GABA Currents Research Articles

Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition.

GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5-10 mM by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca(2+)-induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial-specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain.

Inhibitory effects of insulin on GABAA currents modulated by the GABAA alpha subunit

Insulin, when co-applied with GABA, can cause an inhibition of the induced current at GABAA receptors. This study investigated that inhibitory effect of insulin at a variety of receptor isoforms, concentrating on α1, α2 and α4 containing receptors. Various isoforms were expressed in Xenopus oocytes and currents determined using two-electrode voltage clamp. Submaximal GABA currents at all isoforms studied were inhibited by nanomolar concentrations of insulin. At α2 and α4 containing forms, insulin could inhibit maximal GABA currents. The ability to inhibit maximal currents, and the general potency and effects at submaximal currents paralleled the number of potential MAPK sites on the α subunits. The differences in insulin inhibition of GABA currents at different α containing GABAA receptors could be important in autocrine and paracrine control of hormone secretion in the pancreas, and in control of reward and food intake circuits of the brain.

Central mechanisms of conditioned place preference in rats

The purpose of the investigation was to clear the significance of dopamine, GABA, opioids and sodium influx ionic currents of the nucleus accumbens neurons for the reinforcing effects of a number of psychotropic drugs (opiates, opioids, psychostimulants) on conditioned place preference (CPP) in rats. The microcannules were implanted into the nucleus accumbens (the extended amygdala system) of the Wistar male rats to inject the drugs studied (1 μg in 1 μl in volume for each injection). The rats were learned CPP of a one of narcogenics during 8 days. Some drugs, lidocain, a blocker of sodium influx ionic currents, antagonists of GABAA receptors bicuculline, D1 dopamine receptors SCH23390, D2 dopamine receptors sulpiride and opioid receptors naloxone, administered intrastructurally into the nucleus accumbens, were used for pharmacological analysis. The majority of the blockers studied decreased or abolished the reinforcing effects of amphetamine. Activation of reinforcement by means of fentanyl was reversed with bicuculline, lidocain and naloxone but did not change with dopamine antagonists (SCH23390 and sulpiride). None of the blockers studied effect on CPP of sodium ethaminal excluding bicuculline which reduced it. At last, the leu-enkephaline effects were reversed with naloxone and SCH23390, but strengthened with bicuculline. Sulpiride and lidocain did not effect on CPP of leu-enkephaline. Therefore, the different mechanisms (GABA-, dopamine- and opioidergic) controlling the positive conditioned reinforcement are collected in the nucleus accumbens.

Effects of the volatile anesthetic sevoflurane on tonic GABA currents in the mouse striatum during postnatal development.

The volatile anesthetic sevoflurane, which is widely used in pediatric surgery, has proposed effects on GABAA receptor-mediated extrasynaptic tonic inhibition. In the developing striatum, medium-sized spiny projection neurons have tonic GABA currents, which function in the excitatory/inhibitory balance and maturation of striatal neural circuits. In this study, we examined the effects of sevoflurane on the tonic GABA currents of medium spiny neurons in developing striatal slices. Sevoflurane strongly increased GABAA receptor-mediated tonic conductance at postnatal days 3-35. The antagonist of the GABA transporter-1, 1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride further increased tonic GABA conductance during the application of sevoflurane, thereby increasing the total magnitude of tonic currents. Both GABA (5 μM) and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride, the δ-subunit-containing GABAA receptor agonist, induced tonic GABA currents in medium spiny neurons but not in cholinergic neurons. However, sevoflurane additively potentiated the tonic GABA currents in both cells. Interestingly, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol hydrochloride-sensitive neurons made a large current response to sevoflurane, indicating the contribution of the δ-subunit on sevoflurane-enhanced tonic GABA currents. Our findings suggest that sevoflurane can affect the tone of tonic GABA inhibition in a developing striatal neural network.

