GABA Receptor Currents Research Articles

Qualitatively different scenarios for co-activation of NMDA, AMPA and GABA receptor currents on dopaminergic neuron

We introduced a reduced functional model that qualitatively describes fundamental properties of the dopaminergic (DA) neuron. The neuron is a low-frequency pacemaker, and requires special conditions to increase the frequency of its firing [1]. In more detail, only N-methyl-D-aspartate receptor (NMDAR) current can significantly elevate the frequency (more than 5 fold in comparison with pacemaking). α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) current is not able to evoke such a high frequency. Instead, it suppresses firing at a conductance much lower than NMDAR. γ-Aminobutyric acid receptor (GABAR) activation first decreases the firing frequency, and only then suppresses firing. Thus, the latter property determines type 1 excitability for the DA neuron, whereas the difference in responses to AMPA and NMDA receptor activation make the neuron different from other well-studied types. We focus this report on the co-activation of these receptors. Co-activation of AMPA together with NMDA receptor may obscure the high frequency firing and lead directly to its suppression. There are contradictory experimental data in this case [2,3]. We show that AMPAR can do both: further contribute to the frequency increase produced by NMDAR, or impede the high-frequency firing. We show that the distinction between these two cases is the level of the background NMDAR activation. Co-activation of GABA together with NMDA receptor may also lead directly to the suppression of high-frequency oscillations (e.g. by shunting inhibition), or reduce the frequency first. Experiments clearly show the latter [4]: the frequency can be reduced back to that in control conditions when GABA and NMDA receptor currents are balanced. This property allows us to recalibrate the model so that it retains type 1 excitability not only in control, but also with activated synaptic currents. In general, activation of synaptic currents changes neural excitability, and we investigate the dynamical mechanisms responsible for the transition. As a result, we were able to construct a model of the DA neuron that collects all the properties described above. The model is essential for further research of behaviorally-relevant modulations of the DA neuron activity, which involves simultaneous excitatory and inhibitory inputs to the neuron. Figure 1 Influence of the synaptic currents on the neuron activity. A and B: dependences of the frequency on the AMPAR and NMDAR, GABAR and NMDAR conductances respectively. C: a voltage trace corresponding to the case of balanced GABAR and NMDAR currents.

Propofol Regulates the Surface Expression of GABAA Receptors: Implications in Synaptic Inhibition.

The anesthetic propofol is thought to induce rapid hypnotic sedation by potentiating γ-aminobutyric acid receptor (GABAAR) activity. Little is known about the molecular mechanisms of propofol in modulating inhibitory synaptic transmission. We aimed to investigate the role of propofol in modulating surface expression of GABAARs. C57BL/6 mice received an intraperitoneal injection of propofol. Hippocampal pyramidal neurons were prepared from embryonic day-18 mice and were treated with propofol. Proteins on the plasma membrane were analyzed using cell surface biotinylation, immunoblotting and enzyme-linked immunosorbent assay. Electrophysiological activities were recorded from hippocampal cells in acute brain slices of mice. The interaction between GABAARs and clathrin adaptor protein 2 was assessed by immunoprecipitation. Phosphorylation of GABAARs was shown by in vitro kinase assay. Propofol facilitated membrane accumulation of GABAARβ3 subunits. Propofol mediated phosphorylation of GABAARβ3 by protein kinase Cε which blocked the interaction between GABAARβ3 and the β-adaptin subunit of adaptor protein 2, resulting in an inhibition of the receptor endocytosis in hippocampal pyramidal neurons. Coincident with increased GABAARs surface level, propofol enhanced evoked and miniature synaptic GABA receptor currents. This study offers new insight on the regulatory mechanism of propofol in inhibiting neuronal excitability.

