GABA Spillover Research Articles

Suppressing astrocytic GABA transaminase enhances tonic inhibition and weakens hippocampal spatial memory.

Pharmacological suppression of γ-aminobutyric acid (GABA) transaminase (GABA-T), the sole GABA-degrading enzyme and a potential therapeutic target for treating brain disorders such as epilepsy, increases not only phasic inhibition but also tonic inhibition. However, the specific cellular source, neuromodulatory effects and potential therapeutic benefits of this enhanced tonic inhibition remain unexplored due to the lack of cell-type-specific gene manipulation studies. Here we report that the increase in tonic GABA currents observed after GABA-T suppression is predominantly due to increased tonic GABA release from astrocytes rather than action-potential-dependent synaptic GABA spillover. General GABA-T knockdown (KD) by a short hairpin RNA considerably increased tonic GABA currents in dentate granule cells, thereby enhancing tonic inhibition. An astrocyte-specific rescue of GABA-T following general GABA-T KD normalized the elevated tonic GABA currents to near control levels. Tetrodotoxin-insensitive tonic GABA currents were significantly increased after general GABA-T KD, whereas tetrodotoxin-sensitive tonic GABA currents showed no significant increase, suggesting that this enhanced tonic inhibition is primarily action-potential independent. General GABA-T KD reduced the spike probability of granule cells and impaired dorsal hippocampus-dependent spatial memory, which were fully reversed by astrocyte-specific GABA-T rescue. These findings suggest that suppressing astrocytic GABA-T may be sufficient to influence the excitatory/inhibitory balance in the brain and associated behaviors. Our study implies that the therapeutic benefits of pharmacological GABA-T suppression may be largely attributed to the modulation of astrocytic GABA-T and its impact on tonic GABA release from astrocytes. Here, we report distinct effects of GABA-T suppression depending on cell type; suppressing GABA-T in astrocytes enhances tonic inhibition, while its suppression in GABAergic neurons augments phasic inhibition. Our findings demonstrate that targeted suppression of astrocytic GABA-T not only enhances tonic GABA release from astrocytes but also significantly influences the excitation/inhibition balance in the brain, with consequential effects on behavior. This suggests that astrocytic GABA-T modulation holds promising potential for developing novel therapeutic strategies aimed at treating cognitive and neurological disorders through the regulation of astrocytic GABA metabolism. GAD glutamate decarboxylase, MAO-B monoamine oxidase B, BEST1 bestrophin 1, GABA-T GABA transaminase, GAT GABA transporter, DG dentate gyrus, SSA succinic semialdehyde.

Extrasynaptic δGABAA receptors mediate resistance to migraine-like phenotype in rats

BackgroundGABA, a key inhibitory neurotransmitter, has synaptic and extrasynaptic receptors on the postsynaptic neuron. Background GABA, which spills over from the synaptic cleft, acts on extrasynaptic delta subunit containing GABAA receptors. The role of extrasynaptic GABAergic input in migraine is unknown. We investigated the susceptibility to valid migraine-provoking substances with clinically relevant behavioral readouts in Genetic Absence Epilepsy of Rats Strasbourg (GAERS), in which the GABAergic tonus was altered. Subsequently, we screened relevant GABAergic mechanisms in Wistar rats by pharmacological means to identify the mechanisms.MethodsWistar and GAERS rats were administered nitroglycerin (10 mg/kg) or levcromakalim (1 mg/kg). Mechanical allodynia and photophobia were assessed using von Frey monofilaments and a dark-light box. Effects of GAT-1 blocker tiagabine (5 mg/kg), GABAB receptor agonist baclofen (2 mg/kg), synaptic GABAA receptor agonist diazepam (1 mg/kg), extrasynaptic GABAA receptor agonists gaboxadol (4 mg/kg), and muscimol (0.75 mg/kg), T-type calcium channel blocker ethosuximide (100 mg/kg) or synaptic GABAA receptor antagonist flumazenil (15 mg/kg) on levcromakalim-induced migraine phenotype were screened.ResultsUnlike Wistar rats, GAERS exhibited no reduction in mechanical pain thresholds or light aversion following nitroglycerin or levcromakalim injection. Ethosuximide did not reverse the resistant phenotype in GAERS, excluding the role of T-type calcium channel dysfunction in this phenomenon. Tiagabine prevented levcromakalim-induced mechanical allodynia in Wistar rats, suggesting a key role in enhanced GABA spillover. Baclofen did not alleviate mechanical allodynia. Diazepam failed to mitigate levcromakalim-induced migraine phenotype. Additionally, the resistant phenotype in GAERS was not affected by flumazenil. Extrasynaptic GABAA receptor agonists gaboxadol and muscimol inhibited periorbital allodynia in Wistar rats.ConclusionOur study introduced a rat strain resistant to migraine-provoking agents and signified a critical involvement of extrasynaptic δGABAergic receptors. Extrasynaptic δ GABAA receptors, by mediating constant background inhibition on the excitability of neurons, stand as a novel drug target with a therapeutic potential in migraine.Graphical abstract

