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The aim of the present study was to investigate if basic GABA-mimetics interact with the taurine transporter (TauT, Slc6a6), and to find a suitable cell based model that is robust towards extracellular changes in osmolality during uptake studies. Taurine uptake was measured in human Caco-2 cells, porcine LLC-PK1 cells, and rat SKPT cells using radiolabelled taurine. Hyperosmotic conditions were obtained by incubation with raffinose (final osmolality of 500mOsm) for 24h prior to the uptake experiments. Expression of the taurine transporter, TauT, was investigated at the mRNA level by real-time PCR. Uptake of the GABA-mimetics gaboxadol and vigabatrin was investigated in SKPT cells, and quantified by liquid scintillation or HPLC-MS/MS analysis, respectively. The uptake rate of [(3)H]-taurine was Na(+) and Cl(-) and concentration dependent with taurine with an apparent Vmax of 6.3±1.6pmolcm(-2)min(-1) and a Km of 24.9±15.0μM. β-alanine, nipecotic acid, gaboxadol, GABA, vigabatrin, δ-ALA and guvacine inhibited the taurine uptake rate in a concentration dependent manner. The order of affinity for TauT was β-alanine>GABA>nipecotic acid>guvacine>δ-ALA>vigabatrin>gaboxadol with IC50-values of 0.04, 1.07, 2.02, 4.19, 4.94, 31.4 and 39.9mM, respectively. In conclusion, GABA mimetics inhibited taurine uptake in hyperosmotic rat renal SKPT cells. SKPT cells, which seem to be a useful model for investigating taurine transport in the short-term presence of high concentrations of osmolytes. Furthermore, analogues of β-alanine appear to have higher affinities for TauT than GABA-analogues., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The creatine transporter (CRT) is a member of a large family of sodium-dependent neurotransmitter and amino acid transporters. The CRT is closely related to the gamma-aminobutyric acid (GABA) transporter, GAT-1, yet GABA is not an effective substrate for the CRT. The high resolution structure of a prokaryotic homologue, LeuT has revealed precise details of the substrate binding site for leucine (Yamashita, A., Singh, S. K., Kawate, T., Jin, Y., and Gouaux, E. (2005) Nature 437, 215-223). We have now designed mutations based on sequence comparisons of the CRT with GABA transporters and the LeuT structural template in an attempt to alter the substrate specificity of the CRT. Combinations of two or three amino acid substitutions at four selected positions resulted in the loss of creatine transport activity and gain of a specific GABA transport function. GABA transport by the "gain of function" mutants was sensitive to nipecotic acid, a competitive inhibitor of GABA transporters. Our results show LeuT to be a good structural model to identify amino acid residues involved in the substrate and inhibitor selectivity of eukaryotic sodium-dependent neurotransmitter and amino acid transporters. However, modification of the binding site alone appears to be insufficient for efficient substrate translocation. Additional residues must mediate the conformational changes required for the diffusion of substrate from the binding site to the cytoplasm.

GABAergic inhibition in the brain can be classified as either phasic or tonic. gamma-Aminobutyric acid (GABA) uptake by GABA transporters (GATs) can limit the time course of phasic currents arising from endogenous and exogenous GABA, as well as decrease a tonically active GABA current. GABA transporter subtypes 1 and 3 (GAT-1 and GAT-3) are the most heavily expressed of the four known GAT subtypes. The role of GATs in shaping GABA currents in the neocortex has not been explored. We obtained patch-clamp recordings from layer II/III pyramidal cells and layer I interneurons in rat sensorimotor cortex. We found that selective GAT-1 inhibition with NO711 decreased the amplitude and increased the decay time of evoked inhibitory postsynaptic currents (IPSCs) but had no effect on the tonic current or spontaneous IPSCs (sIPSCs). GAT-2/3 inhibition with SNAP-5114 had no effect on IPSCs or the tonic current. Coapplication of NO711 and SNAP-5114 substantially increased tonic currents and synergistically decreased IPSC amplitudes and increased IPSC decay times. sIPSCs were not resolvable with coapplication of NO711 and SNAP-5114. The effects of the nonselective GAT antagonist nipecotic acid were similar to those of NO711 and SNAP-5114 together. We conclude that synaptic GABA levels in neocortical neurons are controlled primarily by GAT-1, but that GAT-1 and GAT-2/3 work together extrasynaptically to limit tonic currents. Inhibition of any one GAT subtype does not increase the tonic current, presumably as a result of increased activity of the remaining transporters. Thus neocortical GAT-1 and GAT-2/3 have distinct but overlapping roles in modulating GABA conductances.

Neurons in the external and internal segment of the globus pallidus (GPe and GPi, respectively) receive substantial GABAergic inputs from the striatum and through axon collaterals of neighboring pallidal neurons. The effects of GABA on pallidal activity depend on the synaptic localization of GABA receptors and the distribution and activity of GABA transporters (GATs). To explore the contribution of GABA receptors and transporters to pallidal function, we recorded the activity of single neurons in GPe or GPi before, during, and after local microinjections of GABAergic compounds in awake rhesus monkeys. Activation of GABA(A) or GABA(B) receptors with muscimol or baclofen, respectively, inhibited pallidal activity. These effects were reversed by concomitant infusion of the respective GABA receptor antagonists, gabazine and CGP-55845. Given alone, the antagonists were without consistent effect. Application of the selective GAT-1 inhibitor, SKF-89976A, and the semiselective GAT-3 blocker, SNAP-5114, decreased pallidal activity. Both GAT inhibitors increased GABA levels in the pallidum, as measured by microdialysis. Electron microscopic observations revealed that these transporters are located on glial processes and unmyelinated axonal segments, but rarely on terminals. Our results indicate that activation of GABA(A) and GABA(B) receptors inhibits neuronal activity in both segments of the pallidum. GAT-1 and GAT-3 are involved in the modulation of endogenous GABA levels and may be important in regulating the extrasynaptic levels of GABA. Together with previous evidence that a considerable proportion of pallidal GABA receptors are located outside the synaptic cleft, our experiments strongly support the importance of extrasynaptic GABAergic transmission in the primate pallidum.

The relations between apparent affinity for substrates and operating rates have been investigated by two-electrode voltage clamp in the GABA transporter rGAT1 expressed in Xenopus oocytes. We have measured the transport current induced by the presence of GABA, as well as the charge equilibration rate in the absence of the neurotransmitter, in various experimental conditions known to affect the transporter characteristics. The apparent affinities for GABA and for Na(+) were also determined in the same conditions. Two pharmacological actions and three mutated isoforms have been examined. In all cases significant correlations were found between the charge equilibration rates and apparent affinities for both substrates. In particular in the transport process, the apparent affinity for GABA appears to be inversely related to the sum of the unidirectional charge equilibration rates (alpha+beta), while the Na(+) apparent affinity is directly related to their ratio (beta/alpha). Together these observations suggest a kinetic basis for GABA affinity with higher turnover rates resulting in lower affinity, and indicate that an efficient uptake requires a compromise between these two parameters.

