GABA Uptake Blocker Research Articles

Efficacy of background GABA uptake in rat hippocampal slices.

GABA uptake is crucial for the termination of inhibitory synaptic events. In addition, GABA transporters may also control the level of diffusely distributed GABA in the extracellular space. We analysed this function by superfusing rat hippocampal slices with different concentrations of GABA. Whole-cell patch clamp recordings of CA1 pyramidal cells revealed small increases in chloride conductance at 5-10 microM GABA which increased dramatically upon addition of the GABA uptake blocker tiagabine. Tiagabine alone induced a significant chloride conductance indicating that spontaneous release of GABA in hippocampal slices is neutralized by GAT-1, the main hippocampal GABA transporter. Thus, GAT-1 clears the extracellular space in the hippocampus from diffusely distributed GABA with high efficacy.

Stereoselective central nervous system effects of the R‐ and S‐isomers of the GABA uptake blocker N‐(4,4‐di‐(3‐methylthien‐2‐yl)but‐3‐enyl) nipecotic acid in the rat

1. The 'effect compartment' model was applied to characterize the pharmacodynamics of the R- and S-isomers of tiagabine in conscious rats in vivo using increase in the beta activity of the EEG as a pharmacodynamic endpoint. 2. No pharmacokinetic differences in plasma were observed between R- and S-tiagabine. The values for clearance and volume of distribution at steady-state were 103+/-10 versus 90+/-6 ml min(-1) kg(-1) and 1.8+/-0.2 versus 1.6+/-0.2 l kg(-1) for the R- and S-isomer, respectively. In contrast, plasma protein binding showed a statistically significant difference with values of the free fraction of 5.7+/-0.5 and 11.4+/-0.6%. In addition the rate constant for transport to the effect compartment was also different with values of 0.027 versus 0.067 min(-1). 3. For both isomers the relationship between concentration and EEG effect was non-linear and successfully characterized on basis of the Hill equation. A statistically significant difference in the value of EC(50) of 328+/-11 versus 604+/-18 ng ml(-1) was observed for R- and S-tiagabine respectively. The values of the other pharmacodynamic parameters were identical. 4. It is concluded that the differences in in vivo pharmacodynamics of R- and S-tiagabine can be explained by stereoselective differences in both the affinity to the GABA-uptake transporter and the degree of non-specific protein binding in plasma and at the effect site.

Junctional versus Extrajunctional Glycine and GABAAReceptor-Mediated IPSCs in Identified Lamina I Neurons of the Adult Rat Spinal Cord

Colocalization of GABA and glycine in synaptic terminals of the superficial dorsal horn raises the question of their relative contribution to inhibition of different classes of neurons in this area. To address this issue, miniature IPSCs (mIPSCs) mediated via GABA(A) receptors (GABA(A)Rs) and glycine receptors (GlyRs) were recorded from identified laminae I-II neurons in adult rat spinal cord slices. GABA(A)R-mediated mIPSCs had similar amplitude and rise times, but significantly slower decay kinetics than GlyR-mediated mIPSCs. Lamina I neurons appeared to receive almost exclusively GlyR-mediated mIPSCs, even after application of hypertonic solutions. Yet, all neurons responded to exogenous applications of both GABA and glycine, indicating that they expressed both GABA(A)Rs and GlyRs. Given that virtually all glycinergic interneurons also contain GABA, the possibility was examined that GABA(A)Rs may be located extrasynaptically in lamina I neurons. A slow GABA(A)R-mediated component was revealed in large, but not minimally evoked monosynaptic IPSCs. Administration of the benzodiazepine flunitrazepam unmasked a GABA(A)R component to most mIPSCs, suggesting that both transmitters were released from the same vesicle. The isolated GABA(A)R component of these mIPSCs had rising kinetics 10 times slower than that of the GlyR component (or of GABA(A)R mIPSCs in lamina II). The slow GABA(A)R components were prolonged by GABA uptake blockers. It is concluded that, whereas GABA and glycine are likely released from the same vesicle of transmitter in lamina I, GABA(A)Rs appear to be located extrasynaptically. Thus, glycine mediates most of the tonic inhibition at these synapses. This differential distribution of GABA(A)Rs and GlyRs confers distinct functional properties to inhibition mediated by these two transmitters in lamina I.

