GABAA Currents Research Articles

Layer- and cell-type-specific tonic GABAergic inhibition of pyramidal neurons in the rat visual cortex

Tonic inhibition mediated by persistent activation of γ-aminobutyric acidA (GABAA) receptors by ambient GABA plays a crucial role in the regulation of network excitability and neuronal signal processing. Varying degrees in the strength of tonic inhibition were detected across different cell types throughout the brain. Since sensory information flows through cortical layers in a specific order, the characteristics of tonic inhibition in different cortical layers are of interest. Therefore, we examined the properties of tonic inhibition in pyramidal neurons (PyNs) throughout the rat visual cortex. Layer 2/3 PyNs and burst-spiking PyNs in layers 5 and 6 showed prominent tonic GABAA currents. Tonic GABAA currents in layer 4 star PyNs and regular-spiking PyNs in layers 5 and 6 were much weaker. The magnitude of tonic currents correlated well with the inhibition of spike generation. The amplitude of tonic GABAA currents measured with bicuculline and gabazine, the two different GABAA receptor blockers, did not differ. The differences in the expression levels of extrasynaptic GABAA receptors might be the major contributor to the differences in tonic GABAA currents among cell types. Furthermore, α5 subunits might contribute significantly to tonic currents in infragranular burst-spiking PyNs, especially in layer 5. These results suggest that ambient GABA might exert differential effects on the neuronal integration in a layer- and cell-type-specific manner and thus contribute to the processing of sensory properties by selectively tuning the signals flowing through the visual cortex.

Postnatal maturation of GABAergic transmission in the rat basolateral amygdala.

Many psychiatric disorders, including anxiety and autism spectrum disorders, have early ages of onset and high incidence in juveniles. To better treat and prevent these disorders, it is important to first understand normal development of brain circuits that process emotion. Healthy and maladaptive emotional processing involve the basolateral amygdala (BLA), dysfunction of which has been implicated in numerous psychiatric disorders. Normal function of the adult BLA relies on a fine balance of glutamatergic excitation and GABAergic inhibition. Elsewhere in the brain GABAergic transmission changes throughout development, but little is known about the maturation of GABAergic transmission in the BLA. Here we used whole cell patch-clamp recording and single-cell RT-PCR to study GABAergic transmission in rat BLA principal neurons at postnatal day (P)7, P14, P21, P28, and P35. GABAA currents exhibited a significant twofold reduction in rise time and nearly 25% reduction in decay time constant between P7 and P28. This corresponded with a shift in expression of GABAA receptor subunit mRNA from the α2- to the α1-subunit. The reversal potential for GABAA receptors transitioned from depolarizing to hyperpolarizing with age, from around -55 mV at P7 to -70 mV by P21. There was a corresponding shift in expression of opposing chloride pumps that influence the reversal, from NKCC1 to KCC2. Finally, we observed short-term depression of GABAA postsynaptic currents in immature neurons that was significantly and gradually abolished by P28. These findings reveal that in the developing BLA GABAergic transmission is highly dynamic, reaching maturity at the end of the first postnatal month.

R‐ and S‐ mTFD‐MPPB, an Anesthetic and a Convulsant, Have Opposing Effect on γ‐Aminobutyric acid (GABA) type A Receptors

The R‐enantiomer of 1‐methyl‐5‐phenyl‐5‐propyl barbituric acid (MPPB) is an anesthetic whereas the S is a convulsant. We synthesized its trifluoromethyl derivative (mTFD‐MPPB), a novel photoactivable barbiturate. R‐ and S‐mTFD‐MPPB caused loss of righting reflex in tadpoles (EC50 28 μM & 40 μM respectively) but the S‐enantiomer caused excitability. Next we used whole‐cell patch clamp electrophysiology with fast solution exchange (<1 millisecond) on recombinant human α1β3γ2 GABAA receptors expressed in HEK cells and found both enantiomers acted on GABAA currents more potently than MPPB. Currents elicited by low GABA concentration (10 μM) were increased 5‐fold by RmTFD‐MPPB (46μM), but decreased by ~70% by S‐mTFD‐MPPB (46μM). Enhancement was never observed with S‐mTFD‐MPPB at any concentration tested. At high GABA concentration (10 mM), both mTFD‐MPPB enantiomers (46μM) inhibited currents by ~30%. The enantiomers’ actions at low GABA are more consistent with their pharmacology, suggesting that the physiological locus of action is a GABAAR that functions at low GABA occupancy or where GABA is a partial agonist. This might include extrasynaptic receptors such as those containing δ‐subunits. Supported by GM58448.