The identification and subsequent development of the GABA transport inhibitor tiagabine has confirmed the important role that GABA transporters play in the control of CNS excitability. Tiagabine was later demonstrated to be a selective inhibitor of the GABA transporter GAT1. Although selective for GAT1, tiagabine lacks cell type selectivity and is an equipotent inhibitor of neuronal and glial GAT1. To date, four GABA transporters have been cloned, i.e., GAT1-4. The finding that some of these display differential cellular and regional expression patterns suggests that drugs targeting GABA transporters other than GAT1 might offer some therapeutic advantage over GAT1 selective inhibitors. Furthermore, it is particularly interesting that several recently defined GABA transport inhibitors have been demonstrated to display a preferential selectivity for the astrocytic GAT1 transporter. That cellular heterogeneity of GAT1 plays a role in the control of CNS function is confirmed by the demonstration that inhibition of astrocytic GABA uptake is highly correlated to anticonvulsant activity. At the present time, a functional role for the other GABA transporters is less well defined. However, recent findings have suggested a role for the mouse GAT2 (homologous to the human betaine transporter) in the control of seizure activity. In these studies, the non-selective GAT1 and mouse GAT2 transport inhibitor EF1502 (N-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-hydroxy-4-(methylamino)-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol) was found to exert a synergistic anticonvulsant action when tested in combination with the GAT1 selective inhibitors tiagabine and LU-32-176B (N-[4,4-bis(4-fluorophenyl)-butyl]-3-hydroxy-4-amino-4,5,6,7-tetrahydrobenzo[d]isoxazol-3-ol). Additional studies will be required to define a role for the other GABA transporters and to further identify the functional importance of their demonstrated cellular and regional heterogeneity. A summary of these and other issues are discussed in this brief review.

GABA uptake limits GABA actions during synaptic responses when the density of active release sites is high or multiple axons are synchronously activated. GABA transporter-1 (GAT-1) is the main neuronal GABA transporter subtype and is already expressed in the early postnatal rat hippocampus. However, previous studies have demonstrated little functional role for the transporter during this developmental period. We used whole-cell voltage-clamp and field-potential recordings in hippocampal slices of neonatal rats (postnatal day 4-5) to study whether GAT-1 plays a role in GABAergic transmission during spontaneous population oscillations, which are seen as "giant depolarizing potentials" (GDPs) in intracellular recordings. We show that the GDP-associated GABAergic current observed in CA3 pyramidal neurons is strongly enhanced by the GAT-1-specific blocker NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride). Our results indicate a novel role for GAT-1 in the control of endogenous activity of the immature hippocampus.

GABA uptake as well as the distribution of GAT-1, GAT-2 and GAT-3 were investigated in bovine brain membrane fractions. GABA uptake was characterised by kinetic constants and IC50-values for a series of known inhibitors in subcellular fractions of frontal cortex and brainstem obtained by subsequent centrifugations on sucrose gradients. Additionally, the immunoreactivity for rGAT-1, rGAT-2 and rGAT-3 antibodies was studied in these fractions. The pharmacological profile for GABA uptake inhibition as well as results from immunoblotting indicated that GABA uptake in a selected subcellular fraction of frontal cortex (P2B) is almost exclusively due to GAT-1 whereas GABA uptake performed with a selected subcellular fraction of brainstem (P2A) in the presence of NNC 711 is mainly attributable to GAT-3., (Copyright 2004 Elsevier Ireland Ltd.)

Perfusion with high millimolar levels of taurine evoked a long-lasting potentiation (LLP-TAU) of synaptic transmission in the Schaffer-collateral CA1 region of the rat hippocampus. Although LLP-TAU showed some correlations to increases in the total taurine content of hippocampal slices, it could not be blocked by the taurine transport inhibitor guanidinoethanesulfonic acid (GES), which was able to significantly reduce total slice taurine uptake. Inhibition of GABA transport by either nipecotic acid or beta-guanidinopropionate failed to abolish LLP-TAU and had no significant effect on taurine uptake. The combination of GES and nipecotic acid also had no significant effect on LLP-TAU. Experiments with transportable structural analogs of taurine (beta-aminoisobutyric acid, homotaurine, and isethionic acid) suggest that activation of classical taurine transport pathways does not always yield a robust LLP-TAU. Hippocampal LLP-TAU could be significantly attenuated, however, by pre-incubation with submillimolar levels of taurine. In summary, the development of LLP-TAU in the rat hippocampus appears to be associated with the intracellular accumulation rather than the activation of known transporters of taurine, but the precise means of its accumulation remains to be identified.

Taurine application in the CA1 area of rat hippocampal slices induces a long-lasting potentiation of excitatory synaptic transmission that has some mechanistic similitude with the late phase of long-term potentiation (L-LTP). Previous indirect evidence such as temperature and sodium dependence indicated that taurine uptake is one of the primary steps leading to the taurine-induced synaptic potentiation. We show that taurine-induced potentiation is not related to the intracellular accumulation of taurine and is not impaired by 2-guanidinoethanesulphonic acid, a taurine transport inhibitor that is a substrate of taurine transporter. We have found that taurine uptake in hippocampal synaptosomes was inhibited by SKF 89976A, a GABA uptake blocker that is not transportable by GABA transporters. SKF 89976A prevents the induction of synaptic potentiation by taurine application. This effect is neither mimicked by nipecotic acid, a broad inhibitor of GABA transporters that does not affect taurine uptake, nor by NO-711, a specific and potent inhibitor of GABA transporter GAT-1. In addition, L-LTP induced by trains of high-frequency stimulation is also inhibited by SKF 89976A, and taurine, at a concentration that does not change basal synaptic transmission, overcomes such inhibition. We conclude that taurine induces synaptic potentiation through the activation of a system transporting taurine and that taurine uptake is required for the induction of synaptic plasticity phenomena such as L-LTP.

Alpha-latrotoxin evokes massive [3H]GABA release from rat brain synaptosomes by stimulating exocytosis and outflow from non-vesicular pool. In the present study, GABA transporter-mediated [3H]GABA release was shown to be involved in alpha-latrotoxin-triggered release of [3H]GABA from non-vesicular pool. The following agents have been exploited as tools: (1) a protonophore carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazon (FCCP) and bafilomycin A1 for evoking depletion of synaptic vesicle [3H]GABA and enlargement of non-vesicular pool; (2) a non-substrate high-affinity GABA transport blocker NO-711 for determining participation of GABA carrier in the toxin-stimulated GABA release; (3) a competitive inhibitor of GABA reuptake nipecotic acid for heteroexchange [3H]GABA release. As shown by the experiments with nipecotic acid, FCCP and bafilomycin A1 considerably increase the content of non-vesicular [3H]GABA. The treatment of the synaptosomes with these agents modified the response to alpha-latrotoxin, particularly to its subnanomolar concentrations: the lack or substantial lowering of the toxin-evoked release during the first 2 min after the toxin addition and substantial enhancement of release up to the 5th minute were observed. Only the step of enhanced release was sensitive to GABA transporter blocker NO-711. Distinct sensitivity to NO-711 was shown to be characteristic for different steps of alpha-latrotoxin-stimulated [3H]GABA release from the control, untreated synaptosomes: lack of any effect of NO-711 during the first 2 min and powerful inhibition in 10 min after the toxin application. Taken together these data appear to indicate that the toxin non-simultaneously from vesicular and non-vesicular origins releases the neurotransmitter, the first rapid step reflects exocytosis stimulation, and the second tardy step is at least in part due to the release mediated by GABA transporters. The incomplete inhibition with NO-711 of the tardy step of the release evoked by nanomolar toxin concentrations suggests the participation not only of the GABA transporters.

Background and Purpose: The possible neuroprotective effect of endogenous gamma-aminobutyric acid (GABA) on the irreversible electrophysiological changes induced by in vitro ischemia on striatal neurons was investigated. In particular, the aim of the study was the characterization of the neuroprotective action of 2 antiepileptic drugs increasing GABAergic transmission such as tiagabine, a GABA transporter inhibitor, and vigabatrin, an irreversible inhibitor of GABA transaminase., Methods: Extracellular field potential recordings were obtained from rat corticostriatal slice preparations. In vitro ischemia was delivered by switching to an artificial cerebrospinal fluid solution in which glucose was omitted and oxygen was replaced with N(2)., Results: An irreversible loss of the field potentials recorded from striatal neurons was observed after 10 minutes of ischemia in control solution. Conversely, tiagabine and vigabatrin partially prevented the ischemia-induced field potential loss. Surprisingly, both GABA(A) and GABA(B) receptor antagonists blocked these effects. Accordingly, neuroprotection could be obtained only when GABA(A) and GABA(B) receptor agonists were coapplied, but not when a single agonist was given in isolation., Conclusions: Antiepileptic drugs targeting GABAergic transmission can exert neuroprotective effects against ischemia by increasing endogenous GABA levels and via the activation of both GABA(A) and GABA(B) receptors.