Inhibitory postsynaptic currents of rat substantia nigra pars reticulata neurons: role of GABA receptors and GABA uptake

Whole-cell patch-clamp recordings were made from substantia nigra pars reticulata neurons in midbrain slices of young rats to study the characteristics of spontaneous and evoked inhibitory postsynaptic currents and factors which govern their decay kinetics. In the presence of the glutamate receptor antagonists d, l-2-amino-5-phosphonopentanoic acid (20 μM) and 6-cyano-7-nitroquinoxaline-2,3-dione (20 μM), bicuculline-sensitive spontaneous inward inhibitory postsynaptic currents were often observed using high Cl − electrodes. Application of the selective GABA B receptor antagonist CGP55845A (2 μM) did not alter the half decay time of these inhibitory postsynaptic currents, which however was prolonged by the potent GABA uptake blocker tiagabine (1 μM). In addition, the frequencies and amplitudes of the inhibitory postsynaptic currents were significantly reduced by tiagabine but these effects were prevented by CGP55845A. Inhibitory postsynaptic currents with similar sensitivity to bicuculline could also be evoked intranigrally. Similar to the spontaneous currents, the decay time of evoked inhibitory postsynaptic currents was not affected by 2 μM CGP55845A. However, in the absence of CGP55845A, tiagabine shortened the IPSC decay time but had an opposite effect if CGP55845A was present. These data suggest that the spontaneous and evoked inhibitory postsynaptic currents recorded from substantia nigra pars reticulata neurons are mediated mainly by GABA A receptors. Uptake of GABA helps to terminate these currents. When the uptake mechanism is blocked, accumulation of GABA would lead to activation of presynaptic GABA B receptors and reduction in GABA release. The role of postsynaptic GABA B receptors in substantia nigra pars reticulata of young rats seems to be minimal.

Laminar difference in GABA uptake and GAT-1 expression in rat CA1.

1. The axonal plexus of most hippocampal interneurons is restricted to certain strata within the target region. This lamination suggests a possible functional heterogeneity of inhibitory synapses between different interneurons and CA1 pyramidal cells. 2. We therefore compared inhibitory postsynaptic potentials (IPSPs) and currents (IPSCs) in CA1 pyramidal cells, which were evoked from two stimulation sites (stratum oriens and stratum radiatum). Stimulation in stratum oriens yielded faster decaying IPSPs and IPSCs than stimulation in stratum radiatum. 3. IPSP and IPSC kinetics were regulated by GABA uptake in both layers as indicated by the prolongation of the signals under tiagabine, a GAT-1 (neuronal GABA plasma membrane transporter)-specific GABA-uptake blocker. However, the effect of tiagabine was significantly more pronounced following stimulation in stratum radiatum than in stratum oriens (prolongation of IPSC half-decay time by 167 vs. 115 %, respectively). 4. In situ hybridization with antisense mRNA for the GABA-synthesizing enzyme glutamate decarboxylase (GAD65/67) and the GABA transporter GAT-1 showed that the proportion of interneurons expressing GAT-1 was lower in stratum oriens than in stratum radiatum/lacunosum-moleculare. 5. From these functional and molecular data we conclude that the regulation of IPSP and IPSC kinetics in CA1 pyramidal cells by neuronal GABA uptake differs between layers. Our findings suggest that this laminar difference is caused by a lower expression of GAT-1 in interneurons in stratum oriens than in stratum radiatum/lacunosum-moleculare.

An evaluation of the GABA uptake blocker tiagabine in animal models of neuropathic and nociceptive pain

