulting from a change in the reversal potential of GABAA eIPSCs. In adults the GABA uptake blocker tiagabine (20 microM) increased the duration of eIPSCs and the magnitude of GABAB receptor-dependent PPD. In neonates, tiagabine also increased duration of eIPSCs but to a lesser extent than in adult and did not reveal a GABAB receptor-dependent PPD. These results demonstrate that although GABAB receptor-dependent and -independent mechanisms of presynaptic inhibition are present onGABAergic terminals and functional, they do not operate at the level of monosynaptic GABAergic synaptic transmission at early stages of development. Absence of presynaptic autoinhibition of GABA release seems to be due to the small amount of transmitter that can access presynaptic regulatory sites.     

Baclofen inhibition of the hyperpolarization-activated cation current, Ih, in rat substantia nigra zona compacta neurons may be secondary to potassium current activation

1. The properties of the hyperpolarization-activated cation current (Ih), and its modulation by gamma-aminobuturic acid-B (GABAB) receptor activation and protein kinase A, were investigated using whole cell voltage clamp of substantia nigra zona compacta principal neurons in rat midbrain slices in vitro. 2. At 30 degrees C, Ih activated between -75 and -155 mV, with a V1/2 of -115 mV. At 35 degrees C, the activation curve shifted positive by 10 mV. Ih had an estimated reversal potential of -27 mV. Ion substitution experiments showed that the current was carried by Na+ and K+. 3. Application of the GABAB receptor agonist baclofen (30 microM) induced an outward potassium current (GIRK), increased neuronal membrane conductance and inhibited Ih. The inhibition of Ih was voltage independent. Baclofen induced an 11-mV positive shift in the reversal potential of Ih. 4. Extracellular barium (300 microM) markedly reduced the baclofen-evoked outward current and associated increase in membrane conductance due to GIRK activation. There was also very little inhibition of Ih by baclofen in the presence of barium. When cesium was the major intracellular cation, both the increase in membrane conductance due to GIRK activation and the inhibition of Ih evoked by baclofen were reduced by a similar extent. 5. Neither forskolin (10 microM) nor the protein kinase A inhibitor, H89 (10 microM), had any effect on Ih or its inhibition by baclofen. 6. These data suggest that the inhibition of Ih by baclofen is secondary to the activation of GIRK, i.e., due directly to alteration of membrane conductance, rather than a distinct effect, and is not mediated by inhibition of adenylyl cyclase.     

Heterodimerization is required for the formation of a functional GABA(B) receptor

GABA (gamma-aminobutyric acid) is the main inhibitory neurotransmitter in the mammalian central nervous system, where it exerts its effects through ionotropic (GABA(A/C)) receptors to produce fast synaptic inhibition and metabotropic (GABA(B)) receptors to produce slow, prolonged inhibitory signals. The gene encoding a GABA(B) receptor (GABA(B)R1) has been cloned; however, when expressed in mammalian cells this receptor is retained as an immature glycoprotein on intracellular membranes and exhibits low affinity for agonists compared with the endogenous receptor on brain membranes. Here we report the cloning of a complementary DNA encoding a new subtype of the GABAB receptor (GABA(B)R2), which we identified by mining expressed-sequence-tag databases. Yeast two-hybrid screening showed that this new GABA(B)R2-receptor subtype forms heterodimers with GABA(B)R1 through an interaction at their intracellular carboxy-terminal tails. Upon expression with GABA(B)R2 in HEK293T cells, GABA(B)R1 is terminally glycosylated and expressed at the cell surface. Co-expression of the two receptors produces a fully functional GABA(B) receptor at the cell surface; this receptor binds GABA with a high affinity equivalent to that of the endogenous brain receptor. These results indicate that, in vivo, functional brain GABA(B) receptors may be heterodimers composed of GABA(B)R1 and GABA(B)R2.