Ethanol is among the most widely abused drugs in the world. Chronic ethanol consumption leads to ethanol tolerance and addiction, and impairs learning and memory. Na+/Cl- dependent GABA transporters play an important role in controlling the concentration of GABA in the synaptic cleft, and thus they control the intensity and duration of synaptic transmission of GABA. It has been suggested that GABAergic system is involved in ethanol consumption, tolerance and addiction, because chronic ethanol consumption alters the expression of GABAA receptors and drugs on GABA receptors affect ethanol actions. The results of the present study reveal that that activity of GABA transporters in mouse brain after 15-min acute ethanol injection or after chronic ethanol consumption is increased. Moreover, mice pre-injected with a competitive or a noncompetitive antagonist of gamma-aminobutyric acid transporter subtype 1 (GAT1) showed high sensitivity to the sedative/hypnotic effects of ethanol. In contrast, transgenic mice overexpressing GAT1 displayed low sensitivity to ethanol, as shown by the righting reflex test. Mice overexpressing GAT1 survived a lethal dose of ethanol (9 g/kg, i.p.) longer, maintained locomotor activity longer after a sub-lethal dose (1.75 g/kg, i.p.) and exhibited a higher median lethal dose than wild-type littermates. These results suggest that GAT1 plays an important role in sensitivity to ethanol, and might be a therapeutic target for alcoholism prevention and treatment. Acute and chronic ethanol administration resulted in the increase of GABA transporter function. Use of GAT1 selective inhibitors and GAT1 overexpressing mice thus demonstrate that GAT1 should be an important protein mediating sensitivity to ethanol in mice.

In cerebellar granule cells, delta subunit-containing GABA(A) receptors are found exclusively at extrasynaptic sites, but their subcellular distribution in other brain areas is poorly understood. We examined the anatomical localization and physiological activation of these receptors in adult mouse dentate gyrus granule cells. Immunocytochemistry revealed a high density of delta subunits in the molecular layer and a much lower density in the cell body layer. At the ultrastructural level, immunogold-labeled delta subunits were found at the edge of symmetric synapses on granule cell dendrites. Functional correlates of this perisynaptic localization were obtained by comparing inhibitory responses in delta subunit-deficient (delta-/-) and wild-type (wt) mice. In whole-cell recordings at 22 degrees C, the weighted decay time constants (tau(w)) of spontaneous IPSCs (sIPSCs) were significantly longer in wt mice but were similar at 34 degrees C, reflecting the role of temperature-dependent GABA uptake in shaping sIPSC decay. IPSCs evoked by minimal stimulation (eIPSCs) near the somata had similar tau(w) in delta-/- and wt mice, but eIPSCs elicited from dendritic sites decayed significantly more slowly in wt mice, consistent with a higher density of delta subunit-containing receptors in the molecular layer. The tau(w) of dendritic eIPSCs of wt mice were shortened by ZnCl2 (10 microm), reflecting the high Zn2+ sensitivity of delta subunit-containing GABA(A) receptors, and were prolonged by the GAT-1 GABA transporter inhibitor NO711 (10 microm). Our results demonstrate a perisynaptic localization of delta subunit-containing GABA(A) receptors and indicate that these receptors can be activated by GABA overspill in the molecular layer.

Morphine addiction has been shown to result from neural adaptations produced by repeated drug exposure, but the mechanism is still unclear. In the present study, we found that gamma-aminobutyric acid (GABA) uptake was increased in mouse brain 120 min after, but not 20 min after, morphine (10 mg/kg, s.c.) injection. We generated GABA transporter I (GAT1)-overexpressing mice to investigate whether the GABAergic system and GABA transporter are involved in morphine-induced reward effects and withdrawal symptoms. Our results revealed that the rewarding effects induced by morphine were significantly decreased in GAT1-overexpressing mice as measured by the conditioned place preference (CPP) paradigm. Moreover, both somatic and vegetative signs of naloxone-induced morphine withdrawal symptoms were substantially reduced in GAT1-overexpressing mice. In addition, the decreased morphine rewarding in transgenic mice could be recovered when mice were coinjected with NO-711 (a GAT1 selective inhibitor) in the CPP paradigm. These findings suggest that the GABAergic system plays an important role in morphine addiction and point to the possibility of developing drugs that target GAT1 and extend the clinical application of opiates., (Copyright 2003 Wiley-Liss, Inc.)

Aim: The changes of seizure susceptibility of transgenic mice overexpressing GABA transporter-1 (GAT-1) were studied to clarify the possible role of GABAergic transmission in epileptogenesis., Methods: Seizures were induced by intraperitoneal administration of pentylenetetrazol (PTZ), picrotoxin (PIC), or kainic acid (KA) respectively. The anticonvulsant effect of ethyl nipecotate was tested by its intraperitoneal injection 15 min before the administration of the epileptogenic agents., Results: The percentages of occurrence of clonic seizures induced by PTZ 45 mg/kg, PIC 2.5 mg/kg, or KA 20 mg/kg in GAT-1 transgenic mice were 88.9 %, 100 %, and 83.3 % respectively, whereas those in control C57BL/6J mice were 42.9 %, 57.1 %, and 33.3 %. The percentages of occurrence of tonic seizures induced by PTZ 45 mg/kg, PIC 2.5 mg/kg, or KA 20 mg/kg in transgenic mice were 88.9 %, 100 %, and 83.3 % respectively, and whereas those in control mice were 28.6 %, 42.9 %, and 16.7 %. The latencies of both clonic and tonic seizures onset in transgenic mice were markedly shortened compared with those in control animals. The results indicated that GAT-1 transgenic mice showed increased susceptibility to seizures induced by the anti-GABAergic convulsive drugs (PTZ, PIC), as well as glutamic receptor agonist (KA). Ethyl nipecotate, inhibitor of GAT-1, inhibited PTZ-induced seizures in both GAT-1 transgenic and C57BL/6J mice. The incidence of seizures was decreased after the application of ethyl nipecotate, and the latencies to the onset of clonic or tonic seizures were also prolonged., Conclusion: The increase in seizure susceptibility of transgenic mice over-expressing GAT-1 is an evidence for involvement of GABAergic transmission in epileptogenesis, and this transgenic mouse might be a useful animal model for study on the role of GABAergic transmission in epileptogenesis.

The present study focused on the involvement of gamma-aminobutyric acid transporter I (GAT1) in pain. We found that GABA uptake was increased in mouse spinal cord at 20 min and 120 min after formalin injection and in mouse brain at 120 min, but not 20 min, after formalin injection. In addition, the antinociceptive effects of GAT1-selective inhibitors were examined using assays of thermal (tail-flick) and chemical (formalin and acetic acid) nociception in C57BL/6J mice. The GAT1-selective inhibitors, ethyl nipecotate and NO-711, exhibited significant antinociceptive effects in these nociceptive assays. To study further the effects of GAT1 on pain, we used two kinds of GAT1-overexpressing transgenic mice (under the control of a CMV promoter or a NSE promoter) to examine the nociceptive responses in these mice. In the thermal, formalin, and acetic acid assays, both kinds of transgenic mice displayed significant hyperalgesia after nociceptive stimuli. In addition, the micro opioid receptor antagonist naloxone had no influence on nociceptive responses in wild-type and transgenic mice. The results indicate that GAT1 is involved in the regulation of pain processes, and point to the possibility of developing analgesic drugs that target GAT1 other than opioid receptors., (Copyright 2003 Wiley-Liss, Inc.)

