Tonic Inhibition Of Neurons Research Articles

Allosteric potentiation of GABAA receptor single-channel conductance by netrin-1 during neuronal-excitation-induced inhibitory synaptic homeostasis.

As the principal receptor that mediates both synaptic and tonic inhibition of neurons in the brain, the A-type gamma-aminobutyric acid receptor (GABAAR) is functionally important for maintaining the balance between neuronal excitation and inhibition. Here, we report the identification of netrin-1 as an endogenous allosteric modulator of GABAARs. Following increased neuronal excitability, netrin-1 is secreted and binds to the extracellular domains of GABAAR subunits, thereby inducing homeostatic upscaling of GABAAR-mediated synaptic efficacy and currents. Surprisingly, this homeostatic plasticity is primarily mediated by increasing GABAAR single-channel conductance. Our study reveals an important role of netrin-1 as an endogenous GABAAR allosteric modulator in maintaining neuronal excitation-inhibition balance, a fundamental process for brain function and dysfunction.

Prefrontal GABAA(δ)R Promotes Fear Extinction through Enabling the Plastic Regulation of Neuronal Intrinsic Excitability.

Extinguishing the previously acquired fear is critical for the adaptation of an organism to the ever-changing environment, a process requiring the engagement of GABAA receptors (GABAARs). GABAARs consist of tens of structurally, pharmacologically, and functionally heterogeneous subtypes. However, the specific roles of these subtypes in fear extinction remain largely unexplored. Here, we observed that in the medial prefrontal cortex (mPFC), a core region for mood regulation, the extrasynaptically situated, δ-subunit-containing GABAARs [GABAA(δ)Rs], had a permissive role in tuning fear extinction in male mice, an effect sharply contrasting to the established but suppressive role by the whole GABAAR family. First, the fear extinction in individual mice was positively correlated with the level of GABAA(δ)R expression and function in their mPFC. Second, knockdown of GABAA(δ)R in mPFC, specifically in its infralimbic (IL) subregion, sufficed to impair the fear extinction in mice. Third, GABAA(δ)R-deficient mice also showed fear extinction deficits, and re-expressing GABAA(δ)Rs in the IL of these mice rescued the impaired extinction. Further mechanistic studies demonstrated that the permissive effect of GABAA(δ)R was associated with its role in enabling the extinction-evoked plastic regulation of neuronal excitability in IL projection neurons. By contrast, GABAA(δ)R had little influence on the extinction-evoked plasticity of glutamatergic transmission in these cells. Altogether, our findings revealed an unconventional and permissive role of extrasynaptic GABAA receptors in fear extinction through a route relying on nonsynaptic plasticity.SIGNIFICANCE STATEMENT The medial prefrontal cortex (mPFC) is one of the kernel brain regions engaged in fear extinction. Previous studies have repetitively shown that the GABAA receptor (GABAAR) family in this region act to suppress fear extinction. However, the roles of specific GABAAR subtypes in mPFC are largely unknown. We observed that the GABAAR-containing δ-subunit [GABAA(δ)R], a subtype of GABAARs exclusively situated in the extrasynaptic membrane and mediating the tonic neuronal inhibition, works oppositely to the whole GABAAR family and promotes (but does not suppress) fear extinction. More interestingly, in striking contrast to the synaptic GABAARs that suppress fear extinction by breaking the extinction-evoked plasticity of glutamatergic transmission, the GABAA(δ)R promotes fear extinction through enabling the plastic regulation of neuronal excitability in the infralimbic subregion of mPFC. Our findings thus reveal an unconventional role of GABAA(δ)R in promoting fear extinction through a route relying on nonsynaptic plasticity.

Mechanisms Responsible for Genetic Hypertension in Schlager BPH/2 Mice.

