Activation Of Extrasynaptic GABAA Receptors Research Articles

Gene mutations in paediatric epilepsies cause NMDA-pathy, and phasic and tonic GABA-pathy.

The aim of this study was to disentangle mechanisms of epileptogenesis in monogenic epilepsies in children. We reviewed paediatric monogenic epilepsies excluding brain malformation or an inborn error of metabolism, but including the gene function whether there is loss-of-function or gain-of-function, age at gene expression when available, and associated epilepsy syndrome. Genes for which at least five patients with similar epilepsy phenotype had been reported were selected. Three mechanisms are shared by most monogenic epilepsies: (1) excess of N-methyl-d-aspartate (NMDA) transmission activation (NMDA-pathies); (2)abnormal gamma-aminobutyric acid (GABA) transmission with reduced inhibition (phasic GABA-pathies); and (3) tonic activation of extrasynaptic GABAA receptors by extracellular GABA (tonic GABA-pathies). NMDA-pathies comprise early epileptic encephalopathy with suppression-burst, neonatal/infantile benign seizures, West and Lennox-Gastaut syndromes, and encephalopathy with continuous spike waves in slow sleep, thus brief seizures with major interictal spiking. Phasic GABA-pathies comprise mostly generalized epilepsy with febrile seizures plus and Dravet syndrome, thus long-lasting seizures with mild interictal spiking. Tonic GABA-pathies cause epilepsy with myoclonic-atonic seizures and Angelman syndrome, thus major high-amplitude slow-wave activity. This pathophysiological approach to monogenic epilepsies provides diagnostic clues and helps to guide treatment strategy. WHAT THIS PAPER ADDS: In paediatric monogenic epilepsies, electroclinical patterns point to three main mechanisms: NMDA-pathies, and phasic and tonic GABA-pathies. Antiepileptic treatment choice could be guided by each of these mechanisms.

Effects of AMPA receptor antagonist, NBQX, and extrasynaptic GABAA agonist, THIP, on social behavior of adolescent and adult rats.

Adolescence is characterized by high significance of social interactions, along with a propensity to exhibit social facilitating effects of ethanol while being less sensitive than adults to the inhibition of social behavior that emerges at higher doses of ethanol. Among the neural characteristics of adolescence are generally enhanced levels of glutamatergic (especially NMDA receptor) activity relative to adults, whereas the GABA system is still developmentally immature. Activation of NMDA receptors likely plays a role in modulation of social behavior in adolescent animals as well as in socially facilitating and suppressing effects of ethanol. For instance, adolescent and adult rats differ in their sensitivities to the effects of NMDA antagonists and ethanol on social behavior, with adolescents but not adults demonstrating social facilitation at lower doses of both drugs and adults being more sensitive to the socially suppressing effects evident at higher doses of each. The roles of AMPA and extrasynaptic GABAA receptors in modulation of social behavior during adolescence and in adulthood are still unknown. The present study was designed to assess whether pharmacological blockade of AMPA receptors and/or activation of extrasynaptic GABAA receptors results in age-dependent alterations of social behavior. Adolescent and adult male and female Sprague-Dawley rats were injected with an assigned dose of either a selective AMPA antagonist, NBQX (Experiment 1) or extrasynaptic GABAA agonist, THIP (Experiment 2) and placed into a modified social interaction chamber for a 30-min habituation period prior to a 10-min social interaction test with a novel age- and sex-matched partner. Behaviors such as social investigation, contact behavior and play behavior were scored from video recordings of the interaction tests. In Experiment 1, NBQX produced similar social inhibition at higher doses in both age groups. In Experiment 2, THIP induced inhibition in adolescents, but not adults. No social facilitation was evident following low doses of either drug. Therefore, AMPA and extrasynaptic GABAA receptors appear to play little role if any in modulation of peer-directed social behavior in adolescence and adulthood and not likely to contribute to previously observed age differences in the social effects of acute ethanol.

