Compromising the phosphodependent regulation of the GABAAR ß3 subunit reproduces the core phenotypes of autism spectrum disorders.

Alterations in the efficacy of neuronal inhibition mediated by GABAA receptors (GABAARs) containing ß3 subunits are continually implicated in autism spectrum disorders (ASDs). In vitro, the plasma membrane stability of GABAARs is potentiated via phosphorylation of serine residues 408 and 409 (S408/9) in the ß3 subunit, an effect that is mimicked by their mutation to alanines. To assess if modifications in ß3 subunit expression contribute to ASDs, we have created a mouse in which S408/9 have been mutated to alanines (S408/9A). S408/9A homozygotes exhibited increased phasic, but decreased tonic, inhibition, events that correlated with alterations in the membrane stability and synaptic accumulation of the receptor subtypes that mediate these distinct forms of inhibition. S408/9A mice exhibited alterations in dendritic spine structure, increased repetitive behavior, and decreased social interaction, hallmarks of ASDs. ASDs are frequently comorbid with epilepsy, and consistent with this comorbidity, S408/9A mice exhibited a marked increase in sensitivity to seizures induced by the convulsant kainic acid. To assess the relevance of our studies using S408/9A mice for the pathophysiology of ASDs, we measured S408/9 phosphorylation in Fmr1 KO mice, a model of fragile X syndrome, the most common monogenetic cause of ASDs. Phosphorylation of S408/9 was selectively and significantly enhanced in Fmr1 KO mice. Collectively, our results suggest that alterations in phosphorylation and/or activity of ß3-containing GABAARs may directly contribute to the pathophysiology of ASDs.

Psychosocial factors associated with medication adherence in ethnically and socioeconomically diverse patients with epilepsy.

The current study examined psychosocial correlates of medication adherence in a socioeconomically and racially diverse sample of patients with epilepsy. Fifty-five patients with epilepsy completed standardized self-report questionnaires measuring depression, stress, social support, and medication and illness beliefs. Antiepileptic drug (AED) adherence was measured using the 8-item Morisky Medication Adherence Scale 36% reported poor adherence. We tested which psychosocial factors were independently and most strongly associated with AED adherence. Stress and depression were negatively correlated with adherence, while perceived social support was positively correlated with adherence (Ps<.05). When all three of these variables and relevant covariates in a multiple regression model were included, only perceived social support remained a significant predictor of adherence (P=.015). This study is one of the first to suggest the importance of targeting social support in screening and intervention approaches in order to improve AED adherence among low-income, racially/ethnically diverse patients with epilepsy.

The ability of BDNF to modify neurogenesis and depressive-like behaviors is dependent upon phosphorylation of tyrosine residues 365/367 in the GABA(A)-receptor γ2 subunit.

Brain-derived neurotrophic factor (BDNF) is a potent regulator of neuronal activity, neurogenesis, and depressive-like behaviors; however, downstream effectors by which BDNF exerts these varying actions remain to be determined. Here we reveal that BDNF induces long-lasting enhancements in the efficacy of synaptic inhibition by stabilizing γ2 subunit-containing GABA(A) receptors (GABA(A)Rs) at the cell surface, leading to persistent reductions in neuronal excitability. This effect is dependent upon enhanced phosphorylation of tyrosines 365 and 367 (Y365/7) in the GABA(A)R γ2 subunit as revealed using mice in which these residues have been mutated to phenyalanines (Y365/7F). Heterozygotes for this mutation exhibit an antidepressant-like phenotype, as shown using behavioral-despair models of depression. In addition, heterozygous Y365/7F mice show increased levels of hippocampal neurogenesis, which has been strongly connected with antidepressant action. Both the antidepressant phenotype and the increased neurogenesis seen in these mice are insensitive to further modulation by BDNF, which produces robust antidepressant-like activity and neurogenesis in wild-type mice. Collectively, our results suggest a critical role for GABA(A)R γ2 subunit Y365/7 phosphorylation and function in regulating the effects of BDNF.