Gap Junctions May Have A Computational Function In The Cerebellum: A Hypothesis

In the cerebellum, granule cells make parallel fibre contact on (and excite) Golgi cells and Golgi cells inhibit granule cells, forming an open feedback loop. Parallel fibres excite Golgi cells synaptically, each making a single contact. Golgi cells inhibit granule cells in a structure called a glomerulus almost exclusively by GABA spillover acting through extrasynaptic GABAA receptors. Golgi cells are connected dendritically by gap junctions. It has long been suspected that feedback contributes to homeostatic regulation of parallel fibre signals activity, causing the fraction of the population that are active to be maintained at a low level. We present a detailed neurophysiological and computationally-rendered model of functionally grouped Golgi cells which can infer the density of parallel fibre signals activity and convert it into proportional modulation of inhibition of granule cells. The conversion is unlearned and not actively computed; rather, output is simply the computational effect of cell morphology and network architecture. Unexpectedly, the conversion becomes more precise at low density, suggesting that self-regulation is attracted to sparse code, because it is stable. A computational function of gap junctions may not be confined to the cerebellum.

Impact of Developmental Changes of GABAA Receptors on Interneuron-NG2 Glia Transmission in the Hippocampus.

NG2 glia receive synaptic input from neurons, but the functional impact of this glial innervation is not well understood. In the developing cerebellum and somatosensory cortex the GABAergic input might regulate NG2 glia differentiation and myelination, and a switch from synaptic to extrasynaptic neuron-glia signaling was reported in the latter region. Myelination in the hippocampus is sparse, and most NG2 glia retain their phenotype throughout adulthood, raising the question of the properties and function of neuron-NG2 glia synapses in that brain region. Here, we compared spontaneous and evoked GABAA receptor-mediated currents of NG2 glia in juvenile and adult hippocampi of mice of either sex and assessed the mode of interneuron-glial signaling changes during development. With patch-clamp and pharmacological analyses, we found a decrease in innervation of hippocampal NG2 glia between postnatal days 10 and 60. At the adult stage, enhanced activation of extrasynaptic receptors occurred, indicating a spillover of GABA. This switch from synaptic to extrasynaptic receptor activation was accompanied by downregulation of γ2 and upregulation of the α5 subunit. Molecular analyses and high-resolution expansion microscopy revealed mechanisms of glial GABAA receptor trafficking and clustering. We found that gephyrin and radixin are organized in separate clusters along glial processes. Surprisingly, the developmental loss of γ2 and postsynaptic receptors were not accompanied by altered glial expression of scaffolding proteins, auxiliary receptor subunits or postsynaptic interaction proteins. The GABAergic input to NG2 glia might contribute to the release of neurotrophic factors from these cells and influence neuronal synaptic plasticity.

Modeling analysis of subthreshold voltage signaling along hippocampal mossy fiber axons.