Tiagabine HCl [(R]-N-[4,4-bis(3-methyl-2-thienyl]-3-butenyl] nipecotic acid hydrochloride], a potent and selective GABA uptake inhibitor, was evaluated for potential anti-allodynic effects in a rodent model of neuropathic pain and for antinociceptive activity in rodent models of acute and persistent pain. The effect of tiagabine on neuropathic pain was evaluated in rats that developed allodynia after tight ligation of L5 and L6 spinal nerves. The anti-allodynic effects of tiagabine were dose-dependent, with significant increases in response threshold to tactile stimulation occurring at 72.8 μmoles/kg, ip, but not at lower doses of 7.2 and 24.3 μmoles/kg, ip. In the hot-plate test in mice, tiagabine significantly increased foot-licking latency at 7.2 and 24.3 μmoles/kg, ip, and jump latency at 24.3 μmoles/kg, ip. After twice daily dosing with 72.8 μmoles/kg, ip, of tiagabine for 4 days, mice showed a tolerance to the antinociceptive effect of the 7.2 μmoles/kg, ip, dose of tiagabine in the hot-plate test. Tolerance did not occur after twice daily dosing of the 7.2 μmoles/kg, ip, dose of tiagabine. Tiagabine did not have significant effects on the tail-flick latency of rats at doses of 0.7 to 72.8 μmoles/kg, ip. Doses of 7.2 and 24.3 μmoles/kg, ip, of tiagabine significantly reduced the number of acetic acid-induced stretches in mice. In rats, tiagabine significantly decreased the number of paw flinches in the early phase of the formalin test at 24.3 and 72.8 μmoles/kg, ip, and in the late phase of the test at 72.8 μmoles/kg, ip. Tiagabine had no significant effects on carrageenan-induced paw edema at doses of 0.7 to 72.8 μmoles/kg, ip. Taken together, the results of these experiments revealed the potential anti-allodynic and antinociceptive pharmacology of tiagabine. Drug Dev. Res. 44:106–113, 1998. © 1998 Wiley-Liss, Inc.

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.

Synchronous firing of inhibitory interneurons results in saturation of fast GABAA IPSC magnitude but not saturation of fast inhibitory efficacy in rat neocortical pyramidal cells

The kinetic properties of evoked fast inhibitory postsynaptic currents were examined to elucidate factors underlying the limit on the magnitude of fast inhibition in neocortex. Using whole-cell voltage-clamp recordings from layer V pyramidal neurons in slices of rat somatosensory cortex, fast gamma-aminobutyric acid-A (GABA[A])ergic inhibitory postsynaptic currents were selectively recorded by holding cells at potentials equal to excitatory postsynaptic current reversal (approximately 0 mV). As stimulus intensity was increased, the magnitude and duration of the fast inhibitory postsynaptic current increased. Over the range of stimuli applied (2-10 V), fast GABA(A)-mediated inhibitory postsynaptic currents reached a maximum peak conductance of 25.9 +/- 4.2 nS (range 10.5-41.2 nS) at intensities approximately 2-times threshold. As stimulus intensities were increased beyond this point of maximal conductance, the time constant of current decay increased as function of stimulus strength, while rise time remained unaffected. Exposure to nominally magnesium-free solutions did not affect maximal peak conductances of fast inhibitory postsynaptic currents, but did cause an increase in the time constants of current decay by 66.3 +/- 23.6%, resulting in an 85.6 +/- 24.6% increase in the total charge flux carried by single inhibitory postsynaptic currents. This effect may be due to prolonged activation of postsynaptic GABA(A) receptors by excess GABA released in response to increased excitation. Exposure to the GABA uptake blocker, nipecotic acid, similarly prolonged current decay without affecting the maximal peak conductance. Our findings suggest that the limit on recruitment of evoked fast inhibition in neocortical layer V pyramidal cells arises from the saturation of postsynaptic GABA(A) receptors. However, while there is a limit to the peak fast inhibitory postsynaptic conductance which can be recruited with increasing excitation, inhibitory strength may still be modulated by increasing charge flux through the prolongation of fast inhibition.

Different mechanisms regulate IPSC kinetics in early postnatal and juvenile hippocampal granule cells.