Inflammatory mediator-induced modulation of GABAA currents in human sensory neurons

The purpose of the present study was to characterize the properties of A-type GABA receptor (GABAA receptor) currents in human sensory neurons. Neurons were obtained from adult organ donors. GABAA currents were recorded in isolated neurons. Both large inactivating low-affinity currents and smaller persistent high-affinity currents were present in all of the 129 neurons studied from 15 donors. The kinetics of human GABAA currents were slower than those in rat sensory neurons. GABA currents were completely blocked by bicuculline (10μM), and persistent currents were activated by the δ-subunit-preferring agonist, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). The GABA current equilibrium potential was ∼20mV more hyperpolarized than in rat neurons. Both low- and high-affinity currents were increased by inflammatory mediators but via different second messenger pathways. These results highlight potentially important species differences in the properties of ion channels present in their native environment and suggest the use of human sensory neurons may be a valuable tool to test compounds prior to use in humans.

Honeybee Kenyon cells are regulated by a tonic GABA receptor conductance.

The higher cognitive functions of insects are dependent on their mushroom bodies (MBs), which are particularly large in social insects such as honeybees. MB Kenyon cells (KCs) receive multisensory input and are involved in associative learning and memory. In addition to receiving sensory input via excitatory nicotinic synapses, KCs receive inhibitory GABAergic input from MB feedback neurons. Cultured honeybee KCs exhibit ionotropic GABA receptor currents, but the properties of GABA-mediated inhibition in intact MBs are currently unknown. Here, using whole cell recordings from KCs in acutely isolated honeybee brain, we show that KCs exhibit a tonic current that is inhibited by picrotoxin but not by bicuculline. Bath application of GABA (5 μM) and taurine (1 mM) activate a tonic current in KCs, but l-glutamate (0.1-0.5 mM) has no effect. The tonic current is strongly potentiated by the allosteric GABAA receptor modulator pentobarbital and is reduced by inhibition of Ca(2+) channels with Cd(2+) or nifedipine. Noise analysis of the GABA-evoked current gives a single-channel conductance value for the underlying receptors of 27 ± 3 pS, similar to that of resistant to dieldrin (RDL) receptors. The amount of injected current required to evoke action potential firing in KCs is significantly lower in the presence of picrotoxin. KCs recorded in an intact honeybee head preparation similarly exhibit a tonic GABA receptor conductance that reduces neuronal excitability, a property that is likely to contribute to the sparse coding of sensory information in insect MBs.

Opposite actions of alcohol on tonic GABA(A) receptor currents mediated by nNOS and PKC activity.

The molecular mechanisms that mediate genetic variability in response to alcohol are unclear. We find that alcohol has opposite actions (enhancement or suppression) on GABAA receptor (GABAAR) inhibition in granule cells (GCs) of the cerebellum from behaviorally sensitive, low-alcohol consuming Sprague Dawley rats and DBA/2 mice and behaviorally insensitive, high-alcohol consuming C57BL/6 mice, respectively. The impact of alcohol on GC GABAAR inhibition is determined by a balance between two opposing effects: enhanced presynaptic vesicular release of GABA via alcohol inhibition of nitric oxide synthase (NOS), and a direct suppression of the activity of postsynaptic GABAARs. The balance of these two processes is determined by differential expression of neuronal NOS (nNOS) and postsynaptic PKC activity, both of which vary across rodent genotypes. These findings identify opposing molecular processes that differentially control the magnitude and polarity of GABAAR responses to alcohol across rodent genotypes.

Astroglial cells regulate the developmental timeline of human neurons differentiated from induced pluripotent stem cells