Axons are classically thought of as electrically well isolated from other parts of the neurons due to the shape of a long cable-like structure. In contrast to this classical view on axonal compartmentalization, recent studies revealed that subthreshold depolarization of soma and dendrite passively propagates to the axons for a substantial distance, as demonstrated in some experimentally accessible axons including hippocampal mossy fibers and cortical pyramidal cell axons. Passive propagation of subthreshold dendritic EPSPs to the axons, defined as EPreSPs (excitatory presynaptic potentials), has been demonstrated to affect transmitter release from the axon terminals. To further characterize and explore the functional significance of passive subthreshold voltage signaling along the axons, the model of EPreSPs along hippocampal mossy fibers, proposed by Alle and Geiger, was reconstructed on the NEURON simulator. To test the effect of EPreSPs on action potentials and transmitter release from the axon terminals, additional conductances were incorporated into the previous passive propagation model. These include the axonal sodium, potassium, and leak channels as well as presynaptic calcium channels composed of P/Q-, N-, and R-types, which are reconstructed from the properties of those recorded from mossy fiber boutons experimentally. In this revised model, the preceding subthreshold EPreSPs slightly reduced the action potential-evoked presynaptic calcium currents by a decrease in the amplitude of action potentials due to the slow depolarization. It should be mentioned that EPreSPs by themselves elicited small calcium currents during subthreshold depolarization through these high-voltage activated calcium channels. Since the previous experimental study by simultaneous pre and postsynaptic recordings demonstrated that EPreSPs enhanced action potential-evoked transmitter release from the mossy fiber terminals, it has been suggested that different mechanisms from the enhancement of action potential-evoked presynaptic calcium entry may involve enhanced transmitter release by EPreSP. Small calcium entry by subthreshold EPreSPs may enhance transmitter release from the mossy fiber terminals by acting as high-affinity calcium sensors for enhancing transmitter release. Another form of axonal subthreshold voltage signaling, GABA-EPreSPs elicited by a spillover of GABA from surrounding interneurons, was also explored. Functional consequences of the two modes of axonal subthreshold voltage signaling were discussed with the simulation results.

Topographical distance between presynaptic Ca2+ channels and exocytotic Ca2+ sensors contributes to differential facilitatory actions of roscovitine on neurotransmitter release at cerebellar glutamatergic and GABAergic synapses.

Calcium influx into presynaptic terminals through voltage-gated Ca2+ channels triggers univesicular or multivesicular release of neurotransmitters depending on the characteristics of the release machinery. However, the mechanisms underlying multivesicular release (MVR) and its regulation remain unclear. Previous studies showed that in rat cerebellum, the cyclin-dependent kinase inhibitor roscovitine profoundly increases excitatory postsynaptic current (EPSC) amplitudes at granule cell (GC)-Purkinje cell (PC) synapses by enhancing the MVR of glutamate. This compound can also moderately augment the amplitude and prolong the decay time of inhibitory postsynaptic currents (IPSCs) at molecular layer interneuron (MLI)-PC synapses via MVR enhancement and GABA spillover, thus allowing for persistent activation of perisynaptic GABA receptors. The enhanced MVR may depend on the driving force for Cav 2.1 channel-mediated Ca2+ influx. To determine whether the distinct spatiotemporal dynamics of presynaptic Ca2+ influence MVR, we compared the effects of slow and fast Ca2+ chelators, that is, EGTA and BAPTA, respectively, on roscovitine-induced actions at GC-PC and MLI-PC synapses. Membrane-permeable EGTA-AM decreased GC-PC EPSC and MLI-PC IPSC amplitudes to a similar extent but suppressed the roscovitine-induced enhancement of EPSCs. In contrast, BAPTA-AM attenuated the effects of roscovitine on IPSCs. These results suggest that roscovitine augmented glutamate release by activating the release machinery located distally from the Cav 2.1 channel clusters, while it enhanced GABA release in a manner less dependent on those at distal sites. Therefore, the spatial relationships among Ca2+ channels, buffers, and sensors are critical determinants of the differential facilitatory actions of roscovitine on glutamatergic and GABAergic synapses in the cerebellar cortex.

Presynaptic Regulation of Tonic Inhibition by Neuromodulatory Transmitters in the Basal Amygdala.

