Blockade of GABA transporter-1 and GABA transporter-3 in the lateral habenula improves depressive-like behaviors in a rat model of Parkinson's disease.

The hyperactivity of the lateral habenula (LHb) is closely associated with depression. At present, it is unknown how GABA transporter (GAT) in the LHb affects depressive-like behaviors, particularly in Parkinson's disease (PD)-related depression. In this study, unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNc) in rats induced depressive-like behaviors and led to hyperactivity of LHb neurons compared to sham-lesioned rats. Intra-LHb injection of GAT-1 inhibitor NO-711 produced antidepressant-like responses, decreased firing rate of LHb neurons, and increased levels of LHb extracellular GABA in sham-lesioned and the lesioned rats. Further, the dose producing behavioral effects in the lesioned rats was lower than that of sham-lesioned rats. In the lesioned rats, the duration of inhibitory effect on the firing rate and increased levels of the GABA induced by NO-711 was longer than those in sham-lesioned rats, respectively. Intra-LHb injection of GAT-3 inhibitor SNAP-5114 improved depressive-like behaviors and decreased firing rate of LHb neurons in the lesioned rats, but not in sham-lesioned rats. SNAP-5114 increased LHb GABA levels in the lesioned rats, whereas did not alter that in sham-lesioned rats. These changes were involved in the down-regulated expression of LHb GAT-1 and GAT-3 after lesioning the SNc. These findings suggest that GAT-1 plays a major role in transporting LHb GABA under physiological conditions, and depletion of dopamine increases the transport capacity of GAT-3 in the LHb. Further, the study provides evidence that GAT-1 and GAT-3 in the LHb are involved in the regulation of PD-related depression.

Mechanisms underlying the enhancement of γ-aminobutyric acid responses in the external globus pallidus of R6/2 Huntington's disease model mice.

In Huntington's disease (HD), the output of striatal indirect pathway medium-sized spiny neurons (MSNs) is altered in its target region, the external globus pallidus (GPe). In a previous study we demonstrated that selective optogenetic stimulation of indirect pathway MSNs induced prolonged decay time of γ-aminobutyric acid (GABA) responses in GPe neurons. Here we identified the mechanism underlying this alteration. Electrophysiological recordings in slices from symptomatic R6/2 and wildtype (WT) mice were used to evaluate, primarily, the effects of GABA transporter (GAT) antagonists on responses evoked by optogenetic activation of indirect pathway MSNs. In addition, immunohistochemistry (IHC) and Western blots (WBs) were used to examine GAT-3 expression in HD and WT mice. A GAT-3 blocker (SNAP5114) increased decay time of GABA responses in WT and HD GPe neurons, but the effect was significantly greater in WT neurons. In contrast, a GAT-1 antagonist (NO-711) or a GABAB receptor antagonist (CGP 54626) produced small increases in decay time but no differential effects between genotypes. IHC and WBs showed reduction of GAT-3 expression in the GPe of HD mice. Thus, reduced expression or dysfunction of GAT-3 could underlie alterations of GPe responses to GABA inputs from striatum and could be a target for therapeutic intervention.

Exploring the molecular determinants for subtype-selectivity of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid analogs as betaine/GABA transporter 1 (BGT1) substrate-inhibitors.

We have previously identified 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA) as the most potent substrate-inhibitor of the betaine/GABA transporter 1 (BGT1) (IC50 2.5 µM) reported to date. Herein, we characterize the binding mode of 20 novel analogs and propose the molecular determinants driving BGT1-selectivity. A series of N1-, exocyclic-N-, and C4-substituted analogs was synthesized and pharmacologically characterized in radioligand-based uptake assays at the four human GABA transporters (hGATs) recombinantly expressed in mammalian cells. Overall, the analogs retained subtype-selectivity for hBGT1, though with lower inhibitory activities (mid to high micromolar IC50 values) compared to ATPCA. Further characterization of five of these BGT1-active analogs in a fluorescence-based FMP assay revealed that the compounds are substrates for hBGT1, suggesting they interact with the orthosteric site of the transporter. In silico-guided mutagenesis experiments showed that the non-conserved residues Q299 and E52 in hBGT1 as well as the conformational flexibility of the compounds potentially contribute to the subtype-selectivity of ATPCA and its analogs. Overall, this study provides new insights into the molecular interactions governing the subtype-selectivity of BGT1 substrate-inhibitors. The findings may guide the rational design of BGT1-selective pharmacological tool compounds for future drug discovery.