Neurons derived from human induced-pluripotent stem cells (hiPSCs) have been used to model a variety of neurological disorders. Different protocols have been used to differentiate hiPSCs into neurons, but their functional maturation process has varied greatly among different studies. Here, we demonstrate that laminin, a commonly used substrate for iPSC cultures, was inefficient to promote fully functional maturation of hiPSC-derived neurons. In contrast, astroglial substrate greatly accelerated neurodevelopmental processes of hiPSC-derived neurons. We have monitored the neural differentiation and maturation process for up to two months after plating hiPSC-derived neuroprogenitor cells (hNPCs) on laminin or astrocytes. We found that one week after plating hNPCs, there were 21-fold more newly differentiated neurons on astrocytes than on laminin. Two weeks after plating hNPCs, there were 12-fold more dendritic branches in neurons cultured on astrocytes than on laminin. Six weeks after plating hNPCs, the Na+ and K+ currents, as well as glutamate and GABA receptor currents, were 3-fold larger in neurons cultured on astrocytes than on laminin. And two months after plating hNPCs, the spontaneous synaptic events were 8-fold more in neurons cultured on astrocytes than on laminin. These results highlight a critical role of astrocytes in promoting neural differentiation and functional maturation of human neurons derived from hiPSCs. Moreover, our data presents a thorough developmental timeline of hiPSC-derived neurons in culture, providing important benchmarks for future studies on disease modeling and drug screening.

Effect of Somatostatin on Presynaptic and Postsynaptic Glutamate Receptors and Postsynaptic GABA Receptors in the Neurons of Rat Brain

We studied the effect of somatostatin on presinaptic NMDA receptors and postsinaptic GABA, NMDA, and AMPA receptors in rat brain. It was shown that somatostatin inhibits NMDA-induced (45)Ca(2+) uptake into synaptosomes isolated from rat brain cortex (IC50=2.8×10(-11) M). Somatostatin potentiates AMPA receptors and inhibits hippocampal NMDA receptors in the entire range of examined concentrations (10(-14)-10(-7) M); it also potentiates or inhibits GABA receptor currents in a concentration-dependent manner. Our results suggest that somatostatin modulates the function of ionotropic glutamate and GABA receptors and is involved in cognitive and neurodegenerative processes in the mammalian brain.

ROS scavenging mimics anoxia by enhancing GABA receptor‐mediated electrical suppression in anoxia‐tolerant turtle cortex

In anoxic mammalian brain, ATP dependent ion pumping is compromised and the consequent loss of membrane ion gradients elicits hyper‐excitability and cell death. This does not occur in the western painted turtle brain, where γ‐aminobutyric acid (GABA) concentration increases 80 fold and electrical activity decreases 75–95%. During anoxia, electrical suppression results from increased GABA receptor (GABAR)‐mediated postsynaptic currents (PSC) that shunt excitatory current, thereby avoiding excess AP firing. The signaling pathway responsible for initiating GABAergic neuroprotection is unknown but could involve decreases in the concentration of reactive oxygen species (ROS) that occurs following onset of anoxia. Using whole‐cell and perforated patch clamp techniques we determined that, similar to anoxia, ROS scavenging caused: whole cell conductance to increase from 4.5 ±0.4 to 6.6 ±1.1 nS; AP threshold to depolarize from 45 ± 4.2 to 24 ± 2.2 mV; and GABAR‐mediated currents to double in amplitude from 237.8 ± 81.6 to 449.8 ± 136.9 pA. Pharmacological anoxia (normoxic plus cyanide) caused a similar increase in GABAR current amplitude to 407.8 ± 38.5 pA and this was prevented by ROS donors (hydrogen peroxide or xanthine oxidase/xanthine). We conclude that a decrease in mitochondrial ROS production is the signal that increases GABAR currents during anoxic stress. Research supported by OGS scholarship to DWH, NSERC postdoctoral fellowship to MEP and an NSERC Discovery and Accelerator Grant to LTB.

