Extracellular GABA Concentrations Research Articles

Neuron-Astrocyte Interactions and Circadian Timekeeping in Mammals.

Almost every facet of our behavior and physiology varies predictably over the course of day and night, anticipating and adapting us to their associated opportunities and challenges. These rhythms are driven by endogenous biological clocks that, when deprived of environmental cues, can continue to oscillate within a period of approximately 1 day, hence circa-dian. Normally, retinal signals synchronize them to the cycle of light and darkness, but disruption of circadian organization, a common feature of modern lifestyles, carries considerable costs to health. Circadian timekeeping pivots around a cell-autonomous molecular clock, widely expressed across tissues. These cellular timers are in turn synchronized by the principal circadian clock of the brain: the hypothalamic suprachiasmatic nucleus (SCN). Intercellular signals make the SCN network a very powerful pacemaker. Previously, neurons were considered the sole SCN timekeepers, with glial cells playing supportive roles. New discoveries have revealed, however, that astrocytes are active partners in SCN network timekeeping, with their cell-autonomous clock regulating extracellular glutamate and GABA concentrations to control circadian cycles of SCN neuronal activity. Here, we introduce circadian timekeeping at the cellular and SCN network levels before focusing on the contributions of astrocytes and their mutual interaction with neurons in circadian control in the brain.

Neuronal Hyperactivity in the Hippocampus during the Early Stage of Streptozotocin-Induced Type 1 Diabetes in Mice

Introduction: Cognitive dysfunction due to reduced neuronal transmission in the brain is a major emerging complication in diabetes. However, recent neuroimaging studies have demonstrated non-linear changes including hyperactivity in the hippocampus during the early stage of diabetes. This study aimed to determine the changes in neuronal activity at a single-cell level in hippocampal CA1 pyramidal neurons in the early stage of streptozotocin-induced type 1 diabetes in mice. Methods: Whole-cell patch-clamp recordings from acute brain slices were performed in mice over 4 consecutive weeks following the induction of hyperglycaemia using streptozotocin. In addition, microdialysate was collected from CA1 area while the mice were in an arousal state. The concentrations of glutamate and GABA in the microdialysate were then measured using ultra-performance liquid chromatography with mass spectrometry. Results: CA1 neurons in streptozotocin-induced diabetic mice exhibited higher membrane potentials (p = 0.0052), higher frequency of action potentials (p = 0.0052), and higher frequency of spontaneous excitatory post-synaptic currents (p = 0.037) compared with controls during the second week after hyperglycaemia was established. No changes in electrophysiological parameters were observed during the first, the third, and the fourth week. Moreover, the diabetic mice had higher extracellular glutamate concentration in CA1 area compared with controls (p = 0.021) during the second week after the initiation of diabetes. No change in the extracellular GABA concentration was observed. Conclusion: Our study demonstrated a temporary state of neuronal hyperactivity at the single-cell level in the hippocampal CA1 region during the early stage of diabetes. This neuronal hyperactivity might be related to altered glutamate metabolism and provide clues for future brain-target intervention.

Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-‍fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

Hyperammonemia Enhances GABAergic Neurotransmission in Hippocampus: Underlying Mechanisms and Modulation by Extracellular cGMP.

Rats with chronic hyperammonemia reproduce the cognitive and motor impairment present in patients with hepatic encephalopathy. It has been proposed that enhanced GABAergic neurotransmission in hippocampus may contribute to impaired learning and memory in hyperammonemic rats. However, there are no direct evidences of the effects of hyperammonemia on GABAergic neurotransmission in hippocampus or on the underlying mechanisms. The aims of this work were to assess if chronic hyperammonemia enhances the function of GABAA receptors in hippocampus and to identify the underlying mechanisms. Activation of GABAA receptors is enhanced in hippocampus of hyperammonemic rats, as analyzed in a multielectrode array system. Hyperammonemia reduces membrane expression of the GABA transporters GAT1 and GAT3, which is associated with increased extracellular GABA concentration. Hyperammonemia also increases gephyrin levels and phosphorylation of the β3 subunit of GABAA receptor, which are associated with increased membrane expression of the GABAA receptor subunits α1, α2, γ2, β3, and δ. Enhanced levels of extracellular GABA and increased membrane expression of GABAA receptors would be responsible for the enhanced GABAergic neurotransmission in hippocampus of hyperammonemic rats. Increasing extracellular cGMP reverses the increase in GABAA receptors activation by normalizing the membrane expression of GABA transporters and GABAA receptors. The increased GABAergic neurotransmission in hippocampus would contribute to cognitive impairment in hyperammonemic rats. The results reported suggest that reducing GABAergic tone in hippocampus by increasing extracellular cGMP or by other means may be useful to improve cognitive function in hyperammonemia and in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Keeping the Balance: GABAB Receptors in the Developing Brain and Beyond.