Beta-adrenergic receptor activation increases GABA uptake in adolescent mice frontal cortex: Modulation by cannabinoid receptor agonist WIN55,212-2.

GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80 mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.

Pharmacological Characterization of [3H]ATPCA as a Substrate for Studying the Functional Role of the Betaine/GABA Transporter 1 and the Creatine Transporter.

The betaine/γ-aminobutyric acid (GABA) transporter 1 (BGT1) is one of the four GABA transporters (GATs) involved in the termination of GABAergic neurotransmission. Although suggested to be implicated in seizure management, the exact functional importance of BGT1 in the brain is still elusive. This is partly owing to the lack of potent and selective pharmacological tool compounds that can be used to probe its function. We previously reported the identification of 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA), a selective substrate for BGT1 over GAT1/GAT3, but also an agonist for GABAA receptors. With the aim of providing new functional insight into BGT1, we here present the synthesis and pharmacological characterization of the tritiated analogue, [3H]ATPCA. Using traditional uptake assays at recombinant transporters expressed in cell lines, [3H]ATPCA displayed a striking selectivity for BGT1 among the four GATs ( Km and Vmax values of 21 µM and 3.6 nmol ATPCA/(min × mg protein), respectively), but was also found to be a substrate for the creatine transporter (CreaT). In experiments with mouse cortical cell cultures, we observed a Na+-dependent [3H]ATPCA uptake in neurons, but not in astrocytes. The neuronal uptake could be inhibited by GABA, ATPCA, and a noncompetitive BGT1-selective inhibitor, indicating functional BGT1 in neurons. In conclusion, we report [3H]ATPCA as a novel radioactive substrate for both BGT1 and CreaT. The dual activity of the radioligand makes it most suitable for use in recombinant studies.

A Role for Diminished GABA Transporter Activity in the Cortical Discharge Phenotype of MeCP2-Deficient Mice.

Cortical network hyper-excitability is a common phenotype in mouse models lacking the transcriptional regulator methyl-CPG-binding protein 2 (MeCP2). Here, we implicate enhanced GABAB receptor activity stemming from diminished cortical expression of the GABA transporter GAT-1 in the genesis of this network hyper-excitability. We found that administering the activity-dependent GABAB receptor allosteric modulator GS-39783 to female Mecp2(+/-) mice at doses producing no effect in wild-type mice strongly potentiated their basal rates of spontaneous cortical discharge activity. Consistently, administering the GABAB receptor antagonist CGP-35348 significantly decreased basal discharge activity in these mice. Expression analysis revealed that while GABAB or extra-synaptic GABAA receptor prevalence is preserved in the MeCP2-deficient cortex, the expression of GAT-1 is significantly reduced from wild-type levels. This decrease in GAT-1 expression is consequential, as low doses of the GAT-1 inhibitor NO-711 that had no effects in wild-type mice strongly exacerbated cortical discharge activity in female Mecp2(+/-) mice. Taken together, these data indicate that the absence of MeCP2 leads to decreased cortical levels of the GAT-1 GABA transporter, which facilitates cortical network hyper-excitability in MeCP2-deficient mice by increasing the activity of cortical GABAB receptors.

Development of Highly Potent GAT1 Inhibitors: Synthesis of Nipecotic Acid Derivatives by Suzuki-Miyaura Cross-Coupling Reactions.

A new series of potent and selective mGAT1 inhibitors has been identified, featuring a nipecotic acid residue and an N-butenyl linker with a 2-biphenyl residue at the ω-position. Docking, combined with MD calculations, revealed a binding mode for the new compounds similar to that of tiagabine, the only mGAT1 inhibitor currently approved as antiepileptic drug. For the synthesis, a Suzuki-Miyaura cross-coupling reaction was used as a key step by which variously substituted biaryl subunits were assembled. Biological evaluation revealed several compounds that possess binding affinities and inhibitory potencies toward mGAT1, together with subtype selectivities against mGAT2-mGAT4 that were similar to or even higher than those for tiagabine. A derivative carrying the 2',4'-dichloro-2-biphenyl moiety attached to N-but-3-enylnipecotic acid at the terminal position of the linker chain was found to be the most potent binder, with the racemic form of the compound displaying a binding affinity of 8.05±0.13 (pKi ), while the R enantiomer exhibited an affinity value of 8.33±0.06 (pKi ).