In Vivo and In Vitro Pharmacological Studies of Methoxycarbonyl-Carboetomidate

We previously developed 2 etomidate analogs that retain etomidate's favorable hemodynamic properties but whose adrenocortical effects are reduced in duration or magnitude. Methoxycarbonyl (MOC)-etomidate is rapidly metabolized and ultrashort acting whereas (R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (carboetomidate) does not potently inhibit 11β-hydroxylase. We hypothesized that MOC-etomidate's labile ester could be incorporated into carboetomidate to produce a new agent that possesses favorable properties individually found in each agent. We describe the synthesis and pharmacology of MOC-(R)-ethyl 1-(1-phenylethyl)-1H-pyrrole-2-carboxylate (MOC-carboetomidate), a "soft" analog of carboetomidate. MOC-carboetomidate's octanol:water partition coefficient was determined chromatographically and compared with those of etomidate, carboetomidate, and MOC-etomidate. MOC-carboetomidate's 50% effective concentration (EC(50)) and 50% effective dose for loss of righting reflexes (LORR) were measured in tadpoles and rats, respectively. Its effect on γ-aminobutyric acid A (GABA(A)) receptor function was assessed using 2-microelectrode voltage clamp electrophysiological techniques and its metabolic stability was determined in pooled rat blood using high performance liquid chromatography. Its duration of action and effects on arterial blood pressure and adrenocortical function were assessed in rats. MOC-carboetomidate's octanol:water partition coefficient was 3300 ± 280, whereas those for etomidate, carboetomidate, and MOC-etomidate were 800 ± 180, 15,000 ± 3700, and 190 ± 25, respectively. MOC-carboetomidate's EC(50) for LORR in tadpoles was 9 ± 1 μM and its EC(50) for LORR in rats was 13 ± 5 mg/kg. At 13 μM, MOC-carboetomidate enhanced GABA(A) receptor currents by 400% ± 100%. Its metabolic half-life in pooled rat blood was 1.3 min. The slope of a plot of the duration of LORR in rats versus the logarithm of the hypnotic dose was significantly shallower for MOC-carboetomidate than for carboetomidate (4 ± 1 vs 15 ± 3, respectively; P = 0.0004123). At hypnotic doses, the effects of MOC-carboetomidate on arterial blood pressure and adrenocortical function were not significantly different from those of vehicle alone. MOC-carboetomidate is a GABA(A) receptor modulator with potent hypnotic activity that is more rapidly metabolized and cleared from the brain than carboetomidate, maintains hemodynamic stability similar to carboetomidate, and does not suppress adrenocortical function.

Allosteric Modulation of Retinal GABA Receptors by Ascorbic Acid

Ionotropic GABA receptors (GABA(A) and GABA(C)) belong to the Cys-loop receptor family of ligand-gated ion channels. GABA(C) receptors are highly expressed in the retina, mainly localized at the axon terminals of bipolar cells. Ascorbic acid, an endogenous redox agent, modulates the function of diverse proteins, and basal levels of ascorbic acid in the retina are very high. However, the effect of ascorbic acid on retinal GABA receptors has not been studied. Here we show that the function of GABA(C) and GABA(A) receptors is regulated by ascorbic acid. Patch-clamp recordings from bipolar cell terminals in goldfish retinal slices revealed that GABA(C) receptor-mediated currents activated by tonic background levels of extracellular GABA, and GABA(C) currents elicited by local GABA puffs, are both significantly enhanced by ascorbic acid. In addition, a significant rundown of GABA puff-evoked currents was observed in the absence of ascorbic acid. GABA-evoked Cl(-) currents mediated by homomeric ρ(1) GABA(C) receptors expressed in Xenopus laevis oocytes were also potentiated by ascorbic acid in a concentration-dependent, stereo-specific, reversible, and voltage-independent manner. Studies involving the chemical modification of sulfhydryl groups showed that the two Cys-loop cysteines and histidine 141, all located in the ρ(1) subunit extracellular domain, each play a key role in the modulation of GABA(C) receptors by ascorbic acid. Additionally, we show that retinal GABA(A) IPSCs and heterologously expressed GABA(A) receptor currents are similarly augmented by ascorbic acid. Our results suggest that ascorbic acid may act as an endogenous agent capable of potentiating GABAergic neurotransmission in the CNS.

Chronic nicotine treatment differentially modifies acute nicotine and alcohol actions on GABA(A) and glutamate receptors in hippocampal brain slices.