Control of motor coordination by astrocytic tonic GABA release through modulation of excitation/inhibition balance in cerebellum

Tonic inhibition in the brain is mediated through an activation of extrasynaptic GABAA receptors by the tonically released GABA, resulting in a persistent GABAergic inhibitory action. It is one of the key regulators for neuronal excitability, exerting a powerful action on excitation/inhibition balance. We have previously reported that astrocytic GABA, synthesized by monoamine oxidase B (MAOB), mediates tonic inhibition via GABA-permeable bestrophin 1 (Best1) channel in the cerebellum. However, the role of astrocytic GABA in regulating neuronal excitability, synaptic transmission, and cerebellar brain function has remained elusive. Here, we report that a reduction of tonic GABA release by genetic removal or pharmacological inhibition of Best1 or MAOB caused an enhanced neuronal excitability in cerebellar granule cells (GCs), synaptic transmission at the parallel fiber-Purkinje cell (PF-PC) synapses, and motor performance on the rotarod test, whereas an augmentation of tonic GABA release by astrocyte-specific overexpression of MAOB resulted in a reduced neuronal excitability, synaptic transmission, and motor performance. The bidirectional modulation of astrocytic GABA by genetic alteration of Best1 or MAOB was confirmed by immunostaining and in vivo microdialysis. These findings indicate that astrocytes are the key player in motor coordination through tonic GABA release by modulating neuronal excitability and could be a good therapeutic target for various movement and psychiatric disorders, which show a disturbed excitation/inhibition balance.

2-Deoxy-d-glucose enhances tonic inhibition through the neurosteroid-mediated activation of extrasynaptic GABAA receptors.

The inhibition of glycolysis exerts potent antiseizure effects, as demonstrated by the efficacy of ketogenic and low-glucose/nonketogenic diets in the treatment of drug-resistant epilepsy. ATP-sensitive potassium (KATP ) channels have been initially identified as the main determinant of the reduction of neuronal hyperexcitability. However, a plethora of other mechanisms have been proposed. Herein, we report the ability of 2-deoxy-d-glucose (2-DG), a glucose analog that inhibits glycolytic enzymes, of potentiating γ-aminobutyric acid (GABA)ergic tonic inhibition via neurosteroid-mediated activation of extrasynaptic GABAA receptors. Acute effects of 2-DG on the ATP-sensitive potassium currents, GABAergic tonic inhibition, firing activity, and interictal events were assessed in hippocampal slices by whole-cell patch-clamp and local field potential recordings of dentate gyrus granule cells. Acute application of 2-DG activates two distinct outward conductances: a KATP channel-mediated current and a bicuculline-sensitive tonic current. The effect of 2-DG on such GABAergic tonic currents was fully prevented by either finasteride or PK11195, which are specific inhibitors of the neurosteroidogenesis pathway acting via different mechanisms. Moreover, the oxidized form of vitamin C, dehydroascorbic acid, known for its ability to induce neurosteroidogenesis, also activated a bicuculline-sensitive tonic current in a manner indistinguishable from that of 2-DG. Finally, we found that the enhancement of KATP current by 2-DG primarily regulates intrinsic firing rate of granule cells, whereas the increase of the GABAergic tonic current plays a key role in reducing the frequency of interictal events evoked by treatment of hippocampal slices with the convulsive agent 4-aminopyridine. We demonstrated, for the first time, that 2-DG potentiates the extrasynaptic tonic GABAergic current through activation of neurosteroidogenesis. Such tonic inhibition represents the main conductance responsible for the antiseizure action of this glycolytic inhibitor.

Editorial: Plasticity of GABAergic synapses.

Editorial: Plasticity of GABAergic synapses.

Reduction in focal ictal activity following transplantation of MGE interneurons requires expression of the GABAA receptor α4 subunit

Despite numerous advances, treatment-resistant seizures remain an important problem. Loss of neuronal inhibition is present in a variety of epilepsy models and is suggested as a mechanism for increased excitability, leading to the proposal that grafting inhibitory interneurons into seizure foci might relieve refractory seizures. Indeed, transplanted medial ganglionic eminence interneuron progenitors (MGE-IPs) mature into GABAergic interneurons that increase GABA release onto cortical pyramidal neurons, and this inhibition is associated with reduced seizure activity. An obvious conclusion is that inhibitory coupling between the new interneurons and pyramidal cells underlies this effect. We hypothesized that the primary mechanism for the seizure-limiting effects following MGE-IP transplantation is the tonic conductance that results from activation of extrasynaptic GABAA receptors (GABAA-Rs) expressed on cortical pyramidal cells. Using in vitro and in vivo recording techniques, we demonstrate that GABAA-R α4 subunit deletion abolishes tonic currents (Itonic) in cortical pyramidal cells and leads to a failure of MGE-IP transplantation to attenuate cortical seizure propagation. These observations should influence how the field proceeds with respect to the further development of therapeutic neuronal transplants (and possibly pharmacological treatments).

