Neil L. Harrison

Extrasynaptic GABAA Receptors: Form, Pharmacology, and Function

GABA is the principal inhibitory neurotransmitter in the CNS and acts via GABAA and GABAB receptors. Recently, a novel form of GABAA receptor-mediated inhibition, termed “tonic” inhibition, has been described. Whereas synaptic GABAA receptors underlie classical “phasic” GABAA receptor-mediated inhibition (inhibitory postsynaptic currents), tonic GABAA receptor-mediated inhibition results from the activation of extrasynaptic receptors by low concentrations of ambient GABA. Extrasynaptic GABAA receptors are composed of receptor subunits that convey biophysical properties ideally suited to the generation of persistent inhibition and are pharmacologically and functionally distinct from their synaptic counterparts. This mini-symposium review highlights ongoing work examining the properties of recombinant and native extrasynaptic GABAA receptors and their preferential targeting by endogenous and clinically relevant agents. In addition, it emphasizes the important role of extrasynaptic GABAA receptors in GABAergic inhibition throughout the CNS and identifies them as a major player in both physiological and pathophysiological processes.

GABAA receptor α4 subunits mediate extrasynaptic inhibition in thalamus and dentate gyrus and the action of gaboxadol

The neurotransmitter GABA mediates the majority of rapid inhibition in the CNS. Inhibition can occur via the conventional mechanism, the transient activation of subsynaptic GABAA receptors (GABAA-Rs), or via continuous activation of high-affinity receptors by low concentrations of ambient GABA, leading to “tonic” inhibition that can control levels of excitability and network activity. The GABAA-R α4 subunit is expressed at high levels in the dentate gyrus and thalamus and is suspected to contribute to extrasynaptic GABAA-R-mediated tonic inhibition. Mice were engineered to lack the α4 subunit by targeted disruption of the Gabra4 gene. α4 Subunit knockout mice are viable, breed normally, and are superficially indistinguishable from WT mice. In electrophysiological recordings, these mice show a lack of tonic inhibition in dentate granule cells and thalamic relay neurons. Behaviorally, knockout mice are insensitive to the ataxic, sedative, and analgesic effects of the novel hypnotic drug, gaboxadol. These data demonstrate that tonic inhibition in dentate granule cells and thalamic relay neurons is mediated by extrasynaptic GABAA-Rs containing the α4 subunit and that gaboxadol achieves its effects via the activation of this GABAA-R subtype.

Taurine Is a Potent Activator of Extrasynaptic GABAA Receptors in the Thalamus

Taurine is one of the most abundant free amino acids in the brain. In a number of studies, taurine has been reported to activate glycine receptors (Gly-Rs) at moderate concentrations (≥100 μm), and to be a weak agonist at GABAA receptors (GABAA-Rs), which are usually activated at high concentrations (≥1 mm). In this study, we show that taurine reduced the excitability of thalamocortical relay neurons and activated both extrasynaptic GABAA-Rs and Gly-Rs in neurons in the mouse ventrobasal (VB) thalamus. Low concentrations of taurine (10–100 μm) decreased neuronal input resistance and firing frequency, and elicited a steady outward current under voltage clamp, but had no effects on fast inhibitory synaptic currents. Currents elicited by 50 μm taurine were abolished by gabazine, insensitive to midazolam, and partially blocked by 20 μm Zn2+, consistent with the pharmacological properties of extrasynaptic GABAA-Rs (α4β2δ subtype) involved in tonic inhibition in the thalamus. Tonic inhibition was enhanced by an inhibitor of taurine transport, suggesting that taurine can act as an endogenous activator of these receptors. Taurine-evoked currents were absent in relay neurons from GABAA-R α4 subunit knock-out mice. The amplitude of the taurine current was larger in neurons from adult mice than juvenile mice. Taurine was a more potent agonist at recombinant α4β2δ GABAA-Rs than at α1β2γ2 GABAA-Rs. We conclude that physiological concentrations of taurine can inhibit VB neurons via activation of extrasynaptic GABAA-Rs and that taurine may function as an endogenous regulator of excitability and network activity in the thalamus.

