SummaryHistaminergic 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.

The gamma-aminobutyric acid type A (GABA(A)) receptor is a pentameric ligand-gated ion channel responsible for fast synaptic inhibition in the brain. Phosphorylation of the GABA(A) receptor by serine/threonine protein kinases, at residues located in the intracellular loop between the third and fourth transmembrane domains of each subunit, can dynamically modulate receptor trafficking and function. In this study, we have assessed the effect that Ca(2+)-calmodulin-dependent protein kinase-II (CaMK-II) has on GABA(A) receptors. The intracellular application of preactivated CaMK-II failed to modulate the function of alphabeta and alphabetagamma subunit GABA(A) receptors heterologously expressed in human embryonic kidney (HEK)293 cells. However, application of similarly preactivated alpha-CaMK-II significantly potentiated the amplitudes of whole-cell GABA currents recorded from rat cultured cerebellar granule neurons and from recombinant GABA(A) receptors expressed in neuroblastoma, NG108-15, cells. The modulation by alpha-CaMK-II of current amplitude depended upon the subunit composition of GABA(A) receptors. alpha-CaMK-II potentiated GABA currents recorded from alpha1beta3 and alpha1beta3gamma2 GABA(A) receptors, but was unable to functionally modulate beta2 subunit-containing receptors. Similar results were obtained from beta2 -/- mouse cerebellar granule cell cultures and from rat granule cell cultures overexpressing recombinant alpha1beta2 or alpha1beta3 GABA(A) receptors. alpha-CaMK-II had a greater effect on the modulation of GABA responses mediated by alpha1beta3gamma2 compared with alpha1beta3 receptors, indicating a possible role for the gamma2 subunit in CaMK-II-mediated phosphorylation. In conclusion, CaMK-II can upregulate the function of GABA(A) receptors expressed in neurons or a neuronal cell line that is dependent on the beta subunit co-assembled into the receptor complex.

High-affinity extrasynaptic GABA A 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 Ͻ100 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 GABA A 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 GABA A receptors in the treatment of sleep disorders and other neurological conditions.

SummaryThe 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.

Fast synaptic inhibition in the brain is largely mediated by ␥-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 ␥2 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 ␥2 subunit provides an input-specific mechanism that not only regulates the efficacy of synaptic inhibition, but has behavioral consequences.cognition ͉ GABAA receptor ͉ inhibitory synapses G ABA A Rs are the principal sites of fast synaptic inhibition in the adult brain and are the therapeutic sites of action for benzodiazepines and barbiturates (1, 2). In the adult brain, the majority of benzodiazepine synaptic GABA A R subtypes are hetero-pentamers that are primarily assembled from ␣1-3, ␤1-3, and ␥2 subunits (1, 2). A critical determinant for the efficacy of synaptic inhibition is the number of GABA A Rs that are present at inhibitory postsynaptic sites, a process that is subject to dynamic modulation via the phosphorylation of residues within the intracellular domains of individual receptor subunits (3-5, 6, 7). In vitro experiments have revealed that phosphorylation modulates both channel kinetics and receptor membrane trafficking, however the role of GABA A R phosphorylation in shaping the efficacy of synaptic inhibition and affecting behavior remain unknown (5).To begin to explore this issue, we have created a knock-in mouse in which the principal sites of tyrosine phosphorylation within GABA A Rs, residues Y365 and Y367 within the ␥2 subunit, have been mutated to phenylalanines. The resulting animal model provides clear evidence that GABA A R phosphorylation regulates the efficacy of synaptic inhibition by modulating their membrane trafficking in the endocytic pathway in the brain and also influences some aspects of hippocampal-dependent cognition. Results Mutation of Tyrosine Phosphorylation in the ␥2 Subunit Results inEmbryonic Lethality. To explore the role that phosphorylation of GABA A Rs plays in determining the efficacy of synaptic inhibition, we created a mouse in which the principal sites of tyrosine phosphorylation with the ␥2 subunit gene were mutated to phenylalanines (Y365/7F) (Fig. 1A). Linearized DNA was used to ele...

Phosphorylation can affect both the function and trafficking of GABA A receptors with significant consequences for neuronal excitability. Serine/threonine kinases can phosphorylate the intracellular loops between M3-4 of GABA A receptor ␤ and ␥ subunits thereby modulating receptor function in heterologous expression systems and in neurons (1, 2 The ␥-aminobutyric acid type A (GABA A ) 2 receptor is a pentameric ligand-gated ion channel responsible for fast synaptic and tonic inhibition in the brain. The function of GABA A receptors can be modulated by phosphorylation, which affects inhibitory synaptic plasticity and thus has significant consequences for the control of neuronal network excitability (9, 10). Phosphorylation of the intracellular domains between M3-4 of the ␤ and ␥ subunits by serine (Ser)/threonine (Thr) and tyrosine (Tyr) kinases has been shown to modulate receptor function either through a direct effect on receptor properties, such as the probability of channel opening or desensitization, or by regulating trafficking of the receptor to and from the cell surface (1, 2, 9).The use of glutathione-based fusion proteins and site-directed mutagenesis has enabled the sites of phosphorylation within ␤ and ␥ subunits to be identified. The ␤2 subunit has one main site for phosphorylation at Ser 410 (the equivalent of Ser 409

Modulation of GABA A 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 ␣-calcium/calmodulin-dependent protein kinase II (␣-CaMK-II) in cerebellar granule neurons differentially affects populations of IPSCs that correspond to GABA A receptors containing different subtypes of ␤ subunit. By using transgenic mice, we ascertained that ␣-CaMK-II increased IPSC amplitude but not the decay time by acting via ␤2 subunit-containing GABA A receptors. In contrast, IPSC populations whose decay times were increased by ␣-CaMK-II were most likely mediated by ␤3 subunit-containing receptors. Expressing ␣-CaMK-II with mutations that affected kinase function revealed that Ca 2ϩ and calmodulin binding is crucial for ␣-CaMK-II modulation of GABA A receptors, whereas kinase autophosphorylation is not. These findings have significant consequences for understanding the role of synaptic GABA A receptor heterogeneity within neurons and the precise regulation of inhibitory transmission by CaMK-II phosphorylation.

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 Ϫ concentrations than with symmetrical Cl Ϫ solutions. This effect of intracellular Cl Ϫ is due to a direct modulation of the GABA A 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 Ϫ . 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 Ϫ concentrations can be altered.