Activation of extrasynaptic GABA(A) receptors inhibits cyclothiazide-induced epileptiform activity in hippocampal CA1 neurons.

Extrasynaptic GABA(A) receptors (GABA(A)Rs)-mediated tonic inhibition is reported to involve in the pathogenesis of epilepsy. In this study, we used cyclothiazide (CTZ)-induced in vitro brain slice seizure model to explore the effect of selective activation of extrasynaptic GABA(A)Rs by 4,5,6,7-tetrahydroisoxazolo[5,4-c] pyridine-3-ol (THIP) on the CTZ-induced epileptiform activity in hippocampal neurons. Perfusion with CTZ dose-dependently induced multiple epileptiform peaks of evoked population spikes (PSs) in CA1 pyramidal neurons, and treatment with THIP (5 μmol/L) significantly reduced the multiple PS peaks induced by CTZ stimulation. Western blot showed that the δ-subunit of the GABA(A)R, an extrasynaptic specific GABA(A)R subunit, was also significantly down-regulated in the cell membrane 2 h after CTZ treatment. Our results suggest that the CTZ-induced epileptiform activity in hippocampal CA1 neurons is suppressed by the activation of extrasynaptic GABA(A)Rs, and further support the hypothesis that tonic inhibition mediated by extrasynaptic GABA(A)Rs plays a prominent role in seizure generation.

Differences in the reinstatement of ethanol seeking with ganaxolone and gaboxadol

The endogenous neuroactive steroid allopregnanolone (ALLO) has previously been shown to induce reinstatement of ethanol seeking in rodents. ALLO is a positive allosteric modulator at both synaptic and extrasynaptic GABAA receptors. The contribution of each class of GABAA receptors in mediating reinstatement of ethanol seeking is unknown. The first aim of the present study was to determine whether ganaxolone (GAN), a longer-acting synthetic analog of ALLO, also promotes reinstatement of ethanol seeking. The second aim was to examine whether preferentially activating extrasynaptic GABAA receptors with the selective agonist gaboxadol (THIP) was sufficient to reinstate responding for ethanol in mice. Male C57BL/6J mice were trained to lever press for access to a 10% ethanol (v/v) solution (10E), using a sucrose-fading procedure. Following extinction of the lever-pressing behavior, systemic THIP (0, 4 and 6mg/kg) and GAN (0, 10, and 15mg/kg) were tested for their ability to reinstate ethanol-appropriate responding in the absence of 10E access. GAN significantly increased lever pressing on the previously active lever, while THIP did not alter lever-pressing behavior. The results of this study suggest that direct activation of extrasynaptic GABAA receptors at the GABA site is not sufficient to induce ethanol seeking in the reinstatement procedure. Future studies are necessary to elucidate the mechanisms and brain areas by which differences in the pharmacological activity of GAN and THIP at the GABAA receptor contribute to the dissimilarity in their effect on the reinstatement of ethanol seeking. Nonetheless, based on the increased use of these drugs in clinical trials across multiple disease states, the effects of GAN or THIP on alcohol seeking may be an important consideration if these drugs are to be used clinically in a population with a co-occurring alcohol use disorder.

Homeostatic Competition between Phasic and Tonic Inhibition

The GABAA receptors are the major inhibitory receptors in the brain and are localized at both synaptic and extrasynaptic membranes. Synaptic GABAA receptors mediate phasic inhibition, whereas extrasynaptic GABAA receptors mediate tonic inhibition. Both phasic and tonic inhibitions regulate neuronal activity, but whether they regulate each other is not very clear. Here, we investigated the functional interaction between synaptic and extrasynaptic GABAA receptors through various molecular manipulations. Overexpression of extrasynaptic α6β3δ-GABAA receptors in mouse hippocampal pyramidal neurons significantly increased tonic currents. Surprisingly, the increase of tonic inhibition was accompanied by a dramatic reduction of the phasic inhibition, suggesting a possible homeostatic regulation of the total inhibition. Overexpressing the α6 subunit alone induced an up-regulation of δ subunit expression and suppressed phasic inhibition similar to overexpressing the α6β3δ subunits. Interestingly, blocking all GABAA receptors after overexpressing α6β3δ receptors could not restore the synaptic GABAergic transmission, suggesting that receptor activation is not required for the homeostatic interplay. Furthermore, insertion of a gephyrin-binding-site (GBS) into the α6 and δ subunits recruited α6(GBS)β3δ(GBS) receptors to postsynaptic sites but failed to rescue synaptic GABAergic transmission. Thus, it is not the positional effect of extrasynaptic α6β3δ receptors that causes the down-regulation of phasic inhibition. Overexpressing α5β3γ2 subunits similarly reduced synaptic GABAergic transmission. We propose a working model that both synaptic and extrasynaptic GABAA receptors may compete for limited receptor slots on the plasma membrane to maintain a homeostatic range of the total inhibition.

