To elucidate the role of GABAB receptors in the regulation of the electrical activity of magnocellular neurons of the supraoptic nucleus (SON), the effects of GABAB agonist and antagonist on the firing rate of spontaneous action potentials were studied in SON slice preparations of rats by extracellular recordings. In the presence of the gamma-amino butyric acid (GABA)-gated chloride channel blocker, picrotoxin, the selective GABAB agonist, baclofen, reduced the firing rate of action potentials in both phasic and non-phasic neurons in a dose-dependent manner. The reduction in the firing rate induced by baclofen was reversed by the selective GABAB antagonist, 2-hydroxy saclofen (2OH-saclofen), also in a dose-dependent manner. In non-phasic neurons, 2OH-saclofen significantly increased the firing rate and the effect was additive to the effect of picrotoxin. In phasic neurons, 2OH-saclofen alone did not increase the firing rate, but it reversed suppression of the firing induced by increasing extracellular Ca2+ concentration to 2.1 mM. Baclofen also reduced the firing rate of non-phasic neurons of virgin and lactating female rats, indicating that the GABAB receptor-mediated inhibition is not confined to SON neurons of male rats. The evidence indicates that activation of GABAB receptors inhibits electrical activity of SON neurons of both male and female rats and that GABAB receptors may play an important role in the inhibitory regulation of the electrical activity of SON neurons by GABA.

Expression of GAP-43 in the cerebellum and selected regions of the brain has been shown to be developmentally regulated. Localization of GAP-43 mRNA within granule cells of the immature and mature rat cerebellum has been demonstrated by in situ hybridization. Higher levels are detected in the neonate compared to the adult. To determine if the cerebellar neurotransmitters, GABA (gamma-amino-butyric acid) and glutamate are involved in the modulation of GAP-43 expression, cultured cerebellar granule cells were exposed to these transmitters. Cultures were treated with glutamate, GABA, or the agonists/antagonists to their receptors in serum-free media for 5-7 days. Analysis of the levels of GAP-43 mRNA by in situ hybridization indicated that a 7-day exposure to GABA (25 and 50 microM) significantly lowered levels of granule cell GAP-43 mRNA. Specific agonists to the GABAA (muscimol) and GABAB (baclofen) receptors produced a decrease similar to that observed for GABA. Results from these studies also indicated that exposure to non-NMDA (CNQX) and NMDA (CPP, MK-801) glutamate receptor antagonists, and a metabotropic receptor glutamate agonist (ACPD), decreased the level of GAP-43 mRNA. The involvement of GABA and glutamate in the modulation of GAP-43 expression was corroborated by Northern hybridization. These studies revealed that a 5-day exposure to GABA decreased the cellular content of GAP-43 mRNA by 21% whereas exposure to glutamate resulted in a 37% increase. Findings from the studies reported here, using an in vitro cerebellar granule cell model, suggest that levels of GAP-43 mRNA, in vivo, are modulated by input from both excitatory glutamatergic mossy fibers and inhibitory GABAergic Golgi interneurons. Thus, modulation of GAP-43 mRNA by these neurotransmitters may influence granule cell maturation during development in the neonate and neuroplasticity in the adult, possibly at the parallel fiber-Purkinje cell synapse.

Previous studies have shown that: (1) activation of neurons in the dorsomedial hypothalamus (DMH) of the rat by blockade of local GABAA receptors with bicuculline methiodide (BMI) elicits cardiovascular changes resembling those seen in experimental stress, including marked sympathetically-mediated tachycardia, and (2) inhibition of neurons in the same region by local microinjection of the GABAA receptor agonist muscimol can virtually abolish stress-induced tachycardia. This study examined the possibility that GABAB receptors exist in the neural circuitry of the DMH, and that stimulation of these receptors might suppress the cardiovascular response to local disinhibition with BMI. Microinjection of BMI 10 pmol into the DMH in urethane-anesthetized rats resulted in marked tachycardia with little or no effect on arterial pressure. Simultaneous injection of the GABAB receptor agonist baclofen at doses of 2.5, 5.0 and 10 pmol produced dose-related suppression of BMI induced tachycardia. Coinjection of the GABAB receptor antagonist 2-hydroxysaclofen 100 or 200 pmol had no significant effect on the heart rate response to BMI, but reversed the suppression elicited in the presence of baclofen. These findings indicate that (1) functional GABAB receptors exist in the DMH, and (2) stimulation of these receptors inhibits the tachycardia resulting from blockade of local GABAA receptors.