Changes in gene expression were examined in the brain of mice treated with a drug of abuse, 3,4-methylenedioxymethamphetamine (MDMA, also called Ecstasy). Frontal cortex and midbrain mRNA, analyzed by differential display polymerase chain reaction (DD-PCR) method, showed an altered expression of several cDNAs, 11 of which were isolated, cloned and sequenced. The sequence of one MDMA-induced mRNA corresponds (99.3%) to the mouse gamma-amino butyric acid (GABA) transporter 1 (mGAT1). The established involvement of GABA neurotransmission in the activity of several abused drugs prompted us to focus herein on MDMA effect on the GABA transporter gene family. Semi-quantitative PCR analysis with primers selective to the reported mGAT1 sequence confirmed that MDMA treatment increased mGAT1 expression. Time-course study of the expression of the three GABA transporter subtypes showed that MDMA induced a differential temporal activation of mGAT1 and mGAT4, but had no effect on mGAT2. Quantitative real-time PCR further proved the increased expression of mGAT1 and mGAT4 upon MDMA treatment. Western immunoblotting with anti-GAT1 antibodies showed that MDMA also increased GAT1 protein levels, suggesting that neurotransmission of GABA was altered. MDMA effect was also verified in serotonin transporter knockout (-/-) mice that are insensitive behaviorally to MDMA; the drug did not increase GAT1 protein level in these mutants. In mice, tiagabine and NO-711, inhibitors of GABA transporters, restrained MDMA-induced acute toxicity and death. These results should facilitate novel approaches to prevent deleterious effects, including fatality, induced by MDMA and similar abused psychostimulants., (Copyright 2003 Wiley-Liss, Inc.)

Two forms of GABAergic inhibition coexist: fast synaptic neurotransmission and tonic activation of GABA receptors due to ambient GABA. The mechanisms regulating ambient GABA have not been well defined. Here we examined the role of the GABA transporter in the increase in ambient [GABA] induced by the anticonvulsant vigabatrin. Pretreatment of cultured rat hippocampal neurons with vigabatrin (100 microM) for 2-5 days led to a large increase in ambient [GABA] that was measured as the change in holding current induced by bicuculline during patch-clamp recordings. In contrast, there was a decrease in the frequency of spontaneous miniature inhibitory postsynaptic currents mIPSCs with no change in their amplitude distribution, and a decrease in the magnitude of IPSCs evoked by presynaptic stimulation during paired recordings. The increase in ambient [GABA] was not prevented by blockade of vesicular GABA release with tetanus toxin or removal of extracellular calcium. During perforated patch recordings, the increase in ambient [GABA] was prevented by blocking the GABA transporter, indicating that the GABA transporter was continuously operating in reverse and releasing GABA. In contrast, blocking the GABA transporter increased ambient [GABA] during whole cell patch-clamp recordings unless GABA and Na(+) were added to the recording electrode solution, indicating that whole cell recordings can lead to erroneous conclusions about the role of the GABA transporter in control of ambient GABA. We conclude that the equilibrium for the GABA transporter is a major determinant of ambient [GABA] and tonic GABAergic inhibition. We propose that fast GABAergic neurotransmission and tonic inhibition can be independently modified and play complementary roles in control of neuronal excitability.

Local circuits in the spinal cord that generate locomotion are termed central pattern generators (CPGs). These provide coordinated bilateral control over the normal limb alternation that underlies walking. The molecules that organize the mammalian CPG are unknown. Isolated spinal cords from mice lacking either the EphA4 receptor or its ligand ephrinB3 have lost left-right limb alternation and instead exhibit synchrony. We identified EphA4-positive neurons as an excitatory component of the locomotor CPG. Our study shows that dramatic locomotor changes can occur as a consequence of local genetic rewiring and identifies genes required for the development of normal locomotor behavior.

gamma-Amino-butyric acid (GABA) is cleaved from the synaptic cleft by uptake via specific transporters. Inhibition of such transporters increases the effectiveness of physiologically released GABA. Increased GABAergic neurotransmission has an impact on learning and memory. Therefore, effects of tiagabine, a GABA-transporter inhibitor, were investigated on spatial orientation in the Morris water-maze. Rats were given four training trials per day for 4 days and a probe trial without platform on the 5th day. Compared to saline treated rats, rats treated daily with 20 mg/kg tiagabine showed impaired learning during the acquisition trials. Retrieval was impaired in rats treated only at the probe trial with tiagabine. These results further elucidate the role of GABA in learning and memory.

In the intermediate zone of the embryonic rodent neocortex, neurons migrating tangentially from the basal ganglia express both functional amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and gamma-aminobutyric acid (GABA). To test the hypothesis of GABA release triggered by AMPA receptor activation, we used whole-hemisphere cultures prepared from rat embryos (day 15). We observed a marked decrease in the number of detectable GABA-positive cells in the intermediate zone after exposure to T-AMPA. This effect was blocked by coapplying GYKI 53655, an AMPA receptor antagonist. The decrease in GABA immunolabelling induced by T-AMPA did not require extracellular calcium. In contrast, it was abolished after sodium substitution by choline, or after coapplication of nipecotic acid, a GABA transporter inhibitor. Exposure to high potassium reduced the number of detectable GABA-positive cells. These results are compatible with carrier-mediated GABA release consecutive to sodium influx. GABA released from neurons migrating tangentially in the intermediate zone after AMPA receptor activation may influence neighbouring elements including radially migrating postmitotic neurons, proliferating progenitors and possibly the tangential cells themselves.

Previous work has shown that overstimulation of GABA(A) receptors can potentiate neuronal cell damage during excitotoxic or metabolic stress in vitro and that GABA(A) antagonists or GABA transport blockers are neuroprotective under these situations. Malonate, a reversible succinate dehydrogenase/mitochondrial complex II inhibitor, is frequently used in animals to model cell loss in neurodegenerative diseases such as Parkinson's and Huntington's diseases. To determine if GABA transporter blockade during mitochondrial impairment can protect neurons in vivo as compared with in vitro studies, rats received a stereotaxic infusion of malonate (2 micromol) into the left striatum to induce a metabolic stress. The nonsubstrate GABA transport blocker, NO711 (20 nmol) was infused in some rats 30 min before and 3 h following malonate infusion. After 1 week, dopamine and GABA levels in the striata were measured. Malonate caused a significant loss of striatal dopamine and GABA. Blockade of the GABA transporter significantly attenuated GABA, but not dopamine loss. In contrast with several in vitro reports, GABA(A) receptors were not a downstream mediator of protection by NO711. Intrastriatal infusion of malonate (2 micromol) plus or minus the GABA(A) receptor agonist muscimol (1 micromol), the GABA(A) Cl- binding site antagonist picrotoxin (50 nmol) or the GABA(B) receptor antagonist saclofen (33 nmol) did not modify loss of striatal dopamine or GABA when examined 1 week following infusion. These data show that GABA transporter blockade during mitochondrial impairment in the striatum provides protection to GABAergic neurons. GABA transporter blockade, which is currently a pharmacological strategy for the treatment of epilepsy, may thus also be beneficial in the treatment of acute and chronic conditions involving energy inhibition such as stroke/ischemia or Huntington's disease. These findings also point to fundamental differences between immature and adult neurons in the downstream involvement of GABA receptors during metabolic insult.