The effects of volatile anesthetics on synaptic and extrasynaptic GABA-induced neurotransmission

Examination of volatile anesthetic actions at single synapses provides more direct information by reducing interference by surrounding tissue and extrasynaptic modulation. We examined how volatile anesthetics modulate GABA release by measuring spontaneous or miniature GABA-induced inhibitory postsynaptic currents (mIPSCs, sIPSCs) or by measuring action potential-evoked IPSCs (eIPSCs) at individual synapses. Halothane increased both the amplitude and frequency of sIPSCs. Isoflurane and enflurane increased mIPSC frequency while sevoflurane had no effect. These anesthetics did not alter mIPSC amplitudes. Halothane increased the amplitude of eIPSCs, with a decrease in failure rate (Rf) and paired-pulse ratio. In contrast, isoflurane and enflurane decreased the eIPSC amplitude and increased Rf, while sevoflurane decreased the eIPSC amplitude without affecting Rf. Volatile anesthetics did not change kinetics except for sevoflurane, suggesting that presynaptic mechanisms dominate changes in neurotransmission. Each anesthetic showed somewhat different GABA-induced response and these results suggest that GABA-induced synaptic transmission cannot have a uniformly common site of action as suggested for volatile anesthetics. In contrast, all volatile anesthetics concentration-dependently enhanced the GABA-induced extrasynaptic currents. Extrasynaptic receptors containing α4 and α5 subunits are reported to have high sensitivities to volatile anesthetics. Also, inhibition of GABA uptake by volatile anesthetics results in higher extracellular GABA concentration, which may lead to prolonged activation of extrasynaptic GABAA receptors. The extrasynaptic GABA-induced receptors may be major site of volatile anesthetic-induced neurotransmission. This article is part of a Special Issue entitled 'Extrasynaptic ionotropic receptors'.