Enhanced tonic inhibition influences the hypnotic and amnestic actions of the intravenous anesthetics etomidate and propofol.

Intravenous anesthetics exert a component of their actions via potentiating inhibitory neurotransmission mediated by γ-aminobutyric type-A receptors (GABAARs). Phasic and tonic inhibition is mediated by distinct populations of GABAARs, with the majority of phasic inhibition by subtypes composed of α1-3ßγ2 subunits, whereas tonic inhibition is dependent on subtypes assembled from α4-6ßδ subunits. To explore the contribution that these distinct forms of inhibition play in mediating intravenous anesthesia, we have used mice in which tyrosine residues 365/7 within the γ2 subunit are mutated to phenyalanines (Y365/7F). Here we demonstrate that this mutation leads to increased accumulation of the α4 subunit containing GABAARs in the thalamus and dentate gyrus of female Y365/7F but not male Y365/7F mice. Y365/7F mice exhibited a gender-specific enhancement of tonic inhibition in the dentate gyrus that was more sensitive to modulation by the anesthetic etomidate, together with a deficit in long-term potentiation. Consistent with this, female Y365/7F, but not male Y365/7F, mice exhibited a dramatic increase in the duration of etomidate- and propofol-mediated hypnosis. Moreover, the amnestic actions of etomidate were selectively potentiated in female Y365/7F mice. Collectively, these observations suggest that potentiation of tonic inhibition mediated by α4 subunit containing GABAARs contributes to the hypnotic and amnestic actions of the intravenous anesthetics, etomidate and propofol.

Tyrosine phosphorylation regulates the membrane trafficking of the potassium chloride co-transporter KCC2.

The activity of the neuronal-specific potassium chloride co-transporter KCC2 allows neurons to maintain low intracellular Cl(-) concentrations. These low Cl(-) concentrations are critical in mediating fast synaptic inhibition upon the activation of Cl(-)-permeable ligand-gated ion channels such as type A gamma-aminobutyric acid receptors (GABA(A)Rs). Deficits in KCC2 functional expression thus play central roles in the etiology of epilepsy and ischemia. It is emerging that KCC2 is phosphorylated on tyrosine residues, but the molecular substrates for this covalent modification within KCC2 and its functional significance remain poorly understood. Here we demonstrate that in HEK-293 cells the principal sites of tyrosine phosphorylation within KCC2 are residues 903 and 1087 (Y903/1087), which lie within the major C-terminal intracellular domain of KCC2. Phosphorylation of Y903/1087 decreases the cell surface stability of KCC2 principally by enhancing their lysozomal degradation. We further demonstrate that in cultured hippocampal neurons prolonged activation of muscarinic acetylcholine receptors (mAChRs) enhances KCC2 tyrosine phosphorylation and lysozomal degradation. Consistent with our in vitro studies, induction of status epilepticus (SE) in mice using pilocarpine, a mAChR agonist, induces large deficits in the cell surface stability of KCC2 together with enhanced tyrosine phosphorylation. Tyrosine phosphorylation of KCC2 is thus likely to play a key role in regulating the degradation of KCC2, a process that may be responsible for pathological losses of KCC2 function that are evident in SE and other forms of epilepsy.

Impaired GABA(A) receptor endocytosis and its correlation to spatial memory deficits.

GABA(A) receptors mediate the majority of fast synaptic inhibition in the mammalian brain. Mechanisms that regulate GABA(A) function are thus of critical importance in modulating overall synaptic inhibition. Phosphorylation of GABA(A) receptor subunits is one such mechanism that leads to the dynamic modulation of GABA(A) receptor function. In particular, phosphorylation of tyrosine residues 365 and 367 (Y365, Y367) within the gamma2 subunit of GABA(A) receptors has been shown in previous in vitro studies to negatively regulate clathrin-dependent endocytosis of GABA(A) receptors and to enhance the efficacy of synaptic inhibition. With the aim of investigating the impact of this phosphorylation-dependent modulation of GABA(A) receptors on animal behavior, we recently developed a knock-in mouse in which these critical tyrosine residues within the gamma2 subunit have been mutated to phenylalanines (Y365/7F). These animals exhibited enhanced GABA(A) receptor accumulation at postsynaptic inhibitory synaptic specializations on pyramidal neurons within the hippocampus, primarily due to aberrant trafficking within the endocytic pathway. We found that this enhanced inhibition correlated with a specific deficit in spatial memory in these mice, without modifying a number of other behavioral paradigms. Here, we summarize our recently reported observations and further discuss their possible implications.