Tonic inhibition mediated by ambient levels of GABA that activate extrasynaptic GABAA receptors emerges as an essential factor that tunes neuronal network excitability in vitro and shapes behavioral responses in vivo. To address the role of neuromodulatory transmitter systems on this type of inhibition, we employed patch clamp recordings in mouse amygdala slice preparations. Our results show that the current amplitude of tonic inhibition (Itonic) in projection neurons of the basal amygdala (BA) is increased by preincubation with the neurosteroid THDOC, while the benzodiazepine diazepam is ineffective. This suggests involvement of THDOC sensitive δ subunit containing GABAA receptors in mediating tonic inhibition. Moreover, we provide evidence that the neuromodulatory transmitters NE, 5HT, and ACh strongly enhance spontaneous IPSCs as well as Itonic in the BA. As the increase in frequency, amplitude, and charge of sIPSCs by these neuromodulatory transmitters strongly correlated with the amplitude of Itonic, we conclude that spill-over of synaptic GABA leads to activation of Itonic and thereby to dampening of amygdala excitability. Since local injection of THDOC, as a positive modulator of tonic inhibition, into the BA interfered with the expression of contextual fear memory, our results point to a prominent role of Itonic in fear learning.

Impaired GABAB-mediated presynaptic inhibition increases excitatory strength and alters short-term plasticity in synapsin knockout mice.

Synapsins are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes are major epilepsy susceptibility genes in humans. Consistently, synapsin I/II/III triple knockout (TKO) mice are epileptic and exhibit severe impairments in phasic and tonic GABAergic inhibition that precede the appearance of the epileptic phenotype. These changes are associated with an increased strength of excitatory transmission that has never been mechanistically investigated. Here, we observed that an identical effect in excitatory transmission could be induced in wild-type (WT) Schaffer collateral-CA1 pyramidal cell synapses by blockade of GABAB receptors (GABABRs). The same treatment was virtually ineffective in TKO slices, suggesting that the increased strength of the excitatory transmission results from an impairment of GABAB presynaptic inhibition. Exogenous stimulation of GABABRs in excitatory autaptic neurons, where GABA spillover is negligible, demonstrated that GABABRs were effective in inhibiting excitatory transmission in both WT and TKO neurons. These results demonstrate that the decreased GABA release and spillover, previously observed in TKO hippocampal slices, removes the tonic brake of presynaptic GABABRs on glutamate transmission, making the excitation/inhibition imbalance stronger.

Neurotransmitter-Regulated Regeneration in the Zebrafish Retina.

SummaryCurrent efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG)-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA) signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas.

A pharmacological assessment of agonists and modulators at α4β2γ2 and α4β2δ GABAA receptors: The challenge in comparing apples with oranges

Extrasynaptically located γ-aminobutyric acid (GABA) receptors type A are often characterized by the presence of a δ subunit in the receptor complex. δ-Containing receptors respond to low ambient concentrations of GABA, or respond to spillover of GABA from the synapse, and give rise to tonic inhibitory currents. In certain brain regions, e.g. thalamocortical neurons, tonic inhibition is estimated to represent the majority of total GABA-mediated inhibition, which has raised substantial interest in extrasynaptic receptors as potential drug targets. Thalamocortical neurons typically express α4β2/3δ receptors, however, these have proven difficult to study in recombinant in vitro expression systems due to the inherently low current levels elicited in response to GABA.In this study, we sought to characterize a range of agonists and positive allosteric modulators at α4β2δ and α4β2γ2 receptors. All tested agonists (GABA, THIP, muscimol, and taurine) displayed between 8 and 22 fold increase in potency at the α4β2δ receptor. In contrast, modulatory potencies of steroids (allopregnanolone, THDOC and alfaxalone), anesthetics (etomidate, pentobarbital) and Delta-Selective agents 1 and 2 (DS1 and DS2) were similar at α4β2δ and α4β2γ2 receptors. When evaluating modulatory efficacies, the neurosteroids and anesthetics displayed highest efficacy at α4β2γ2 receptors whereas DS1 and in particular DS2 had highest efficacy at α4β2δ receptors. Overall, several key messages emerged: (i) none of the tested compounds displayed significant selectivity and a great need for identifying new δ-selective compounds remains; (ii) α4β2δ and α4β2γ2 receptors have such divergent intrinsic activation properties that valid comparisons of modulator efficacies are at best challenging.

Excitation by Axon Terminal GABA Spillover in a Sound Localization Circuit.

