Catriona M. Houston

Are Extrasynaptic GABAA Receptors Important Targets for Sedative/Hypnotic Drugs?

High-affinity extrasynaptic GABAA receptors are persistently activated by the low ambient GABA levels that are known to be present in extracellular space. The resulting tonic conductance generates a form of shunting inhibition that is capable of altering cellular and network behavior. It has been suggested that this tonic inhibition will be enhanced by neurosteroids, antiepileptics, and sedative/hypnotic drugs. However, we show that the ability of sedative/hypnotic drugs to enhance tonic inhibition in the mouse cerebellum will critically depend on ambient GABA levels. For example, we show that the intravenous anesthetic propofol enhances tonic inhibition only when ambient GABA levels are <100 nm. More surprisingly, the actions of the sleep-promoting drug 4,5,6,7-tetrahydroisothiazolo-[5,4-c]pyridin-3-ol (THIP) are attenuated at ambient GABA levels of just 20 nm. In contrast, our data suggest that neurosteroid enhancement of tonic inhibition will be greater at high ambient GABA concentrations. We present a model that takes into account realistic estimates of ambient GABA levels and predicted extrasynaptic GABAA receptor numbers when considering the ability of sedative/hypnotic drugs to enhance tonic inhibition. These issues will be important when considering drug strategies designed to target extrasynaptic GABAA receptors in the treatment of sleep disorders and other neurological conditions.

Wakefulness Is Governed by GABA and Histamine Cotransmission

Summary Histaminergic neurons in the tuberomammilary nucleus (TMN) of the hypothalamus form a widely projecting, wake-active network that sustains arousal. Yet most histaminergic neurons contain GABA. Selective siRNA knockdown of the vesicular GABA transporter (vgat, SLC32A1) in histaminergic neurons produced hyperactive mice with an exceptional amount of sustained wakefulness. Ablation of the vgat gene throughout the TMN further sharpened this phenotype. Optogenetic stimulation in the caudate-putamen and neocortex of “histaminergic” axonal projections from the TMN evoked tonic (extrasynaptic) GABAA receptor Cl− currents onto medium spiny neurons and pyramidal neurons. These currents were abolished following vgat gene removal from the TMN area. Thus wake-active histaminergic neurons generate a paracrine GABAergic signal that serves to provide a brake on overactivation from histamine, but could also increase the precision of neocortical processing. The long range of histamine-GABA axonal projections suggests that extrasynaptic inhibition will be coordinated over large neocortical and striatal areas.

CaMKII phosphorylation of the GABAA receptor: receptor subtype- and synapse-specific modulation

As a major inhibitory neurotransmitter, GABA plays a vital role in the brain by controlling the extent of neuronal excitation. This widespread role is reflected by the ubiquitous distribution of GABAA receptors throughout the central nervous system. To regulate the level of neuronal inhibition requires some endogenous control over the release of GABA and/or its postsynaptic response. In this context, Ca2+ ions are often used as primary or secondary messengers frequently resulting in the activation of protein kinases and phosphatases. One such kinase, Ca2+/calmodulin‐dependent protein kinase II (CaMKII), can target the GABAA receptor to cause its phosphorylation. Evidence is now emerging, which is reviewed here, that GABAA receptors are indeed substrates for CaMKII and that this covalent modification alters the expression of cell surface receptors and their function. This type of regulation can also feature at inhibitory synapses leading to long‐term inhibitory synaptic plasticity. Most recently, CaMKII has now been proposed to differentially phosphorylate particular isoforms of GABAA receptors in a synapse‐specific context.

Intracellular chloride ions regulate the time course of GABA-mediated inhibitory synaptic transmission.

The time-dependent integration of excitatory and inhibitory synaptic currents is an important process for shaping the input–output profiles of individual excitable cells, and therefore the activity of neuronal networks. Here, we show that the decay time course of GABAergic inhibitory synaptic currents is considerably faster when recorded with physiological internal Cl− concentrations than with symmetrical Cl− solutions. This effect of intracellular Cl− is due to a direct modulation of the GABAA receptor that is independent of the net direction of current flow through the ion channel. As a consequence, the time window during which GABAergic inhibition can counteract coincident excitatory inputs is much shorter, under physiological conditions, than that previously measured using high internal Cl−. This is expected to have implications for neuronal network excitability and neurodevelopment, and for our understanding of pathological conditions, such as epilepsy and chronic pain, where intracellular Cl− concentrations can be altered.

Distinct Regulation of β2 and β3 Subunit-Containing Cerebellar Synaptic GABAA Receptors by Calcium/Calmodulin-Dependent Protein Kinase II

Modulation of GABAA receptor function and inhibitory synaptic transmission by phosphorylation has profound consequences for the control of synaptic plasticity and network excitability. We have established that activating α-calcium/calmodulin-dependent protein kinase II (α-CaMK-II) in cerebellar granule neurons differentially affects populations of IPSCs that correspond to GABAA receptors containing different subtypes of β subunit. By using transgenic mice, we ascertained that α-CaMK-II increased IPSC amplitude but not the decay time by acting via β2 subunit-containing GABAA receptors. In contrast, IPSC populations whose decay times were increased by α-CaMK-II were most likely mediated by β3 subunit-containing receptors. Expressing α-CaMK-II with mutations that affected kinase function revealed that Ca2+ and calmodulin binding is crucial for α-CaMK-II modulation of GABAA receptors, whereas kinase autophosphorylation is not. These findings have significant consequences for understanding the role of synaptic GABAA receptor heterogeneity within neurons and the precise regulation of inhibitory transmission by CaMK-II phosphorylation.