Activity patterns in the prefrontal cortex and hippocampus during and after awakening from etomidate anesthesia.

BACKGROUND: The anesthetic properties of etomidate are largely mediated by gamma-aminobutyric acid type A receptors. There is evidence for the existence of gamma-aminobutyric acid type A receptor subtypes in the brain, which respond to small concentrations of etomidate. After awakening from anesthesia, these subtypes are expected to cause cognitive dysfunction for a yet unknown period of time. The corresponding patterns of brain electrical activity and the molecular identity of gamma-aminobutyric acid type A receptors contributing to these actions remain to be elucidated. METHODS: Anesthesia was induced in wild-type and beta3(N265M) knock-in mice by intravenous injection of 10 mg/kg etomidate. Local field potentials were recorded simultaneously in the prefrontal cortex and hippocampus using chronically implanted electrode arrays. Local field potentials were sampled before, during, and after anesthesia. RESULTS: In the prefrontal cortex and hippocampus of wild-type mice, intravenous bolus injection of etomidate evoked isoelectric baselines and subsequent burst suppression. These effects were largely attenuated by the beta3(N265M) mutation. During emergence from anesthesia, power density in the theta band (5-15 Hz) transiently increased in the hippocampus of wild types, but not in the mutants, indicating that this action was caused by the receptors harboring beta3 subunits. In both genotypes, etomidate produced a long-lasting (> 1 h after recovery of righting reflexes) decrease in theta-peak frequency. Significant slowing of theta activity was apparent in the hippocampus and prefrontal cortex. CONCLUSIONS: Etomidate-induced patterns of brain activity during deep anesthesia mostly involve actions at beta3 containing gamma-aminobutyric acid type A receptors. During the postanesthesia period, altered theta-band activity indicates ongoing anesthetic action.

Fyn kinase contributes to tyrosine phosphorylation of the GABA(A) receptor gamma2 subunit.

Phosphorylation of GABA(A) receptors is an important mechanism for dynamically modulating inhibitory synaptic function in the mammalian brain. In particular, phosphorylation of tyrosine residues 365 and 367 (Y365/7) within the GABA(A) receptor gamma2 subunit negatively regulates the endocytosis of GABA(A) receptors and enhances synaptic inhibition. Here we show that Fyn, a Src family kinase (SFK), interacts with the gamma2 subunit in a phosphorylation-dependent manner. Furthermore, we demonstrate that Fyn binds within a region of the gamma2 intracellular domain that is centered on residues Y365/7, with the phosphorylation of Y367 being particularly important for mediating this interaction. Tyrosine phosphorylation of the gamma2 subunit is significantly reduced in the hippocampus of Fyn knockout mice, suggesting that Fyn is an important kinase that contributes to the phosphorylation of this subunit in vivo. Tyrosine phosphorylation of the gamma2 subunit is not completely abolished in Fyn kinase mice, suggesting that other SFKs, such as Src, also contribute to maintaining and regulating the endogenous phosphorylation level of gamma2-containing GABA(A) receptors. In summary, we demonstrate Fyn as one of the SFKs that binds to and phosphorylates the gamma2 subunit of the GABA(A) receptor. This has important implications for the regulation of synaptic GABA(A) receptors via signaling pathways that lead to the activation of Fyn kinase.