Tobacco and alcohol are often co-abused producing interactive effects in the brain. Although nicotine enhances memory while ethanol impairs it, variable cognitive changes have been reported from concomitant use. This study was designed to determine how nicotine and alcohol interact at synaptic sites to modulate neuronal processes. Acute effects of nicotine, ethanol, and both drugs on synaptic excitatory glutamatergic and inhibitory GABAergic transmission were measured using whole-cell recording in hippocampal CA1 pyramidal neurons from brain slices of mice on control or nicotine-containing diets. Acute nicotine (50 nM) enhanced both GABAergic and glutamatergic synaptic transmission; potentiated GABA(A) receptor currents via activation of α7* and α4β2* nAChRs, and increased N-methyl-D-aspartate (NMDA) and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor currents through α7* receptors. While ethanol (80 mM) also increased GABA(A) currents, it inhibited NMDA currents. Although ethanol had no effect on AMPA currents, it blocked nicotine-induced increases in NMDA and AMPA currents. Following chronic nicotine treatment, acute nicotine or ethanol did not affect NMDA currents, while the effects of GABAergic responses were not altered. Acute ethanol ingestion selectively attenuated nicotine enhancement of excitatory glutamatergic NMDA and AMPA receptor function, suggesting an overall reduction in excitatory output from the hippocampus. It also indicated that ethanol could decrease the beneficial effects of nicotine on memory performance. In addition, chronic nicotine treatment produced tolerance to the effects of nicotine and cross-tolerance to the effects of ethanol on glutamatergic activity, leading to a potential increase in the use of these drugs.

Identification and characterization of Cs+‐permeable K+ channel current in mouse cerebellar Purkinje cells in lobules 9 and 10 evoked by molecular layer stimulation

The mouse cerebellum consists of 10 lobules, which are distinguishable by their anatomical and functional properties. However, the differences in the slow postsynaptic currents (sPSCs) of Purkinje cells between lobules have not been well studied. We recorded the sPSCs of lobules 3, 9 and 10 evoked by tetanic stimulation of the molecular layer in cerebellar slices, and found a novel outward sPSC mediated by the GABA(B) receptor in loblues 9 and 10 but hardly at all in lobule 3. We showed that the lobule-specific difference is at least partly attributable to differences in the density of GABAergic neurons (higher in lobule 10 than in lobules 3 and 9), and the functional expression level of postsynaptic GABA(B) receptor currents (larger in lobules 9 and 10 than in lobule 3). The G-protein-coupled inward rectifying K(+) channel (GIRK) is known to be activated by GABA(B) receptors; however, the outward sPSC was not blocked by a GIRK blocker, was not sensitive to Cs(+) block, and was observed when Cs(+) was used as a charge carrier. These results suggest that a K(+) channel other than GIRK could be activated by GABA(B) receptors. KCNK13 is a Cs(+)-permeable K(+) channel that shows intense expression of mRNA in Purkinje cells. KCNK13 current was enhanced by co-expression of G(βγ) subunits and was observed when Cs(+) was used as a charge carrier in heterologous expression systems, and the amino acids critical for these features were identified by mutagenesis. Taken together, these results show that KCNK13 is a legitimate candidate for the Cs(+)-permeable K(+) channel activated by GABA(B) receptors, presumably via G(βγ) subunits in Purkinje cells.

Oxcarbazepine, not its active metabolite, potentiates GABAA activation and aggravates absence seizures

Studies in genetic absence epileptic rats from Strasbourg (GAERS) indicate that enhancement of gamma aminobutyric acid (GABA(A)) receptor activity is a critical mechanism in the aggravation of seizures by carbamazepine (CBZ). We examined whether structural analogs of CBZ, oxcarbazepine (OXC), and its active metabolite, monohydroxy derivative (MHD), also potentiate GABA(A) receptor current and aggravate seizures. In vitro studies in Xenopus oocytes compared the three drugs' effect on GABA(A) receptor currents. In vivo studies compared seizure activity in GAERS after intraperitoneal drug administration. OXC potentiated GABA(A) receptor current and aggravated seizures in GAERS, similarly to the effect of CBZ. Conversely, MHD showed only a minor potentiation of GABA(A) receptor current and did not aggravate seizures. A hydroxyl group at the C-10 position on the CBZ tricyclic structure in MHD reduces GABA(A) receptor potentiation and seizure aggravation. Reports of the aggravation of absence seizures in patients taking OXC may result from circulating unmetabolized OXC rather than MHD.