Developmental Regulation and Activity-Dependent Maintenance of GABAergic Presynaptic Inhibition onto Rod Bipolar Cell Axonal Terminals

Presynaptic inhibition onto axons regulates neuronal output, but how such inhibitory synapses develop and are maintained invivo remains unclear. Axon terminals of glutamatergic retinal rod bipolar cells (RBCs) receive GABAA and GABAC receptor-mediated synaptic inhibition. We found that perturbing GABAergic or glutamatergic neurotransmission does not prevent GABAergic synaptogenesis onto RBC axons. But, GABA release is necessary for maintaining axonal GABA receptors. This activity-dependent process is receptor subtype specific: GABAC receptors are maintained, whereas GABAA receptors containing α1, but not α3, subunits decrease over time in mice with deficient GABA synthesis. GABAA receptor distribution on RBC axons is unaffected in GABAC receptor knockout mice. Thus, GABAA and GABAC receptor maintenance are regulated separately. Although immature RBCs elevate their glutamate release when GABA synthesis is impaired, homeostatic mechanisms ensure that the RBC output operates within its normal range after eye opening, perhaps to regain proper visual processing within the scotopic pathway.

Coupling Between GABA-A Receptor and Chloride Transporter Underlies Ionic Plasticity in Cerebellar Purkinje Neurons

Ionic plasticity, a form of synaptic plasticity unique to inhibitory neurotransmission, can be induced in cerebellar Purkinje neurons by brain-derived neurotrophic factor (BDNF). It is expressed as a decrease in synaptic strength of GABA-A transmission onto Purkinje neurons due to reduced transmembrane chloride gradient. By making whole-cell recordings, we found that the effect of BDNF is mediated by neuronal potassium and chloride transporter KCC2 because it is blocked by inhibitors of KCC2 or by raising the intracellular chloride concentration. Under these conditions in which KCC2 activity is reduced, BDNF augments evoked GABA-A currents suggesting a direct facilitatory effect of BDNF on GABA-A receptor. We also found that the effect of BDNF is highly localized at the GABA-A synapse and is secured by physical coupling between GABA-A receptor and KCC2, as revealed by coimmunoprecipitation studies. Based on these results, we hypothesize that the interaction between KCC2 and specific subunit of GABA-A receptor represents a fundamental mechanism rendering the rapid induction of ionic plasticity in individual or input-specific GABA synapses possible. Such a mechanism may be important for the function of Purkinje neurons that are known to express GABA-A receptors with different subunit compositions.

Metabotropic regulation of extrasynaptic GABAA receptors

A large body of work now shows the importance of GABAA receptor-mediated tonic inhibition in regulating CNS function. However, outside of pathological conditions, there is relatively little evidence that the magnitude of tonic inhibition is itself under regulation. Here we review the mechanisms by which tonic inhibition is known to be modulated, and outline the potential behavioral consequences of this modulation. Specifically, we address the ability of protein kinase A and C to phosphorylate the extrasynaptic receptors responsible for the tonic GABAA current, and how G-protein coupled receptors can regulate tonic inhibition through these effectors. We then speculate about the possible functional consequences of regulating the magnitude of the tonic GABAA current.