Cerebellar Golgi cell inhibition gets slowly more complicated

The cellular architecture of the cerebellum that was so beautifully described by Ramon y Cajal (1899) offers one of the most complete systems for understanding how networks of excitatory and inhibitory neurons interact with one another to control the flow of sensory information in the mammalian brain. However, despite decades of anatomical and electrophysiological studies, this relatively simple and well-defined neuronal circuit still holds a few surprises. For example, in a recent issue of The Journal of Physiology, Holtzman and colleagues (2011) demonstrate some unusual features of cerebellar Golgi cell function. Our view of cerebellar Golgi cells has, in many respects, changed very little since Eccles first described the basic features of feed-forward and feed-back inhibition of granule cells by Golgi cell interneurons in the 1960s (Eccles, 1964). Cerebellar Golgi cells are one of the few cell types that can control granule cell firing within the cerebellar cortex due to their ability to release the inhibitory neurotransmitter γ-aminobutyric acid (GABA) onto both synaptic and extrasynaptic GABAA receptors that are expressed on granule cells (Brickley et al. 1996; Crowley et al. 2009). The current study is concerned with the excitatory drive onto Golgi cells that triggers the release of this GABA onto granule cells. Excitatory drive onto Golgi cells arises from glutamatergic mossy fibre inputs that convey sensory information into the cerebellar cortex (Kanichay & Silver, 2008), and from the glutamate released from granule cell axons that form the parallel fibre inputs onto Golgi cell dendrites (Dieudonne, 1998). It is this feedback loop involving the mossy-fibre → granule cell → Golgi cell → granule cell circuit that has been the focus of attention for the current study. Holtzmann et al. used in vivo recording techniques to monitor action potential firing patterns from Golgi cells during parallel fibre stimulation. In this way they confirmed an observation that was first made in an acute in vitro slice preparation of the cerebellum (Watanbe & Nakanishi, 2003) that the release of glutamate from granule cells leads to a decrease in Golgi cell firing through activation of mGluR2 receptors. This contradicts the classical view that granule cells serve only to excite Golgi cells. The decrease in Golgi firing that occurs following parallel fibre input lasts for hundreds of milliseconds, resulting in a conspicuous disinhibition of granule cells. This sustained depression of Golgi cell firing has been reported previously in vivo (Vos et al. 1999; Holtzman et al. 2006), but the authors have now confirmed the involvement of mGluR2 activation in this process. Moreover, the authors now show that it is possible to trigger this mGluR2-mediated disinhibition by modulating granule cell excitability through an enhancement of the tonic inhibition that is present on granule cells. In this study, tonic inhibition of granule cells was enhanced using the application of the GABA agonist gaboxadol (or THIP) that is known to have a selective action on extrasynaptic GABAA receptors that contain the delta subunit when used at sub-micromolar concentrations. Obviously, in an in vivo study of this type it is difficult to establish the effective concentration of the drug within the cerebellum, but it would appear that increased glutamate release from granule cells can lead to mGluR2 activation and, therefore, inhibition of Golgi cell firing. The authors further speculate that a spillover-type mechanism is involved in this glutamate activation of mGluR receptors on Golgi cells in an analogous manner to the GABA activation of extrasynaptic GABAA receptors. Spillover refers to the fact that glutamate diffuses out of the synaptic cleft to activate mGluR2 receptors located some distance away from the synapse. Binding of this ‘escaped’ glutamate to mGluR2 receptors is assumed to reduce Golgi cell firing due to an increased potassium permeability associated with activation of this G-protein-coupled receptor. However, an involvement of other mechanisms such as electrical coupling via gap-junctions cannot be ruled out at this stage (e.g. Vervaeke et al. 2010). Nevertheless, it is interesting to speculate that this form of slow intercellular signalling may support oscillations in granule cell firing such that modulation of granule cell activity levels over such slow time-scales are matched to incoming sensory inputs of the type observed during sleep and certain types of behaviour (Dugueet al. 2009; Roset al. 2009). Given the importance of the cerebellar cortex in the acquisition of new motor skills, an important challenge for the future will be to determine how these types of intercellular signalling mechanisms may impact upon granule cell firing during motor learning tasks.

The mechanisms of the strong inhibitory modulation of long-term potentiation in the rat dentate gyrus

The hippocampus is essential for the formation of certain types of memory, and synaptic plasticity such as long-term potentiation (LTP) is widely accepted as a cellular basis of hippocampus-dependent memory. Although LTP in both perforant path-dentate gyrus (DG) granule cell and CA3-CA1 pyramidal cell synapses is similarly dependent on activation of postsynaptic N-methyl-D-aspartate receptors, several reports suggest that modulation of LTP by γ-aminobutyric acid (GABA) receptor-mediated inhibitory inputs is stronger in perforant path-DG granule cell synapses. However, little is known about how different the mechanism and physiological relevance of the GABAergic modulation of LTP induction are among different brain regions. We confirmed that the action of GABA(A) receptor antagonists on LTP was more prominent in the DG, and explored the mechanism introducing such difference by examining two types of GABA(A) receptor-mediated inhibition, i.e. synaptic and tonic inhibition. As synaptic inhibition, we compared inhibitory vs. excitatory monosynaptic responses and their summation during an LTP-inducing stimulus, and found that the balance of the summated postsynaptic currents was biased toward inhibition in the DG. As tonic inhibition, or sustained activation of extrasynaptic GABA(A) receptors by ambient GABA, we measured the change in holding currents of the postsynaptic cells induced by GABA(A) receptor antagonists, and found that the tonic inhibition was significantly stronger in the DG. Furthermore, we found that tonic inhibition was associated with LTP modulation. Our results suggest that both the larger tonic inhibition and the larger inhibitory/excitatory summation balance during conditioning are involved in the stronger inhibitory modulation of LTP in the DG.