1. Monosynaptic inhibitory postsynaptic currents (IPSCs) were recorded from early postnatal and juvenile dentate granule cells in rat brain slices at room temperature. The focally evoked currents were mediated by gamma-aminobutyric acid-A (GABAA) receptors. 2. IPSCs were characterized by a steep rising phase and a slower, monoexponential decay time course. The decay time constant was potential dependent and average values ranged from 33 ms at a holding potential of -60 mV to 58 ms at a holding potential of +40 mV. 3. IPSCs were studied in tissue from animals between postnatal day (p) 3 and p25. All kinetic parameters as well as the mean current amplitude were unchanged during this ontogenetic period. 4. In juvenile granule cells from animals aged 13-16 days, addition of the GABA uptake blocker (R)-N-[4,4-bis (3-methyl-2-thienyl) but-3-en1-yl] nipecotic acid (tiagabine) (10 microM) prolonged the decaying phase of the IPSCs. The current decay remained monoexponential but the time constant increased to 250% of control values. Mean current amplitudes remained largely unchanged. 5. In contrast, tiagabine had no effect on IPSCs in early postnatal tissuĕ. The decay time constant remained unchanged in cells recorded from animals aged p4-p6. Other uptake blockers were also ineffective during the first postnatal week, whereas beta-alanine, NNC-711, and L-2,3-diaminoproprionic acid enhanced the decay time constant in the older tissue (p13-p16). 6. Hypoosmolaric extracellular solution was applied to restrict the extracellular space. In juvenile tissue (p13-p16), IPSCs were not affected by this treatment, whereas early postnatal granule cells (p4-p6) displayed clearly prolonged IPSC decay time constants (165% of control). 7. We conclude that the mechanism governing the kinetics of evoked IPSCs in granule cells changes during ontogenesis. Whereas in early postnatal tissue the transmitter leaves the postsynaptic site by diffusion, GABA uptake becomes time limiting after 2 wk of postnatal development.

208 Developmental changes in GABAergic ipscs recorded from rat thalamic neurons in slice

Rats anesthetized with urethane had stimulating and recording electrodes placed in the perforant pathway and in the dentate gyrus. They were then exposed to increasing doses of either the vehicle control dimethylsulfoxide (DMSO) or one of two γ-aminobutyric acid (GABA)-uptake blockers (SKF-100330A or SKF-89976A). Analysis of evoked field potentials from dentate granule cells indicated that the only effect of the GABA uptake blockers was to increase the threshold for evoking the field population spikes (PS). No other measure of excitatory postsynaptic potentials (EPSPs) or PS's were significantly affected. The lack of effect on evoked EPSP by these drugs suggests no direct effect on transmitter release at this synapse, while the increase in PS threshold suggests a slight decrease in granule cell excitability. The effects of the two GABA-uptake blockers on synaptically mediated facilitation and inhibition was tested by using paired-pulse paradigms. Both GABA-uptake blockers increased early GABA-mediated inhibition to a greater extent than they reduced synaptically mediated facilitation. Neither GABA uptake blocker appeared to effect the late inhibition seen at paired-pulse intervals of 400–1000 ms which is presumably associated with calcium-activated increases in potassium conductance. These effects on granule cell responses occurred at doses found previously not to be associated with side effects and yet to be anticonvulsant in unanesthetized rats. These data confirm in vivo that SKF-100330A and SKF-89976A increase GABA-mediated inhibition. The effect on granule cell excitability and late inhibition are minimal. Although facilitation was reduced by exposure to these drugs, the mechanism of this reduction (direct or prolongation of early inhibition) cannot be determined.

STRUCTURAL, METABOLIC AND IONIC REQUIREMENTS FOR THE UPTAKE OF1-CARNITINE BY PRIMARY RAT CORTICAL CELLS

L-Carnitine (L-C) is involved in the transport of acyl groups into mitochondria for β-oxidation, although its role in the adult brain is still uncertain. We have shown before that the uptake of L-carnitine into cultured rat cortical neurones was dependent on temperature as well as the Na gradient and is inhibited by compounds resembling its structure, like ‰-aminobutyric acid (GABA), but most potently by specific GABA uptake blockers. In this study we have characterised this uptake process further. We have shown that the uptake of L-carnitine may be dependent on Cl ions, in addition to Na ions, but non on Ca ions. The L-C uptake was inhibited by substituent anions in the order gluconate (83%) >isethionate (32%), with propionate being ineffective, whereas GABA uptake was inhibited most potently by propionate substitution (79%) and equally by isethionate and gluconate (67%). This L-C uptake process was not affected by the amino acids, glutamine or lysine, up to 1mM concentration, although β-alanine at 500μ Mcaused a 38% inhibition. The uptake of L-C was also significantly inhibited by structurally-related compounds, with a carbon chain length of three to six atoms, possessing an amine group and/or a carboxyl group. At a concentration of 500μ M, 3-aminopropane sulphonic acid (53%), ‰-butyrobetaine (31%), ‰-hydroxybutyric acid (34%) and 4 methylaminobutyric acid (33%). Other compounds were effective only at the lower concentration of 10μ M, such as butyric acid (25%), nicotinic acid (26%), isonicotinic acid (26%), hexanoic acid (23%) and at 100μ M, like 6-aminocapric acid (22%). Drugs suggested to affect membrane properties, such as chlorpromazine, was without effect at 1 or 10μ M, whereas flunarizine (FLU) at 1μ Minhibited both L-C (24%) and GABA uptake (17%). Other drugs like the cholinesterase inhibitors, tacrine and eserine, also had a small inhibitory effect on L-C uptake, reducing it at 1μ Mby 22 and 21% respectively, although higher concentrations were toxic (>100μ M). Pretreatment of the cells with neuraminidase (50Uml −1, 10min) reduced the subsequent uptake of both L-C (18%) and GABA (42%). Hypoxia (3h) also significantly attenuated L-C uptake (42%), however part of these effects were related to the loss of cell viability. In summary, L-C uptake occurs by a complex mechanism which at least in part may occur by a Na/Cl cotransport mechanism, which could be similar, to that of GABA or may even in part occur via the GABA transporter.