Two GABAA responses with distinct kinetics in a sound localization circuit

The temporal characteristics and functional diversity of GABAergic inhibition are determined by the spatiotemporal neurotransmitter profile, intrinsic properties of GABAA receptors, and other factors. Here, we report two distinct GABAA responses and the underlying mechanisms in neurons of the chicken nucleus laminaris (NL), the first encoder of interaural time difference for sound localization in birds. The time course of the postsynaptic GABAA currents in NL neurons, recorded with whole-cell voltage clamp, differed between different characteristic frequency (CF) regions. Compared to low-CF (LF) neurons, middle/high-CF (MF/HF) neurons had significantly slower IPSCs, with a 2.6-fold difference in the decay time constants of spontaneous IPSCs and a 5.3-fold difference in the decay of IPSCs elicited by single-pulse stimulus. Such differences were especially dramatic when IPSCs were elicited by train stimulations at physiologically relevant frequencies, and at high stimulus intensities. To account for these distinct GABAA responses, we showed that MF/HF neurons exhibited more prominent asynchronous release of GABA. Supporting this observation, replacement of extracellular Ca2+ with Sr2+ increased the decay of IPSCs in LF neurons, and EGTA-AM reduced the decay of IPSCs in MF/HF neurons. Furthermore, pharmacological evidence suggests that GABA spillover plays a greater role in prolonging the IPSCs of MF/HF neurons. Consequently, under whole-cell current clamp, synaptically released GABA produced short- and long-lasting suppression of the neuronal excitability of LF and MF/HF neurons, respectively. Taken together, these results suggest that the GABAergic inputs to NL neurons may exert a dynamic modulation of interaural time difference (ITD) coding in a CF-dependent manner.

Selective modulation of different GABAA receptor isoforms by diazepam and etomidate in hippocampal neurons

Diazepam modulation of native γ2-containing GABAA (γGABAA) receptors increases channel conductance by facilitating protein interactions involving the γ2-subunit amphipathic (MA) region, which is found in the cytoplasmic loop between transmembrane domains 3 and 4 (Everitt et al., 2009). However, many drugs, predicted to act on different GABAA receptor subtypes, increase channel conductance leading us to hypothesize that conductance variation in GABAA receptors may be a general property, mediated by protein interactions involving the cytoplasmic MA stretch of amino acids. In this study we have tested this hypothesis by potentiating extrasynaptic GABAA currents with etomidate and examining the ability of peptides mimicking either the γ2- or δ-subunit MA region to affect conductance. In inside-out hippocampal patches from newborn rats the general anesthetic etomidate potentiated GABA currents, producing either an increase in open probability and single-channel conductance or an increase in open probability, as described previously (Seymour et al., 2009). In patches displaying high conductance channels application of a δ(392–422) MA peptide, but not a scrambled version or the equivalent γ2(381–403) MA peptide, reduced the potentiating effects of etomidate, significantly reducing single-channel conductance. In contrast, when GABA currents were potentiated by the γ2-specific drug diazepam the δ MA peptide had no effect. These data reveal that diazepam and etomidate potentiate different extrasynaptic GABAA receptor subtypes but both drugs modulate conductance similarly. One interpretation of the data is that these drugs elicit potentiation through protein interactions and that the MA peptides compete with these interactions to disrupt this process.

Inflammatory mediators potentiate high affinity GABAA currents in rat dorsal root ganglion neurons

Following acute tissue injury action potentials may be initiated in afferent processes terminating in the dorsal horn of the spinal cord that are propagated back out to the periphery, a process referred to as a dorsal root reflex (DRR). The DRR is dependent on the activation of GABAA receptors. The prevailing hypothesis is that DRR is due to a depolarizing shift in the chloride equilibrium potential (ECl) following an injury-induced activation of the Na+–K+–Cl−-cotransporter. Because inflammatory mediators (IM), such as prostaglandin E2 are also released in the spinal cord following tissue injury, as well as evidence that ECl is already depolarized in primary afferents, an alternative hypothesis is that an IM-induced increase in GABAA receptor mediated current (IGABA) could underlie the injury-induced increase in DRR. To test this hypothesis, we explored the impact of IM (prostaglandin E2 (1μM), bradykinin (10μM), and histamine (1μM)) on IGABA in dissociated rat dorsal root ganglion (DRG) neurons with standard whole cell patch clamp techniques. IM potentiated IGABA in a subpopulation of medium to large diameter capsaicin insensitive DRG neurons. This effect was dependent on the concentration of GABA, manifest only at low concentrations (<10μM). THIP evoked current were also potentiated by IM and GABA (1μM) induced tonic currents enhanced by IM were resistant to gabazine (20μM). The present data are consistent with the hypothesis that an acute increase in IGABA contributes to the emergence of injury-induced DRR.