Neurons in the subfornical organ (SFO) sense both neurotransmitters and circulating humoral factors such as angiotensin II (AII) and atrial natriuretic peptide (ANP), and regulate multiple physiological functions including drinking behavior. We recently reported that AII at nanomolar concentrations induced a persistent [Ca2+]i increase in acutely dissociated SFO neurons and that this effect of AII was reversibly inhibited by GABA. In the present study, we studied the inhibitory mechanism of GABA using Ca2+ imaging and patch-clamp electrophysiology. The AII-induced persistent [Ca2+]i increase was inhibited by GABA in more than 90% of AII-responsive neurons and by other two SFO inhibitory ligands, ANP and galanin, in about 60 and 30% of neurons respectively. The inhibition by GABA was mimicked by the GABAA and GABAB receptor agonists muscimol and baclofen. The involvement of both GABA receptor subtypes was confirmed by reversal of the GABA-mediated inhibition only when the GABAA and GABAB receptors antagonists bicuculline methiodide and CGP55845 were both present. The GABAB agonist baclofen rapidly and reversibly inhibited voltage-gated Ca2+ channel (VGCC) currents recorded in response to depolarizing pulses in voltage-clamp electrophysiology using Ba2+ as a charge carrier (IBa). Baclofen inhibition of IBa was antagonized by CGP55845, confirming GABAB receptor involvement; was reduced by N-ethylmaleimide, suggesting downstream Gi-mediated actions; and was partially removed by a large prepulse, indicating voltage-dependency. The magnitude of IBa inhibition by baclofen was reduced by the application of selective blockers for N-, P/Q-, and L-type VGCCs (ω-conotoxin GVIA, ω-agatoxin IVA, and nifedipine respectively). Overall, our study indicates that GABA inhibition of the AII-induced [Ca2+]i increase is mediated by both GABAA and GABAB receptors, and that GABAB receptors associated with Gi proteins suppress Ca2+ entry through VGCCs in SFO neurons.

The role of gamma-aminobutyric acid (GABA) as an inhibitory neurotransmitter in the rostral, gustatory zone of the nucleus of the solitary tract (rNST) was examined using whole cell recordings in brain slices of the adult rat medulla. Superfusion of GABA resulted in a concentration-dependent reduction in input resistance in 68% of the neurons in rNST. The change in input resistance was often accompanied by membrane hyperpolarization. The effect of GABA was a direct action on the postsynaptic membrane since it could be elicited when synaptic transmission was blocked by tetrodotoxin or in a low Ca2+ and high Mg2+ perfusing solution. The mean reversal potential of the GABA effect was about -60 mV, determined by applying GABA at different holding potentials, or from the intersection of current-voltage curves measured in control saline and saline containing GABA. When neurons were separated into groups based on intrinsic membrane properties, some neurons in each group responded to GABA. Superfusion of the slices with either the GABAA agonist, muscimol, or the GABAB agonist, baclofen, caused a decrease in input resistance accompanied by membrane hyperpolarization. The GABAA antagonist bicuculline either totally or partially blocked the neuronal response to GABA and blocked the response to muscimol but did not antagonize responses to baclofen. Superfusion of the GABAB antagonist phaclofen depressed the membrane responses to GABA. The use of the GABAA and GABAB agonists and antagonists demonstrates that some neurons in rNST have both GABAA and GABAB receptors. Since most rNST neurons studied respond to GABA, inhibition probably plays a major role in sensory processing by the rNST.