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 gamma-aminobutyric acid receptors (GABA(A)R). While the pharmacological manipulation of GABA(A)R 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 GABA(A)Rs gamma2 subunit, a posttranslational modification that is critical for their functional modulation, has been ablated. These animals exhibited enhanced GABA(A)R 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 GABA(A)R function involving just two tyrosine residues in the gamma2 subunit provides an input-specific mechanism that not only regulates the efficacy of synaptic inhibition, but has behavioral consequences.

Distinct actions of etomidate and propofol at beta3-containing gamma-aminobutyric acid type A receptors.

Etomidate and propofol have clearly distinguishable effects on the central nervous system. However, studies in knock-in mice provided evidence that these agents produce anesthesia via largely overlapping molecular targets, namely GABA(A) receptors containing beta3 subunits. Here the authors address the question as to whether etomidate and propofol are targeting different subpopulations of beta3 subunit containing GABA(A) receptors. The effects of etomidate and propofol (0.5 muM and 1.0 muM) on spontaneous activity of neocortical neurons were investigated in organotypic slice cultures from wild-type and beta3(N265M) knock-in mice. Firing patterns were characterized by mean burst length and number of action potentials per burst. Additionally, etomidate and propofol actions on GABA(A) receptor-mediated currents were investigated by whole-cell voltage clamp recordings. On the network level, the duration of spontaneously occurring bursts of action potentials was decreased by etomidate but increased by propofol in the wild-type. The effects of etomidate were abolished in beta3(N265M) mutant slices while those of propofol were qualitatively inverted. On the receptor level, GABA(A) receptor-mediated inhibition of cortical neurons was modulated by etomidate and propofol in different ways. Again, drug-specific actions of etomidate and propofol were largely attenuated by the beta3(N265M) mutation. Etomidate and propofol alter the firing patterns and GABA(A) receptor-mediated inhibition of neocortical neurons in different ways. This suggests that etomidate and propofol act via non-uniform molecular targets. Because the major effects induced by these anesthetics were attenuated by the beta3(N265M) mutation, different subpopulations of beta3-containing GABA(A) receptors are likely to be involved.

The involvement of hypothalamic sleep pathways in general anesthesia: testing the hypothesis using the GABAA receptor beta3N265M knock-in mouse.

The GABA(A) receptor has been identified as the single most important target for the intravenous anesthetic propofol. How effects at this receptor are then translated into a loss of consciousness, however, remains a mystery. One possibility is that anesthetics act on natural sleep pathways. Here, we test this hypothesis by exploring the anesthetic sensitivities of GABAergic synaptic currents in three specific brain nuclei that are known to be involved in sleep. Using whole-cell electrophysiology, we have recorded GABAergic IPSCs from the tuberomammillary nucleus (TMN), the perifornical area (Pef), and the locus ceruleus (LC) in brain slices from both wild-type mice and mice that carry a specific mutation in the GABA(A) receptor beta(3) subunit (N265M), which greatly reduces their sensitivity to propofol, but not to the neurosteroid alphaxalone. We find that this in vivo pattern of anesthetic sensitivity is mirrored in the hypothalamic TMN and Pef nuclei, consistent with their role as direct anesthetic targets. In contrast, anesthetic sensitivity in the LC was unaffected by the beta(3)N265M mutation, ruling out this nucleus as a major target for propofol. In support of the hypothesis that orexinergic neurons in the Pef are involved in propofol anesthesia, we further show that these neurons are selectively inhibited by GABAergic drugs in vivo during anesthesia, and that a modulation in the activity of Pef neurons alone can affect loss of righting reflex. Overall, our results support the idea that GABAergic anesthetics such as propofol exert their effects, at least in part, by modulating hypothalamic sleep pathways.

Alpha4-containing GABAA receptors in the nucleus accumbens mediate moderate intake of alcohol.