Synaptic and Extrasynaptic Origin of the Excitation/Inhibition Imbalance in the Hippocampus of Synapsin I/II/III Knockout Mice

Synapsins (Syn I, Syn II, and Syn III) are a family of synaptic vesicle phosphoproteins regulating synaptic transmission and plasticity. SYN1/2 genes have been identified as major epilepsy susceptibility genes in humans and synapsin I/II/III triple knockout (TKO) mice are epileptic. However, excitatory and inhibitory synaptic transmission and short-term plasticity have never been analyzed in intact neuronal circuits of TKO mice. To clarify the generation and expression of the epileptic phenotype, we performed patch-clamp recordings in the CA1 region of acute hippocampal slices from 1-month-old presymptomatic and 6-month-old epileptic TKO mice and age-matched controls. We found a strong imbalance between basal glutamatergic and γ-aminobutyric acid (GABA)ergic transmission with increased evoked excitatory postsynaptic current and impaired evoked inhibitory postsynaptic current amplitude. This imbalance was accompanied by a parallel derangement of short-term plasticity paradigms, with enhanced facilitation of glutamatergic transmission in the presymptomatic phase and milder depression of inhibitory synapses in the symptomatic phase. Interestingly, a lower tonic GABA(A) current due to the impaired GABA release is responsible for the more depolarized resting potential found in TKO CA1 neurons, which makes them more susceptible to fire. All these changes preceded the appearance of epilepsy, indicating that the distinct changes in excitatory and inhibitory transmission due to the absence of Syns initiate the epileptogenic process.

Upregulation of high-affinity GABAA receptors in cultured rat dorsal root ganglion neurons

Despite evidence that high-affinity GABAA receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low-affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high-affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABAA currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 h (acute) and 24 h (cultured) after plating with whole-cell patch-clamp techniques, Western blot, and semiquantitative reverse transcriptase polymerase chain reaction (sqRT-PCR) analysis. GABAA current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50's of 0.25±0.01 μM and 3.2±0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16±10.1 μM to 9.56±1.3 μM with time in culture. A fraction of the high-affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute, but the membrane fraction in cultured, DRG neurons, with no detectable change in δ-subunit mRNA. However, the emergence of a high-affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2- and ρ3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit-containing GABAA receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution.

Gamma Oscillations Are Generated Locally in an Attention-Related Midbrain Network

Gamma-band (25-140 Hz) oscillations are a hallmark of sensory processing in the forebrain. The optic tectum (OT), a midbrain structure implicated in sensorimotor processing and attention, also exhibits gamma oscillations. However, the origin and mechanisms of these oscillations remain unknown. We discovered that in acute slices of the avian OT, persistent (>100 ms) epochs of large amplitude gamma oscillations can be evoked that closely resemble those recorded in vivo. We found that cholinergic, glutamatergic, and GABAergic mechanisms differentially regulate the structure of the oscillations at various timescales. These persistent oscillations originate in the multisensory layers of the OT and are broadcast to visual layers via the cholinergic nucleus Ipc, providing a potential mechanism for enhancing the processing of visual information within the OT. The finding that the midbrain contains an intrinsic gamma-generating circuit suggests that the OT could use its own oscillatory code to route signals to forebrain networks.