Identification of embryonic stem cell-derived midbrain dopaminergic neurons for engraftment.

Embryonic stem cells (ESCs) represent a promising source of midbrain dopaminergic (DA) neurons for applications in Parkinson disease. However, ESC-based transplantation paradigms carry a risk of introducing inappropriate or tumorigenic cells. Cell purification before transplantation may alleviate these concerns and enable identification of the specific DA neuron stage most suitable for cell therapy. Here, we used 3 transgenic mouse ESC reporter lines to mark DA neurons at 3 stages of differentiation (early, middle, and late) following induction of differentiation using Hes5::GFP, Nurr1::GFP, and Pitx3::YFP transgenes, respectively. Transplantation of FACS-purified cells from each line resulted in DA neuron engraftment, with the mid-stage and late-stage neuron grafts being composed almost exclusively of midbrain DA neurons. Mid-stage neuron cell grafts had the greatest amount of DA neuron survival and robustly induced recovery of motor deficits in hemiparkinsonian mice. Our data suggest that the Nurr1+ stage (middle stage) of neuronal differentiation is particularly suitable for grafting ESC-derived DA neurons. Moreover, global transcriptome analysis of progeny from each of the ESC reporter lines revealed expression of known midbrain DA neuron genes and also uncovered previously uncharacterized midbrain genes. These data demonstrate remarkable fate specificity of ESC-derived DA neurons and outline a sequential stage-specific ESC reporter line paradigm for in vivo gene discovery.

The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subunits.

Background Previous studies have shown that specific amino acid residues in the putative second transmembrane segment (TM2) of the &ggr;-aminobutyric acid receptor type A (GABAA) receptor play a critical role in the enhancement of GABAA receptor function by halothane, enflurane, and isoflurane. However, very little is known about the actions of sevoflurane and desflurane on recombinant GABAA receptors. The aim of this study was to examine the effects of sevoflurane and desflurane on potentiation of GABA-induced responses in the wild-type GABAA receptor and in receptors mutated in TM2 of the &agr;1, &agr;2, or &bgr;2 subunits. Methods GABAA receptor &agr;1 or &agr;2, &bgr;2 or &bgr;3, and &ggr;2s subunit cDNAs were expressed for pharmacologic study by transfection of human embryonic kidney 293 cells and assayed using the whole cell voltage clamp technique. Concentration-response curves and EC50 values for agonist were determined in the wild-type &agr;1&bgr;2&ggr;2s and &agr;2&bgr;3&ggr;2s receptors, and in receptors harboring mutations in TM2, such as &agr;1(S270W)&bgr;2&ggr;2s, &agr;1&bgr;2(N265W)&ggr;2s, and &agr;2(S270I)&bgr;3&ggr;2s. The actions of clinically relevant concentration of volatile anesthetics (isoflurane, sevoflurane, and desflurane) on GABA activated Cl− currents were compared in the wild-type and mutant GABAA receptors. Results Both sevoflurane and desflurane potentiated submaximal GABA currents in the wild-type GABAA &agr;1&bgr;2&ggr;2s receptor and &agr;2&bgr;3&ggr;2s receptor. Substitution of Ser270 in TM2 of the &agr; subunit by a larger amino acid, tryptophan (W) or isoleucine (I), as in &agr;1(S270W)&bgr;2&ggr;2s and &agr;2(S270I)&bgr;3&ggr;2s, completely abolished the potentiation of GABA-induced currents by these anesthetic agents. In contrast, mutation of Asn265 in TM2 of the &bgr; subunit to tryptophan (W) did not prevent potentiation of GABA-induced responses. The actions of sevoflurane and desflurane in the wild-type receptor and in mutated receptors were qualitatively and quantitatively similar to those observed for isoflurane. Conclusions Positions Ser270 of the GABAA &agr;1 and &agr;2 subunits, but not Asn265 in the TM2 of the &bgr;2 subunit, are critical for regulation of the GABAA receptor by sevoflurane and desflurane, as well as isoflurane, consistent with the idea that these three volatile anesthetics share a common site of actions on the &agr; subunit of the GABAA receptor.