Alcohol has subjective and behavioral effects at the pharmacological levels typically reached during the consumption of one or two alcoholic drinks. Here we provide evidence that an alpha4-subunit-containing GABA(A) receptor contributes to the consumption of low-to-moderate levels of alcohol. Using viral-mediated RNA interference (RNAi), we found that reduced expression of the alpha4 subunit in the nucleus accumbens (NAc) shell of rats decreased their free consumption of and preference for alcohol. The time course for the reduced alcohol intake paralleled the time course of alpha4 mRNA reductions achieved after viral-mediated RNAi for alpha4. Furthermore, the reduction in drinking was region- and alcohol-specific: there was no effect of reductions in alpha4 expression in the NAc core on alcohol intake, and reductions in alpha4 expression in the NAc shell did not alter sucrose or water intake. These results indicate that the GABA(A) receptor alpha4 subunit in the NAc shell mediates alcohol intake.

GABA(A) receptor trafficking and its role in the dynamic modulation of neuronal inhibition.

GABA (gamma-aminobutyric acid) type A receptors (GABA(A)Rs) mediate most fast synaptic inhibition in the mammalian brain, controlling activity at both the network and the cellular levels. The diverse functions of GABA in the CNS are matched not just by the heterogeneity of GABA(A)Rs, but also by the complex trafficking mechanisms and protein-protein interactions that generate and maintain an appropriate receptor cell-surface localization. In this Review, we discuss recent progress in our understanding of the dynamic regulation of GABA(A)R composition, trafficking to and from the neuronal surface, and lateral movement of receptors between synaptic and extrasynaptic locations. Finally, we highlight a number of neurological disorders, including epilepsy and schizophrenia, in which alterations in GABA(A)R trafficking occur.

Mind bomb-2 is an E3 ligase that ubiquitinates the N-methyl-D-aspartate receptor NR2B subunit in a phosphorylation-dependent manner.

The N-methyl-D-aspartate receptor (NMDAR) plays a critical role in synaptic plasticity. Post-translational modifications of NMDARs, such as phosphorylation, alter both the activity and trafficking properties of NMDARs. Ubiquitination is increasingly being recognized as another post-translational modification that can alter synaptic protein composition and function. We identified Mind bomb-2 as an E3 ubiquitin ligase that interacts with and ubiquitinates the NR2B subunit of the NMDAR in mammalian cells. The protein-protein interaction and the ubiquitination of the NR2B subunit were found to be enhanced in a Fyn phosphorylation-dependent manner. Immunocytochemical studies reveal that Mind bomb-2 is localized to postsynaptic sites and colocalizes with the NMDAR in apical dendrites of hippocampal neurons. Furthermore, we show that NMDAR activity is down-regulated by Mind bomb-2. These results identify a specific E3 ubiquitin ligase as a novel interactant with the NR2B subunit and suggest a possible mechanism for the regulation of NMDAR function involving both phosphorylation and ubiquitination.

Modulation of presynaptic beta3-containing GABAA receptors limits the immobilizing actions of GABAergic anesthetics.

Intravenous GABAergic anesthetics are potent hypnotics but are rather ineffective in depressing movements. Immobility is mediated, in part, by the ventral horn of the spinal cord. We hypothesized that the efficacy of these anesthetics in producing immobility is compromised by the activation of GABA(A) receptors located presynaptically, which modulate GABA release onto neurons in the ventral horn. Because anesthetics acting by modulation of GABA(A) receptor function require GABA to be present at its binding site, a decrease in GABA release would abate their efficacy in reducing neuronal excitability. Here we report that in organotypic spinal cord slices, the efficacy of the intravenous anesthetic etomidate to depress network activity of ventral horn neurons is limited to approximately 60% at concentrations greater than 1 microM that produce immobility. Depression of spinal network activity was almost abolished in spinal slices from beta3(N265M) knock-in mice. In the wild type, etomidate prolonged decay times of GABA(A) receptor-mediated inhibitory postsynaptic currents (IPSCs) and concomitantly reduced the frequency of action potential-dependent IPSCs. Etomidate prolonged the decay time of GABA(A) receptors at all tested concentrations. At concentrations greater than 1.0 microM, anesthetic-induced decrease of GABA release via modulation of presynaptic GABA(A) receptors and enhancement of postsynaptic GABA(A) receptor-function compensated for each other. The results suggest that the limited immobilizing efficacy of these agents is probably due to a presynaptic mechanism and that GABAergic agents with a specificity for post-versus presynaptic receptors would probably have much stronger immobilizing actions, pointing out novel avenues for drug development.