Guanine nucleotide-binding regulatory proteins (G proteins) play a pivotal role in receptor-mediated transmembrane signal transduction, and have been implicated in modes of action of psychotropic drugs as well as in pathogenesis of psychiatric disorders. In the present investigation, functional activation of G proteins coupled with several receptors, in particular with GABAB receptors, was assessed by agonist-induced stimulation of high-affinity GTPase, an enzyme that is intrinsic to alpha subunit of G protein, in postmortem human frontal cortical membranes. High-affinity GTPase activity was stimulated by GABA as well as (+/-)-baclofen, a selective GABAB receptor agonist, with EC50 values of 60-150 and 10-40 microM, respectively, in a Mg(2+)-dependent manner. The (+/-)-baclofen-stimulated response was antagonized by the selective GABAB receptor antagonist, 2-hydroxy-saclofen, in a competitive manner with a KB value of 59 microM. Although the maximal percent increase above basal value (% Emax) for GABAB receptor-mediated high-affinity GTPase activity was varied from subject to subject, % Emax values for both agonists were highly correlated with each other, and replicable and stable in a given subject, indicating that this measure is trustworthy as an index of functional coupling between receptors and G proteins in future studies at the aim of elucidating possible alteration of receptor/G protein interaction in psychiatric disorders. The % Emax values for GABAB receptor-mediated responses were correlated inversely with brain storage duration, which should be critically considered in postmortem studies. The increases in high-affinity GTPase activity stimulated by several agonists other than GABAB receptor agonists seemed too low to quantify for making a comparison in future studies.

El mouse has been found to be characteristics with hippocampal disinhibition, and has been suggested decrease in GABAergic synaptic transmission [Ono et al., Brain Res. 745 (1997) 165-172; Fueta et al. , Brain Res. 779 (1998) 324-328]. The efficacy of GABAergic synapses can be modulated in response to trains of low frequency stimulation. The frequency potentiation of a population spike (PS) and the field excitatory postsynaptic potential (fEPSP) induced by a low frequency stimulation (2 Hz for 15 s) were recorded for the CA3 subfield, and PS alone for the CA1 subfield and dentate gyrus. PS frequency potentiation was greater in El mice than in non-epileptic control ddY mice. Especially the CA3 subfield exhibited a high PS frequency potentiation (300+/-73%) compared to age-matched ddY mice (64+/-24%). However, EPSP frequency potentiation was similar in El and ddY mice. The degree of PS frequency potentiation in CA3 was decreased by the reduction of extracellular Ca2+ from 2 to 1 mM in both strains, suggesting presynaptic involvement. The potentiation in El mice was suppressed by AMPA/kainate type receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dion (CNQX), but more than half of the control value remained at 5 microM, whereas the potentiation in ddY mice was abolished at this concentration. N-methyl-d-aspartate (NMDA) type receptor antagonist 3-3 (2-carboxypiperazine-4-yl) propyl-1-phosphonate (10 microM; CPP) did not affect the potentiation. Bicuculline (5 microM), GABAA receptor antagonist, did not increase the amplitude of PS during stimulation but induced epileptic (multiple PSs) potentials. High PS frequency potentiation of El mice was mimicked to the degree of that in ddY mice by a low dose of GABAB receptor agonist baclofen (3 microM). The suppression by baclofen was partially reversed by the antagonist saclofen (500 microM). The large frequency potentiation in young El mice, which do not have seizure-susceptibility, indicates an intrinsic property in El mice. It is suggested that the high synchronization of CA3 neurons in El mice is due to a little activation of GABAB receptor activation and also to enhancement of non-NMDA type synaptic transmission.

Galanin-mediated modulation of the arcuate nucleus (Arc) neurons is thought to be involved in the regulation of feeding behavior, hormone secretion, and reproduction. We previously reported that galanin perfusion significantly hyperpolarized the resting membrane potential and suppressed the spontaneous firing in the Arc neurons in slice preparation. In this study, we focused on the cellular and molecular mechanisms underlying the galanin effect. The galanin action is mediated by the galanin receptors (GAL1/2/3R). We found that activation of galanin receptors alone is not sufficient to mediate the galanin effect on resting membrane potential and spontaneous firing; co-activation of GABA(B) receptors is required for galanin to accomplish its modulation on the membrane properties of Arc neurons. In more details, the effect of galanin on the membrane properties of Arc neurons is blocked by either lowering the extracellular Ca(2+) or the inhibition of GABA(B) receptors with the selective GABA(B) antagonist, saclofen. In addition, activation of GABA(B) receptors by baclofen restored the galanin effect under low Ca (2+) conditions. These results suggest that GABA(B) receptors may serve as a molecular gate for galanin signaling, and thus can be targeted to manipulate the galanin-mediated physiological and behavioral responses.