NMDA and non-NMDA receptor-mediated release of [3H]GABA from granule cell dendrites of rat olfactory bulb.

We have studied the effect of glutamate and the glutamatergic agonists N-methyl-D-aspartate (NMDA), kainate, and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) on [3H]GABA release from the external plexiform layer of the olfactory bulb. The GABA uptake blocker nipecotic acid significantly increased the basal [3H]GABA release and the release evoked by a high K+ concentration, glutamate, and kainate. The glutamate uptake blocker pyrrolidine-2,4-dicarboxylate (2,4-PDC) inhibited by 50% the glutamate-induced [3H]GABA release with no change in the basal GABA release. The glutamatergic agonists NMDA, kainate, and AMPA also induced a significant [3H]GABA release. The presence of glycine and the absence of Mg2+ have no potentiating effect on NMDA-stimulated release; however, when the tissue was previously depolarized with a high K+ concentration, a significant increase in the NMDA response was observed that was potentiated by glycine and inhibited by the NMDA receptor antagonist 2-amino-5-phosphonoheptanoic acid (AP-7). The kainate and AMPA effects were antagonized by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) but not by AP-7. The glutamate effect was also inhibited by CNQX but not by the NMDA antagonist 2-amino-5-phosphonopentanoic acid (AP-5); nevertheless, in the presence of glycine, [3H]GABA release evoked by glutamate was potentiated, and this response was significantly antagonized by AP-5. Tetrodotoxin inhibited glutamate- and kainate-stimulated [3H]GABA release but not the NMDA-stimulated release.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of l-carnitine uptake into primary rat cortical cell cultures by gaba and gaba uptake blockers

l-carnitine plays a central role in mitochondrial function and is found to be differentially distributed in the brain. We have shown before that the uptake of l-carnitine into cultured rat cortical neurones was temperature-dependent, as well as potently inhibited by factors affecting the sodium gradient as well as by molecules resembling its structure, e.g. d-carnitine, acetyl- l-carnitine and γ-aminobutyric acid (GABA). GABA was the most potent inhibitor of l-carnitine uptake. In the present study we have found that specific GABA uptake blockers, nipecotic acid, cis-4-hydroxynipecotic (HNA), guvacine, 2,4-diaminobutyric acid (DABA) and NO 711 inhibit l-carnitine uptake even more potently than GABA. However, apart from NO 711, they caused about the same maximal inhibition, 67.4% at 50 μM for guvacine, compared to 60.5% by GABA. NO 711 was extremely potent and blocked 80.5% of the l-carnitine uptake. In contrast, the GABA A receptor agonists, isonipecotic acid and isoguvacine, or the antagonist bicuculline, at similar concentrations (50 μM) did not significantly inhibit the uptake of the l-carnitine. However, bicuculline at relatively high concentration (500 μM) was inhibitory (38%). The GABA B receptor agonist, baclofen, or antagonist, phaclofen, were ineffective, although 5-aminovaleric acid did significantly inhibit uptake at both 50 and 500 μM) causing 22 and 48% inhibition respectively. Like bicuculline, it was not as effective as GABA or the specific GABA uptake blockers. The results indicate that the uptake of l-carnitine by rat cortical neurones occurs in part by a process that can be potently inhibited by GABA and GABA uptake blockers.