Altered spontaneous synaptic inhibition in an animal model of cerebral heterotopias

We have investigated spontaneous synaptic transmission in hippocampal nodular heterotopias in rats exposed to methylazoxymethanol (MAM) in utero. Pregnant Wistar rats were injected with MAM at E16. Acute hippocampal slices were prepared from the rat pups P14 to P40. Whole-cell voltage-clamp recordings were made from visually identified neurons using IR-DIC video microscopy. Synaptic events were recorded from either heterotopic neurons in the CA1 region or “slice-matched” normotopic CA1 pyramidal neurons. Both the spontaneous inhibitory (sIPSC) and excitatory synaptic transmission (sEPSC) to the same neurons were recorded. We found a profound reduction in the frequency of sIPSCs in the heterotopic neurons vs. normotopic neurons. No significant differences in the frequency of sEPSCs were found. We also found a profound reduction in the frequency of spontaneous IPSCs in normotopic neurons following application of the GABA reuptake blocker, NO-711, even in the presence of a GABAB receptor antagonist (CGP 55845). Preferentially blocking extrasynaptic GABAA receptors caused an increased frequency of sIPSCs in the heterotopic neurons. Our data suggest that there is a predominant change in inhibitory synaptic transmission, as measured by changes in sIPSCs, with no change in excitatory synaptic transmission to heterotopic neurons in hippocampus of rats exposed to MAM in utero. We suggest that this change is caused by an increase in the extracellular concentration of GABA but is not mediated via activation of presynaptic GABAB receptors. Rather, we propose that the increased extracellular GABA concentration in the heterotopias dampens the activity in inhibitory neurons via activation of extrasynaptic GABAA receptors.

Postnatal Switch from Synaptic to Extrasynaptic Transmission between Interneurons and NG2 Cells

NG2 cells, oligodendrocyte precursors, play a critical role in myelination during postnatal brain maturation, but a pool of these precursors is maintained in the adult and recruited to lesions in demyelinating diseases. NG2 cells in immature animals have recently been shown to receive synaptic inputs from neurons, and these have been assumed to persist in the adult. Here, we investigated the GABAergic synaptic activity of NG2 cells in acute slices of the barrel cortex of NG2-DsRed transgenic mice during the first postnatal month, which corresponds to the period of active myelination in the neocortex. Our data demonstrated that the frequency of spontaneous and miniature GABAergic synaptic activity of cortical NG2 cells dramatically decreases after the second postnatal week, indicating a decrease in the number of synaptic inputs onto NG2 cells during development. However, NG2 cells still receive GABAergic inputs from interneurons in the adult cortex. These inputs do not rely on the presence of functional synapses but involve a form of GABA spillover. This GABA volume transmission allows interneurons to induce phasic responses in target NG2 cells through the activation of extrasynaptic GABA(A) receptors. Hence, after development is complete, volume transmission allows NG2 cells to integrate neuronal activity patterns at frequencies occurring during in vivo sensory stimulation.

Abnormal adult neurogenesis in the dentate gyrus of GABA(A)R δ subunit knockout mice