It has been 45 years since Gunther Schlager used a cross breeding program in mice to develop inbred strains with high, normal, and low blood pressure (BPH/2, BPN/3, and BPL/1 respectively). Thus, it is timely to gather together the studies that have characterized and explored the mechanisms associated with the hypertension to take stock of exactly what is known and what remains to be determined. Growing evidence supports the notion that the mechanism of hypertension in BPH/2 mice is predominantly neurogenic with some of the early studies showing aberrant brain noradrenaline levels in BPH/2 compared with BPN/3. Analysis of the adrenal gland using microarray suggested an association with the activity of the sympathetic nervous system. Indeed, in support of this, there is a larger depressor response to ganglion blockade, which reduced blood pressure in BPH/2 mice to the same level as BPN/3 mice. Greater renal tyrosine hydroxylase staining and greater renal noradrenaline levels in BPH/2 mice suggest sympathetic hyperinnervation of the kidney. Renal denervation markedly reduced the blood pressure in BPH/2 but not BPN/3 mice, confirming the importance of renal sympathetic nervous activity contributing to the hypertension. Further, there is an important contribution to the hypertension from miR-181a and renal renin in this strain. BPH/2 mice also display greater neuronal activity of amygdalo-hypothalamic cardiovascular regulatory regions. Lesions of the medial nucleus of the amygdala reduced the hypertension in BPH/2 mice and abolished the strain difference in the effect of ganglion blockade, suggesting a sympathetic mechanism. Further studies suggest that aberrant GABAergic inhibition may play a role since BPH/2 mice have low GABAA receptor δ, α4 and β2 subunit mRNA expression in the hypothalamus, which are predominantly involved in promoting tonic neuronal inhibition. Allopregnanolone, an allosteric modulator of GABAA receptors, which increase the expression of these subunits in the amygdala and hypothalamus, is shown to reduce the hypertension and sympathetic nervous system contribution in BPH/2 mice. Thus far, evidence suggests that BPH/2 mice have aberrant GABAergic inhibition, which drives neuronal overactivity within amygdalo-hypothalamic brain regions. This overactivity is responsible for the greater sympathetic contribution to the hypertension in BPH/2 mice, thus making this an ideal model of neurogenic hypertension.

Effect of GABRA1 Cytoplasmic Peptide on a Regulatory Protein

The pentameric neurotransmitter-gated chloride channel, GABA (type A) receptor, is well-recognized for phasic and tonic neuronal inhibition. The phasic response at the synapse is due to receptors typically containing two alpha, two beta, and one gamma subunit. Half of all GABA (type A) receptors in adult human brain have the alpha (type 1), or alpha-1, subunit (gene ID: GABRA1). Each consists of an extracellular ligand-binding domain and four transmembrane helices (TM1-4), both of which are highly conserved, as well as a large intracellular loop domain (ILD), whose structure is unknown. The human receptor is crystallized without the ILD, which connects TM3 and TM4, that is engineered out. The topology and function of the 86-residue alpha-1 ILD remains to be fully elucidated. GAPDH (d-glyceraldehyde-3-phosphate dehydrogenase) phosphorylates specific ILD residues, regulating receptor function. In this study, we examined the effect of a synthesized ILD peptide (residues 335 to 364 from GABRA1) on the oxidoreductase activity of GAPDH. The custom peptide, containing the first 30 cytosolic residues, was pre-incubated with GAPDH, and enzyme activity measured by NADH absorbance (340nm) upon oxidation of the substrate d-glyceraldehyde. When pre-incubated for 1hr at 20degC, the ILD peptide inhibited enzyme activity in a concentration-dependent manner showing an exponential non-linear function (y=a∗e ˆ (b/x+c)). The half-maximum inhibition occurred at a [peptide] = 8.7micromolar. When pre-incubated for 48hr at 4degC, the ILD peptide further enhanced inhibition of oxidoreductase activity, suggesting that hydrophobic forces play a role in the interaction between the ILD peptide and GAPDH. Molecular modeling experiments indicate that the ILD binding site on GAPDH is in close proximity to the phosphatidylserine-binding site on GAPDH. The findings may contribute to our understanding of receptor function and point to possible treatments in conditions, such as Alzheimer's dementia, where GABA receptor pharmacology is shown to play a role.