Synapses from neurons of the medial nucleus of the trapezoid body (MNTB) onto neurons of the lateral superior olive (LSO) in the auditory brainstem are glycinergic in maturity, but also GABAergic and glutamatergic in development. The role for this neurotransmitter cotransmission is poorly understood. Here we use electrophysiological recordings in brainstem slices from P3-P21 mice to demonstrate that GABA release evoked from MNTB axons can spill over to neighboring MNTB axons and cause excitation by activating GABAAR. This spillover excitation generates patterns of staggered neurotransmitter release from different MNTB axons resulting in characteristic "doublet" postsynaptic currents in LSO neurons. Postembedding immunogold labeling and electron microscopy provide evidence that GABAARs are localized at MNTB axon terminals. Photolytic uncaging of p-hydroxyphenacyl (pHP) GABA demonstrates backpropagation of GABAAR-mediated depolarizations from MNTB axon terminals to the soma, some hundreds of microns away. These somatic depolarizations enhanced somatic excitability by increasing the probability of action potential generation. GABA spillover excitation between MNTB axon terminals may entrain neighboring MNTB neurons, which may play a role in the developmental refinement of the MNTB-LSO pathway. Axonal spillover excitation persisted beyond the second postnatal week, suggesting that this mechanism may play a role in sound localization, by providing new avenues of communication between MNTB neurons via their distal axonal projections. Significance statement: In this study, a new mechanism of neuronal communication between auditory synapses in the mammalian sound localization pathway is described. Evidence is provided that the inhibitory neurotransmitter GABA can spill over between axon terminals to cause excitation of nearby synapses to further stimulate neurotransmitter release. Excitatory GABA spillover between inhibitory axon terminals may have important implications for the development and refinement of this auditory circuit and may play a role in the ability to precisely localize sound sources.

Spillover transmission is mediated by the excitatory GABA receptor LGC-35 in C. elegans.

Under most circumstances, GABA activates chloride-selective channels and thereby inhibits neuronal activity. Here, we identify a GABA receptor in the nematode Caenorhabditis elegans that conducts cations and is therefore excitatory. Expression in Xenopus oocytes demonstrates that LGC-35 is a homopentameric cation-selective receptor of the cys-loop family exclusively activated by GABA. Phylogenetic analysis suggests that LGC-35 evolved from GABA-A receptors, but the pore-forming domain contains novel molecular determinants that confer cation selectivity. LGC-35 is expressed in muscles and directly mediates sphincter muscle contraction in the defecation cycle in hermaphrodites, and spicule eversion during mating in the male. In the locomotory circuit, GABA release directly activates chloride channels on the muscle to cause muscle relaxation. However, GABA spillover at these synapses activates LGC-35 on acetylcholine motor neurons, which in turn cause muscles to contract, presumably to drive wave propagation along the body. These studies demonstrate that both direct and indirect excitatory GABA signaling plays important roles in regulating neuronal circuit function and behavior in C. elegans.

Regulation of output spike patterns by phasic inhibition in cerebellar granule cells.

The complex interplay of multiple molecular mechanisms taking part to synaptic integration is hard to disentangle experimentally. Therefore, we developed a biologically realistic computational model based on the rich set of data characterizing the cerebellar glomerulus microcircuit. A specific issue was to determine the relative role of phasic and tonic inhibition in dynamically regulating granule cell firing, which has not been clarified yet. The model comprised the excitatory mossy fiber—granule cell and the inhibitory Golgi cell—granule cell synapses and accounted for vesicular release processes, neurotransmitter diffusion and activation of different receptor subtypes. Phasic inhibition was based on stochastic GABA release and spillover causing activation of two major classes of postsynaptic receptors, α1 and α6, while tonic inhibition was based on steady regulation of a Cl− leakage. The glomerular microcircuit model was validated against experimental responses to mossy fiber bursts while metabotropic receptors were blocked. Simulations showed that phasic inhibition controlled the number of spikes during burst transmission but predicted that it specifically controlled time-related parameters (firing initiation and conclusion and first spike precision) when the relative phase of excitation and inhibition was changed. In all conditions, the overall impact of α6 was larger than that of α1 subunit-containing receptors. However, α1 receptors controlled granule cell responses in a narrow ±10 ms band while α6 receptors showed broader ±50 ms tuning. Tonic inhibition biased these effects without changing their nature substantially. These simulations imply that phasic inhibitory mechanisms can dynamically regulate output spike patterns, as well as calcium influx and NMDA currents, at the mossy fiber—granule cell relay of cerebellum without the intervention of tonic inhibition.