The main neurotransmitter in the brain responsible for the inhibition of neuronal activity is γ-aminobutyric acid (GABA). It plays a crucial role in circuit formation during development, both via its primary effects as a neurotransmitter and also as a trophic factor. The GABAB receptors (GABABRs) are G protein-coupled metabotropic receptors; on one hand, they can influence proliferation and migration; and, on the other, they can inhibit cells by modulating the function of K+ and Ca2+ channels, doing so on a slower time scale and with a longer-lasting effect compared to ionotropic GABAA receptors. GABABRs are expressed pre- and post-synaptically, at both glutamatergic and GABAergic terminals, thus being able to shape neuronal activity, plasticity, and the balance between excitatory and inhibitory synaptic transmission in response to varying levels of extracellular GABA concentration. Furthermore, given their subunit composition and their ability to form complexes with several associated proteins, GABABRs display heterogeneity with regard to their function, which makes them a promising target for pharmacological interventions. This review will describe (i) the latest results concerning GABABRs/GABABR-complex structures, their function, and the developmental time course of their appearance and functional integration in the brain, (ii) their involvement in manifestation of various pathophysiological conditions, and (iii) the current status of preclinical and clinical studies involving GABABR-targeting drugs.

Vasoactive intestinal peptide (VIP) conducts the neuronal activity during absence seizures: GABA seems to be the main mediator of VIP

Absence epilepsy is classified as a childhood generalized epilepsy syndrome with distinctive electroencephalographic patterns. The Wistar Albino Glaxo originating from Rijswijk (WAG/Rij) strain is a very well validated animal model of absence epilepsy that also shows behavioral deficits. In addition to the gastrointestinal system, VIP is highly expressed throughout numerous brain regions, and it plays crucial roles as a neurotransmitter and as a neuromodulatory, neurotrophic and neuroprotective factor in both the central and peripheral nervous systems.In this study, adult WAG/Rij rats were divided into two groups (n = 10): a group that was administered VIP (25 ng/kg i.p.) every 2 days for 15 days and an age-matched control group that was administered physiological saline. Electrical brain activity and behavior (depressive- like behavior, learning and memory and anxiety) were investigated in both groups. In addition, the extracellular concentrations of GABA and glutamate and the GABA/glutamate ratio were measured by high-performance liquid chromatography in microdialysate samples collected from the somatosensorial cortex of WAG/Rij rats.Our results demonstrated that VIP treatment significantly suppressed the total duration and number of spike wave discharges in WAG/Rij rats. However, VIP had no significant effect on behavior. VIP increased the extracellular concentration of GABA and the GABA/glutamate ratio in the somatosensory cortex. In conclusion, VIP has suppressive effects on absence seizures, possibly by increasing the GABA concentration and inducing the transformation of glutamate to GABA in the somatosensory cortex of WAG/Rij rats.

Volume-Transmitted GABA Waves Drive Epileptiform Rhythms in the Hippocampal Network

Synchronised rhythmic activity of the brain is thought to arise from neuronal network behaviours that arise from synaptic signalling between individual cells. This notion has been a basis to explain periodic epileptiform discharges that are driven by interneuronal networks. However, interneuronal spiking not only engages cell-cell GABAergic transmission but also controls the extracellular GABA concentration ([GABA] e ) and thus tonic GABA A receptor conductance ( G tonic ). Furthermore, the firing activity of interneuron populations shows a bell-shaped dependence on G tonic , suggesting an innate susceptibility to self-sustained oscillations. Here, we employ a patch-clamp GABA 'sniffer' and fast two-photon excitation imaging of a GABA sensor to show that periodic epileptiform discharges are preceded by a region-wide, rising wave of extracellular GABA. Neural network simulations based on such observations reveal that it is the volume-transmitted, extrasynaptic actions of GABA targeting multiple off-target cells that drives synchronised interneuronal spiking prompting periodic epileptiform bursts. We validate this hypothesis using simultaneous patch-clamp recordings from multiple nerve cells and selective optogenetic stimulation of fast-spiking interneurons. Our findings thus unveil to a key role of volume-transmitted GABA actions in enabling and pacing regenerative rhythmic activity in brain networks.