Caffeine potentiates the release of GABA mediated by NMDA receptor activation: Involvement of A1 adenosine receptors.

Caffeine, a methylated derivative of xanthine and widely consumed psychoactive substance, acts in several targets in the nervous system. We investigated its role in retinal explants of chick embryo analyzing the role of purinergic receptors in [(3)H]-GABA release induced by d-aspartate (d-asp). d-Asp increases GABA-release 4.5-fold when compared to basal levels from 13-day-old chick embryo retinal explants. Caffeine 500µM elevated d-asp-induced GABA release in 60%. The release was inhibited in the presence of NNC-711, a GABA transporter-1 (GAT-1) blocker or by MK-801, an N-methyl-d-aspartate receptor (NMDAR) antagonist. Caffeine did not modify [(3)H]-GABA uptake carried out for 5, 10, 30 and 60min and did not increase the release of d-asp or glutamate at basal or stimulated conditions. The caffeine effect was mimicked by the adenosine A1 receptor antagonist DPCPX and by the adenylyl cyclase (AC) activator forskolin. It was also blocked by the protein kinase A (PKA) inhibitor H-89, tyrosine kinase inhibitor genistein or by the src family kinase (SFK) inhibitor PP1. Forskolin-stimulated cyclic adenosine monophosphate (cAMP) levels were reduced in the presence of the A1 receptor agonist CHA. Western blot analysis revealed that 500µM caffeine increased phosphoGluN2B expression levels in approximately 60% when compared to total GluN2B levels in embryonic E13 retina. The GluN2B subunit-containing NMDAR antagonist ifenprodil inhibited the caffeine effect. Our results suggest that caffeine potentiates d-asp-induced GABA release, which is mediated by GAT-1, via inhibition of adenosine A1 receptor and activation of the PKA pathway. Regulation of NMDAR by phosphorylation of GluN2B subunit by a SFK may also be involved in the effect promoted by caffeine.

Antiallodynic action of 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), a betaine/GABA transporter inhibitor.

The GABAergic system in the spinal cord has been shown to participate in neuropathic pain in various animal models. GABA transporters (GATs) play a role in controlling the synaptic clearance of GABA; however, their role in neuropathic pain remains unclear. In the present study, we compared the betaine/GABA transporter (BGT-1) with other GAT subtypes to determine its participation in neuropathic pain using a mouse model of sciatic nerve ligation. 1-(3-(9H-Carbazol-9-yl)-1-propyl)-4-(2-methyoxyphenyl)-4-piperidinol (NNC05-2090), an inhibitor that displays moderate selectivity for BGT-1, had an antiallodynic action on model mice treated through both intrathecally and intravenous administration routes. On the other hand, SKF89976A, a selective GAT-1 inhibitor, had a weak antiallodynic action, and (S)-SNAP5114, an inhibitor that displays selectivity for GAT-3, had no antiallodynic action. Systemic analysis of these compounds on GABA uptake in CHO cells stably expressing BGT-1 revealed that NNC05-2090 not only inhibited BGT-1, but also serotonin, noradrenaline, and dopamine transporters, using a substrate uptake assay in CHO cells stably expressing each transporter, with IC50: 5.29, 7.91, and 4.08 µM, respectively. These values were similar to the IC50 value at BGT-1 (10.6 µM). These results suggest that the antiallodynic action of NNC05-2090 is due to the inhibition of both BGT-1 and monoamine transporters.

GABA release provoked by disturbed Na(+), K(+) and Ca(2+) homeostasis in cerebellar nerve endings: roles of Ca(2+) channels, Na(+)/Ca(2+) exchangers and GAT1 transporter reversal.