Extracellular GABA concentrations in rat supraoptic nucleus during lactation and following haemodynamic changes: An in vivo microdialysis study

Morphological and pharmacological evidence suggest that the dense GABAergic innervation of the supraoptic nucleus is important for regulating the electrical activity of vasopressin and oxytocin neurons. We have employed the technique of intracranial microdialysis to examine extracellular GABA concentrations in the supraoptic nucleus of the anaesthetized rat and questioned whether differences exist in the dynamics of GABA release between virgin and lactating rats, and if events during lactation or following blood pressure manipulation alter endogenous GABA levels in this nucleus. No significant differences were detected between virgin and lactating animals in either basal or 100 mM potassium ion-evoked GABA release. The inclusion of the GABA uptake blocker nipecotic acid (0.5 mM) into the dialysate resulted in a six- to eight-fold increase ( P < 0.01) in GABA outflow in both groups of animals. In lactating rats, GABA outflow measured at 4 min intervals was not altered during a 60 min period of suckling by a full litter of pups and no significant change in GABA outflow was detected in relation to individual milk ejections. In virgin rats, removal of 1.5–2 ml of blood resulted in a 30–60 mmHg fall in blood pressure and a non-significant decline in GABA outflow. Replacement of blood resulted in an abrupt 50 mmHg increase in blood pressure and a significant 22% increase in GABA outflow ( P < 0.01), but no change in aspartate or methionine concentrations. Repeated intravenous injections of the α-adrenoceptor agonist, metaraminol, similarly evoked approximately 50 mmHg increments in blood pressure and a 26% increase in GABA outflow ( P < 0.05). Electrical stimulation of the diagonal band of Broca for 10 min produced a two-fold increase in GABA outflow from the supraoptic nucleus ( P < 0.05). These results show that the overall profile of basal and potassium-stimulated GABA concentrations in the supraoptic nucleus is not substantially different between lactating and virgin rats. In lactating animals we have found that GABA levels are not altered in response to suckling or at the time of high-frequency firing by oxytocin neurons to induce milk ejection. In contrast, our data further support the hypothesis that GABA inputs to supraoptic neurons are part of a baroreceptor reflex, relaying through the diagonal band of Broca, to signal periods of acute hypertension and inhibit the firing of vasopressin neurons. Such observations suggest the physiological importance of GABA inputs to the supraoptic nuclei and indicate that GABA may be used in a stimulus-specific manner to influence the activity of magnocellular neurons.

GABA synapses formed in vitro by local axon collaterals of nucleus accumbens neurons

GABAergic medium-spiny neuron axons not only form the principal projections of the nucleus accumbens (nAcc) but also branch locally in a dense network overlapping their own dendrites, suggesting that their recurrent synapses mediate the major information processing functions of the nAcc. We used postnatal nAcc cultures to study these synapses individually. In culture, as in the intact nAcc, medium-spiny neurons account for over 95% of the cells and are GABAergic. Strikingly, these neurons showed a spike afterhyperpolarization (AHP) that was largely blocked by the GABAA antagonist bicuculline. The bicuculline-sensitive AHP occurred without or with latency, and met criteria for monosynapticity; consistent with this, dye fills showed the presence of recurrent axons and a low incidence of dye coupling. Blockade of Ca2+ influx eliminated this autaptic PSP, while TTX almost completely eliminated it, indicating that it is due to exocytic GABA release principally at axodendritic contacts. While blocking GABAB receptors had no direct effect on the autaptic PSP, activating these receptors with baclofen produced presynaptic inhibition, as well as directly mediated hyperpolarization; together, these actions increased the signal-to-noise ratio in the cellular response to synaptic inputs. Bicuculline also increased the signal-to-noise ratio; in addition, it induced burst firing and depolarization inactivation. In contrast, the indirect GABA agonist flurazepam and the GABA uptake blocker nipecotic acid each enhanced autaptic PSPs. Since autapses formed in vitro appear to be functionally equivalent to synapses between neighboring medium-spiny neurons that receive similar inputs, these results bear on the function of intrinsic GABA synapses in the intact nAcc. Thus, intrinsic GABA synapses are likely to regulate the signal-to-noise ratio in nAcc information processing and may be important targets for the modulatory actions of endogenous neurotransmitters and drugs.