Syntaxin 1B regulates synaptic GABA release and extracellular GABA concentration, and is associated with temperature-dependent seizures.

De novo heterozygous mutations in the STX1B gene, encoding syntaxin 1B, cause a familial, fever-associated epilepsy syndrome. Syntaxin 1B is an essential component of the pre-synaptic neurotransmitter release machinery as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein that regulates the exocytosis of synaptic vesicles. It is also involved in regulating the functions of the SLC6 family of neurotransmitter transporters that reuptake neurotransmitters, including inhibitory neurotransmitters, such as γ-aminobutyric acid (GABA) and glycine. The purpose of the present study was to elucidate the molecular mechanisms underlying the development of febrile seizures by examining the effects of syntaxin 1B haploinsufficiency on inhibitory synaptic transmission during hyperthermia in a mouse model. Stx1b gene heterozygous knockout (Stx1b+/- ) mice showed increased susceptibility to febrile seizures and drug-induced seizures. In cultured hippocampal neurons, we examined the temperature-dependent properties of neurotransmitter release and reuptake by GABA transporter-1 (GAT-1) at GABAergic neurons using whole-cell patch-clamp recordings. The rate of spontaneous quantal GABA release was reduced in Stx1b+/- mice. The hyperthermic temperature increased the tonic GABAA current in wild-type (WT) synapses, but not in Stx1b+/- synapses. In WT neurons, recurrent bursting activities were reduced in a GABA-dependent manner at hyperthermic temperature; however, this was abolished in Stx1b+/- neurons. The blockade of GAT-1 increased the tonic GABAA current and suppressed recurrent bursting activities in Stx1b+/- neurons at the hyperthermic temperature. These data suggest that functional abnormalities associated with GABA release and reuptake in the pre-synaptic terminals of GABAergic neurons may increase the excitability of the neural circuit with hyperthermia.

Blocking glycine receptors reduces neuroinflammation and restores neurotransmission in cerebellum through ADAM17-TNFR1-NF-\u03ba\u03b2 pathway

BackgroundChronic hyperammonemia induces neuroinflammation in cerebellum, with glial activation and enhanced activation of the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway. Hyperammonemia also increases glycinergic neurotransmission. These alterations contribute to cognitive and motor impairment. Activation of glycine receptors is reduced by extracellular cGMP, which levels are reduced in cerebellum of hyperammonemic rats in vivo.We hypothesized that enhanced glycinergic neurotransmission in hyperammonemic rats (1) contributes to induce neuroinflammation and glutamatergic and GABAergic neurotransmission alterations; (2) is a consequence of the reduced extracellular cGMP levels. The aims were to assess, in cerebellum of hyperammonemic rats, (a) whether blocking glycine receptors with the antagonist strychnine reduces neuroinflammation; (b) the cellular localization of glycine receptor; (c) the effects of blocking glycine receptors on the TNFR1-NF-kB-glutaminase-glutamate-GABA pathway and microglia activation; (d) whether adding extracellular cGMP reproduces the effects of strychnine.MethodsWe analyzed in freshly isolated cerebellar slices from control or hyperammonemic rats the effects of strychnine on activation of microglia and astrocytes, the content of TNFa and IL1b, the surface expression of ADAM17, TNFR1 and transporters, the phosphorylation levels of ERK, p38 and ADAM17. The cellular localization of glycine receptor was assessed by immunofluorescence. We analyzed the content of TNFa, IL1b, HMGB1, glutaminase, and the level of TNF-a mRNA and NF-κB in Purkinje neurons. Extracellular concentrations of glutamate and GABA were performed by in vivo microdialysis in cerebellum. We tested whether extracellular cGMP reproduces the effects of strychnine in ex vivo cerebellar slices.ResultsGlycine receptors are expressed mainly in Purkinje cells. In hyperammonemic rats, enhanced glycinergic neurotransmission leads to reduced membrane expression of ADAM17, resulting in increased surface expression and activation of TNFR1 and of the associated NF-kB pathway. This increases the expression in Purkinje neurons of TNFa, IL-1b, HMGB1, and glutaminase. Increased glutaminase activity leads to increased extracellular glutamate, which increases extracellular GABA. Increased extracellular glutamate and HMGB1 potentiate microglial activation. Blocking glycine receptors with strychnine or extracellular cGMP completely prevents the above pathway in hyperammonemic rats.ConclusionsGlycinergic neurotransmission modulates neuroinflammation. Enhanced glycinergic neurotransmission in hyperammonemia would be due to reduced extracellular cGMP. These results shed some light on possible new therapeutic target pathways for pathologies associated to neuroinflammation.