Event Abstract Back to Event Abnormal adult neurogenesis in the dentate gyrus of GABA(A)R δ subunit knockout mice Shira Rosenzweig1*, E. Czerwinska1, Beverley A. Orser2 and Martin J. Wojtowicz1 1 University of Toronto, Department of Physiology, Canada 2 University of Toronto, Physiology and Anesthesia, Canada Background: During adult neurogenesis, cells are born in the sub-granular zone (SGZ) of the dentate gyrus (DG) and subsequently undergo differentiation, migration and maturation. The mechanisms that regulate the various stages of adult neurogenesis remain poorly understood. One known regulator is the neurotransmitter GABA, which depolarizes young DG cells before they become responsive to glutamate. This early depolarization initially occurs via activation of extrasynaptic GABAA receptors (GABAARs). As most extrasynaptic GABAARs in the DG contain the delta (δ) subunit, we hypothesized that this receptor subtype is involved in the early depolarization and developmental regulation of young DG neurons. To test this hypothesis we characterized and compared neurogenesis in the DG of mice that are homozygous null for the GABAA receptor δ gene (Garbd-/-) and wild type (WT) C57BL/6J mice. Garbd-/- mice exhibit an increased susceptibility to seizures and attenuated responses to neuroactive steroids which modulate δ-containing GABAARs. Methods: Two months old WT and Garbd-/- mice were injected intra-peritoneally with the mitotic indicator BrdU and sacrificed two weeks later. Hippocampal sections were immunostained for Ki-67, a marker for proliferating cells, doublecortin (DCX), a marker for immature neurons, and BrdU. Labeled cells were counted using a confocal microscope. Results: There were no genotype-related differences in the number of Ki-67+ and two-week old (BrdU+) cells. The total number of immature (DCX+) neurons and the fraction of immature BrdU+ cells were 20% higher in Garbd-/- compared with WT mice (p<0.03 and p<0.01, n=3). Analysis of the position of BrdU+ cells across the granule cell layer (GCL) revealed that in WT mice, 22% of the cells had migrated from the SGZ deeper into the GCL by two weeks. In contrast, only 9.8% of the cells had migrated in Garbd-/- mice (p<0.01, n=3). Conclusions: These results indicate that proliferation and two-week survival are unchanged in Garbd-/- mice. In contrast, a greater number of cells remain immature at two weeks, and their normal migration is disrupted in Garbd-/- mice compared to WT mice. The abnormalities observed in the Garbd-/- mice suggest that δ-containing GABAARs play an important role in the normal development of adult-born DG granule neurons. To fully characterize survival, differentiation and maturation of adult-born neurons in the absence of δ subunits, ongoing experiments will examine more advanced time points of neuronal development. Conference: B.R.A.I.N. platform in Physiology poster day 2009, Toronto, ON, Canada, 16 Dec - 16 Dec, 2009. Presentation Type: Oral Presentation Topic: Oral presentations Citation: Rosenzweig S, Czerwinska E, Orser BA and Wojtowicz MJ (2009). Abnormal adult neurogenesis in the dentate gyrus of GABA(A)R δ subunit knockout mice. Front. Neurosci. Conference Abstract: B.R.A.I.N. platform in Physiology poster day 2009. doi: 10.3389/conf.neuro.03.2009.17.049 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 18 Dec 2009; Published Online: 18 Dec 2009. * Correspondence: Shira Rosenzweig, University of Toronto, Department of Physiology, Toronto, Canada, shirarosen@outlook.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz Google Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz Google Scholar Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz PubMed Shira Rosenzweig E. Czerwinska Beverley A Orser Martin J Wojtowicz Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Taurine Is a Potent Activator of Extrasynaptic GABAAReceptors in the Thalamus

Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (> or = 100 microM), and to be a weak agonist at GABA(A) receptors (GABA(A)-Rs), which are usually activated at high concentrations (> or = 1 mM). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABA(A)-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10-100 microM) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 microM taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 microM Zn2+, consistent with the pharmacological properties of extrasynaptic GABA(A)-Rs (alpha4beta2delta subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABA(A)-R alpha4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant alpha4beta2delta GABA(A)-Rs than at alpha1beta2gamma2 GABA(A)-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABA(A)-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

S.03.03 Paracapsular cells of the amygdala: an inhibitory gate modulated by D1 dopamine receptors

The endogenous neuroactive steroid allopregnanolone (ALLO) has previously been shown to induce reinstatement of ethanol seeking in rodents. ALLO is a positive allosteric modulator at both synaptic and extrasynaptic GABAA receptors. The contribution of each class of GABAA receptors in mediating reinstatement of ethanol seeking is unknown. The first aim of the present study was to determine whether ganaxolone (GAN), a longer-acting synthetic analog of ALLO, also promotes reinstatement of ethanol seeking. The second aim was to examine whether preferentially activating extrasynaptic GABAA receptors with the selective agonist gaboxadol (THIP) was sufficient to reinstate responding for ethanol in mice. Male C57BL/6J mice were trained to lever press for access to a 10% ethanol (v/v) solution (10E), using a sucrose-fading procedure. Following extinction of the lever-pressing behavior, systemic THIP (0, 4 and 6 mg/kg) and GAN (0, 10, and 15 mg/kg) were tested for their ability to reinstate ethanol-appropriate responding in the absence of 10E access. GAN significantly increased lever pressing on the previously active lever, while THIP did not alter lever-pressing behavior. The results of this study suggest that direct activation of extrasynaptic GABAA receptors at the GABA site is not sufficient to induce ethanol seeking in the reinstatement procedure. Future studies are necessary to elucidate the mechanisms and brain areas by which differences in the pharmacological activity of GAN and THIP at the GABAA receptor contribute to the dissimilarity in their effect on the reinstatement of ethanol seeking. Nonetheless, based on the increased use of these drugs in clinical trials across multiple disease states, the effects of GAN or THIP on alcohol seeking may be an important consideration if these drugs are to be used clinically in a population with a co-occurring alcohol use disorder.