Striatal grafts in infarct striatopallidum increase GABA release, reorganize GABA A receptor and improve water-maze learning in the rat

We grafted fetal striatal cells in ischemic rat models, and investigated graft survival/growth, GABA release, GABA A receptor reorganization and functional recovery. One hour intraluminal occlusion of the middle cerebral artery (MCA) induced ischemic infarct in the lateral part of the striatum and adjacent cortex. In ischemic rats, the acquisition of Morris' water-maze learning was significantly slower than that of control rats. In these animals GABA level in the globus pallidus, detected by microdialysis, was about the half of that of controls. However, after the grafts of fetal striatal cells in the Striatopallidum, the acquisition was improved, thus no difference was observed in the time course of learning curves in control and grafted animals. GABA level recovered to almost normal level by the graft. It further increased by the treatment of a GABA uptake blocker (nipecotic acids) in the perfusion. In the grafts, GABA A receptor organization detected by autoradiography using [ 3H] labeled SR95531 was restored for more than 1 year after the graft. Data suggest that fetal striatal cell grafts in infarct striatum may partially reconstruct striatopallidal GABA projection and reorganize GABA A receptor. This might be a basis of improvement of function.

GABA uptake into isolated retinal Müller glial cells of the guinea-pig detected electrophysiologically.

In order to investigate GABA uptake into Müller glial cells electrophysiologically, we used the whole-cell voltage-clamp technique. Since Müller cells were insensitive to application of muscimol and baclofen, the expression of GABAA and GABAB receptors can be excluded. Therefore, the observed GABA current must be due to an electrogenic GABA transporter. This transporter is driven by the transmembrane Na+ gradient, as replacement of the extracellular Na+ by choline inhibits the GABA current. Currents evoked by cis-4-aminocrotonic acid, a GABAC specific agonist, are also inhibited by replacement of extracellular Na+, indicating that this compound is a substrate for the uptake. The competitive GABA uptake blockers beta-alanine and nipecotic acid are substrates of the transporter as well, and produce 42% and 65% of the currents elicited by the same GABA concentration, respectively. Affinity of the transporter for GABA is high, the Km value being 5 microM at -80 mV.

Frequency-dependent enhancement of hippocampal inhibition by GABA uptake blockers

The effects of GABA uptake inhibitors, SKF 89976A and SKF 100330A, on recurrent inhibition were studied in the rat hippocampal slice preparation by the antidromic-orthodromic stimulation test. Population spikes evoked orthodromically by stimulation of the stratum radiatum and recorded in the CA1 pyramidal cell body layer were inhibited antidromically by stimulation of the alveus by a single pulse or by a train of pulses, either at low or at high frequency. Low frequency train conditioning produced less inhibition than a single pulse. The uptake blockers had no effect or slightly enhanced the inhibition produced by single stimuli or low frequency trains. High frequency train conditioning produced more and much longer inhibition than a single pulse. This inhibition was further substantially enhanced and prolonged by the drugs. Frequency-dependent enhancement of inhibition may be responsible for suppression of epileptiform discharges by GABA uptake blockers.

Membrane currents evoked by excitatory amino acid agonists in ON bipolar cells of the mudpuppy retina.