Extracellular GABA waves regulate coincidence detection in excitatory circuits.

Key points Rapid changes in neuronal network activity trigger widespread waves of extracellular GABA in hippocampal neuropil.Elevations of extracellular GABA narrow the coincidence detection window for excitatory inputs to CA1 pyramidal cells.GABA transporters control the effect of extracellular GABA on coincidence detection.Small changes in the kinetics of dendritic excitatory currents amplify when reaching the soma. Coincidence detection of excitatory inputs by principal neurons underpins the rules of signal integration and Hebbian plasticity in the brain. In the hippocampal circuitry, detection fidelity is thought to depend on the GABAergic synaptic input through a feedforward inhibitory circuit also involving the hyperpolarisation‐activated Ih current. However, afferent connections often bypass feedforward circuitry, suggesting that a different GABAergic mechanism might control coincidence detection in such cases. To test whether fluctuations in the extracellular GABA concentration [GABA] could play a regulatory role here, we use a GABA 'sniffer' patch in acute hippocampal slices of the rat and document strong dependence of [GABA] on network activity. We find that blocking GABAergic signalling strongly widens the coincidence detection window of direct excitatory inputs to pyramidal cells whereas increasing [GABA] through GABA uptake blockade shortens it. The underlying mechanism involves membrane‐shunting tonic GABAA receptor current; it does not have to rely on Ih but depends strongly on the neuronal GABA transporter GAT‐1. We use dendrite‐soma dual patch‐clamp recordings to show that the strong effect of membrane shunting on coincidence detection relies on nonlinear amplification of changes in the decay of dendritic synaptic currents when they reach the soma. Our results suggest that, by dynamically regulating extracellular GABA, brain network activity can optimise signal integration rules in local excitatory circuits.

Reducing variability in motor cortex activity at a resting state by extracellular GABA for reliable perceptual decision-making.

Interaction between sensory and motor cortices is crucial for perceptual decision-making, in which intracortical inhibition might have an important role. We simulated a neural network model consisting of a sensory network (NS) and a motor network (NM) to elucidate the significance of their interaction in perceptual decision-making in association with the level of GABA in extracellular space: extracellular GABA concentration. Extracellular GABA molecules acted on extrasynaptic receptors embedded in membranes of pyramidal cells and suppressed them. A reduction in extracellular GABA concentration either in NS or NM increased the rate of errors in perceptual decision-making, for which an increase in ongoing-spontaneous fluctuations in subthreshold neuronal activity in NM prior to sensory stimulation was responsible. Feedback (NM-to-NS) signaling enhanced selective neuronal responses in NS, which in turn increased stimulus-evoked neuronal activity in NM. We suggest that GABA in extracellular space contributes to reducing variability in motor cortex activity at a resting state and thereby the motor cortex can respond correctly to a subsequent sensory stimulus. Feedback signaling from the motor cortex improves the selective responsiveness of the sensory cortex, which ensures the fidelity of information transmission to the motor cortex, leading to reliable perceptual decision-making.

Oxygen-sensitive interneurons exhibit increased activity and GABA release during ROS scavenging in the cerebral cortex of the western painted turtle.

The western painted turtle (Chrysemys picta bellii) has the unique ability of surviving several months in the absence of oxygen, which is termed anoxia. One major protective strategy that the turtle employs during anoxia is a reduction in neuronal electrical activity, which may result from a natural reduction in reactive oxygen species (ROS). We previously linked a reduction in ROS levels to an increase in γ-amino butyric acid (GABA) receptor currents. The purpose of this study is to understand how fast-spiking, GABA-releasing neurons respond to reductions in ROS and how this affects GABA release. Using a fluorescence-coupled enzymatic microplate assay for GABA, we found that anoxia, the ROS scavenger N-(2-mercaptopriopionyl)glycine (MPG), or the mitochondria-specific ROS scavenger MitoTEMPO resulted in a 2.5-, 2.0-, and 2.5-fold increase in extracellular GABA concentration, respectively. This phenomenon could be blocked with TTX, indicating that it is activity dependent. Using whole cell patch-clamping techniques, we found that fast-spiking, burst-firing GABAergic turtle neurons increase the duration and number of action potentials per burst by 26% and 42%, respectively, in response to ROS scavenging via MPG. These results suggest that the reduction in mitochondrially produced ROS that occurs during anoxia leads to increased GABA release, which promotes postsynaptic inhibitory activity through activation of GABA receptors.NEW & NOTEWORTHY This is a novel study examining the response of cerebral cortical stellate interneurons to anoxia and mitochondrial reactive oxygen species (ROS) scavenging with MitoTEMPO. Under both conditions burst firing increases in these cells, and we show that extracellular GABA release increases in the presence of the ROS scavenger. We conclude that in the anoxia-tolerant painted turtle brain, a decrease in ROS levels is an important low oxygen signal.