Acute GABA transporter inhibition can induce anxiolytic-like behaviors. The present analysis addressed whether chronic treatment (23 days via drinking water) with a GABA transporter inhibitor affects rat behavior similar to acute treatment and interferes with additional benzodiazepine-receptor agonistic treatment. Seventy-one rats divided into seven groups were acutely treated with either vehicle, diazepam (2 mg/kg), zolpidem (0.05 mg/kg), tiagabine (19 mg/kg) or chronically with tiagabine with or without acute diazepam or zolpidem. Animals were behaviorally characterized in an elevated plus-maze. None of the treatments induced changes in the activity of the animals. Acute and chronic treatment with tiagabine induced anxiolytic-like effects, similar to acute doses of diazepam. Acute diazepam did not enhance chronic tiagabine effects, whereas acute zolpidem attenuated the anxiolytic-like effects of chronic tiagabine. It is concluded that anxiolytic effects of acute GABA-uptake inhibition by tiagabine persist under chronic treatment and are sensitive to concomitant use of benzodiazepine receptor ligands.

Several ion channels and pumps are regulated by syntaxin 1A, a component of the synaptic vesicle docking and fusion apparatus. One such regulated protein is the rat brain gamma-aminobutyric acid (GABA) transporter GAT1. The N-terminal cytoplasmic domain of GAT1 directly interacts with syntaxin 1A; this interaction induces a decrease in the rate at which GABA and associated ions are transported. GAT1 function also is regulated by transporter substrates, raising the possibility that substrates mediate at least some of their effects by regulating the interaction between GAT1 and syntaxin 1A. In oocytes expressing GAT1 and syntaxin 1A, superfusion of transporter substrates increases the GAT1 transport rate. The substrate-induced rate change (i) is prevented by coapplication of GAT1 antagonists, (ii) does not occur in oocytes expressing GAT1 alone, and (iii) does not occur in oocytes expressing interaction-deficient syntaxin 1A mutants. In oocytes, and in hippocampal neurons that endogenously express both GAT1 and syntaxin 1A, substrate application results in a decrease in the fraction of syntaxin 1A that is bound to GAT1 on a time-scale comparable to the substrate-induced change in transport rates. These data suggest that substrate translocation regulates GAT1-syntaxin 1A interactions and provide a mechanism by which GABA transport can be increased during times of rising synaptic GABA concentrations.

Inhibition is mediated by two classes of ionotropic receptors in the retina, GABA(A) and GABA(C) receptors. We used the GABA transport blocker NO-711 to examine the role of GABA transporters in shaping synaptic responses mediated by these two receptors in the salamander retinal slice preparation. Focal applications (puffs) of GABA onto GABA(C) receptors on bipolar cells terminals or GABA(A) receptors on ganglion cells elicited currents that were enhanced by NO-711, demonstrating the presence of transporters in the inner plexiform layer (IPL). IPSCs were evoked in bipolar and ganglion cells by puffing kainate into the IPL. NO-711 enhanced the IPSCs only in bipolar cells, suggesting that, when GABA uptake was blocked, the GABA(C) receptors were more strongly activated by spillover transmission than the GABA(A) receptors on ganglion cells. NO-711 enhanced the light-evoked IPSCs mediated by GABA(C) receptors on bipolar cell axon terminals, which resulted in reduced transmission between bipolar and ganglion cells. NO-711 also shifted the intensity-response relationship of the ganglion cell, reducing its sensitivity to light. Surround illumination has been shown by others to produce similar shifts in ganglion cell light sensitivity. Our results show that GABA transporters limit the extent of inhibitory transmission at the inner retina during light-evoked signal processing.

Postmortem samples from individuals with schizophrenia (n = 13) and control subjects (n = 10) were investigated for binding of [(3)H]tiagabine to GABA transporter-1 GAT-1. The binding was analyzed in the cingulate cortex and the caudate nucleus. There were no differences in binding affinity between the groups in any of the investigated areas. The maximum number of binding sites (B(max)) was elevated in the schizophrenic cingulate cortex compared to controls (1,264 +/- 96 vs. 860 +/- 123 fmol/mg of protein). The B(max) in the caudate nucleus for schizophrenics (426 +/- 40 fmol/mg of protein) was the same as for controls (495 +/- 69 fmol/mg of protein). The increase in GAT-1 in schizophrenia could be explained by a modulatory upregulation in the cingulate cortex., (Copyright 2002 S. Karger AG, Basel)

1. Bi-directional GABA-transport was studied by performing uptake and superfusion experiments in human embryonic kidney 293 cells stably expressing the rat GABA transporter rGAT-1. 2. K(M) and V(max) values for [(3)H]-GABA uptake were 11.7+/-1.8 microM and 403+/-55 pmol min(-1) 10(-6) cells (n=9), respectively. 3. Kinetic analysis of outward transport was performed by pre-labelling the cells with increasing concentrations of [(3)H]-GABA and triggering outward transport with 333 microM GABA. Approximate apparent K(M) and V(max) values were 12 mM and 50 pmol min(-1) 10(-6) cells, respectively. 4. GABA re-uptake inhibitors (RI; e.g. tiagabine), as well as, substrates of the rGAT-1 (e.g. GABA, nipecotic acid) concentration dependently decreased [(3)H]-GABA uptake and increased efflux of [(3)H]-GABA from pre-labelled cells. The IC(50) values for inhibiting uptake and the EC(50) values for increasing efflux were significantly correlated (r(2)=0.99). 5. On superfusion, RI antagonized the efflux-enhancing effect of the substrates. The effect of the latter was markedly augmented in the presence of ouabain (100 microM), whereas the effect of RI remained unchanged. The most likely explanation for the release enhancing effect of RI is interruption of ongoing re-uptake. 6. The structural GABA-analogue 2,4-diamino-n-butyric acid (DABA) exhibited a bell-shaped concentration response curve on [(3)H]-GABA efflux with the maximum at 1 mM, and displayed a deviation from the sigmoidal inhibition curve in uptake experiments in the same concentration range. At concentrations below 1 mM, DABA inhibited [(3)H]-GABA uptake non-competitively, while at 1 mM and above the inhibition of uptake followed a competitive manner. 7. The results provide information of GABA inward and outward transport, and document a complex interaction of the rGAT-1 with its substrate DABA.

Neurotransmitter transporters regulate synaptic transmitter levels and are themselves functionally regulated by a number of different signal transduction cascades. A common theme in transporter regulation is redistribution of transporter protein between intracellular stores and the plasma membrane. The triggers and mechanisms underlying this regulation are important in the control of extracellular transmitter concentrations and hence synaptic signaling. Previously, we demonstrated that the gamma-aminobutyric acid transporter GAT1 is regulated by direct tyrosine phosphorylation, resulting in an up-regulation of transporter expression on the plasma membrane. In the present report, we show that two tyrosine residues on GAT1 contribute to the phosphorylation and transporter redistribution. Tyrosine phosphorylation is concomitant with a decrease in the rate of transporter internalization from the plasma membrane. A decrease in GAT internalization rates also occurs in the presence of GAT1 substrates, suggesting the hypothesis that tyrosine phosphorylation is required for the substrate-induced up-regulation of GAT1 surface expression. In support of this hypothesis, incubation of GAT1-expressing cells with transporter ligands alters the amount of GAT1 tyrosine phosphorylation, and substrate-induced surface expression is unchanged in a GAT1 mutant lacking tyrosine phosphorylation sites. These data suggest a model in which substrates permit the phosphorylation of GAT1 on tyrosine residues and that the phosphorylated state of the transporter is refractory for internalization.