Previous studies have shown that chronic i.v. treatment with morphine or heroin decreased mu opioid receptor activation of G-proteins in specific brain regions. The present study examined the effect of intrathecal (i.t.) morphine administration on receptor/G-protein coupling in the spinal cord. In spinal cord membranes, [35S]GTP gamma S binding was stimulated by agonists of several G-protein-coupled receptors, including mu opioid (DAMGO), delta opioid (DPDPE), GABA(B) (baclofen), cannabinoid CB(1) (WIN 55,212-2), muscarinic cholinergic (carbachol) and adenosine A(1) (PIA). [35S]GTP gamma S autoradiography revealed that most of this agonist activation of G-proteins was localized to laminae I and II of dorsal horn. To determine the effects of chronic morphine on these receptor activities, rats were treated for 7 days with 0.11 mg/kg/day i.t. morphine, and receptor activation of G-proteins was determined by [35S]GTP gamma S autoradiography of brain and spinal cord. In spinal cord sections, chronic morphine treatment decreased DAMGO-stimulated [35S]GTP gamma S binding in laminae I and II at all levels of spinal cord examined. There were no effects of morphine treatment on [35S]GTP gamma S stimulation in spinal cord by other receptor systems examined (Adenosine A(1) and GABA(B)), and no significant effects of chronic i.t. morphine treatment were observed in brain sections. These data show that homologous desensitization of mu receptor/G-protein coupling occurs specifically in spinal cord following chronic morphine administration.

Experiments were undertaken to examine whether once daily i.p. administration of either of two antidepressants used for the treatment of neuropathic pain, amitriptyline (10 mg/kg) and fluoxetine (5 mg/kg), to rats for 7 days modifies GABA(B) receptor function and subunit expression in the lumbar spinal cord. The results indicate that, as previously reported for desipramine, both amitriptyline and fluoxetine increase the pain threshold to a thermal stimulus, the expression of GABA(B(1)) subunits, and baclofen-stimulated [35S]GTPgammaS binding, a measure of GABA(B) receptor function. The effects of antidepressant administration on GABA(B(1b)) and GABA(B(2)) subunit expression in spinal cord are more variable than for GABA(B(1a)). It was also discovered that repeated daily exposure to a thermal stimulus or immobilization stress increases GABA(B(1a)) expression in the lumbar spinal cord, with no commensurate change in thermal pain threshold or GABA(B) receptor sensitivity. These results support a relationship between GABA(B) receptors and the action of antidepressants. The findings demonstrate that drug-induced increases in GABA(B) receptor function can occur independently of any change in GABA(B) receptor subunit expression and are consistent with the notion that GABA(B) receptor subunits have multiple functions, only one of which is dimerization to form GABA(B) receptors. The data also suggest that GABA(B) subunit gene expression may serve as a preclinical marker of antidepressant efficacy and of drug- or stress-induced modifications in central nervous system activity.