Spontaneously opening GABAA receptors play a significant role in neuronal signal filtering and integration

Continuous (tonic) charge transfer through ionotropic receptors of γ-aminobutyric acid (GABAARs) is an important mechanism of inhibitory signalling in the brain. The conventional view has been that tonic GABA-ergic inhibitory currents are mediated by low concentrations of ambient GABA. Recently, however, it was shown that the GABA-independent, spontaneously opening GABAARs (s-GABAARs), may contribute significantly to the tonic GABAAR current. One of the common approaches to temporal lobe epilepsy (TLE) therapy is an increase of GABA concentration in the cerebrospinal fluid to augment tonic current through GABAARs. Such an increase, however, generates multiple side effects, which impose significant limitations on the use of correspondent drugs. In contrast, activation/deactivation of s-GABAARs in a GABA-independent manner may provide a mechanism of regulation of tonic conductance without modification of extracellular GABA concentration, thus avoiding connected side effects. Although s-GABAARs have been detected in our earlier work, it is unclear whether they modulate neural signalling, or, due to their independence from the neurotransmitter, they provide just a stable background effect without much impact on neural crosstalk dynamics. Here, we focused on the causal relationship between s-GABAAR activity and signal integration in the rat’s dentate gyrus granule cells to find that s-GABAARs play an important role in neural signal transduction. s-GABAARs shape the dynamics of phasic inhibitory responses, regulate the action potential generation machinery and control the coincidence detection window pertinent to excitatory input summation. Our results demonstrate that tonic inhibition delivered by s-GABAARs contributes to the key mechanisms that ensure implementation of neural signal filtering and integration, in a GABA-independent manner. This makes s-GABAAR a new and important actor in the regulation of long-term neural plasticity and a perspective target for TLE therapy.

GAT-3 Dysfunction Generates Tonic Inhibition in External Globus Pallidus Neurons in Parkinsonian Rodents

The external globus pallidus (GP) is a key GABAergic hub in the basal ganglia (BG) circuitry, a neuronal network involved in motor control. In Parkinson's disease (PD), the rate and pattern of activity of GPneurons are profoundly altered and contribute to the motor symptoms of the disease. In rodent models of PD, the striato-pallidal pathway is hyperactive, and extracellular GABA concentrations are abnormally elevated in the GP, supporting the hypothesis of an alteration of neuronal and/or glial clearance of GABA. Here, we discovered the existence of persistent GABAergic tonic inhibition in GP neurons of dopamine-depleted (DD) rodent models. We showed that glial GAT-3 transporters are downregulated while neuronal GAT-1 function remains normal in DD rodents. Finally, we showed that blocking GAT-3 activity invivo alters the motor coordination of control rodents, suggesting that GABAergic tonic inhibition in the GP contributes to the pathophysiology of PD.

Propionate enters GABAergic neurons, inhibits GABA transaminase, causes GABA accumulation and lethargy in a model of propionic acidemia.