Cross talk between synaptic receptors mediates NMDA-induced suppression of inhibition

Past research has shown that calcium influx through NMDA receptors (NMDARs) depresses GABA(A) currents. We examined upstream triggers of this suppression, including involvement of target synaptic GABA(A) receptors and the NMDARs triggering suppression. In hippocampal neurons, conditioning with 20 μM NMDA for 20 s caused 50% suppression of GABA responses. The suppression was delayed by ≈ 60 s following NMDA application and persisted for at least 5 min following conditioning. Pharmacology experiments suggested a shift in both the sensitivity to GABA and a loss of functional receptors. NMDA conditioning strongly suppressed inhibitory postsynaptic currents and speeded decay kinetics. Synaptic NMDAR conditioning was necessary to suppress GABA current in pyramidal neurons; extrasynaptic NMDAR activation did not suppress, even when matched to synaptic activation. We found no evidence that specific synaptic NMDAR subunits mediate depression of GABA responses. Although physical colocalization of glutamate and GABA(A) receptors is mostly likely in extrasynaptic regions, our evidence suggests that NMDAR-induced suppression of GABA responsiveness prominently affects precise, moment-to-moment signaling from synaptic receptors to synaptic receptors.

Modulation of inhibitory strength and kinetics facilitates regulation of persistent inward currents and motoneuron excitability following spinal cord injury

Spasticity is commonly observed after chronic spinal cord injury (SCI) and many other central nervous system disorders (e.g., multiple sclerosis, stroke). SCI-induced spasticity has been associated with motoneuron hyperexcitability partly due to enhanced activation of intrinsic persistent inward currents (PICs). Disrupted spinal inhibitory mechanisms also have been implicated. Altered inhibition can result from complex changes in the strength, kinetics, and reversal potential (E(Cl(-))) of γ-aminobutyric acid A (GABA(A)) and glycine receptor currents. Development of optimal therapeutic strategies requires an understanding of the impact of these interacting factors on motoneuron excitability. We employed computational methods to study the effects of conductance, kinetics, and E(Cl(-)) of a dendritic inhibition on PIC activation and motoneuron discharge. A two-compartment motoneuron with enhanced PICs characteristic of SCI and receiving recurrent inhibition from Renshaw cells was utilized in these simulations. This dendritic inhibition regulated PIC onset and offset and exerted its strongest effects at motoneuron recruitment and in the secondary range of the current-frequency relationship during PIC activation. Increasing inhibitory conductance compensated for moderate depolarizing shifts in E(Cl(-)) by limiting PIC activation and self-sustained firing. Furthermore, GABA(A) currents exerted greater control on PIC activation than glycinergic currents, an effect attributable to their slower kinetics. These results suggest that modulation of the strength and kinetics of GABA(A) currents could provide treatment strategies for uncontrollable spasms.

Progesterone blocks multiple routes of ion flux

The administration of progesterone as a neuroprotective agent following traumatic brain injury has recently entered phase III clinical trials. Previous work has demonstrated that therapeutic concentrations of progesterone decrease excitotoxicity through direct inhibition of voltage-gated calcium channels, an action independent of the nuclear progesterone receptor. Here we report using cultured rat striatal neurons that these same concentrations of progesterone also block voltage-gated potassium channels, sodium channels and GABAA currents. The actions of progesterone act at the surface membrane of neurons in a steroid specific, voltage-independent, concentration-dependent manner. Notably, these broad actions of progesterone on ion channel and neurotransmitter receptor function mirror those of dihydropyridines, and indicate potential shared mechanisms of action, the prospective of additional therapeutic applications, and possibly, untoward effects.

Striatal origin of the pathologic beta oscillations in Parkinson's disease

Enhanced oscillations at beta frequencies (8-30 Hz) are a signature neural dynamic pathology in the basal ganglia and cortex of Parkinson's disease patients. The mechanisms underlying these pathological beta oscillations remain elusive. Here, using mathematical models, we find that robust beta oscillations can emerge from inhibitory interactions between striatal medium spiny neurons. The interaction of the synaptic GABAa currents and the intrinsic membrane M-current promotes population oscillations in the beta frequency range. Increased levels of cholinergic drive, a condition relevant to the parkinsonian striatum, lead to enhanced beta oscillations in the striatal model. We show experimentally that direct infusion of the cholinergic agonist carbachol into the striatum, but not into the neighboring cortex, of the awake, normal rodent induces prominent beta frequency oscillations in the local field potential. These results provide evidence for amplification of normal striatal network dynamics as a mechanism responsible for the enhanced beta frequency oscillations in Parkinson's disease.