Combination of fluoxetine and extinction treatments forms a unique synaptic protein profile that correlates with long-term fear reduction in adult mice

The antidepressant fluoxetine induces synaptic plasticity in the visual and fear networks and promotes the structural remodeling of neuronal circuits, which is critical for experience-dependent plasticity in response to an environmental stimulus. We recently demonstrated that chronic fluoxetine administration together with extinction training in adult mice reduced fear in a context-independent manner. Fear conditioning and extinction alter excitatory and inhibitory transmissions within the fear circuitry. In this study, we investigated whether fluoxetine, extinction or their combination produced distinct long-lasting changes in the synaptic protein profile in the amygdala, hippocampus and prefrontal cortex of conditioned mice. We determined that extinction induced synaptophysin expression and down-regulated the GluA1:GluA2 ratio throughout the fear network in water- and fluoxetine-treated mice, suggesting a common fluoxetine-independent mechanism for increased synaptic transmission and re-arrangement of AMPA-receptors by extinction training. In contrast to common changes, the presynaptic vesicular neurotransmitter transporters VGAT and Vglut1 were upregulated after extinction in water- and fluoxetine-treated mice, respectively. The cortical levels of the GABA transporter Gat1 were reduced in high-freezing water-drinking mice, suggesting a maladaptive increase of GABA spillover at cortical inhibitory synapses. Fear conditioning decreased, and extinction induced the expression of GABA-receptor alpha1 and alpha2 subunits in water- and fluoxetine-treated mice, respectively. Only a combination of fluoxetine with extinction enhanced GluN2A expression in the amygdala and hippocampus, emphasizing the role of this NMDA-receptor subunit in the successful erasure of fear memories. Our finding provides novel data that may become helpful in developing beneficial pharmacological fear-reducing treatment strategies.

Activation of Axonal Receptors by GABA Spillover Increases Somatic Firing

Axons can be depolarized by ionotropic receptors and transmit subthreshold depolarizations to the soma by passive electrical spread. This raises the possibility that axons and axonal receptors can participate in integration and firing in neurons. Previously, we have shown that exogenous GABA depolarizes cerebellar granule cell axons through local activation of GABA(A) receptors (GABA(A)Rs) and the soma through electrotonic spread of the axonal potential resulting in increased firing. We show here that excitability of granule cells is also increased by release of endogenous GABA from molecular layer interneurons (MLIs) and spillover activation of parallel fiber GABA(A)Rs in mice and rats. Changes in granule cell excitability were assessed by excitability testing after activation of MLIs with channelrhodopsin or electrical stimulation in the molecular layer. In granule cells lacking an axon, excitability was not changed, suggesting that axonal receptors are required. To determine the distance over which subthreshold potentials may spread, we estimated the effective axonal electrical length constant (520 μm) by excitability testing and focal uncaging of RuBi-GABA on the axon at varying distances from the soma. These data suggest that GABA(A)R-mediated axonal potentials can participate in integration and firing of cerebellar granule cells.

Cl− homeodynamics in gap junction‐coupled astrocytic networks on activation of GABAergic synapses

The electrophysiological properties and functional role of GABAergic signal transmission from neurons to the gap junction-coupled astrocytic network are still unclear. GABA-induced astrocytic Cl− flux has been hypothesized to affect the driving force for GABAergic transmission by modulating [Cl−]o. Thus, revealing the properties of GABA-mediated astrocytic responses will deepen our understanding of GABAergic signal transmission. Here, we analysed the Cl− dynamics of neurons and astrocytes in CA1 hippocampal GABAergic tripartite synapses, using Cl− imaging during GABA application, and whole cell recordings from interneuron–astrocyte pairs in the stratum lacunosum-moleculare. Astrocytic [Cl−]i was adjusted to physiological conditions (40 mm). Although GABA application evoked bidirectional Cl− flux via GABAA receptors and mouse GABA transporter 4 (mGAT4) in CA1 astrocytes, a train of interneuron firing induced only GABAA receptor-mediated inward currents in an adjacent astrocyte. A GAT1 inhibitor increased the interneuron firing-induced currents and induced bicuculline-insensitive, mGAT4 inhibitor-sensitive currents, suggesting that synaptic spillover of GABA predominantly induced the astrocytic Cl− efflux because GABAA receptors are localized near the synaptic clefts. This GABA-induced Cl− efflux was accompanied by Cl− siphoning via the gap junctions of the astrocytic network because gap junction inhibitors significantly reduced the interneuron firing-induced currents. Thus, Cl− efflux from astrocytes is homeostatically maintained within astrocytic networks. A gap junction inhibitor enhanced the activity-dependent depolarizing shifts of reversal potential of neuronal IPSCs evoked by repetitive stimulation to GABAergic synapses. These results suggest that Cl− conductance within the astrocytic network may contribute to maintaining GABAergic synaptic transmission by regulating [Cl−]o.