Effects of short-term synaptic plasticity mechanisms on the dynamics of the network conductances

In this work, we analyzed the effects that different levels of short-term synaptic facilitation and depression cause on the dynamics of the conductances in a network of excitatory and inhibitory neurons. For this purpose, we added the short-term plasticity mechanisms for depression and facilitation [2] to the biophysical network model described in [1]. This model is made up of a population of excitatory and inhibitory multi-compartment neurons containing different membrane channels modeled according to the Hodgkin-Huxley formalism. Neurons have been spatially arranged on a line to emulate the connectivity rule experimentally observed in visual cortex. The synaptic transmission has been mediated by excitatory AMPA and NMDA, and inhibitory GABA currents. Depending on the level of depression, the dynamics of the network presents two different behaviors: (i) a regime of up and down states; (ii) tonic activity [3]. By plotting the firing rate of excitatory versus inhibitory neurons, our results show that, without depression, the model seems to present a periodic orbit closed to one saddle node and one attractor. The possible saddle node, which corresponds to the (0,0) point is reached and maintained for a couple of seconds by the spontaneous dynamics causing down states. On the contrary, with respect to the possible attractor, by increasing the level of depression, the trajectory turns around it inducing oscillations in the firing rate scenario. For these levels, the trajectory reaches the saddle causing the tonic firing. We defined as critical value the minimum level of depression that switch the network behavior. The critical value of the depression plays an important role in the changes of the conductances. As the depression level decreases we observe that the excitatory and inhibitory conductances exponentially increase (the goodness of fit presents an R2 coefficient greater than 0.8) for all levels of depression which are not close to the critical value. On the other hand, the network shows a very different dynamics under the presence of short-term facilitation. In this case, we observed that for a low probability of release the network is active only for few seconds with a low firing rate. By increasing the level of facilitation of the network, we increased the probability to achieve up states with higher firing rate. These two different behaviors could be explained by observing that for low values of facilitation the AMPA conductances are almost zero. No claims can be inferred in the conductance values by changing the facilitation level since the data cannot be fitted by any kind of curve. Finally, both facilitation and depression were treated together to see the joint effects on the network dynamics.

GABAB receptors in maintenance of neocortical circuit function

Activation of metabotropic GABAB receptors (GABABRs) enhances tonic GABA current and substantially increases the frequency of spontaneous seizures. Despite the and pro-epileptic consequences of GABABR activation, mice lacking functional GABAB receptors (GABAB1R KO mice) exhibit clonic and rare absence seizures. To examine these mutant mice further, we recorded excitatory and inhibitory synaptic inputs and tonic mutant GABA currents from Layer 2 neocortical pyramidal neurons of GABAB1R WT and KO mice (P30–40). Tonic current was increased while the frequency of synaptic inputs was unchanged in KO mice relative to WT littermates. The neocortical laminar distribution of interneuron subtypes derived from the medial ganglionic eminence (MGE) was also not statistically different in KO mice relative to WT while the number of calretinin-positive, caudal GE-derived cells in Layer 1 was reduced. Transplantation of MGE progenitors obtained from KO mice lacking functional GABAB1R did not increase tonic inhibition in the host brain above that of media-injected controls. Taken together, these results suggest a complex role for GABAB receptors in mediating neocortical circuit function.

Mutation of a single threonine in the cytoplasmic NH2 terminus disrupts trafficking of renal betaine-GABA transporter 1 during hypertonic stress.

Betaine is an important osmolyte and is, compared with other organs, much more abundant in the kidneys, where it enters cells in the medulla by betaine-GABA transporter 1 (BGT1) to balance osmoregulation in the countercurrent system. In wild-type (wt-)BGT1-expressing oocytes, GABA-mediated currents were diminished by preincubation of oocytes with 100 nM PMA or 5 μM dioctanoyl-sn-glycerol, activators of PKC, whereas the application of staurosporine before the application of dioctanoyl-sn-glycerol restored the response to GABA. Four potential phosphorylation sites on BGT1 were mutated to alanine by site-directed mutagenesis. Three mutants (T235A, S428A, and S564A) evoked GABA currents comparable in magnitude to currents observed in wt-BGT1-expressing oocytes, whereas GABA currents in T40A were barely detectable. Uptake of [(3)H]GABA was also determined in human embryonic kidney-293 cells expressing enhanced green fluorescent protein (EGFP)-tagged BGT1 with the same mutations. T235A, S428A, and S564A showed upregulation of GABA uptake after hypertonic stress and downregulation by PMA similar to EGFP-wt-BGT1. In contrast, T40A did not respond to either hypertonicity or PMA. Confocal microscopy of the EGFP-BGT1 mutants expressed in Madin-Darby canine kidney cells revealed that T40A was present in the cytoplasm after 24 h of hypertonic stress. whereas the other mutants and EGFP-wt-BGT1 were in the plasma membrane. All mutants, including T40A, comigrated with wt-BGT1 on Western blots, suggesting that they are full-length proteins. T40A, however, cannot be phosphorylated, as revealed using a specific anti-phosphoantibody, and, therefore, T40 may be important for the trafficking and insertion of BGT1 in the plasma membrane.