Thalamic δ-subunit containing GABAA receptors promote electrocortical signatures of deep non-REM sleep but do not mediate the effects of etomidate at the thalamus in vivo.

Extrasynaptic δ-subunits containing GABAA receptors (δGABAARs) are sensitive targets for several commonly used hypnotic agents and mediate tonic neuronal inhibition. δGABAARs are highly expressed within the thalamus and their activation promotes a switch from tonic to burst firing in vitro. Here we test two hypotheses in vivo. (1) Activation of thalamic δGABAARs will elicit electrocortical signatures consistent with widespread thalamocortical burst firing such as increased delta oscillations (1-4 Hz) and reciprocal changes in spindle-like oscillations (7-14 Hz). (2) These signatures will be recapitulated by the general anesthetic etomidate, if the electrocortical effects of etomidate at the thalamus are mediated by δGABAARs. Microperfusion of the δGABAAR-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP; 10 and 50 μM) into the ventrobasal complex produced significant effects on electrocortical activity in wild-type mice, but not in mice lacking δGABAARs (Gabrd(-/-)), i.e., the effects with THIP were dependent on δGABAARs. THIP (1) increased 1-4 Hz power in wakefulness and nonrapid-eye movement (NREM) sleep; (2) reduced spindle-like oscillations in NREM sleep; and (3) increased the speed of stable transitions into NREM sleep, indicating effects on state-space dynamics. In contrast, microperfusion of etomidate (10 and 30 μM) into the ventrobasal complex produced effects on electrocortical activity that were independent of δGABAARs, i.e., effects occurred in wild-type and Gabrd(-/-) mice. Etomidate (1) decreased 1-4 Hz power, increased 8-12 Hz, and/or 12-30 Hz power in all sleep-wake states; (2) increased spindle-like oscillations; and (3) increased REM sleep expression. These results indicate that thalamic δGABAARs promote electrocortical signatures of deep NREM sleep, but do not mediate the effects of etomidate at the thalamus in vivo.

Abstract W MP41: Potentiation of Gaba-Mediated Synaptic Inhibition in the Recovery Phase: A Novel Therapeutic Target for Stroke

Background: Stroke is a major cause of disability yet pharmacotherapy targeting the recovery phase is lacking. Cortical circuit reorganization adjacent to the stroke site promotes recovery, thus elucidating mechanisms that promote this plasticity could lead to new therapeutics. Tonic neuronal inhibition, mediated by extrasynaptic GABA A receptors,inhibits post-stroke recovery. However, effects of phasic (synaptic) GABA signaling - which promotes plasticity during development - are unknown. Here we use a combined approach of i) array tomography to determine the composition of GABA synapses in the post-stroke mouse brain, ii) electrophysiology to determine whether stroke leads to functional changes in GABA-mediated phasic inhibition, and (iii) treatment with zolpidem, an FDA-approved GABA agonist, to modulate recovery. Results: We found, using array tomography, a 1.7-fold increase in the number of GABAergic synapses containing the α1 receptor subunit in layer 5 of the peri-infarct cortex (synapse number/μm 3 : 0.039±0.006 (control) vs 0.064±0.006 (stroke); P<0.01), but not in layer 2/3. There was an associated increase in spontaneous inhibitory post-synaptic currents (sIPSC) specific to layer 5 pyramidal neurons (sIPSC charge (fC): -403±27.8 (control) vs -724±166 (stroke); p=0.03). This effect was transient, occurring during the onset of functional recovery. To test whether the increased phasic inhibitory GABAergic signaling promotes stroke recovery, we treated animals with zolpidem, an agonist with high affinity for α1 subunit-containing GABA A receptors. Low dose zolpidem increased GABA A phasic signaling in layer 5 pyramidal cells and notably increased the rate and extent of behavioral recovery without altering infarct size. Conclusions: These data provide the first evidence that enhanced GABA A -mediated synaptic activity during the recovery phase improves stroke outcome. These data identify modulation of phasic GABA signaling as a novel therapeutic strategy for stroke, indicate zolpidem as a potential drug to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA inhibition in stroke recovery.