Ischemic insult to cerebellar Purkinje cells causes diminished GABAA receptor function and allopregnanolone neuroprotection is associated with GABAA receptor stabilization

Cerebellar Purkinje cells (PC) are particularly vulnerable to ischemic injury and excitotoxicity, although the molecular basis of this sensitivity remains unclear. We tested the hypothesis that ischemia causes rapid down-regulation of GABA(A) receptors in cerebellar PC, thereby increasing susceptibility to excitotoxicity. Oxygen-glucose deprivation (OGD) caused a decline in functional GABA(A) receptors, within the first hour of re-oxygenation. Decreased amplitude of miniature inhibitory post-synaptic potentials confirmed that OGD caused a significant decrease in functional synaptic GABA(A) receptors and quantitative Western blot analysis demonstrated the loss of GABA(A) receptor current was associated with a decline in total receptor protein. Interestingly, the potent neuroprotectant allopregnanolone (ALLO) prevented the decline in GABA(A) receptor current and protein. Consistent with our in vitro data, global ischemia in mice caused a significant decline in total cerebellar GABA(A) receptor protein and PC specific immunoreactivity. Moreover, ALLO provided strong protection of PC and prevented ischemia-induced decline in GABA(A) receptor protein. Our findings indicate that ischemia causes a rapid and sustained loss of GABA(A) receptors in PC, whereas ALLO prevents the decline in GABA(A) receptors and protects against ischemia-induced damage. Thus, interventions which prevent ischemia-induced decline in GABA(A) receptors may represent a novel neuroprotective strategy.

Postsynaptic dopamine D3 receptor modulation of evoked IPSCs via GABAA receptor endocytosis in rat hippocampus

Dopamine is known to be an important modulator of learning and memory processes, but its mechanisms of action at the cellular level are diverse and are not fully characterized. In the hippocampus, pharmacologically isolated monosynaptic IPSCs were measured using the whole-cell voltage-clamp recording technique. Both electrically evoked and spontaneous miniature GABA(A) receptor currents were recorded from CA1 pyramidal neurons in slices obtained from mature rats in the presence of the D3-selective agonist PD128907. The activation of D3 receptors inhibited synaptic GABAergic input without affecting presynaptic function or passive membrane properties. Inhibition of IPSCs evoked from stratum radiatum occurred via regulation of dynamin-dependent trafficking of the GABA(A) receptor, as inclusion of dynamin inhibitory peptide (50 microM) in the recording solution prevented the inhibitory effects of PD128907 (1 microM). This effect of D3 receptor activation could be prevented by intracellular application of either an inhibitor of protein kinase A (PKI, 20 microM) or an activator of protein kinase A (8-OH-cAMP, 50 microM). Neither synchronous IPSCs evoked from the stratum oriens nor asynchronous miniature IPSCs recorded from the stratum radiatum were affected by D3 agonist. The induction of long-term potentiation (LTP) of the extracellular field response in both the stratum radiatum and stratum oriens demonstrated that only potentiation in the stratum radiatum was significantly enhanced by PD128907 (1 microM). Our results suggest that the activation of D3 receptors can modulate GABA(A) receptor endocytosis in the hippocampus in a lamina specific manner, and thereby alter the efficacy of GABAergic transmission in the stratum radiatum of the CA1 region through a postsynaptic mechanism of action.