Interview with Cristina Roseti, 2013 Morris-Coole/Epilepsia prize winner (interview by Gary Mathern and Astrid Nehlig).

The Morris-Coole Trust and the International League Against Epilepsy (ILAE) congratulate Cristina Roseti as the winner of the 2013 Morris-Coole/Epilepsia prize for the best study published in Epilepsia by a new investigator. Papers published in the 2013 calendar year are nominated by Associate Editors and Editorial Board members of Epilepsia, and then selected by the editors in conjunction with the ILAE president, Emilio Perucca. We wanted to know more about Cristina, her research, and the impact of this prize in this interview. We hope other prospective new investigators in epilepsy research can be recognized through this prize. I got my degree in Pharmacy in 2005 at University of Padua; then I moved to Rome where I received my PhD in Neurophysiology from University of Rome “Sapienza” in 2010, working on the role of adenosine receptor antagonists in the modulation of GABAA receptors in human tissues from patients with temporal lobe epilepsy. Now, I am working as a post-doc in the group directed by Prof. Eleonora Palma, at Department of Physiology and Pharmacology of University of Rome “Sapienza,” where I am using mainly electrophysiologic techniques. My interest in epilepsy research arose for two simple reasons: (1) about 1% of population in the world is affected by this disease and even in the cases where symptoms are well controlled by drugs, the quality of the patient's life is extremely impaired; and (2) besides up to date, there are many papers studying animal models of epilepsy but very few on epileptic patients. Now my research interests focus on understanding the role of GABAA receptors in human epilepsies and on identifying possible approaches to ameliorate the dysfunction of the inhibitory transmission in drug-resistant temporal lobe epilepsy patients. Temporal lobe epilepsy (TLE) is the most common form of focal epilepsy in adults, characterized by recurrent seizures, neuronal loss, and microglial activation. Recently, it has been shown that inflammatory mechanisms have a role in the pathogenesis of epilepsy and that seizure activity rapidly increases the synthesis of inflammatory mediators involved in the initiation and propagation of neuronal hyperexcitability. The chemokine fractalkine (CX3CL1) and its receptor CX3CR1 are widely expressed in the central nervous system. It is reported that fractalkine modulates glutamatergic currents, microglial neurotoxicity, and neuron survival. We have previously shown that in patients with drug-resistant epilepsy the inhibitory GABAergic transmission is strongly defective, increasing the bad effects of the disease and rendering not easy the pharmacologic approach. Our goal in this work was to investigate whether CX3CL1 can have a modulatory effect on GABAergic transmission. We investigated the role of CX3CL1 through electrophysiologic recordings performed in Xenopus oocytes microtransplanted with membranes isolated from brain tissues of epileptic patients. In addition we performed electrophysiologic recordings (patch-clamp) on the pyramidal neurons in epileptic human slices. In collaboration with Dr. Eleonora Aronica, immunohistochemical staining and double-labeling studies were carried out on the same brain tissues to analyze if CX3CR1 expression was altered. We found for the first time that CX3CL1 modulates GABA currents in human epileptic brain tissue. In particular, CX3CL1 reduces the loss of function of GABAA receptors affecting GABA current amplitude both in oocytes and native human neurons. Of interest, CX3CL1 does not influence GABA currents in oocytes injected with nonepileptic tissues. Consistent with a specific effect of CX3CL1 on tissues from patients with TLE, CX3CR1 immunoreactivity is higher in TLE sclerotic hippocampi than in control tissues, with a prominent expression in activated microglial cells. These findings suggest that CX3CR1 increase may be part of the inflammatory process present in epileptic hippocampus playing a role in epileptogenesis. Although we cannot demonstrate from our data that the increase of CX3CR1 precedes or follows the onset of epilepsy, our results on both epileptic hippocampus and cortex would suggest a potential neuroprotective role for the chemokine CX3CL1 in TLE. I would like to continue in the study of the modulation of neurotransmission in epilepsy by chemokines and cytokines and generally on the role of inflammation in the disease. Most important, I would like to continue my studies on human tissues because my motivation is increased by the fact that this can imply the real application of scientific results to the patients, and this makes my work even more fascinating. I feel honored to receive this prestigious award so important for me and for my laboratory. That is a big satisfaction for the job and research done until now and it is a strong encouragement to go eagerly on with commitment. I would like to thank all members of the team; only their precious contribution allowed me to reach this important result. This is the first prize I received for my job and I'm particularly proud and thrilled, and I hope this will push further my passion and career!