In this study, GABA efflux transport from brain to blood was estimated by using the brain efflux index (BEI) method. [3H]GABA microinjected into parietal cortex area 2 (Par2) of the rat brain was eliminated from the brain with an apparent elimination half-life of 16.9 min. The blood-brain barrier (BBB) efflux clearance of [3H]GABA was at least 0.153 mL/min/g brain, which was calculated from the elimination rate constant (7.14 x 10(-2) x min(-1)) and the distribution volume in the brain (2.14 mL/g brain). Direct comparison of the apparent BBB influx clearance [3H]GABA (9.29 microL/min/g brain) and the apparent efflux clearance (153 microL/min/g brain) indicated that the efflux clearance was at least 16-fold greater than the influx clearance. In order to reduce the effect of metabolism in the neuronal cells following intracerebral microinjection, we determined the apparent efflux of [3H]GABA in the presence of nipecotic acid, a GABA transport inhibitor in parenchymal cells, using the BEI method. Under such conditions, the elimination of [3H]GABA across the BBB showed saturation and inhibition by probenecid in the presence of nipecotic acid. Furthermore, the uptake of [3H]GABA by MBEC4 cells was inhibited by GABA, taurine, beta-alanine and nipecotic acid in a concentration-dependent manner. It is likely that GABA inhibits the first step in the abluminal membrane uptake by brain endothelial cells, and that probenecid selectively inhibits the luminal membrane efflux transport process from the brain capillary endothelial cells based on the in vivo and in vitro evidence. The BBB acts as the efflux pump for GABA to reduce the brain interstitial fluid concentration.

Whole-cell patch recordings were made from dopamine-containing neurons in the ventral tegmental area (VTA) and substantia nigra zona compacta (SNC). Isoguvacine evoked an outward current (at -60 mV) in a concentration-dependent manner with an EC50 of 62+/-8 microM. The gamma-aminobutyric acid (GABA) uptake inhibitor 1-(2(((diphenylmethylene)imino)oxy)ethyl)-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO 711) (3 microM) shifted the isoguvacine concentration-response curve to the left, with a new EC50 of 22+/-4 microM. L-Arginine (3 mM) also shifted the isoguvacine concentration-response curve to the left, with a new EC50 of 29+/-5 microM. L-Arginine (3 mM) increased the currents evoked by GABA (100 microM) and muscimol (1 microM) by 208% and 261%, respectively. The GABA uptake inhibitor 4,5,6,7,-tetrahydroisoxazolo[4,5-c]-pyridin-3-ol hydrobromide (THPO) (300 microM) not only mimicked but also occluded the ability of L-arginine (3 mM) to potentiate currents evoked by isoguvacine. Equimolar replacement of Na+ with choline increased GABA-evoked currents, suggesting that a low Na+ concentration has an inhibitory effect on GABA transport. Low Na+ concentration (25 mM) inhibited isoguvacine currents but still occluded the potentiating effects of L-arginine. We conclude that GABA uptake inhibitors potentiate the actions of the GABA(A) receptor agonists, isoguvacine and muscimol, probably because they are effective substrates for GABA transporters in the ventral midbrain.

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.

The GABA transporter can reverse with depolarization, causing nonvesicular GABA release. However, this is thought to occur only under pathological conditions. Patch-clamp recordings were made from rat hippocampal neurons in primary cell cultures. Inhibition of GABA transaminase with the anticonvulsant gamma-vinyl GABA (vigabatrin; 0.05-100 microm) resulted in a large leak current that was blocked by bicuculline (50 microm). This leak current occurred in the absence of extracellular calcium and was blocked by the GABA transporter antagonist SKF-89976a (5 microm). These results indicate that vigabatrin induces spontaneous GABA efflux from neighboring cells via reversal of GABA transporters, subsequently leading to the stimulation of GABA(A) receptors on the recorded neuron. The leak current increased slowly over 4 d of treatment with 100 microm vigabatrin, at which time it reached an equivalent conductance of 9.0 +/- 4.9 nS. Blockade of glutamic acid decarboxylase with semicarbazide (2 mm) decreased the leak current that was induced by vigabatrin by 47%. In untreated cells, carrier-mediated GABA efflux did not occur spontaneously but was induced by an increase in [K(+)](o) from 3 to as little as 6 mm. Vigabatrin enhanced this depolarization-evoked nonvesicular GABA release and also enhanced the heteroexchange release of GABA induced by nipecotate. Thus, the GABA transporter normally operates near its equilibrium and can be easily induced to reverse by an increase in cytosolic [GABA] or mild depolarization. We propose that this transporter-mediated nonvesicular GABA release plays an important role in neuronal inhibition under both physiological and pathophysiological conditions and is the target of some anticonvulsants.

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH-group reagents (N-ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4-aminobutyrate (GABA) in a competitive way (Ki = 20.9 +/- 2.4 mM). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1-[2-([(diphenylmethylene)amino]oxy)ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride (NO-711) acid decreased carnitine accumulation while betaine, taurine and beta-alanine had no effect. The GABA transporters expressed in Xenopus laevis oocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nałecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nałecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.

In vivo dialysis and in vitro electrophysiological studies suggest that GABA uptake is altered in the dentate gyrus of human temporal lobe epileptics characterized with mesial temporal sclerosis (MTLE). Concordantly, anatomical studies have shown that the pattern of GABA-transporter immunoreactivity is also altered in this region. This decrease in GABA uptake, presumably due to a change in the GABA transporter system, may help preserve inhibitory tone interictally. However, transporter reversal can also occur under several conditions, including elevations in [K(+)]o, which occurs during seizures. Thus GABA transporters could contribute to seizure termination and propagation through heterotransport. To test whether GABA transport is compromised in both the forward (uptake) and reverse (heterotransport) direction in the sclerotic epileptic dentate gyrus, the physiological effects of microapplied GABA and nipecotic acid (NPA; a compound that induces heterotransport) were examined in granule cells in hippocampal slices from kainate (KA)-induced epileptic rats and patients with temporal lobe epilepsy (TLE). GABA- and NPA-induced responses were prolonged in granule cells from epileptic rats versus controls (51.3 and 31.3% increase, respectively) while the conductance change evoked with NPA microapplication was reduced by 40%. Furthermore the ratio of GABA/NPA conductance, but not duration, was significantly >1 in epileptic rats but not controls, suggesting a compromise in transporter function in both directions. Similar changes were observed in tissue resected from epileptic patients with medial temporal sclerosis but not in those without the anatomical changes associated with MTLE. These data suggest that the GABA transporter system is functionally compromised in both the forward and reverse directions in the dentate gyrus of chronically epileptic tissue characterized by mesial temporal sclerosis. This alteration may enhance inhibitory tone interically yet be permissive for seizure propagation due to a decreased probability for GABA heterotransport during seizures.

Correlated spiking activity and associated Ca(2+) waves in the developing retina are important in determining the connectivity of the visual system. Here, we show that GABA, via GABA(B) receptors, regulates the temporal characteristics of Ca(2+) waves occurring before synapse formation in the embryonic chick retina. Blocking ionotropic GABA receptors did no affect these Ca(2+) transients. However, when these receptors were blocked, GABA abolished the transients, as did the GABA(B) agonist baclofen. The action of baclofen was prevented by the GABA(B) antagonist p-3-aminopropyl-p-diethoxymethyl phosphoric acid (CGP35348). CGP35348 alone increased the duration of the transients, showing that GABA(B) receptors are tonically activated by endogenous GABA. Blocking the GABA transporter GAT-1 with 1-(4,4-diphenyl-3-butenyl)-3-piperidine carboxylic acid (SKF89976A) reduced the frequency of the transients. This reduction was prevented by CGP35348 and thus resulted from activation of GABA(B) receptors by an increase in external [GABA]. The effect of GABA(B) receptor activation persisted in the presence of activators and blockers of the cAMP-PKA pathway. Immunocytochemistry showed GABA(B) receptors and GAT-1 transporters on ganglion and amacrine cells from the earliest times when Ca(2+) waves occur (embryonic day 8). Patch-clamp recordings showed that K(+) channels on ganglion cell layer neurons are not modulated by GABA(B) receptors, whereas Ca(2+) channels are; however, Ca(2+) channel blockade with omega-conotoxin-GVIA or nimodipine did not prevent Ca(2+) waves. Thus, the regulation of Ca(2+) waves by GABA(B) receptors occurs independently of N- and L-type Ca(2+) channels and does not involve K(+) channels of the ganglion cell layer. GABA(B) receptors are likely to be of key importance in regulating retinal development.