Functional segregation of synaptic GABAA and GABAC receptors in goldfish bipolar cell terminals

The transmission of light responses to retinal ganglion cells is regulated by inhibitory input from amacrine cells to bipolar cell (BC) synaptic terminals. GABA(A) and GABA(C) receptors in BC terminals mediate currents with different kinetics and are likely to have distinct functions in limiting BC output; however, the synaptic properties and localization of the receptors are currently poorly understood. By recording endogenous GABA receptor currents directly from BC terminals in goldfish retinal slices, I show that spontaneous GABA release activates rapid GABA(A) receptor miniature inhibitory postsynaptic currents (mIPSCs) (predominant decay time constant (tau(decay)), 1.0 ms) in addition to a tonic GABA(C) receptor current. The GABA(C) receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) has no effect on the amplitude or kinetics of the rapid GABA(A) mIPSCs. In addition, inhibition of the GAT-1 GABA transporter, which strongly regulates GABA(C) receptor currents in BC terminals, fails to reveal a GABA(C) component in the mIPSCs. These data suggest that GABA(A) and GABA(C) receptors are highly unlikely to be synaptically colocalized. Using non-stationary noise analysis of the mIPSCs, I estimate that GABA(A) receptors in BC terminals have a single-channel conductance (gamma) of 17 pS and that an average of just seven receptors mediates a quantal event. From noise analysis of the tonic current, GABA(C) receptor gamma is estimated to be 4 pS. Identified GABA(C) receptor mIPSCs exhibit a slow decay (tau(decay), 54 ms) and are mediated by approximately 42 receptors. The distinct properties and localization of synaptic GABA(A) and GABA(C) receptors in BC terminals are likely to facilitate their specific roles in regulating the transmission of light responses in the retina.

Modulation of GABAA Receptor Phosphorylation and Membrane Trafficking by Phospholipase C-related Inactive Protein/Protein Phosphatase 1 and 2A Signaling Complex Underlying Brain-derived Neurotrophic Factor-dependent Regulation of GABAergic Inhibition

Brain-derived neurotrophic factor (BDNF) modulates several distinct aspects of synaptic transmission, including GABAergic transmission. Exposure to BDNF alters properties of GABA(A) receptors and induces changes in the expression level at the cell surface. Although phospholipase C-related inactive protein-1 (PRIP-1) plays an important role in GABA(A) receptor trafficking and function, its role in BDNF-dependent modulation of these receptors, together with the role of PRIP-2, was investigated using neurons cultured from PRIP double knock-out mice. The BDNF-dependent inhibition of whole cell GABA-evoked currents observed in wild type neurons was not detected in neurons cultured from knock-out mice. Instead, a gradual increase in GABA-evoked currents in these neurons correlated with a gradual increase in phosphorylation of GABA(A) receptor beta3 subunit in response to BDNF. To characterize the specific role(s) that PRIP plays as components of underlying molecular machinery, we examined the recruitment of protein phosphatase(s) to GABA(A) receptors. We demonstrate that PRIP associates with phosphatases as well as with beta subunits. PRIP was found to colocalize with GABA(A) receptor clusters in cultured neurons and with recombinant GABA(A) receptors when co-expressed in HEK293 cells. Importantly, a peptide mimicking a domain of PRIP involved in binding to beta subunits disrupted the co-localization of these proteins in HEK293 cells and potently inhibited the BDNF-mediated attenuation of GABA(A) receptor currents in wild type neurons. Together, the results suggest that PRIP plays an important role in BDNF-dependent regulation of GABA(A) receptors by mediating the specific association between beta subunits of these receptors with protein phosphatases.