Development of dendritic tonic GABAergic inhibition regulates excitability and plasticity in CA1 pyramidal neurons.

Synaptic plasticity rules change during development: while hippocampal synapses can be potentiated by a single action potential pairing protocol in young neurons, mature neurons require burst firing to induce synaptic potentiation. An essential component for spike timing-dependent plasticity is the backpropagating action potential (BAP). BAP along the dendrites can be modulated by morphology and ion channel composition, both of which change during late postnatal development. However, it is unclear whether these dendritic changes can explain the developmental changes in synaptic plasticity induction rules. Here, we show that tonic GABAergic inhibition regulates dendritic action potential backpropagation in adolescent, but not preadolescent, CA1 pyramidal neurons. These developmental changes in tonic inhibition also altered the induction threshold for spike timing-dependent plasticity in adolescent neurons. This GABAergic regulatory effect on backpropagation is restricted to distal regions of apical dendrites (>200 μm) and mediated by α5-containing GABA(A) receptors. Direct dendritic recordings demonstrate α5-mediated tonic GABA(A) currents in adolescent neurons which can modulate BAPs. These developmental modulations in dendritic excitability could not be explained by concurrent changes in dendritic morphology. To explain our data, model simulations propose a distally increasing or localized distal expression of dendritic α5 tonic inhibition in mature neurons. Overall, our results demonstrate that dendritic integration and plasticity in more mature dendrites are significantly altered by tonic α5 inhibition in a dendritic region-specific and developmentally regulated manner.

Comparing the high affinity benzodiazepine binding site with the homologous “CGS 9895” site in GABA‐A receptors (1059.1)

GABA‐A receptors are the major inhibitory transmitter receptors in the brain and the site of action of many clinically important drugs. CGS 9895 and many other pyrazoloquinolinones are high affinity null modulators at the benzodiazepine (Bz) binding site and at higher concentrations also modulate the receptors via the α+β‐ interface. To gain insights into the interactions of CGS 9895 with both binding sites, we used our homology models to select γ2‐ side amino acid residues important for Bz binding and to incorporate them into the β3‐ side by site directed mutagenesis. These mutated β3 subunits were co‐expressed with α subunits. The recombinant receptors were investigated by the two electrode voltage clamp method in Xenopus laevis oocytes and by radioligand binding assays. With only four mutations we were able to introduce a high affinity binding site for CGS 9895 into αβ3 receptors. Furthermore, GABA currents of several recombinant αβ3(mut) receptors could be modulated by the tested pyrazoloquinolinones and by other ligands of the high affinity Bz binding site, but not by diazepam. We thus introduced a subsite of the high affinity Bz binding site into the α+β3‐ interface.Grant Funding Source: Supported by FWF W1232 Molecular Drug Targets

Carbon monoxide excitatory effect in vasopressin neurons of heart failure rats involves nitric oxide/GABA signaling (686.24)