Ethanol modulates synaptic and extrasynaptic GABAA receptors in the thalamus.

Drinking alcohol is associated with the disturbance of normal sleep rhythms, and insomnia is a major factor in alcoholic relapse. The thalamus is a brain structure that plays a pivotal role in sleep regulation and rhythmicity. A number of studies have implicated GABAA receptors (GABAA-Rs) in the anxiolytic, amnestic, sedative, and anesthetic effects of ethanol. In the present study, we examined the effects of ethanol on both synaptic and extrasynaptic GABAA-Rs of relay neurons in the thalamus. We found that ethanol (≥50 mM) elicits a sustained current in thalamocortical relay neurons from the mouse ventrobasal thalamus, and this current is associated with a decrease in neuronal excitability and firing rate in response to depolarization. The steady current induced by ethanol was totally abolished by gabazine and was absent in relay neurons from GABAA-R α4 subunit knockout mice, indicating that the effect of ethanol is to enhance tonic GABA-mediated inhibition. Ethanol (50 mM) enhanced the amplitude of tonic inhibition by nearly 50%. On the other hand, ethanol had no effect on spontaneous or evoked inhibitory postsynaptic currents (IPSCs) at 50 mM but did prolong IPSCs at 100 mM. Ethanol had no effect on the paired-pulse depression ratio, suggesting that the release of GABA from presynaptic terminals is insensitive to ethanol. We conclude that ethanol, at moderate (50 mM) but not low (10 mM) concentrations, can inhibit thalamocortical relay neurons and that this occurs mainly via the actions of ethanol at extrasynaptic GABAA-Rs containing GABAA-R α4 subunits.

Alcohol regulates gene expression in neurons via activation of heat shock factor 1.

Drinking alcohol causes widespread alterations in gene expression that can result in long-term physiological changes. Although many alcohol-responsive genes (ARGs) have been identified, the mechanisms by which alcohol alters transcription are not well understood. To elucidate these mechanisms, we investigated Gabra4, a neuron-specific gene that is rapidly and robustly activated by alcohol (10–60 mm), both in vitro and in vivo. Here we show that alcohol can activate elements of the heat shock pathway in mouse cortical neurons to enhance the expression of Gabra4 and other ARGs. The activation of Gabra4 by alcohol or high temperature is dependent on the binding of heat shock factor 1 (HSF1) to a short downstream DNA sequence, the alcohol response element (ARE). Alcohol and heat stimulate the translocation of HSF1 from the cytoplasm to the nucleus and the induction of HSF1-dependent genes, Hsp70 and Hsp90, in cultured neurons and in the mouse cerebral cortex in vivo. The reduction of HSF1 levels using small interfering RNA prevented the stimulation of Gabra4 and Hsp70 by alcohol and heat shock. Microarray analysis showed that many ARGs contain ARE-like sequences and that some of these genes are also activated by heat shock. We suggest that alcohol activates phylogenetically conserved pathways that involve intermediates in the heat shock cascade and that sequence elements similar to the ARE may mediate some of the changes in gene expression triggered by alcohol intake, which could be important in a variety of pathophysiological responses to alcohol.