The paraventricular nucleus and heart failure

What is the topic of this review? This review gives an update on the cellular and molecular mechanisms within the autonomic nervous system involved in non-pathological and pathological cardiovascular regulation. What advances does it highlight? For cardiovascular homeostasis in non-pathological conditions to be maintained, discrete neural networks using specified signalling mechanisms at both cellular and molecular levels are required. In heart failure, the cell signalling protein partners CAPON and PIN decrease the bioavailability of nitric oxide by inhibiting neuronal nitric oxide synthase activity, leading to the removal of tonic neuronal inhibition. Following a myocardial infarction, pro-inflammatory cytokines in the paraventricular nucleus and the subsequent generation of reactive oxygen species, via angiotensin II activation of the angiotensin II type 1 receptor, increase neuronal excitability further, leading to sympathetic excitation. A pathological feature of heart failure is abnormal control of the sympathetic nervous system. The paraventricular nucleus of the hypothalamus (PVN) is one of the most important central sites involved in regulating sympathetic tone and is, in part, responsible for the dysregulation of the sympathetic nervous system evident in heart failure. Generation of sympathetic tone in response to fluctuations in cardiovascular regulation uses discrete anatomical pathways and neurochemical modulators. Direct and indirect projections from the PVN pre-autonomic neurons innervate the sympathetic preganglionic neurons in the spinal cord, which in turn innervate sympathetic ganglia that give rise to the sympathetic nerves. Pre-autonomic neurons of the PVN themselves receive an afferent input arising from the nucleus tractus solitarii, and viscerosensory receptors convey cardiovascular fluctuations to the nucleus tractus solitarii. The PVN contains excitatory and inhibitory neurons, whose balance determines the sympathetic tone. In non-pathological conditions, the tonic inhibition of the PVN pre-autonomic neurons is mediated by GABA- and NO-releasing neurons. In heart failure, the pre-autonomic neurons are disinhibited by the actions of the excitatory neurotransmitters glutamate and angiotensin II, leading to increased sympathetic activity. A key feature of the disinhibition is a reduction in the bioavailability of NO as a consequence of disrupted CAPON and PIN signalling mechanisms within the neuron. Another critical feature that contributes to increased neuronal excitation within the PVN is the production of pro-inflammatory cytokines immediately following a myocardial infarction, the activation of the angiotensin II type 1 receptor and the production of reactive oxygen species. By examining the changes associated with the sympathetic nervous system pathway, we will progress our understanding of sympathetic regulation in heart failure, identify gaps in our knowledge and suggest new therapeutic strategies.

Astrocytes convert network excitation to tonic inhibition of neurons

BackgroundGlutamate and γ-aminobutyric acid (GABA) transporters play important roles in balancing excitatory and inhibitory signals in the brain. Increasing evidence suggest that they may act concertedly to regulate extracellular levels of the neurotransmitters.ResultsHere we present evidence that glutamate uptake-induced release of GABA from astrocytes has a direct impact on the excitability of pyramidal neurons in the hippocampus. We demonstrate that GABA, synthesized from the polyamine putrescine, is released from astrocytes by the reverse action of glial GABA transporter (GAT) subtypes GAT-2 or GAT-3. GABA release can be prevented by blocking glutamate uptake with the non-transportable inhibitor DHK, confirming that it is the glutamate transporter activity that triggers the reversal of GABA transporters, conceivably by elevating the intracellular Na+ concentration in astrocytes. The released GABA significantly contributes to the tonic inhibition of neurons in a network activity-dependent manner. Blockade of the Glu/GABA exchange mechanism increases the duration of seizure-like events in the low-[Mg2+] in vitro model of epilepsy. Under in vivo conditions the increased GABA release modulates the power of gamma range oscillation in the CA1 region, suggesting that the Glu/GABA exchange mechanism is also functioning in the intact hippocampus under physiological conditions.ConclusionsThe results suggest the existence of a novel molecular mechanism by which astrocytes transform glutamatergic excitation into GABAergic inhibition providing an adjustable, in situ negative feedback on the excitability of neurons.