The physiological significance and subcellular distribution of voltage dependent calcium channels was defined using calcium channel blockers to inhibit potassium induced rises in cytosolic calcium concentration in cultured mouse neocortical neurons. The cytosolic calcium concentration was measured using the fluorescent calcium chelator fura-2. The types of calcium channels present at the synaptic terminal were determined by the inhibitory action of calcium channel blockers on potassium-induced [3H]GABA release in the same cell preparation. L-, N-, P-, Q- and R-/T-type voltage dependent calcium channels were differentially distributed in somata, neurites and nerve terminals. omega-conotoxin MVIIC (omega-CgTx MVIIC) inhibited approximately 40% of the Ca(2+)-rise in both somata and neurites and 60% of the potassium induced [3H]GABA release, indicating that the Q-type channel is the quantitatively most important voltage dependent calcium channel in all parts of the neuron. After treatment with thapsigargin the increase in cytosolic calcium was halved, indicating that calcium release from thapsigargin sensitive intracellular calcium stores is an important component of the potassium induced rise in cytosolic calcium concentration. The results of this investigation demonstrate that pharmacologically distinct types of voltage dependent calcium channels are differentially localized in cell bodies, neurites and nerve terminals of mouse cortical neurons but that the Q-type calcium channel appears to predominate in all compartments.

In this study we explored the effect of the stimulation of nicotinic acetylcholine receptors located on interneurons by measuring 4-amino-n-[2,3-(3)H]butyric acid ([(3)H]GABA) release and monitoring [Ca (2+)](i) in superfused hippocampal slices. In the presence of 6-cyano-7-nitroquinoxaline-2,3-dione, (+/-)-2-amino-5-phosphonopentanoic acid, and atropine, i.e., under the blockade of N-methyl-D-aspartate and non-N-methyl-D-aspartate glutamate and muscarinic receptors, nicotine did not alter the spontaneous outflow of [(3)H]GABA, but significantly increased the stimulation-evoked [(3)H]GABA efflux. This effect of nicotine depended on the time interval between nicotine treatment and electrical stimulus, the concentration of nicotine (1-100 microM), and the parameters of electrical depolarization. Acetylcholine (0.03-3 mM), and the alpha 7 subtype-selective agonist choline (0.1-10 mM), also potentiated stimulus-evoked release of [(3)H]GABA, whereas 1,1-dimethyl-4-phenilpiperazinium iodide failed to increase the tritium outflow significantly. The effect of nicotine treatment was prevented by tetrodotoxin (1 microM) and by the nicotinic acetylcholine receptor antagonist mecamylamine (10 microM), and the alpha 7 subtype-selective antagonists alpha-bungarotoxin (100 nM) and methyllycaconitine (10 nM), whereas dihidro-beta-erythroidine (20 nM) was without effect. Perfusion of 100 microM nicotine caused a [Ca(2+)](i) transient in about one-third of the tested interneurons; however, the response to subsequent electrical stimulation remained unchanged. Inhibition of the GABA transporter system by nipecotic acid (1 mM) or by decreasing the bath temperature to 12 degrees C abolished completely the effect of nicotine to potentiate the stimulation-evoked release of GABA. These findings indicate that the activation of alpha 7-type nicotinic receptors of hippocampal interneurons results in a long-lasting ability of these cells to respond to depolarization with an increased release of GABA mediated by the transporter system.

At central synapses, a large and fast spike of neurotransmitter efficiently activates postsynaptic receptors. However, low concentrations of transmitter can escape the cleft and activate presynaptic and postsynaptic receptors. We report here that low concentrations of GABA reduce IPSCs in hippocampal neurons by preferentially desensitizing rather than opening GABA(A) channels. GABA transporter blockade also caused desensitization by locally elevating GABA to approximately 1 microm. Recovery of the IPSC required several seconds, mimicking recovery of the channel from slow desensitization. These results indicate that low levels of GABA can regulate the amplitude of IPSCs by producing a slow form of receptor desensitization. Accumulation of channels in this absorbing state allows GABA(A) receptors to detect even a few molecules of GABA in the synaptic cleft.

The present analysis addressed behavioral changes after treatment with 4.5 mg/kg or 18.5 mg/kg of the GABA-uptake inhibitor tiagabine combined with either the benzodiazepine diazepam (1.5 mg/kg) or the imidazopyridine zolpidem (0.05 mg/kg), the latter two acting differentially on GABA(A) receptor subtypes. The study included 97 male PVG/OIaHsd rats. A standard open field, an enriched open field, and an elevated plus-maze was used to study rat behavior. Treatment with the low dose of tiagabine alone induced no specific behavioral effects, whereas the high dose had an anxiolytic-like potential. Furthermore, diazepam but not zolpidem displayed anxiolytic-like effects. Combination of each benzodiazepine receptor agonist with tiagabine at the low dose decreased explorative activity. Diazepam plus the high dose of tiagabine increased the activity in the open-field test. Zolpidem together with 18.5 mg/kg tiagabine had an angiogenic-like effect compared to pure tiagabine treatment. These results provide evidence for a pharmacodynamic interaction between the GABA-uptake inhibitor tiagabine and diazepam or zolpidem. The interaction might be relevant in the clinic when combining the anticonvulsant tiagabine and a benzodiazepine receptor agonist.

Epileptic syndromes frequently start at childhood and therefore it is crucial to test new anticonvulsants at immature stages of the nervous system. We compared the effects of the gamma-aminobutyric acid (GABA) uptake inhibitor tiagabine [(R)-N-(4, 4-bis(3-methyl-2-thienyl)but)3-en-1-yl nipecotic acid] on low-Mg(2+)-induced epileptic discharges in brain slices from rat pups (p 5-8) and juvenile animals (p 15-20). In tissue from rat pups, tiagabine slightly reduced epileptiform activity in hippocampal area CA1 but had no effect in the entorhinal cortex. In juvenile rats, epileptiform discharges were unaffected in CA1 but suppressed by 60% in the entorhinal cortex. While tiagabine increases its efficacy with age, in-situ hybridisation and PCR analysis show that mRNA coding for the neuronal GABA-transporter GAT-1 is already present at p 5. We therefore conclude that the increasing efficacy of tiagabine during ontogenesis is due to functional maturation of GABAergic synapses rather than to up-regulation of GAT-1 expression.