Indiplon Is a High-Affinity Positive Allosteric Modulator with Selectivity for α1 Subunit-Containing GABAAReceptors

Indiplon (NBI 34060) is a novel pyrazolopyrimidine currently in development for the treatment of insomnia. We have previously shown that indiplon exhibits high-affinity binding to native GABA(A) receptors from rat brain and acts as a positive allosteric modulator of GABA(A) receptor currents in cultured rat neurons (Sullivan et al., 2004). In this study, we examined the GABA(A) receptor alpha subunit selectivity of indiplon using electrophysiological techniques to record GABA-activated chloride currents from recombinant rodent GABA(A) receptors expressed in human embryonic kidney 293 cells. Indiplon potentiated the GABA-activated chloride current in recombinant GABA(A) receptors in a dose-dependent and reversible manner and was approximately 10-fold selective for alpha1 subunit-containing receptors over GABA(A) receptors containing alpha2, alpha3, or alpha5 subunits. The EC(50) values were 2.6, 24, 60, and 77 nM for alpha1beta2gamma2, alpha2beta2gamma2, alpha3beta3gamma2, and alpha5beta2gamma2 receptors, respectively. Indiplon was approximately 10 times more potent than zolpidem and zopiclone and >100 times more potent than zaleplon. Moreover, indiplon, up to 1 microM, did not potentiate GABA(A) receptors composed of alpha4beta2gamma2 and alpha6beta2gamma2 subunits. This mechanism of action is proposed to underlie the sedative-hypnotic effects of indiplon in animals and humans.

Nonsynaptic GABA signaling in postnatal subventricular zone controls proliferation of GFAP-expressing progenitors

In the postnatal subventricular zone (SVZ), local cues or signaling molecules released from neuroblasts limit the proliferation of glial fibrillary acidic protein (GFAP)-expressing progenitors thought to be stem cells. However, signals between SVZ cells have not been identified. We show that depolarization of neuroblasts induces nonsynaptic SNARE-independent GABA(A) receptor currents in GFAP-expressing cells, the time course of which depends on GABA uptake in acute mouse slices. We found that GABA(A) receptors are tonically activated in GFAP-expressing cells, consistent with the presence of spontaneous depolarizations in neuroblasts that are sufficient to induce GABA release. These data demonstrate the existence of nonsynaptic GABAergic signaling between neuroblasts and GFAP-expressing cells. Furthermore, we show that GABA(A) receptor activation in GFAP-expressing cells limits their progression through the cell cycle. Thus, as GFAP-expressing cells generate neuroblasts, GABA released from neuroblasts provides a feedback mechanism to control the proliferation of GFAP-expressing progenitors by activating GABA(A) receptors.

Cultured Hippocampal Pyramidal Neurons Express Two Kinds of GABAAReceptors

We combined a study of the subcellular distribution of the alpha1, alpha2, alpha4, beta1, beta2/3, gamma2, and delta subunits of the GABAA receptor with an electrophysiological analysis of GABAA receptor currents determine the to types of receptors expressed on cultured hippocampal pyramidal neurons. The immunocytochemistry study demonstrated that alpha1, alpha2, beta2/3, and gamma2 subunits formed distinct clusters of various sizes, which were colocalized with clusters of glutamate decarboxylase (GAD) immunoreactivity at rates ranging from 22 to 58%. In contrast, alpha4, beta1, and delta subunits were distributed diffusely over the cell soma and neuronal processes of cultured neurons and did not colocalize with the synaptic marker GAD. Whole-cell GABA receptor currents were moderately sensitive to GABAA and were modulated by diazepam. The whole-cell currents were also enhanced by the neurosteroid allopregnanolone (10 nM). Tonic currents, measured as changes in baseline current and noise, were sensitive to Zn2+, furosemide, and loreclezole; they were insensitive to diazepam. These studies suggest that two kinds of GABAA receptors are expressed on cultured hippocampal neurons. One kind of receptor formed clusters, which were present at GABAergic synapses and in the extrasynaptic membrane. The alpha1, alpha2, beta2/3, and gamma2 subunits were contained in clustered receptors. The second kind was distributed diffusely in the extrasynaptic membrane. The alpha4, beta1, and delta subunits were contained in these diffusely distributed receptors. The properties of tonic currents recorded from these neurons were similar to those from recombinant receptors containing alpha4, beta1, and delta subunits.