Insulin Reduces Neuronal Excitability by Turning on GABAA Channels that Generate Tonic Current

Insulin signaling to the brain is important not only for metabolic homeostasis but also for higher brain functions such as cognition. GABA (γ-aminobutyric acid) decreases neuronal excitability by activating GABAA channels that generate phasic and tonic currents. The level of tonic inhibition in neurons varies. In the hippocampus, interneurons and dentate gyrus granule cells normally have significant tonic currents under basal conditions in contrast to the CA1 pyramidal neurons where it is minimal. Here we show in acute rat hippocamal slices that insulin (1 nM) “turns on” new extrasynaptic GABAA channels in CA1 pyramidal neurons resulting in decreased frequency of action potential firing. The channels are activated by more than million times lower GABA concentrations than synaptic channels, generate tonic currents and show outward rectification. The single-channel current amplitude is related to the GABA concentration resulting in a single-channel GABA affinity (EC50) in intact CA1 neurons of 17 pM with the maximal current amplitude reached with 1 nM GABA. They are inhibited by GABAA antagonists but have novel pharmacology as the benzodiazepine flumazenil and zolpidem are inverse agonists. The results show that tonic rather than synaptic conductances regulate basal neuronal excitability when significant tonic conductance is expressed and demonstrate an unexpected hormonal control of the inhibitory channel subtypes and excitability of hippocampal neurons. The insulin-induced new channels provide a specific target for rescuing cognition in health and disease.

Increased network activity induces tonic inhibition by emerging a glial GABA pool

Event Abstract Back to Event Increased network activity induces tonic inhibition by emerging a glial GABA pool O. Kékesi1*, L. Héja1, G. Nyitrai1, Á. Dobolyi2, P. Szabó3, M. Palkovits2 and J. Kardos1 1 Hungarian Academy of Sciences, Department of Neurochemistry, Hungary 2 Semmelweis University and Hungarian Academy of Sciences, Hungary 3 Hungarian Academy of Sciences, Department of Pharmacobiochemistry, Hungary Maintenance of the balance between excitation and inhibition is of crucial importance in the brain. Increasing evidence suggest that astroglial glutamate/Na+ symporters induce the reverse action of extrasynaptic astroglial γ-amino-butyric acid/Na+ symporter subtypes GAT-2 and GAT-3, generating tonic inhibition in neurons. The source of the released γ-amino-butyric acid (GABA), however, is uncertain, because GABA is synthesized mainly in neurons from Glu by the enzyme Glu decarboxylase. Here we show that an alternative pathway for the GABA synthetic route exists in astrocytes and this pathway is responsible for the synthesis of the released GABA. We used in vitro hippocampal slices under low-[Mg2+] conditions to measure GABA currents by whole cell patch clamp method in the absence and presence of SNAP-5114, a non-transportable blocker of GAT-2/3. Under control conditions SNAP-5114 application reveals the GAT-2/3 mediated tonic current component. Incubation of the slices with monoamine and diamine oxidase inhibitors (deprenyl and aminoguanidine, respectively) that prevent GABA formation from putrescine eliminated this tonic current component, demonstrating that under low-[Mg2+] conditions astrocytes utilize the polyamine pathway to synthesize GABA for maintaining tonic inhibition. We envision that the discovery of this mechanism will support the development of new pathomechanism-specific treatments for pathological conditions characterized by sustained excitation. Keywords: Neurophysiology, Neuroscience Conference: 13th Conference of the Hungarian Neuroscience Society (MITT), Budapest, Hungary, 20 Jan - 22 Jan, 2011. Presentation Type: Abstract Topic: Neurophysiology Citation: Kékesi O, Héja L, Nyitrai G, Dobolyi Á, Szabó P, Palkovits M and Kardos J (2011). Increased network activity induces tonic inhibition by emerging a glial GABA pool. Front. Neurosci. Conference Abstract: 13th Conference of the Hungarian Neuroscience Society (MITT). doi: 10.3389/conf.fnins.2011.84.00152 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: 03 Mar 2011; Published Online: 23 Mar 2011. * Correspondence: Dr. O. Kékesi, Hungarian Academy of Sciences, Department of Neurochemistry, Budapest, Hungary, kekesi.orsolya@chemres.hu 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 O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos Google O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos Google Scholar O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos PubMed O. Kékesi L. Héja G. Nyitrai Á. Dobolyi P. Szabó M. Palkovits J. Kardos 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.