We showed that the gas neurotransmitter carbon monoxide (CO) regulates vasopressin (VP) neuronal activity. Neurohumoral activation in the hypothalamic paraventricular nucleus (PVN), characterized by elevated VP secretion, occurs during congestive heart failure (HF). Moreover, blunted nitric oxide (NO)/GABA inhibitory signaling pathways within PVN are major contributing factors. In this study we determined whether PVN expression of heme‐oxygenase‐1 (HO‐1), a CO‐synthesizing enzyme, was changed in HF; whether CO effects on the firing rate (FR) of VP neurons was altered, and whether CO effects were mediated by NO/GABA function. Immunofluorescence and western blots studies showed an increased PVN HO‐1 protein levels during HF (P<0.05). Additionally, patch‐clamp recordings obtained from identified VP neurons in transgenic eGFP‐VP rats, showed that tin‐protoporphyrin IX (20µM, a HO inhibitor) diminished FR in 75% VP responsive cells in HF rats, compared with no effects in 62% VP cells in sham rats. Besides, tricarbonyldichlororuthenium dimer (30µM, a CO donor) increased FR in 71% and 75% VP responsive neurons in HF and sham rats, respectively. Interesting, the majority of VP neurons in HF rats were non responsive to the CO donor after previous application of a NO blocker (62%) or a GABA blocker (80%). In addition, the excitatory CO effect was accompanied by a reduction of GABA currents in 93% of VP neurons in HF rats. Taken together, our results showed overexpression of PVN HO‐1 and excitatory CO responses on VP neurons in HF rats, likely involving changes in NO/GABA‐mediated signaling.Grant Funding Source: AHA 12POST12060540

Alcohol Selectivity of β3-Containing GABAA Receptors: Evidence for a Unique Extracellular Alcohol/Imidazobenzodiazepine Ro15-4513 Binding Site at the α+β- Subunit Interface in αβ3δ GABAA Receptors

GABAA receptors (GABARs) have long been the focus for acute alcohol actions with evidence for behaviorally relevant low millimolar alcohol actions on tonic GABA currents and extrasynaptic α4/6, δ, and β3 subunit-containing GABARs. Using recombinant expression in oocytes combined with two electrode voltage clamp, we show with chimeric β2/β3 subunits that differences in alcohol sensitivity among β subunits are determined by the extracellular N-terminal part of the protein. Furthermore, by using point mutations, we show that the β3 alcohol selectivity is determined by a single amino acid residue in the N-terminus that differs between GABAR β subunits (β3Y66, β2A66, β1S66). The β3Y66 residue is located in a region called "loop D" which in γ subunits contributes to the imidazobenzodiazepine (iBZ) binding site at the classical α+γ2- subunit interface. In structural homology models β3Y66 is the equivalent of γ2T81 which is one of three critical residues lining the benzodiazepine binding site in the γ2 subunit loop D, opposite to the "100H/R-site" benzodiazepine binding residue in GABAR α subunits. We have shown that the α6R100Q mutation at this site leads to increased alcohol-induced motor in-coordination in alcohol non-tolerant rats carrying the α6R100Q mutated allele. Based on the identification of these two amino acid residues α6R100 and β66 we propose a model in which β3 and δ containing GABA receptors contain a unique ethanol site at the α4/6+β3- subunit interface. This site is homologous to the classical benzodiazepine binding site and we propose that it not only binds ethanol at relevant concentrations (EC50-17mM), but also has high affinity for a few selected benzodiazepine site ligands including alcohol antagonistic iBZs (Ro15-4513, RY023, RY024, RY80) which have in common a large moiety at the C7 position of the benzodiazepine ring. We suggest that large moieties at the C7-BZ ring compete with alcohol for its binding pocket at a α4/6+β3- EtOH/Ro15-4513 site. This model reconciles many years of alcohol research on GABARs and provides a plausible explanation for the competitive relationship between ethanol and iBZ alcohol antagonists in which bulky moieties at the C7 position compete with ethanol for its binding site. We conclude with a critical discussion to suggest that much of the controversy surrounding this issue might be due to fundamental species differences in alcohol and alcohol antagonist responses in rats and mice.