GABA release provoked by ion dysregulations typical of some neuropathological conditions was analyzed in purified cerebellar synaptosomes pre-labeled with [(3)H]GABA and exposed in superfusion to KCl, 4-aminopyridine (4-AP) or veratridine. The overflows caused by relatively low concentrations of the releasers were almost totally external Ca(2+)-dependent. Higher concentrations of KCl or veratridine, but not 4-AP, involved also external Ca(2+)-independent mechanisms. The GABA overflows evoked by veratridine and, less so, the overflow evoked by high K(+), occurred in part by reversal of the GAT1 transporter. None of the depolarizing agents activated store-operated or transient receptor potential or L-type Ca(2+) channels. Only the overflow caused by 4-AP occurred in part by N- and P/Q-type voltage-sensitive calcium channel-dependent exocytosis. Significant portions of the external Ca(2+)-dependent overflows evoked by the three releasers involved reversal of plasmalemmal Na(+)/Ca(2+) exchangers. The overflows evoked by high K(+) or veratridine, but not by 4-AP were evoked by Ca(2+) originated through mitochondrial Na(+)/Ca(2+) exchangers. Ca(2+)-induced Ca(2+) release mediated by inositoltrisphosphate receptors participated exclusively in the GABA release stimulated by high KCl which also occurred in a modest portion through anion channels. Important differences could be observed between the release mechanisms of GABA here described and those previously reported for glycine, in spite of the abundant vesicular co-localization of the two transmitters in cerebellar interneurons.

Evaluation of anxiolytic-like, anticonvulsant, antidepressant-like and antinociceptive properties of new 2-substituted 4-hydroxybutanamides with affinity for GABA transporters in mice.

PURPOSE: The inhibition of plasma membrane GABA transporters (GATs) is responsible for anxiolytic-like, anticonvulsant, antinociceptive and antidepressant-like effects in mice. It also influences animals' motor coordination and their sensitivity to ethanol. The aim of this study was to assess the pharmacological activity of two novel 2-substituted 4-hydroxybutanamides (BM 130 and BM 131) in some screening models. An attempt has been made to establish the relationship between the inhibition of GAT subtype and the observed in vivo activity. METHODS: The affinity for GAT subtypes was evaluated by means of [(3)H]GABA uptake assay. It indicated that BM 130 inhibited GAT1 and GAT2, whereas BM 131 inhibited GAT1 and GAT3. In mice anxiolytic-like, antidepressant-like, anticonvulsant and antinociceptive properties of the test compounds were assessed. Their influence on motor coordination, locomotor activity and the ability to potentiate effects of subnarcotic doses of ethanol was also tested. RESULTS: Both compounds administered intraperitoneally exerted a significant anxiolytic-like effect in the four plate test with ED50 values 3.4 and 7.9 mg/kg, respectively. At 30 mg/kg they reduced duration of immobility in the forced swim test for 33% and 19%, respectively. They had no effect on electroconvulsive threshold or pain reactivity in the hot plate assay but they were antinociceptive in the acetic acid-induced writhing test (ED50 values were 12.7 and 18.6 mg/kg, respectively) and in both phases of the formalin test (ED50 values in the first phase were 10.2 and 2.1 mg/kg for BM 130 and BM 131, respectively). No motor adverse effects were observed in mice pretreated with the test compounds in the rotarod or chimney tests but BM 131 caused a transient but statistically significant decrease of animals' locomotor activity. CONCLUSIONS: In mice BM 130 and BM 131 have anxiolytic-like, antidepressant-like and antinociceptive properties which can be attributed to their affinity for not only mGAT1 but also mGAT2-4.

The antinociceptive effect of SNAP5114, a gamma-aminobutyric acid transporter-3 inhibitor, in rat experimental pain models.