Reducing excessive GABA-mediated tonic inhibition promotes functional recovery after stroke

Stroke is a leading cause of disability; but no pharmacological therapy is currently available for promoting recovery. The brain region adjacent to stroke damage, the peri-infarct zone, is critical for rehabilitation, as it exhibits heightened neuroplasticity, allowing sensorimotor functions to re-map from damaged areas1–3. Thus, understanding the neuronal properties constraining this plasticity is important to developing new treatments. Here we show that after a stroke in mice, tonic neuronal inhibition is increased in the peri-infarct zone. This increased tonic inhibition is mediated by extrasynaptic GABAA receptors (GABAARs) and is caused by an impairment in GABA transporter (GAT-3/4) function. To counteract the heightened inhibition, we administered in vivo a benzodiazepine inverse agonist specific for the α5-subunit-containing extrasynaptic GABAARs at a delay after stroke. This treatment produced an early and sustained recovery of motor function. Genetically lowering the number of α5 or δ-subunit-containing GABAARs responsible for tonic inhibition also proved beneficial for post-stroke recovery, consistent with the therapeutic potential of diminishing extrasynaptic GABAAR function. Together, our results identify new pharmacological targets and provide the rationale for a novel strategy to promote recovery after stroke and possibly other brain injuries.

Channel-Mediated Tonic GABA Release from Glia

Synaptic inhibition is based on both tonic and phasic release of the inhibitory transmitter γ-aminobutyric acid (GABA). Although phasic GABA release arises from Ca(2+)-dependent exocytosis from neurons, the mechanism of tonic GABA release is unclear. Here we report that tonic inhibition in the cerebellum is due to GABA being released from glial cells by permeation through the Bestrophin 1 (Best1) anion channel. We demonstrate that GABA directly permeates through Best1 to yield GABA release and that tonic inhibition is eliminated by silencing of Best1. Glial cells express both GABA and Best1, and selective expression of Best1 in glial cells, after preventing general expression of Best1, fully rescues tonic inhibition. Our results identify a molecular mechanism for tonic inhibition and establish a role for interactions between glia and neurons in mediating tonic inhibition.

GABA Transporter Preserving Ongoing Spontaneous Neuronal Activity at Firing Subthreshold

There has been compelling evidence that the GABA transporter is crucial not only for removing gamma-aminobutyric acid (GABA) from but also releasing it into extracellular space, thereby clamping ambient GABA (GABA in extracellular space) at a certain level. The ambient GABA is known to activate extrasynaptic GABA receptors and provide tonic inhibitory current into neurons. We investigated how the transporter regulates the level of ambient GABA, mediates tonic neuronal inhibition, and influences ongoing spontaneous neuronal activity. A cortical neural network model is proposed in which GABA transporters on lateral (L) and feedback (F) inhibitory (GABAergic) interneurons are functionally made. Principal (P) cell assemblies participate in expressing information about elemental sensory features. At membrane potentials below the reversal potential, there is net influx of GABA, whereas at membrane potentials above the reversal potential, there is net efflux of GABA. Through this transport mechanism, ambient GABA concentration is kept within a submicromolar range during an ongoing spontaneous neuronal activity time period. Here we show that the GABA transporter on L cells regulates the overall level of ambient GABA across cell assemblies, and that on F cells it does so within individual cell assemblies. This combinatorial regulation of ambient GABA allows P cells to oscillate near firing threshold during the ongoing time period, thereby reducing their reaction time to externally applied stimuli. We suggest that the GABA transporter, with its forward and reverse transport mechanism, could regulate the ambient GABA. This transporter-mediated ambient GABA regulation may contribute to establishing an ongoing subthreshold neuronal state by which the network can respond rapidly to subsequent sensory input.