Selective modulation of tonic and phasic inhibitions in dentate gyrus granule cells.

In some nerve cells, activation of GABA(A) receptors by GABA results in phasic and tonic conductances. Transient activation of synaptic receptors generates phasic inhibition, whereas tonic inhibition originates from GABA acting on extrasynaptic receptors, like in cerebellar granule cells, where it is thought to result from the activation of extrasynaptic GABA(A) receptors with a specific subunit composition (alpha(6)beta(x)delta). Here we show that in adult rat hippocampal slices, extracellular GABA levels are sufficiently high to generate a powerful tonic inhibition in delta subunit-expressing dentate gyrus granule cells. In these cells, the mean tonic current is approximately four times larger than that produced by spontaneous synaptic currents occurring at a frequency of approximately 10 Hz. Antagonizing the GABA transporter GAT-1 with NO-711 (2.5 microM) selectively enhanced tonic inhibition by 330% without affecting the phasic component. In contrast, by prolonging the decay of inhibitory postsynaptic currents (IPSCs), the benzodiazepine agonist zolpidem (0.5 microM) augmented phasic inhibition by 66%, while leaving the mean tonic conductance unchanged. These results demonstrate that a tonic GABA(A) receptor-mediated conductance can be recorded from dentate gyrus granule cells of adult rats in in vitro slice preparations. Furthermore, we have identified distinct pharmacological tools to selectively modify tonic and phasic inhibitions, allowing future studies to investigate their specific roles in neuronal function.

Post-episode depression of GABAergic transmission in spinal neurons of the chick embryo.

Whole cell recordings were obtained from ventral horn neurons in spontaneously active spinal cords isolated from the chick embryo [embryonic days 10 to 11 (E10-E11)] to examine the post-episode depression of GABAergic transmission. Spontaneous activity occurred as recurrent, rhythmic episodes approximately 60 s in duration with 10- to 15-min quiescent inter-episode intervals. Current-clamp recording revealed that episodes were followed by a transient hyperpolarization (7 +/- 1.2 mV, mean +/- SE), which dissipated as a slow (0.5-1 mV/min) depolarization until the next episode. Local application of bicuculline 8 min after an episode hyperpolarized spinal neurons by 6 +/- 0.8 mV and increased their input resistance by 13%, suggesting the involvement of GABAergic transmission. Gramicidin perforated-patch recordings showed that the GABAa reversal potential was above rest potential (E(GABAa) = -29 +/- 3 mV) and allowed estimation of the physiological intracellular [Cl(-)] = 50 mM. In whole cell configuration (with physiological electrode [Cl(-)]), two distinct types of endogenous GABAergic currents (I(GABAa)) were found during the inter-episode interval. The first comprised TTX-resistant, asynchronous miniature postsynaptic currents (mPSCs), an indicator of quantal GABA release (up to 42% of total mPSCs). The second (tonic I(GABAa)) was complimentary to the slow membrane depolarization and may arise from persistent activation of extrasynaptic GABAa receptors. We estimate that approximately 10 postsynaptic channels are activated by a single quantum of GABA release during an mPSC and that about 30 extrasynaptic GABAa channels are required for generation of the tonic I(GABAa) in ventral horn neurons. We investigated the post-episode depression of I(GABAa) by local application of GABA or isoguvacine (100 microM, for 10-30 s) applied before and after an episode at holding potentials (V(hold)) -60 mV. The amplitude of the evoked I(GABA) was compared after clamping the cell during the episode at one of three different V(hold): -60 mV, below E(GABAa) resulting in Cl(-) efflux; -30 mV, close to E(GABAa) with minimal Cl(-) flux; and 0 mV, above E(GABAa) resulting in Cl(-) influx during the episode. The amplitude of the evoked I(GABA) changed according to the direction of Cl(-) flux during the episode: at -60 mV a 41% decrease, at -30 mV a 4% reduction, and at 0 mV a 19% increase. These post-episode changes were accompanied by shifts of E(GABAa) of -10, -1.2, and +7 mV, respectively. We conclude that redistribution of intracellular [Cl(-)] during spontaneous episodes is likely to be an important postsynaptic mechanism involved in the post-episode depression of GABAergic transmission in chick embryo spinal neurons.