A functional role for both -aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus.

Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K+-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.

Pathophysiological power of improper tonic GABAA conductances in mature and immature models

High-affinity extrasynaptic gamma-aminobutyric acid A (GABAA) receptors are tonically activated by low and consistent levels of ambient GABA, mediating chronic inhibition against neuronal excitability (tonic inhibition) and the modulation of neural development. Synaptic (phasic) inhibition is spatially and temporally precise compared with tonic inhibition, which provides blunt yet strong integral inhibitory force by shunting electrical signaling. Although effects of acute modification of tonic inhibition are known, its pathophysiological significance remains unclear because homeostatic regulation of neuronal excitability can compensate for long-term deficit of extrasynaptic GABAA receptor activation. Nevertheless, tonic inhibition is of great interest for its pathophysiological involvement in central nervous system (CNS) diseases and thus as a therapeutic target. Together with the development of experimental models for various pathological states, recent evidence demonstrates such pathological involvements of tonic inhibition in neuronal dysfunction. This review focuses on the recent progress of tonic activation of GABAA conductance on the development and pathology of the CNS. Findings indicate that neuronal function in various brain regions are exacerbated with a gain or loss of function of tonic inhibition by GABA spillover. Disturbance of tonic GABAA conductance mediated by non-synaptic ambient GABA may result in brain mal-development. Therefore, various pathological states (epilepsy, motor dysfunctions, psychiatric disorders, and neurodevelopmental disorders) may be partly attributable to abnormal tonic GABAA conductances. Thus, the tone of tonic conductance and level of ambient GABA may be precisely tuned to maintain the regular function and development of the CNS. Therefore, receptor expression and factors for regulating the ambient GABA concentration are highlighted to gain a deeper understanding of pathology and therapeutic strategy for CNS diseases.

Neurogliaform cells of amygdala: a source of slow phasic inhibition in the basolateral complex

Synaptic inhibition in the amygdala actively participates in processing emotional information. To improve the understanding of interneurons in amygdala networks it is necessary to characterize the GABAergic cell types, their connectivity and physiological roles. We used a mouse line expressing a green fluorescent protein (GFP) under the neuropeptide Y (NPY) promoter. Paired recordings between presynaptic NPY-GFP-expressing (+) cells and postsynaptic principal neurons (PNs) of the basolateral amygdala (BLA) were performed. The NPY-GFP+ neurons displayed small somata and short dendrites embedded in a cloud of highly arborized axon, suggesting a neurogliaform cell (NGFC) type. We discovered that a NPY-GFP+ cell evoked a GABA(A) receptor-mediated slow inhibitory postsynaptic current (IPSC) in a PN and an autaptic IPSC. The slow kinetics of these IPSCs was likely caused by the low concentration and spillover of extracellular GABA. We also report that NGFCs of the BLA fired action potentials phase-locked to hippocampal theta oscillations in anaesthetized rats. When this firing was re-played in NPY+-NGFCs in vitro, it evoked a transient depression of the IPSCs. Presynaptic GABA(B) receptors and functional depletion of synaptic vesicles determined this short-term plasticity. Synaptic contacts made by recorded NGFCs showed close appositions, and rarely identifiable classical synaptic structures. Thus, we report here a novel interneuron type of the amygdala that generates volume transmission of GABA. The peculiar functional mode of NGFCs makes them unique amongst all GABAergic cell types of the amygdala identified so far.

Inhibitory synaptic release properties are topographically distributed in auditory circuitry

Inhibitory synaptic release properties are topographically distributed in auditory circuitry