BACKGROUND: Gamma-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the mammalian central nervous system. GABAergic transmission has an important role in regulating nociception at the spinal dorsal horn. It is terminated by rapid uptake of the neurotransmitter from the synaptic cleft into neurons and glial cells, via specific GABA transporters (GATs). Among the 4 GATs, GAT-3 has the greatest expression in central nervous system regions closely associated with nociceptive transmission, including the spinal cord. In this study, we examined the antinociceptive effect of intrathecal administration of a selective GAT-3 inhibitor, SNAP5114, on acute, inflammatory, and neuropathic pain in experimental models. METHODS: Male Sprague-Dawley rats were used to assess thermal, mechanical, and chemical nociception in the tail flick and hotplate tests, the paw pressure test, and the formalin test. A rotarod test was performed to assess motor function. Chronic constriction injury to the sciatic nerve was induced in the rats. The electronic von Frey test and the plantar test were then performed to assess mechanical allodynia and thermal hyperalgesia. SNAP5114 (10, 50, 100, or 200 µg) was administered intrathecally to examine antinociceptive activity. To confirm whether the action of SNAP5114 was mediated by GABAergic transmission, the GABAA receptor antagonist bicuculline (0.3 µg) or the GABAB receptor antagonist CGP35348 (30 µg) was administered intrathecally before 200 µg of SNAP5114 in the tail flick test, the formalin test, and the electronic von Frey test. RESULTS: Spinally applied SNAP5114 in normal rats dose-dependently prolonged withdrawal latencies in the tail flick test and suppressed the late-phase response in the formalin test. SNAP5114 did not affect motor performance. In the chronic constriction injury rats, SNAP5114 inhibited mechanical allodynia dose-dependently. The antinociceptive action of SNAP5114 was partially reversed by bicuculline or CGP35348 at doses at which the antagonist alone did not affect baseline behavioral responses. CONCLUSIONS: These results suggest that SNAP5114 exerts antinociceptive effects by activating GABAA and GABAB receptors in the spinal cord. The GAT-3 inhibitor may prove useful in treatment of various painful conditions.

Selective mGAT2 (BGT-1) GABA uptake inhibitors: design, synthesis, and pharmacological characterization.

ß-Amino acids sharing a lipophilic diaromatic side chain were synthesized and characterized pharmacologically on mouse GABA transporter subtypes mGAT1-4. The parent amino acids were also characterized. Compounds 13a, 13b, and 17b displayed more than 6-fold selectivity for mGAT2 over mGAT1. Compound 17b displayed anticonvulsive properties inferring a role of mGAT2 in epileptic disorders. These results provide new neuropharmacological tools and a strategy for designing subtype selective GABA transport inhibitors.

A combined approach using transporter-flux assays and mass spectrometry to examine psychostimulant street drugs of unknown content.

The illicit consumption of psychoactive compounds may cause short and long-term health problems and addiction. This is also true for amphetamines and cocaine, which target monoamine transporters. In the recent past, an increasing number of new compounds with amphetamine-like structure such as mephedrone or 3,4-methylenedioxypyrovalerone (MDPV) entered the market of illicit drugs. Subtle structural changes circumvent legal restrictions placed on the parent compound. These novel drugs are effectively marketed "designer drugs" (also called "research chemicals") without any knowledge of the underlying pharmacology, the potential harm or a registration of the manufacturing process. Accordingly new entrants and their byproducts are identified postmarketing by chemical analysis and their pharmacological properties inferred by comparison to compounds of known structure. However, such a heuristic approach fails, if the structures diverge substantially from a known derivative. In addition, the understanding of structure-activity relations is too rudimentary to predict detailed pharmacological activity. Here, we tested a combined approach by examining the composition of street drugs using mass spectrometry and by assessing the functional activity of their constituents at the neuronal transporters for dopamine, serotonin, and norepinephrine. We show that this approach is superior to mere chemical analysis in recognizing novel and potentially harmful street drugs.

Age-dependent changes in the susceptibility to thiopental anesthesia in mice: analysis of the relationship to the functional expression of GABA transporter.

The potency of anesthetics changes during development, probably due not only to pharmacokinetic factors such as differential distribution and/or metabolism, but also to pharmacodynamic factors such as changes to the GABAergic system in the brain. To explore the latter mechanism, we focused on the GABA transporter (GAT), the uptake system for GABA, which participates in the synaptic clearance of GABA. Thiopental-induced anesthesia, as assessed by the onset and duration of loss of the righting reflex, was more pronounced in 3-week-old mice than in 7-week-old mice. Both NO-711 and SKF89976A, selective GAT-1 inhibitors, significantly enhanced the anesthesia in the 7-week-old but not in the 3-week-old mice. In synaptosomes prepared from the cerebral cortex, the kinetics of GABA transport was similar between the two age groups, as assessed by [(3)H]GABA uptake assay. In addition, expression of GAT mRNA was similar between the two age groups, as assessed by quantitative RT-PCR. Thiopental reduced [(3)H]GABA uptake only at high concentrations in a similar manner at both ages. Conversely, the ability of SKF89976A to inhibit [(3)H]GABA uptake was greater in the 7-week-old mice than in the 3-week-old mice. Based on these results, GAT seems unlikely to contribute to the greater susceptibility to thiopental anesthesia in 3-week-old mice, while the increased ability of GABA uptake inhibitors to enhance thiopental-induced anesthesia in 7-week-old mice is at least partly due to higher sensitivity of GAT to the inhibitors.