Effect of membrane cholesterol enrichment on the potentiation of GABAA currents by neurosteroids

Effect of membrane cholesterol enrichment on the potentiation of GABAA currents by neurosteroids

Drug safety is a barrier to the discovery and development of new androgen receptor antagonists

Androgen receptor (AR) antagonists are part of the standard of care for prostate cancer. Despite the almost inevitable development of resistance in prostate tumors to AR antagonists, no new AR antagonists have been approved for over a decade. Treatment failure is due in part to mutations that increase activity of AR in response to lower ligand concentrations as well as to mutations that result in AR response to a broader range of ligands. The failure to discover new AR antagonists has occurred in the face of continued research; to enable progress, a clear understanding of the reasons for failure is required. Non-clinical drug safety studies and safety pharmacology assays were performed on previously approved AR antagonists (bicalutamide, flutamide, nilutamide), next generation antagonists in clinical testing (MDV3100, BMS-641988), and a pre-clinical drug candidate (BMS-501949). In addition, non-clinical studies with AR mutant mice, and EEG recordings in rats were performed. Non-clinical findings are compared to disclosures of clinical trial results. As a drug class, AR antagonists cause seizure in animals by an off-target mechanism and are found in vitro to inhibit GABA-A currents. Clinical trials of candidate next generation AR antagonists identify seizure as a clinical safety risk. Non-clinical drug safety profiles of the AR antagonist drug class create a significant barrier to the identification of next generation AR antagonists. GABA-A inhibition is a common off-target activity of approved and next generation AR antagonists potentially explaining some side effects and safety hazards of this class of drugs.

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Fourth Conference on Epileptogenesis, May 23–26, 2007, Pisa, Italy

Inhibition by α‐Tetrahydrodeoxycorticosterone (THDOC) of Pre‐Sympathetic Parvocellular Neurones in the Paraventricular Nucleus of Rat Hypothalamus

alpha-tetrahydrodeoxycorticosterone (THDOC) is an endogenous neuroactive steroid which increases in plasma and brain concentration during stress. It has both positive and negative modulatory effects on GABA activated GABAA currents, dependent upon the dose. We investigated the effects of THDOC on spinally-projecting "pre-sympathetic" neurones in the parvocellular subnucleus of the hypothalamic paraventricular nucleus (PVN), to determine whether it activates or inhibits these neurones, and by what mechanism. Rat spinally-projecting (parvocellular) PVN neurones were identified by retrograde labelling and the action of THDOC investigated with three modes of patch-clamp: cell-attached action current, whole-cell voltage-clamp and cell-attached single-channel recording. In cell-attached patch mode, parvocellular neurones fired action potentials spontaneously with an average frequency of 3.6 +/- 1.1 Hz. Bath application of THDOC reduced this with an EC50 of 67 nM (95% confidence limits: 54 to 84 nM), Hill coefficient 0.8 +/- 0.04, n = 5. In whole-cell patch-clamp mode, pressure ejection of GABA evoked inward currents. These were clearly GABAA currents, since they were inhibited by the GABAA receptor antagonist bicuculline, and reversed near the chloride equilibrium potential. THDOC significantly potentiated GABAA currents (1 microM THDOC: 148 +/- 15% of control, n = 5, p < or = 0.05, ANOVA). Single-channel analysis showed no differences in conductance or corrected mean open times in the presence of 1 microM THDOC. THDOC inhibited parvocellular neuronal activity without showing any evidence of the bidirectional activity demonstrated previously with cultured hypothalamic neurones. Our data are consistent with the hypothesis that THDOC acts by potentiating the post-synaptic activity of endogenously released GABA.