Etomidate and propofol-hyposensitive GABAA receptor beta3(N265M) mice show little changes in acute alcohol sensitivity but enhanced tolerance and withdrawal.

Gamma-aminobutyric acid-A (GABAA) receptors are ligand-gated ion channels comprised of subunits from several classes (alpha, beta, gamma, delta). Recent studies have clearly demonstrated that the functional properties of GABAA receptors are altered following chronic ethanol administration that could provide the molecular basis for the previously proposed role of these receptors in ethanol tolerance and dependence. Because the subunit composition of GABAA receptors determines receptor pharmacology, the present study was devoted to assess if the behavioral sensitivity after acute and chronic ethanol exposure depends on beta3-containing GABAA receptors. In the present study, we used knock-in mice harboring a point mutation (N265M) in the second transmembrane region of the beta3 subunit of the GABAA receptor in order to study acute and chronic behavioral effects of ethanol. More specifically, we tested tolerance to loss of righting reflex (LORR) and the development of withdrawal signs after chronic ethanol exposure using ethanol vapor chambers. Our results show that the beta3(N265M) mutation does not play a major modulatory role of acute ethanol-induced LORR. However, following repeated LORR testing, enhanced tolerance to the intoxicating effects of ethanol was observed--a finding which was unrelated to the pharmacokinetics of ethanol as both genotypes had the same blood alcohol concentrations following repeated LORR testing. In addition, following chronic alcohol vapor exposure, mouse mutants displayed increased handling-induced convulsions during withdrawal. The results of the present study suggest that the alcohol effects abolished by the beta3(N265M) mutation do not play a dominant role in acute alcohol intoxication but influence ethanol tolerance and withdrawal.

Mapping the contribution of beta3-containing GABAA receptors to volatile and intravenous general anesthetic actions.

BACKGROUND: Agents belonging to diverse chemical classes are used clinically as general anesthetics. The molecular targets mediating their actions are however still only poorly defined. Both chemical diversity and substantial differences in the clinical actions of general anesthetics suggest that general anesthetic agents may have distinct pharmacological targets. It was demonstrated previously that the immobilizing action of etomidate and propofol is completely, and the immobilizing action of isoflurane partly mediated, by beta3-containing GABAA receptors. This was determined by using the beta3(N265M) mice, which carry a point mutation known to decrease the actions of general anesthetics at recombinant GABAA receptors. In this communication, we analyzed the contribution of beta3-containing GABAA receptors to the pharmacological actions of isoflurane, etomidate and propofol by means of beta3(N265M) mice. RESULTS: Isoflurane decreased core body temperature and heart rate to a smaller degree in beta3(N265M) mice than in wild type mice, indicating a minor but significant role of beta3-containing GABAA receptors in these actions. Prolonged time intervals in the ECG and increased heart rate variability were indistinguishable between genotypes, suggesting no involvement of beta3-containing GABAA receptors. The anterograde amnesic action of propofol was indistinguishable in beta3(N265M) and wild type mice, suggesting that it is independent of beta3-containing GABAA receptors. The increase of heart rate variability and prolongation of ECG intervals by etomidate and propofol were also less pronounced in beta3(N265M) mice than in wild type mice, pointing to a limited involvement of beta3-containing GABAA receptors in these actions. The lack of etomidate- and propofol-induced immobilization in beta3(N265M) mice was also observed in congenic 129X1/SvJ and C57BL/6J backgrounds, indicating that this phenotype is stable across different backgrounds. CONCLUSION: Our results provide evidence for a defined role of beta3-containing GABAA receptors in mediating some, but not all, of the actions of general anesthetics, and confirm the multisite model of general anesthetic action. This pharmacological separation of anesthetic endpoints also suggests that subtype-selective substances with an improved side-effect profile may be developed.