Protein Kinase Cϵ Regulates γ-Aminobutyrate Type A Receptor Sensitivity to Ethanol and Benzodiazepines through Phosphorylation of γ2 Subunits

Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.

Graded response to GABA by native extrasynaptic GABAA receptors

GABA is the main inhibitory neurotransmitter in the mammalian CNS. GABA in the brain is commonly associated with a fast, point-to-point form of signalling called synaptic transmission (phasic inhibition), but there is growing evidence that GABA participates in another, slower and more diffuse form of signalling often referred to as tonic inhibition. Unresolved questions regarding tonic neuronal inhibition concern activation and functional properties of extrasynaptic GABAA receptors (GABARex) present on neurones. Extrasynaptic receptors are exposed to submicromolar GABA concentrations and may modulate the overall excitability of neurones and neuronal networks. Here, we examined GABA-activated single-channel currents in dentate gyrus granule neurones in rat hippocampal slices. We activated three types (I, II, III) of GABARex channels by nanomolar GABA concentrations (EC50 I: 27 +/- 12; II: 4 +/- 3; III: 43 +/- 19 nm). The channels opened after a delay and the single-channel conductance was graded (gammamax I: 61 +/- 3; II: 85 +/- 8, III: 40 +/- 3 pS). The channels were differentially modulated by 1 microm diazepam, 200 nm zolpidem, 1 microm flumazenil and 50 nm THDOC (3alpha, 21-dihydroxy-5alpha-pregnan-20-one), consistent with the following minimal subunit composition of GABARex I alpha1betagamma2, GABARex II alpha4betagamma2 and GABARex III alphabetadelta channels.

Role of the GABA transporter in epilepsy.

The GABA transporter plays a well-established role in reuptake of GABA after synaptic release. The anticonvulsant effect of tiagabine appears to result largely from blocking this reuptake. However, there is another side to the GABA transporter, contributing to GABA release by reversing in response to depolarization. We have recently shown that this form of GABA release is induced by even small increases in extracellular [K+], and has a powerful inhibitory effect on surrounding neurons. This transporter-mediated GABA release is enhanced by the anticonvulsants gabapentin and vigabatrin. The latter drug also potently increases ambient [GABA], inducing tonic inhibition of neurons. Here we review the evidence in support of a physiological role for GABA transporter reversal, and the evidence that it is increased by high-frequency firing. We postulate that the GABA transporter is a major determinant of the level of tonic inhibition, and an important source of GABA release during seizures. These recent findings indicate that the GABA transporter plays a much more dynamic role in control of brain excitability than has previously been recognized. Further defining this role may lead to a better understanding of the mechanisms of epilepsy and new avenues for treatment.

α1 and α6 subunits specify distinct desensitization, deactivation and neurosteroid modulation of GABA A receptors containing the δ subunit