Propionic acidemia is the accumulation of propionate in blood due to dysfunction of propionyl-CoA carboxylase. The condition causes lethargy and striatal degeneration with motor impairment in humans. How propionate exerts its toxic effect is unclear. Here, we show that intravenous administration of propionate causes dose-dependent propionate accumulation in the brain and transient lethargy in mice. Propionate, an inhibitor of histone deacetylase, entered GABAergic neurons, as could be seen from increased neuronal histone H4 acetylation in the striatum and neocortex. Propionate caused an increase in GABA (γ-amino butyric acid) levels in the brain, suggesting inhibition of GABA breakdown. In vitro propionate inhibited GABA transaminase with a Ki of ∼1 mmol/l. In isolated nerve endings, propionate caused increased release of GABA to the extracellular fluid. In vivo, propionate reduced cerebral glucose metabolism in both striatum and neocortex. We conclude that propionate-induced inhibition of GABA transaminase causes accumulation of GABA in the brain, leading to increased extracellular GABA concentration, which inhibits neuronal activity and causes lethargy. Propionate-mediated inhibition of neuronal GABA transaminase, an enzyme of the inner mitochondrial membrane, indicates entry of propionate into neuronal mitochondria. However, previous work has shown that neurons are unable to metabolize propionate oxidatively, leading us to conclude that propionyl-CoA synthetase is probably absent from neuronal mitochondria. Propionate-induced inhibition of energy metabolism in GABAergic neurons may render the striatum, in which >90% of the neurons are GABAergic, particularly vulnerable to degeneration in propionic acidemia.

GABA Uptake Inhibition Reduces In Vivo Extraction Fraction in the Ventral Tegmental Area of Long Evans Rats Measured by Quantitative Microdialysis Under Transient Conditions.

Inhibitory signaling in the ventral tegmental area (VTA) is involved in the mechanism of action for many drugs of abuse. Although drugs of abuse have been shown to alter extracellular γ-aminobutyric acid (GABA) concentration in the VTA, knowledge on how uptake mechanisms are regulated in vivo is limited. Quantitative (no-net-flux) microdialysis is commonly used to examine the extracellular concentration and clearance of monoamine neurotransmitters, however it is unclear whether this method is sensitive to changes in clearance for amino acid neurotransmitters such as GABA. The purpose of this study was to determine whether changes in GABA uptake are reflected by in vivo extraction fraction within the VTA. Using quantitative (no-net-flux) microdialysis adapted for transient conditions, we examined the effects of local perfusion with the GABA uptake inhibitor, nipecotic acid, in the VTA of Long Evans rats. Basal extracellular GABA concentration and in vivo extraction fraction were 44.4 ± 1.9nM (x-intercepts from 4 baseline regressions using a total of 24 rats) and 0.19 ± 0.01 (slopes from 4 baseline regressions using a total of 24 rats), respectively. Nipecotic acid (50μM) significantly increased extracellular GABA concentration to 170 ± 4nM and reduced in vivo extraction fraction to 0.112 ± 0.003. Extraction fraction returned to baseline following removal of nipecotic acid from the perfusate. Conventional microdialysis substantially underestimated the increase of extracellular GABA concentration due to nipecotic acid perfusion compared with that obtained from the quantitative analysis. Together, these results show that inhibiting GABA uptake mechanisms within the VTA alters in vivo extraction fraction measured using microdialysis and that in vivo extraction fraction may be an indirect measure of GABA clearance.

Improved Perceptual Learning by Control of Extracellular GABA Concentration by Astrocytic Gap Junctions.

Learning of sensory cues is believed to rely on synchronous pre- and postsynaptic neuronal firing. Evidence is mounting that such synchronicity is not merely caused by properties of the underlying neuronal network but could also depend on the integrity of gap junctions that connect neurons and astrocytes in networks too. In this perspective, we set out to investigate the effect of astrocytic gap junctions on perceptual learning, introducing a model for coupled neuron-astrocyte networks. In particular, we focus on the fact that astrocytes are rich of GABA transporters (GATs) which can either uptake or release GABA depending on the astrocyte membrane potential, which is a function of local neural activity. We show that GABAergic signaling is a crucial component of intracolumnar neuronal synchronization, thereby promoting learning by neurons in the same cell assembly that are activated by a shared sensory cue. At the same time, we show that this effect can critically depend on astrocytic gap junctions insofar as these latter could synchronize extracellular GABA levels around many neurons and throughout entire cell assemblies. These results are supported by extensive computational arguments and predict that astrocytic gap junctions could improve perceptual learning by controlling extracellular GABA.

GABA transporters regulate tonic and synaptic GABAA receptor-mediated currents in the suprachiasmatic nucleus neurons.