To analyze mediatory mechanisms underlying attention-deficit hyperactivity disorder (ADHD) and their association with epilepsy, the electroencephalogram (EEG) responses to various centrally applied neurotransmitter agonists were studied in spontaneously hypertensive (SH), kainate-treated (KA), and normotensive (control) rats, with chronically implanted electrodes into the frontal cortex and hippocampus and a cannula into the lateral cerebral ventricle. In SH rats, the baseline EEG showed increased delta and beta2 activity in the hippocampus and decreased alpha/beta1 activity in both brain areas. In KA rats, these delta and alpha/beta1 effects were observed 2 weeks post-kainate, while the beta2 activity increase occurred after 5 weeks in the hippocampus and, to a greater extent, 9 weeks post-injection in both brain areas. In SH rats, NMDA increased delta and decreased alpha/beta1 activity, similar to KA rats 5 weeks post-injection. In SH rats, clonidine augmented theta/beta2 increase in the cortex and alpha suppression in both brain areas, in parallel with induction of beta2 activity in the hippocampus. These beta2 effects were observed 5 and 9 weeks post-kainate. In SH rats, baclofen produced robust delta/theta enhancement and alpha/beta1 suppression in both brain areas, with additional beta2 activity increase in the hippocampus, while muscimol was ineffective in both groups of rats. In KA rats, EEG responses to GABA agonists were similar to those in control. Our results demonstrate sensitization of NMDA receptors and α2-adrenoceptors both in SH and KA rats and that of GABAb receptors specifically in SH rats.

Little is known about the chronology of expression, cellular localization and function of GABA(B) subunits in the developing rat spinal cord. In the present study, in situ hybridization, immunohistochemistry and quantitative RT-PCR analysis were used to examine this issue. At embryonic day 18, in situ hybridization reveals that all three transcripts, GABA(B(1a)), GABA(B(1b)), and GABA(B(2)), are present throughout the gray matter. At postnatal day (PN) 2, while overall expression appears to decrease, it becomes more highly concentrated in motoneurons of the ventral horn. By PN 7, distinct subpopulations of cells expressing the transcripts become heavily expressed in motoneurons. Immunohistochemical analysis revealed that, unlike mRNA, GABA(B(1)) protein is more highly concentrated in the dorsal horn as compared to the motoneurons. Analysis using RT-PCR demonstrates that in spinal cord GABA(B(1a)) mRNA expression remains constant throughout development, GABA(B(1b)) increases from PN 2 to adult, and GABA(B(2)) decreases from PN 2 to adult. The distribution of functional GABA(B) receptors, as measured by baclofen-stimulated [35S]GTPgammaS binding, in the spinal cord during development generally follows the distribution of subunit expression, being widely distributed throughout the gray matter in embryonic spinal cord slices and becoming more concentrated in the dorsal horn during postnatal development, similar to the distribution of subunit proteins as measured by immunohistochemistry. These findings suggest that spinal cord GABA(B(1a)), GABA(B(1b)), and GABA(B(2)) transcripts are differentially regulated during development with the chronology of this expression suggesting that GABA(B) receptor subunits, in addition to forming functional GABA(B) receptors, may have a trophic function or participate in synaptogenesis.

Whole-cell and intracellular recordings were made in coronal hypothalamic slices prepared from ovariectomized female guinea pigs. 62% of preoptic area (POA) neurons fired action potentials in a bursting manner, and exhibited a significantly greater afterhyperpolarization (AHP) than did non-bursting POA neurons. The majority (70%) of POA neurons (n=76) displayed a time-dependent inward rectification (I(h)) that was blocked by CsCl (3 mM) or by ZD 7288 (30 microM). In addition, 51% of the cells expressed a low-threshold spike (LTS) associated with a transient inward current (I(T)) that was blocked by NiCl(2) (200 microM). A smaller percentage of POA neurons (29%) expressed a transient outward, A-type K(+) current that was antagonized by a high concentration of 4-aminopyridine (3 mM). Moreover, POA neurons responded to bath application of the mu-opioid receptor agonist DAMGO (93%) or the GABA(B) receptor agonist baclofen (83%) with a membrane hyperpolarization or an outward current. These responses were accompanied by a decrease in input resistance or an increase in conductance, respectively, and were attenuated by BaCl(2) (100 microM). In addition, the reversal potential for these responses closely approximated the Nernst equilibrium potential for K(+). These results suggest that POA neurons endogenously express to varying degrees an AHP, an I(h), an I(T) and an A-type K(+) current. The vast majority of these neurons also are inhibited upon mu-opioid or GABA(B) receptor stimulation via the activation of an inwardly-rectifying K(+) conductance. Such intrinsic and transmitter-activated conductances likely serve as important determinants of the firing patterns of POA neurons.