Striatal D2 Receptors Regulate Dendritic Morphology of Medium Spiny Neurons via Kir2 Channels

Structural plasticity in the adult brain is essential for adaptive behaviors and is thought to contribute to a variety of neurological and psychiatric disorders. Medium spiny neurons of the striatum show a high degree of structural plasticity that is modulated by dopamine through unknown signaling mechanisms. Here, we demonstrate that overexpression of dopamine D2 receptors in medium spiny neurons increases their membrane excitability and decreases the complexity and length of their dendritic arbors. These changes can be reversed in the adult animal after restoring D2 receptors to wild-type levels, demonstrating a remarkable degree of structural plasticity in the adult striatum. Increased excitability and decreased dendritic arborization are associated with downregulation of inward rectifier potassium channels (Kir2.1/2.3). Downregulation of Kir2 function is critical for the neurophysiological and morphological changes in vivo because virally mediated expression of a dominant-negative Kir2 channel is sufficient to recapitulate the changes in D2 transgenic mice. These findings may have important implications for the understanding of basal ganglia disorders, and more specifically schizophrenia, in which excessive activation of striatal D2 receptors has long been hypothesized to be of pathophysiologic significance.

Isoflurane Is a Potent Modulator of Extrasynaptic GABAA Receptors in the Thalamus

Volatile anesthetics are used clinically to produce analgesia, amnesia, unconsciousness, blunted autonomic responsiveness, and immobility. Previous work has shown that the volatile anesthetic isoflurane, at concentrations that produce unconsciousness (250–500 μM), enhances fast synaptic inhibition in the brain mediated by GABAA receptors (GABAA-Rs). In addition, isoflurane causes sedation at concentrations lower than those required to produce unconsciousness or analgesia. In this study, we found that isoflurane, at low concentrations (25–85 μM) associated with its sedative actions, elicits a sustained current associated with a conductance increase in thalamocortical neurons in the mouse ventrobasal (VB) nucleus. These isoflurane-evoked currents reversed polarity close to the Cl– equilibrium potential and were totally blocked by the GABAA-R antagonist gabazine. Isoflurane (25–250 μM) produced no sustained current in VB neurons from GABAA-R α4-subunit knockout (Gabra4–/–) mice, although 250 μM isoflurane enhanced synaptic inhibition in VB neurons from both wild-type and Gabra4–/– mice. These data indicate an obligatory requirement for α4-subunit expression in the generation of the isoflurane-activated current. In addition, isoflurane directly activated α4β2δ GABAA-Rs expressed in human embryonic kidney 293 cells, and it was more potent at α4β2δ than at α1β2γ2 receptors (the presumptive extrasynaptic and synaptic GABAA-R subtypes in VB neurons). We conclude that the extrasynaptic GABAA-Rs of thalamocortical neurons are sensitive to low concentrations of isoflurane. In view of the crucial role of the thalamus in sensory processing, sleep, and cognition, the modulation of these extrasynaptic GABAA-Rs by isoflurane may contribute to the sedation and hypnosis associated with low doses of this anesthetic agent.

A Highly Conserved Aspartic Acid Residue in the Signature Disulfide Loop of the α1 Subunit Is a Determinant of Gating in the Glycine Receptor

Ligand-gated ion channels (LGICs) mediate rapid chemical neurotransmission. This gene superfamily includes the nicotinic acetylcholine, GABAA/C, 5-hydroxytryptamine type 3, and glycine receptors. A signature disulfide loop (Cys loop) in the extracellular domain is a structural motif common to all LGIC member subunits. Here we report that a highly conserved aspartic acid residue within the Cys loop at position 148 (Asp-148) of the glycine receptor α1 subunit is critical in the process of receptor activation. Mutation of this acidic residue to the basic amino acid lysine produces a large decrease in the potency of glycine, produces a decrease in the Hill slope, and converts taurine from a full agonist to a partial agonist; these data are consistent with a molecular defect in the receptor gating mechanism. Additional mutation of Asp-148 shows that alterations in the EC50 for agonists are dependent upon the charge of the side chain at this position and not molecular volume, polarity, or hydropathy. This study implicates negative charge at position Asp-148 as a critical component of the process in which agonist binding is coupled to channel gating. This finding adds to an emerging body of evidence supporting the involvement of the Cys loop in the gating mechanism of the LGICs.