(3R)-1-[4,4-bis(3-methyl-2-thienyl)-3-butenyl]-3-piperidinecarboxylic acid 1 (tiagabine, Gabitril) is a potent and selective gamma-aminobutyric acid (GABA) uptake inhibitor with proven anticonvulsant efficacy in humans. This drug, which has a unique mechanism of action among marketed anticonvulsant agents, has been launched for add-on treatment of partial seizures with or without secondary generalization in patients >12 years of age. Using this new agent as a benchmark, we have designed two series of novel GABA uptake inhibitors of remarkable potency, using a putative new model of ligand interaction at the GABA transporter type 1 (GAT-1) uptake site. This model involves the postulated interaction of an electronegative region in the GABA uptake inhibitor with a positively charged domain in the protein structure of the GAT-1 site. These two novel series of anticonvulsant agents contain diaryloxime or diarylvinyl ether functionalities linked to cyclic amino acid moieties and were derived utilizing the new model, via a series of design steps from the known 4,4-diarylbutenyl GABA uptake inhibitors. The new compounds are potent inhibitors of [(3)H]-GABA uptake in rat brain synaptosomes in vitro, and their antiepileptic potential was demonstrated in vivo by their ability to protect against seizures induced by the benzodiazepine receptor inverse agonist methyl 4-ethyl-6,7-dimethoxy-beta-carboline-3-carboxylate (DMCM) in mice. From structure-activity studies of these new GABA uptake inhibitors, we have shown that insertion of an ether oxygen in conjugation with the double bond in tiagabine (K(i) = 67 nM) improves in vitro potency by 5-fold to 14 nM.

The antiepileptic agents sodium valproate (VPA), vigabatrin (VGB) and tiagabine (TGB) have been proposed to exert their effects, at least in part, by an action on the transport of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). This information has, however, been gleaned from studies employing experimental systems derived from animal tissues. We have conducted preliminary studies of the effects of VPA, VGB and TGB on the transport of GABA into primary cultures of human astrocytes, derived from both adult and foetal tissues. Astrocytes were prepared from cerebral cortical tissue obtained from patients undergoing surgery for intractable epilepsy, and from spontaneously aborted foetuses (16-24 weeks gestation). The cells were isolated via a series of enzymatic digestions, grown under standard culture conditions for around 21 days and then assayed for GABA uptake activity. VPA (1,000 microM), VGB (100 microM) and TGB (200 nM) all significantly (p < 0.05) reduced the uptake of GABA into primary cultures of human adult astrocytes following a one hour exposure. VPA (1,000 microM) and VGB (100 microM) similarly reduced GABA uptake into astrocytes derived from human foetal tissue, while TGB (200 and 500 nM) was without effect. The results of these preliminary studies suggest that VPA and VGB reduce GABA transport into both adult- and foetally-derived human astrocytes, whereas TGB appears active only in cells cultured from adult brain. Delayed development of the GAT-1 transporter in foetal tissue could explain this observation.

1. We have expressed the GABA transporter (GAT1) of mouse brain in Xenopus oocytes and have investigated the effects of four antiepileptic drugs, tiagabine (TGB), vigabatrin (VGB), gabapentin (GBP) and valproate (VAL), on GAT1 transporter function by measurements of 3H-labelled GABA uptake and GAT1-mediated currents. 2. Not only TGB, a well-known inhibitor of GAT1-mediated transport, but also the other drugs efficiently inhibit the uptake of [3H]-GABA by GAT1. Inhibition at 50% is obtained for VGB, TGB, GBP, and VAL at concentrations of about 1 nM, 1 microM, 50 microM and 100 microM, respectively. 3. However, GAT1-mediated steady-state and transient currents are nearly unaffected by VGB, GBP, and VAL at even five times higher concentrations. Only TGB blocks the uptake and steady-state and transient currents at micromolar concentrations. 4. VGB exhibits a complex interaction with GAT1; at concentrations about 1 nM, the inhibition of uptake is released, but at millimolar concentrations the uptake is inhibited again, and also the GAT1-mediated current is finally inhibited at these concentrations with a KI value of 0.5 mM. The concentration dependency of inhibition of uptake can be explained by two interaction sites with different affinities, a blocking site and a transport site. 5. The differences in effects of VAL, GBP, and VGB on uptake and currents can be attributed to the fact that GAT1 has the capability to operate in an electrogenic mode without uptake of GABA. We suggest that inhibition occurs only when GAT1 operates in the GABA-uptake mode. 6. The inhibition of GABA uptake by these four drugs will result in an elevation of the GABA concentration in the synaptic cleft, which will enhance synaptic inhibition and thereby contribute to their antiepileptic effects.

1. The rabbit Na+-glucose (SGLT1) and the human Na+-Cl--GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive water permeabilities (Lp) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 Lp values were obtained by measuring Lp in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the Lp values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the Lp of control oocytes. Passive Na+ transport (Na+ leak) was obtained from the blocker-sensitive Na+ currents in the absence of substrates (glucose and GABA). 2. Passive Na+ and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant (Ki), 3-5 microM). When Na+ was replaced with Li+, phlorizin also inhibited Li+ and water transport, but with a lower affinity (Ki, 100 microM). When Na+ was replaced by choline, which is not transported, the SGLT1 Lp was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 3. The activation energies (Ea) for passive Na+ and water transport through SGLT1 were 21 and 5 kcal mol-1, respectively. The high Ea for Na+ transport is comparable to that of Na+-glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low Ea for water transport is similar to that of water channels (aquaporins). 4. GAT1 also behaved as an SKF89976A-sensitive water channel. We did not observe passive Na+ transport through GAT1. 5. We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across the intestinal brush border membrane.

The electrophysiological effects of GABA and the GABA uptake inhibitor, NO-711, were investigated on cortical slices prepared from adult audiogenic seizure-prone DBA/2 mice. GABA, perfused for 1 min, elicited depolarizing responses which had a mean duration of 80-100 s and were concentration-dependent (0.1-32 mM). NO-711 (25 microM), perfused for 15 min, produced depolarizations with a mean duration of 50-60 s and these persisted for 4-5 h. The responses to both compounds were blocked by the GABA(A) receptor antagonist, bicuculline. Pre-treatment of the slices with NO-711 potentiated the responses to GABA and moved the concentration-response curve to the left. The EC50 to GABA following pre-treatment with NO-711 was 111+/-24 microM from a control value of 1.17+/-0.19 mM. These results demonstrate that in cortical slices GABA has a depolarizing action rather than the conventional one of hyperpolarization and that this response is potentiated by inhibition of GABA reuptake.

The behavioural consequences of inhibition of gamma-aminobutyric acid (GABA) uptake were studied. Two GABA uptake inhibitors, tiagabine and SKF 89976-A, were administered to rats, and behaviour was analysed 30 min later in a standard open field, an enriched open field, and an elevated plus-maze. Eight groups of animals received either saline (0.9%), tiagabine, or SKF 89976-A. At a dose of 18.5 mg/kg, tiagabine, an established antiseizure drug, impaired motor coordination, enhanced exploratory activity and reduced anxiety related behaviour. SKF 89976-A exhibited minimal effects over the dose range tested. These results indicate that inhibition of GABA uptake might be a pharmacological strategy to treat not only epilepsy, but also anxiety disorders.

Gabitril (tiagabine) is a potent selective inhibitor of the principal neuronal gamma-aminobutyric acid (GABA) transporter (GAT-1) in the cortex and hippocampus. By slowing the reuptake of synaptically-released GABA, it prolongs inhibitory postsynaptic potentials. In animal models of epilepsy, tiagabine is particularly effective against kindled (limbic) seizures and against reflexly-induced generalized convulsive seizures. These data are predictive of its efficacy in complex partial seizures in humans. Possible clinical applications outside the field of epilepsy include bipolar disorder and pain.

The presynaptically located gamma-aminobutyric acid (GABA) transporter (GAT-1) was studied in a group of patients with Alzheimer's disease (AD) and in a control group using the GAT-1 selective radioligand [3H]tiagabine. Post mortem brain tissue from frontal cortex, temporal cortex, and caudate nucleus from 18 AD patients and 23 age-matched controls were studied. The binding was saturable (Kd 26 nM) and region specific. There were no significant differences between the groups with respect to the binding capacity (Bmax) and binding affinity (Kd). The unaltered [3H]tiagabine binding to GAT-1 protein indicates that intrinsic GABA neurons are spared in Alzheimer's disease.

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.