Deficits in spatial memory correlate with modified {gamma}-aminobutyric acid type A receptor tyrosine phosphorylation in the hippocampus

Fast synaptic inhibition in the brain is largely mediated by γ-aminobutyric acid receptors (GABAAR). While the pharmacological manipulation of GABAAR function by therapeutic agents, such as benzodiazepines can have profound effects on neuronal excitation and behavior, the endogenous mechanisms neurons use to regulate the efficacy of synaptic inhibition and their impact on behavior remains poorly understood. To address this issue, we created a knock-in mouse in which tyrosine phosphorylation of the GABAARs γ2 subunit, a posttranslational modification that is critical for their functional modulation, has been ablated. These animals exhibited enhanced GABAAR accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the CA3 subdomain of the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. This enhanced inhibition correlated with a specific deficit in spatial object recognition, a behavioral paradigm dependent upon CA3. Thus, phospho-dependent regulation of GABAAR function involving just two tyrosine residues in the γ2 subunit provides an input-specific mechanism that not only regulates the efficacy of synaptic inhibition, but has behavioral consequences.

Identification of the Sites for CaMK-II-dependent Phosphorylation of GABAA Receptors

Phosphorylation can affect both the function and trafficking of GABAA receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABAA receptor β and γ subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2). Specifically, CaMK-II has been demonstrated to phosphorylate the M3-4 loop of GABAA receptor subunits expressed as GST fusion proteins (3, 4). It also increases the amplitude of GABAA receptor-mediated currents in a number of neuronal cell types (5-7). To identify which substrate sites CaMK-II might phosphorylate and the consequent functional effects, we expressed recombinant GABAA receptors in NG108-15 cells, which have previously been shown to support CaMK-II modulation of GABAA receptors containing the β3 subunit (8). We now demonstrate that CaMK-II mediates its effects on α1β3 receptors via phosphorylation of Ser383 within the M3-4 domain of the β subunit. Ablation of β3 subunit phosphorylation sites for CaMK-II revealed that for αβγ receptors, CaMK-II has a residual effect on GABA currents that is not mediated by previously identified sites of CaMK-II phosphorylation. This residual effect is abolished by mutation of tyrosine phosphorylation sites, Tyr365 and Tyr367, on the γ2S subunit, and by the tyrosine kinase inhibitor genistein. These results suggested that CaMK-II is capable of directly phosphorylating GABAA receptors and activating endogenous tyrosine kinases to phosphorylate the γ2 subunit in NG108-15 cells. These findings were confirmed in a neuronal environment by expressing recombinant GABAA receptors in cerebellar granule neurons.

Copper Block of Extrasynaptic GABAA Receptors in the Mature Cerebellum and Striatum

Inhibition of GABAA receptors by Cu2+ has been appreciated for some time, but differences between synaptic and extrasynaptic GABAA receptors have not been explored. We show that Cu2+ potently blocks steady-state GABA currents mediated by extrasynaptic δ subunit-containing GABAA receptors (δ-GABAARs) with an IC50 of 65 nm. This compares with an IC50 of 85 μm for synaptic γ subunit-containing GABAARs (γ-GABAARs). To test the significance of this subunit selectivity, we examined the blocking action of Cu2+ on neurons of the mouse cerebellum and striatum, brain regions that are known to express both types of receptor. Cu2+ was shown to significantly reduce tonic inhibition mediated by extrasynaptic δ-GABAARs with little action on phasic inhibition mediated by conventional synaptic γ-GABAARs. We speculate on the implications of these observations for conditions, such as Wilsons disease, that can involve raised Cu2+ levels in the brain.

The contribution of δ subunit-containing GABAA receptors to phasic and tonic conductance changes in cerebellum, thalamus and neocortex.

We have made use of the δ subunit-selective allosteric modulator DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide) to assay the contribution of δ-GABAARs to tonic and phasic conductance changes in the cerebellum, thalamus and neocortex. In cerebellar granule cells, an enhancement of the tonic conductance was observed for DS2 and the orthosteric agonist THIP (4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol). As expected, DS2 did not alter the properties of GABAA receptor-mediated inhibitory postsynaptic synaptic conductances (IPSCs) supporting a purely extrasynaptic role for δ-GABAARs in cerebellar granule cells. DS2 also enhanced the tonic conductance recorded from thalamic relay neurons of the visual thalamus with no alteration in IPSC properties. However, in addition to enhancing the tonic conductance DS2 also slowed the decay of IPSCs recorded from layer II/III neocortical neurons. A slowing of the IPSC decay also occurred in the presence of the voltage-gated sodium channel blocker TTX. Moreover, under conditions of reduced GABA release the ability of DS2 to enhance the tonic conductance was attenuated. These results indicate that δ-GABAARs can be activated following vesicular GABA release onto neocortical neurons and that the actions of DS2 on the tonic conductance may be influenced by the ambient GABA levels present in particular brain regions.

Excitatory Pathways from the Lateral Habenula Enable Propofol-Induced Sedation

Summary The lateral habenula has been widely studied for its contribution in generating reward-related behaviors [1, 2]. We have found that this nucleus plays an unexpected role in the sedative actions of the general anesthetic propofol. The lateral habenula is a glutamatergic, excitatory hub that projects to multiple targets throughout the brain, including GABAergic and aminergic nuclei that control arousal [3, 4, 5]. When glutamate release from the lateral habenula in mice was genetically blocked, the ability of propofol to induce sedation was greatly diminished. In addition to this reduced sensitivity to propofol, blocking output from the lateral habenula caused natural non-rapid eye movement (NREM) sleep to become highly fragmented, especially during the rest (“lights on”) period. This fragmentation was largely reversed by the dual orexinergic antagonist almorexant. We conclude that the glutamatergic output from the lateral habenula is permissive for the sedative actions of propofol and is also necessary for the consolidation of natural sleep.