Allosteric modulation of GABAA receptors by extracellular ATP

BackgroundThe γ-aminobutyric acid type A receptor (GABAAR) is the primary receptor mediating fast synaptic inhibition in the brain and plays a critical role in modulation of neuronal excitability and neural networks. Previous studies have demonstrated that ATP and its nucleotide analogs may regulate the function of GABAARs via Ca2+-dependent intracellular mechanisms, which require activation of purinergic 2 (P2) receptors or cross-talk between two receptors.ResultsHere, we report a potentiation of GABAARs by extracellular ATP via a previously un-recognized allosteric mechanism. Using cultured hippocampal neurons as well as HEK293 cells transiently expressing GABAARs, we demonstrate that extracellular ATP potentiates GABAAR mediated currents in a dose-dependent manner with an EC50 of 2.1 ± 0.2 mM. The potentiation was mediated by a postsynaptic mechanism that was not dependent on activation of either ecto-protein kinase or P2 receptors. Single channel recordings from cell-free excised membrane patches under outside-out mode or isolated membrane patches under cell-attached mode suggest that the ATP modulation of GABA currents is achieved through a direct action of ATP on the channels themselves and manifested by increasing the single channel open probability without alteration of its conductance. Moreover, this ATP potentiation of GABAAR could be reconstituted in HEK293 cells that transiently expressed recombinant rat GABAARs.ConclusionsOur data strongly suggest that extracellular ATP allosterically potentiates GABAAR-gated chloride channels. This novel mode of ATP-mediated modulation of GABAARs may play an important role in regulating neuronal excitability and thereby in fine-tuning the excitation-inhibition balance under conditions where a high level of extracellular ATP is ensured.

Allosteric Modulation of GABA-A Receptors by Different Drugs

Understanding how drugs allosterically modulate GABA-A receptor (GABAAR) channel function remains a challenge. Anesthetic drugs like etomidate, propofol and pentobarbital bind at transmembrane domain (TMD) inter-subunit interfaces at different locations. Benzodiazepines bind at the αγ interface in the extracellular domain (ECD) and neurosteroids are believed to bind within the α-subunit TMD helix bundle. A common feature of these drugs is that they potentiate GABA-induced currents. Here, we examined whether structural mechanisms underlying their functional effects are distinct. We monitored motions induced individually by these drugs at each inter-subunit TMD interface and also tested whether modulation caused by pairs of drugs binding to distinct sites are additive or super-additive. We individually introduced a cysteine in the M1 helices of each subunit at α1I227, β2L223 and γ2LI238 to probe the different inter-subunit interfaces. We expressed wild-type and mutant α1β2γ2L GABAARs in Xenopus oocytes and measured rates of modification of the substituted cysteines in the absence and presence of GABA and different allosteric modulators: the anesthetics pentobarbital (PB) and etomidate, the neurosteroid THDOC and the benzodiazepine flurazepam. GABA activation significantly increased MTSEA modification of αI227C, βL223C and γI238C. PB activation increased modification of αI227C and γI238C but not βL223C. Modulating concentrations of PB or etomidate had no effects. THDOC significantly increased the rate of modification of βL223C whereas flurazepam increased γI238C modification. We found that potentiation of EC10 GABA currents by combining modulating concentrations of THDOC and flurazepam was the sum of the potentiation produced by the two drugs individually. Combining PB and THDOC produced potentiation more than the sum of the potentiation produced by the two drugs individually. Our data suggests that drugs binding to different sites act via distinct mechanisms. Further analysis is underway to determine if these mechanisms are independent or coupled.

Postnatal maternal separation enhances tonic GABA current of cortical layer 5 pyramidal neurons in juvenile rats and promotes genesis of GABAergic neurons in neocortical molecular layer and subventricular zone in adult rats

Postnatal maternal separation (PMS) has been shown to be associated with an increased vulnerability to psychiatric illnesses in adulthood. However, the underlying neurological mechanisms are not well understood. Here we evaluated its effects on neurogenesis and tonic GABA currents of cortical layer 5 (L5) pyramidal neurons. PMS not only increased cell proliferation in the subventricular zone, cortical layer 1 and hippocampal dentate gyrus in the adult brain, but also promoted the newly generated cells to differentiate into GABAergic neurons, and PMS adult brain maintained higher ratios of GABAergic neurons in the survival of newly generated cells within 5 days immediately post PMS. Additionally, PMS increased the tonic currents at P7–10 and P30–35 in cortical L5 pyramidal cells. Our results suggest that the newly generated GABAergic neurons and the low GABA concentration-activated tonic currents may be involved in the development of psychiatric disorders after PMS.