1. Whole-cell patch-clamp recordings were obtained from ON bipolar cells in a retinal slice preparation of the mudpuppy, Necturus maculosus. The effects of excitatory amino acid (EAA) agonists applied in the presence of cobalt (2-5 mM) were examined. 2. At the holding potential of -50 mV, L-2-amino-4-phosphonobutanoic acid (L-AP4, 5-10 microM) evoked an outward current accompanied by a conductance decrease. The zero current potential of the L-AP4-evoked current was near 0 mV independent of whether the intracellular Ringer solution contained CsCl or CsCH3SO4. The currents evoked by light were also accompanied by a conductance decrease and reversed near 0 mV. Replacing external sodium with choline or N-methyl-D-glucamine generated an outward current and suppressed the response to L-AP4. The response to L-AP4 was enhanced by removing extracellular calcium and suppressed by increasing extracellular calcium. These results indicate that L-AP4 closes nonspecific cation channels that are blocked by extracellular calcium. 3. In 2 mM cobalt, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA, 50-100 microM) evoked membrane currents that were accompanied by a conductance increase. AMPA-evoked currents exhibited a significant chloride dependence and were suppressed by gamma-aminobutyric acid-A (GABAA) antagonists bicuculline and picrotoxin; a GABA uptake blocker, nipecotic acid; and a glycine antagonist, strychnine. AMPA-induced currents were virtually absent in the presence of 5 mM cobalt and nominally 0 mM extracellular calcium. These results indicate that the conductance increase induced by AMPA in the presence of 2 mM cobalt is largely the result of calcium-dependent synaptic inputs onto GABAA and glycine receptors of ON bipolar cells. 4. N-methyl-D-aspartic acid (250 microM) was ineffective when applied in the presence of 100 microM cadmium or 2 mM cobalt. 5. 1S,3R/1R,3S-1-aminocyclopentane-1,3-dicarboxylic acid (100-200 microM) evoked an outward current accompanied by a conductance decrease and appears to be an agonist at the L-AP4 receptor. 6. The findings of this study suggest that the only type of EAA receptor in mudpuppy ON bipolar cells is the L-AP4 receptor and that L-AP4 receptor activation results in the closing of nonspecific cation channels that are blocked by extracellular calcium.

Extracellular γ-aminobutyric acid levels in the rat caudate-putamen: Monitoring the neuronal and glial contribution by intracerebral microdialysis

Intracerebral microdialysis with high pressure liquid chromatography (HPLC) coupled to electrochemical detection was employed to characterize γ-aminobutyric acid (GABA) release and the effects induced by a preceding neuron-depleting ibotenic acid (IBO) lesion in the rat caudate-putamen (CPu). Extracellular GABA overflow was monitored in the intact and excitotoxically lesioned CPu, either 7–10 days (acute) or more than 3 months post-lesioning (chronic), using loop type dialysis probes perfused at a rate of 2 μ/min. In the intact CPuu, basal GABA levels were 0.97 pmol/30 μl of dialysate in the awake animals and 0.76 pmol/30 μl under halothane anaesthesia. In both the acute and chronic IBO lesioned CPu the extracellular GABA levels were reduced by 80% and 67%, respectively, under halothane anaesthesia. KCl added to the perfusion fluid at a concentration of 100 mM resulted in dramatic increases in GABA overflow from baseline levels in the intact CPu (60- to 70-fold), which were almost totally abolished (> 95%) in the excitotoxicity lesioned CPu. Veratridine administered at 75 μM, produced a 45-fold increase in GABA overflow in the intact CPu, but failed to produce any effect in the lesioned CPu. The addition of nipecotic acid (0.5 mM), a GABA uptake blocker, increased basal extracellular GABA levels 6–15-fold in the intact CPu, while GABA overflow in either the acute or chronic lesioned CPu was not significantly altered. Although Ca 2+-free conditions (with 20 mM Mg 2+ added) or tetrodotoxin (TTX, 1 μM) did not alter the basal GABA overflow in the intact CPU under halothane anaesthesia, the omission of Ca 2+ resulted in a 47% reduction in basal extracellular GABA levels in awake, freely moving animals. Nipecotic acid-induced GABA overflow was reduced by 22% under Ca 2+-free conditions, and by 33% in the presence of 1 μM TTX. Moreover, KCl-evoked GABA overflow was reduced by 86% in Ca 2+-free conditions and by 40% when administered in the presence of 1 μM TTX. These results indicate that the extracellular GABA levels recorded by intracerebral microdialysis in the CPu are derived predominantly from neuronal sources. Under baseline resting conditions only a small fraction (up to 20–30%) of the neuronal release was Ca 2+-dependent and TTX-sensitive (i.e. possessing the characteristics of impulse-dependent vesicular release). While glial contributions (as assessed from the excitotoxic lesioned CPu), may account for as much as 20–40% of the basal extracellular GABA levels, more than 95% of the evoked release (i.e. after application of KCl and veratridine) is neuronal in origin. The KCl-evoked GABA release was likely vesicular in nature since removal of Ca 2+ blocked the response by almost 90%. The reductions in basal and evoked extracellular GABA levels seen in the excitotoxically lesioned CPu persisted also after long survival periods, suggesting that functional recovery of GABA release does not occur.