GABA A receptor α subunit subtypes have distinct CNS distributions and confer different pharmacological and biophysical properties to αβγ receptor isoforms. However, the α subtype-dependent properties of αβδ receptor isoforms that may be targeted to extrasynaptic sites remain poorly understood. We investigated the properties of α1β3δ and α6β3δ receptor currents evoked by concentration jumps using a saturating GABA concentration (1 mM). α1β3δ receptor currents desensitized slowly, deactivated rapidly and displayed voltage-dependence only of peak amplitude. In contrast, α6β3δ receptor currents had voltage-dependent increased desensitization and slower deactivation, but did not show rectification. The neurosteroid THDOC (1 μM) enhanced α1β3δ more than α6β3δ currents, but increased the extent of desensitization and prolonged deactivation for both receptor isoforms. α1–α6 and α6–α1 chimeras (spliced in transmembrane domain 1) suggested that differences in deactivation rate and its voltage-dependence correlated with N-terminal domains, while the extent of desensitization and its voltage-dependence correlated with C-terminal domains. Both chimeras showed outward rectification. α1 subunit-like THDOC enhancement was observed with the α1–α6 chimera, but the α6–α1 chimera did not confer α6 subunit-like enhancement, suggesting that multiple α1 subunit domains contributed to neurosteroid efficacy. Thus, α subunit subtypes may regulate the kinetic and pharmacological properties of tonic neuronal inhibition.

Tonic neuronal inhibition by AII revealed by iontophoretic application of losartan, a specific antagonist of AII type-1 receptors

Short-term low-dose mineralocorticoid pretreatment enhances subsequent neuronal activity in the medial septum/preoptic region and in the stria terminalis/posterior amygdala of urethane anaesthetised male Wistar rats and sensitises these neurons to angiotensin II (AII). We have investigated the effect of iontophoretic application of Losartan, a specific nonpeptidergic AII type-1 receptor antagonist, on the background activity of spontaneously active neurons in these regions using a seven-barrelled microiontophoretic electrode sealed to a recording electrode. The influence of Losartan on the effects of iontophoretically applied AH in deoxycorticosterone acetate (DOCA) pretreated and nonpretreated rats was also investigated. Iontophoretically applied Losartan was observed to block the excitatory effect of AII in some neurons. In other spontaneously active neurons Losartan was seen to stimulate (or inhibit) immediately, this effect being greater in nonpretreated than in DOCA pretreated rats. Losartan was also observed to provoke persistent excitation of some spontaneously active neurons only in the nonpretreated rats. These results suggest that there exists a tonic inhibition by AII on the neurons in this area of the forebrain and that there may exist at least two subtypes of the AII type-1 receptor.

Halothane enhances tonic neuronal inhibition of elevating intracellular calcium

Whether the major action of anesthetics is to depress the central nervous system (CNS) by reducing excitation or enhancing inhibition remains unknown. Using whole cell patch-clamp recording in hippocampal slices, halothane and pentobarbital were found to prolong the decay time constant (TAU D) of GABA A-mediated spontaneous inhibitory postsynaptic (sIPSCs). Intracellular administration of the Ca 2+ chelator BAPTA or the Ca 2+ release inhibitor dantrolene significantly (ANOVA, P < 0.005) reduced halothane's effect; in contrast, the pentobarbital effect was unchanged. Halothane induced depression of population spike amplitude was blocked by the GABA A antagonist bicuculline. Together, these findings suggest that a major depressant effect of halothane involves enhancement of GABA A-mediated inhibition through release of intraneuronally stored Ca 2+.

Epithelial and mucosal preparations of canine proximal colon in ussing chambers: Comparison of responses

A novel epithelial preparation from proximal canine colon, devoid of its underlying muscularis mucosa, was studied in Ussing chambers and its responses to added agonists compared with those of a conventional full mucosal preparation. We assumed that the responses elicited from the former would reflect direct action of agonists on epithelial cells, and from the latter both direct and indirect effects mediated by functioning nerves in the attached submucosal plexus. Responses of the two preparations to tetrodotoxin, ouabain, scorpion venom, and field stimulation were different, the differences being explicable on the basis of functioning nerves in the mucosal preparation. Histamine stimulated both preparations; the increase in short-circuit current seen with the mucosal but not the epithelial preparation was inhibited by tetrodotoxin. Serotonin was a better stimulant of the epithelial preparation. Basal absorptions of both Na + and Cl − were seen in both mucosa and epithelium, but net absorptions were significantly greater in the latter, suggesting tonic neuronal inhibition of absorption in the mucosa. These two preparations would prove useful in analysing direct and indirect effects of agonists on colonic transport.