GABA is a principal neurotransmitter in the hypothalamic suprachiasmatic nucleus (SCN) that contributes to intercellular communication between individual circadian oscillators within the SCN network and the stability and precision of the circadian rhythms. GABA transporters (GAT) regulate the extracellular GABA concentration and modulate GABAA receptor (GABAAR)-mediated currents. GABA transport inhibitors were applied to study how GABAAR-mediated currents depend on the expression and function of GAT. Nipecotic acid inhibits GABA transport and induced an inward tonic current in concentration-dependent manner during whole cell patch-clamp recordings from SCN neurons. Application of either the selective GABA transporter 1 (GAT1) inhibitors NNC-711 or SKF-89976A, or the GABA transporter 3 (GAT3) inhibitor SNAP-5114, produced only small changes of the baseline current. Coapplication of GAT1 and GAT3 inhibitors induced a significant GABAAR-mediated tonic current that was blocked by gabazine. GAT inhibitors decreased the amplitude and decay time constant and increased the rise time of spontaneous GABAAR-mediated postsynaptic currents. However, inhibition of GAT did not alter the expression of either GAT1 or GAT3 in the hypothalamus. Thus GAT1 and GAT3 functionally complement each other to regulate the extracellular GABA concentration and GABAAR-mediated synaptic and tonic currents in the SCN. Coapplication of SKF-89976A and SNAP-5114 (50 µM each) significantly reduced the circadian period of Per1 expression in the SCN by 1.4 h. Our studies demonstrate that GAT are important regulators of GABAAR-mediated currents and the circadian clock in the SCN.NEW & NOTEWORTHY In the suprachiasmatic nucleus (SCN), the GABA transporters GAT1 and GAT3 are expressed in astrocytes. Inhibition of these GABA transporters increased a tonic GABA current and reduced the circadian period of Per1 expression in SCN neurons. GAT1 and GAT3 showed functional cooperativity: inhibition of one GAT increased the activity but not the expression of the other. Our data demonstrate that GABA transporters are important regulators of GABAA receptor-mediated currents and the circadian clock.

A Theoretical Study on the Role of Astrocytic Activity in Neuronal Hyperexcitability by a Novel Neuron-Glia Mass Model.

Recent experimental evidence on the clustering of glutamate and GABA transporters on astrocytic processes surrounding synaptic terminals pose the question of the functional relevance of the astrocytes in the regulation of neural activity. In this perspective, we introduce a new computational model that embeds recent findings on neuron–astrocyte coupling at the mesoscopic scale intra- and inter-layer local neural circuits. The model consists of a mass model for the neural compartment and an astrocyte compartment which controls dynamics of extracellular glutamate and GABA concentrations. By arguments based on bifurcation theory, we use the model to study the impact of deficiency of astrocytic glutamate and GABA uptakes on neural activity. While deficient astrocytic GABA uptake naturally results in increased neuronal inhibition, which in turn results in a decreased neuronal firing, deficient glutamate uptake by astrocytes may either decrease or increase neuronal firing either transiently or permanently. Given the relevance of neuronal hyperexcitability (or lack thereof) in the brain pathophysiology, we provide biophysical conditions for the onset identifying different physiologically relevant regimes of operation for astrocytic uptake transporters.

GABAB receptors in the hippocampal dentate gyrus are involved in spatial learning and memory impairment in a rat model of vascular dementia.

The roles of γ-aminobutyric acid (GABA) and GABAB receptors of the hippocampal dentate gyrus (DG) in spatial learning and memory impairment were investigated in a rat model of vascular dementia (VaD) established by permanent bilateral carotid occlusion. The extracellular concentration of GABA in the DG was determined by in vivo microdialysis and HPLC, and spatial learning and memory were assessed by the Morris water maze (MWM) test. Next, the possible involvement of GABAB receptors in spatial learning and memory impairments of VaD rats was examined by microinjection of its antagonist into the DG region. In VaD group rats, the extracellular concentration of GABA in the DG was significantly increased, and during MWM test, the escape latency was increased in place navigation trial and the percentage of time spent in target quadrant and the number of platform crossings were decreased in spatial probe trial, compared with the sham group. In sham-operated rats, the extracellular concentrations of glutamate (Glu) and glycine (Gly) in the DG were significantly increased during place navigation trial of MWM test, and these responses were inhibited in VaD rats. Saclofen (an antagonist of GABAB receptor) significantly attenuated the spatial learning and memory impairment in VaD rats, and partly reversed the inhibitory effects of VaD in responses of Glu and Gly in the DG during MWM test. Our results suggest that GABA and GABAB receptors in the hippocampal DG are involved in spatial learning and memory impairment in VaD rats, in part by attenuating the responses of Glu and Gly during spatial learning.