GABA transporter subtype 1 and GABA transporter subtype 3 modulate glutamatergic transmission via activation of presynaptic GABA(B) receptors in the rat globus pallidus.

The intra-pallidal application of γ-aminobutyric acid (GABA) transporter subtype 1 (GAT-1) or GABA transporter subtype 3 (GAT-3) transporter blockers [1-(4,4-diphenyl-3-butenyl)-3-piperidinecarboxylic acid hydrochloride (SKF 89976A) or 1-[2-[tris(4-methoxyphenyl)methoxy]ethyl]-(S)-3-piperidinecarboxylic acid (SNAP 5114)] reduces the activity of pallidal neurons in monkey. This effect could be mediated through the activation of presynaptic GABA(B) heteroreceptors in glutamatergic terminals by GABA spillover following GABA transporter (GAT) blockade. To test this hypothesis, we applied the whole-cell recording technique to study the effects of SKF 89976A and SNAP 5114 on evoked excitatory postsynaptic currents (eEPSCs) in the presence of gabazine, a GABA(A) receptor antagonist, in rat globus pallidus slice preparations. Under the condition of postsynaptic GABA(B) receptor blockade by the intra-cellular application of N-(2,6-dimethylphenylcarbamoylmethyl)-triethylammonium bromide (OX314), bath application of SKF 89976A (10 µM) or SNAP 5114 (10 µM) decreased the amplitude of eEPSCs, without a significant effect on its holding current and whole cell input resistance. The inhibitory effect of GAT blockade on eEPSCs was blocked by (2S)-3-[[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl](phenylmethyl)phosphinic acid, a GABA(B) receptor antagonist. The paired-pulse ratio of eEPSCs was increased, whereas the frequency, but not the amplitude, of miniature excitatory postsynaptic currents was reduced in the presence of either GAT blocker, demonstrating a presynaptic effect. These results suggest that synaptically released GABA can inhibit glutamatergic transmission through the activation of presynaptic GABA(B) heteroreceptors following GAT-1 or GAT-3 blockade. In conclusion, our findings demonstrate that presynaptic GABA(B) heteroreceptors in putative glutamatergic subthalamic afferents to the globus pallidus are sensitive to increases in extracellular GABA induced by GAT inactivation, thereby suggesting that GAT blockade represents a potential mechanism by which overactive subthalamopallidal activity may be reduced in parkinsonism.

GABA transporters mediate glycine release from cerebellum nerve endings: roles of Ca(2+)channels, mitochondrial Na(+)/Ca(2+) exchangers, vesicular GABA/glycine transporters and anion channels.

GABA transporters accumulate GABA to inactivate or reutilize it. Transporter-mediated GABA release can also occur. Recent findings indicate that GABA transporters can perform additional functions. We investigated how activation of GABA transporters can mediate release of glycine. Nerve endings purified from mouse cerebellum were prelabeled with [(3)H]glycine in presence of the glycine GlyT1 transporter inhibitor NFPS to label selectively GlyT2-bearing terminals. GABA was added under superfusion conditions and the mechanisms of the GABA-evoked [(3)H]glycine release were characterized. GABA stimulated [(3)H]glycine release in a concentration-dependent manner (EC(50) = 8.26 µM). The GABA-evoked release was insensitive to GABA(A) and GABA(B) receptor antagonists, but it was abolished by GABA transporter inhibitors. About 25% of the evoked release was dependent on external Ca(2+) entering the nerve terminals through VSCCs sensitive to ω-conotoxins. The external Ca(2+)-independent release involved mitochondrial Ca(2+), as it was prevented by the Na(+)/Ca(2+) exchanger inhibitor CGP37157. The GABA uptake-mediated increases in cytosolic Ca(2+) did not trigger exocytotic release because the [(3)H]glycine efflux was insensitive to clostridial toxins. Bafilomycin inhibited the evoked release likely because it reduced vesicular storage of [(3)H]glycine so that less [(3)H]glycine can become cytosolic when GABA taken up exchanges with [(3)H]glycine at the vesicular inhibitory amino acid transporters shared by the two amino acids. The GABA-evoked [(3)H]glycine efflux could be prevented by niflumic acid or NPPB indicating that the evoked release occurred essentially by permeation through anion channels. In conclusion, GABA uptake into GlyT2-bearing cerebellar nerve endings triggered glycine release which occurred essentially by permeation through Ca(2+)-dependent anion channels. Glial GABA release mediated by anion channels was proposed to underlie tonic inhibition in the cerebellum; the present results suggest that glycine release by neuronal anion channels also might contribute to tonic inhibition.