Seizure-induced alterations in fast-spiking basket cell GABA currents modulate frequency and coherence of gamma oscillation in network simulations

Gamma frequency oscillations have been proposed to contribute to memory formation and retrieval. Fast-spiking basket cells (FS-BCs) are known to underlie development of gamma oscillations. Fast, high amplitude GABA synapses and gap junctions have been suggested to contribute to gamma oscillations in FS-BC networks. Recently, we identified that, apart from GABAergic synapses, FS-BCs in the hippocampal dentate gyrus have GABAergic currents mediated by extrasynaptic receptors. Our experimental studies demonstrated two specific changes in FS-BC GABA currents following experimental seizures [Yu et al., J. Neurophysiol. 109, 1746 (2013)]: increase in the magnitude of extrasynaptic (tonic) GABA currents and a depolarizing shift in GABA reversal potential (E(GABA)). Here, we use homogeneous networks of a biophysically based model of FS-BCs to examine how the presence of extrasynaptic GABA conductance (g(GABA-extra)) and experimentally identified, seizure-induced changes in g(GABA-extra) and E(GABA) influence network activity. Networks of FS-BCs interconnected by fast GABAergic synapses developed synchronous firing in the dentate gamma frequency range (40-100 Hz). Systematic investigation revealed that the biologically realistic range of 30 to 40 connections between FS-BCs resulted in greater coherence in the gamma frequency range when networks were activated by Poisson-distributed dendritic synaptic inputs rather than by homogeneous somatic current injections, which were balanced for FS-BC firing frequency in unconnected networks. Distance-dependent conduction delay enhanced coherence in networks with 30-40 FS-BC interconnections while inclusion of gap junctional conductance had a modest effect on coherence. In networks activated by somatic current injections resulting in heterogeneous FS-BC firing, increasing g(GABA-extra) reduced the frequency and coherence of FS-BC firing when E(GABA) was shunting (-74 mV), but failed to alter average FS-BC frequency when E(GABA) was depolarizing (-54 mV). When FS-BCs were activated by biologically based dendritic synaptic inputs, enhancing g(GABA-extra) reduced the frequency and coherence of FS-BC firing when E(GABA) was shunting and increased average FS-BC firing when E(GABA) was depolarizing. Shifting E(GABA) from shunting to depolarizing potentials consistently increased network frequency to and above high gamma frequencies (>80 Hz). Since gamma oscillations may contribute to learning and memory processing [Fell et al., Nat. Neurosci. 4, 1259 (2001); Jutras et al., J. Neurosci. 29, 12521 (2009); Wang, Physiol. Rev. 90, 1195 (2010)], our demonstration that network oscillations are modulated by extrasynaptic inhibition in FS-BCs suggests that neuroactive compounds that act on extrasynaptic GABA receptors could impact memory formation by modulating hippocampal gamma oscillations. The simulation results indicate that the depolarized FS-BC GABA reversal, observed after experimental seizures, together with enhanced spillover extrasynaptic GABA currents are likely to promote generation of focal high frequency activity associated with epileptic networks.

Ethanol Untangles the Amygdala‐Anxiety Circuit Through Tonic GABA Inhibition

The central amygdala (CeA) has a unique role in integrating stress and the rewarding effects of ethanol (EtOH) and plays a major role in the development of EtOH dependence via signaling of corticotropin-releasing factor (CRF). A recent report by Herman and colleagues (2013) entitled "Novel Subunit-Specific Tonic GABA Currents and Differential Effects of Ethanol in the Central Amygdala of CRF Receptor-1 Reporter Mice" is the first study to investigate inhibitory tonic currents in relation to CRF signaling in the CeA. The findings of that study significantly enhance our understanding of inhibitory tonic currents in the CeA and give insight into how EtOH may differentially affect CRF signaling within the CeA, leading to the development of EtOH dependence. This commentary will focus on the recent findings of Herman and colleagues and will discuss the effects of EtOH on the entire anxiety/emotion circuitry.

Aβ40 modulates GABAA receptor α6 subunit expression and rat cerebellar granule neuron maturation through the ERK/mTOR pathway

In addition to their neurotoxic role in Alzheimer's disease (AD), β-amyloid peptides (Aβs) are also known to play physiological roles. Here, we show that recombinant Aβ40 significantly increased the outward current of the GABA(A) receptor containing (GABA(A)α6) in rat cerebellar granule neurons (CGNs). The Aβ40-mediated increase in GABA(A)α6 current was mediated by an increase in GABA(A)α6 protein expression at the translational rather than the transcriptional level. The exposure of CGNs to Aβ40 markedly induced the phosphorylation of ERK (pERK) and mammalian target of rapamycin (pmTOR). The increase in GABA(A)α6 current and expression was attenuated by specific inhibitors of ERK or mTOR, suggesting that the ERK and mTOR signaling pathways are required for the effect of Aβ40 on GABA(A)α6 current and expression in CGNs. A pharmacological blockade of the p75 neurotrophin receptor (p75(NTR)), but not the insulin or α7-nAChR receptors, abrogated the effect of Aβ40 on GABA(A)α6 protein expression and current. Furthermore, the expression of GABA(A)α6 was lower in CGNs from APP(-/-) mice than in CGNs from wild-type mice. Moreover, the internal granule layer (IGL) in APP(-/-) mice was thinner than the IGL in wild-type mice. The injection of Aβ40 into the cerebellum reversed this effect, and the application of p75(NTR) blocking antibody abolished the effects of Aβ40 on cerebellum morphology in APP(-/-) mice. Our results suggest that low concentrations of Aβ40 play a role in regulating CGN maturation through p75(NTR).

Unexpected Properties of δ-Containing GABAA Receptors in Response to Ligands Interacting with the α+ β− Site

GABAA receptors are the major inhibitory neurotransmitter receptors in the central nervous system and are the targets of many clinically important drugs, which modulate GABA induced chloride flux by interacting with separate and distinct allosteric binding sites. Recently, we described an allosteric modulation occurring upon binding of pyrazoloquinolinones to a novel binding site at the extracellular α+ β- interface. Here, we investigated the effect of 4-(8-methoxy-3-oxo-3,5-dihydro-2H-pyrazolo[4,3-c]quinolin-2-yl)benzonitrile (the pyrazoloquinolinone LAU 177) at several αβ, αβγ and αβδ receptor subtypes. LAU 177 enhanced GABA-induced currents at all receptors investigated, and the extent of modulation depended on the type of α and β subunits present within the receptors. Whereas the presence of a γ2 subunit within αβγ2 receptors did not dramatically change LAU 177 induced modulation of GABA currents compared to αβ receptors, we observed an unexpected threefold increase in modulatory efficacy of this compound at α1β2,3δ receptors. Steric hindrance experiments as well as inhibition by the functional α+ β- site antagonist LAU 157 indicated that the effects of LAU 177 at all receptors investigated were mediated via the α+ β- interface. The stronger enhancement of GABA-induced currents by LAU 177 at α1β3δ receptors was not observed at α4,6β3δ receptors. Other experiments indicated that this enhancement of modulatory efficacy at α1β3δ receptors was not observed with another α+ β- modulator, and that the efficacy of modulation by α+ β- ligands is influenced by all subunits present in the receptor complex and by structural details of the respective ligand.

Multifunctional aspects of allopregnanolone in stress and related disorders

Allopregnanolone (3α-hydroxy-5α-pregnan-20-one) is a major cholesterol-derived neurosteroid in the central nervous system and is synthesized from progesterone by steroidogenic enzymes, 5α-reductase (the rate-limiting enzyme) and 3α-hydroxysteroid dehydrogenase. The pathophysiological role of allopregnanolone in neuropsychiatric disorders has been highlighted in several investigations. The changes in neuroactive steroid levels are detected in stress and stress-related disorders including anxiety, panic and depression. The changes in allopregnanolone in response to acute stressor tend to restore the homeostasis by dampening the hyper-activated HPA axis. However, long standing stressors leading to development of neuropsychiatric disorders including depression and anxiety are associated with decrease in the allopregnanolone levels. GABAA receptor complex has been considered as the primary target of allopregnanolone and majority of its inhibitory actions are mediated through GABA potentiation or direct activation of GABA currents. The role of progesterone receptors in producing the late actions of allopregnanolone particularly in lordosis facilitation has also been described. Moreover, recent studies have also described the involvement of other multiple targets including brain-derived neurotrophic factor (BDNF), glutamate, dopamine, opioids, oxytocin, and calcium channels. The present review discusses the various aspects of allopregnanolone in stress and stress-related disorders including anxiety, depression and panic.