Deletion of the betaine-GABA transporter (BGT1; slc6a12) gene does not affect seizure thresholds of adult mice.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian brain. Once released, it is removed from the extracellular space by cellular uptake catalyzed by GABA transporter proteins. Four GABA transporters (GAT1, GAT2, GAT3 and BGT1) have been identified. Inhibition of the GAT1 by the clinically available anti-epileptic drug tiagabine has been an effective strategy for the treatment of some patients with partial seizures. Recently, the investigational drug EF1502, which inhibits both GAT1 and BGT1, was found to exert an anti-convulsant action synergistic to that of tiagabine, supposedly due to inhibition of BGT1. The present study addresses the role of BGT1 in seizure control and the effect of EF1502 by developing and exploring a new mouse line lacking exons 3-5 of the BGT1 (slc6a12) gene. The deletion of this sequence abolishes the expression of BGT1 mRNA. However, homozygous BGT1-deficient mice have normal development and show seizure susceptibility indistinguishable from that in wild-type mice in a variety of seizure threshold models including: corneal kindling, the minimal clonic and minimal tonic extension seizure threshold tests, the 6Hz seizure threshold test, and the i.v. pentylenetetrazol threshold test. We confirm that BGT1 mRNA is present in the brain, but find that the levels are several hundred times lower than those of GAT1 mRNA; possibly explaining the apparent lack of phenotype. In conclusion, the present results do not support a role for BGT1 in the control of seizure susceptibility and cannot provide a mechanistic understanding of the synergism that has been previously reported with tiagabine and EF1502.

Contribution of astrocytic glutamate and GABA uptake to corticostriatal information processing.

The astrocytes, active elements of the tripartite synapse, remove most of the neurotransmitter that spills over the synaptic cleft. Neurotransmitter uptake operated by astrocytes contributes to the strength and timing of synaptic inputs. The striatum, the main input nucleus of basal ganglia, extracts pertinent cortical signals from the background noise and relays cortical information toward basal ganglia output structures. We investigated the role of striatal astrocytic uptake in the shaping of corticostriatal transmission.We performed dual patch-clamp recordings of striatal output neuron (the medium-sized spiny neurons, MSNs)­astrocyte pairs while stimulating the somatosensory cortex. Cortical activity evoked robust synaptically activated transporter-mediated currents (STCs) in 78% of the recorded astrocytes. STCs originated equally from the activities of glutamate transporters and GABA transporters (GATs). Astrocytic STCs reflected here a presynaptic release of neurotransmitters. STCs displayed a large magnitude associated with fast kinetics, denoting an efficient neurotransmitter clearance at the corticostriatal pathway. Inhibition of glutamate transporters type-1 (GLT-1) and GATs decreased the corticostriatal synaptic transmission, through, respectively, desensitization of AMPA receptors and activation of GABAA receptor. STCs displayed a bidirectional short-term plasticity (facilitation for paired-pulse intervals less than 100 ms and depression up to 1 s).We report a genuine facilitation of STCs for high-frequency cortical activity, which could strengthen the detection properties of cortical activity operated by MSNs. MSN EPSCs showed a triphasic short-term plasticity, which was modified by the blockade of GLT-1 or GATs. We show here that neurotransmitter uptake by astrocytes plays a key role in the corticostriatal information processing.