Ammonia induces amyloidogenesis in astrocytes by promoting amyloid precursor protein translocation into the endoplasmic reticulum.

Hyperammonemia is known to cause various neurological dysfunctions such as seizures and cognitive impairment. Several studies have suggested that hyperammonemia may also be linked to the development of Alzheimer's disease (AD). However, the direct evidence for a role of ammonia in the pathophysiology of AD remains to be discovered. Herein, we report that hyperammonemia increases the amount of mature amyloid precursor protein (mAPP) in astrocytes, the largest and most prevalent type of glial cells in the central nervous system that are capable of metabolizing glutamate and ammonia, and promotes amyloid beta (Aß) production. We demonstrate the accumulation of mAPP in astrocytes was primarily due to enhanced endocytosis of mAPP from the plasma membrane. A large proportion of internalized mAPP was targeted not to the lysosome, but to the endoplasmic reticulum, where processing enzymes ß-secretase BACE1 (beta-site APP cleaving enzyme 1) and γ-secretase presenilin-1 are expressed, and mAPP is cleaved to produce Aß. Finally, we show the ammonia-induced production of Aß in astrocytic endoplasmic reticulum was specific to Aß42, a principal component of senile plaques in AD patients. Our studies uncover a novel mechanism of Aß42 production in astrocytes and also provide the first evidence that ammonia induces the pathogenesis of AD by regulating astrocyte function.

Direct Interaction of PP2A Phosphatase with GABAB Receptors Alters Functional Signaling.

Addictive drugs usurp the brain's intrinsic mechanism for reward, leading to compulsive and destructive behaviors. In the ventral tegmental area (VTA), the center of the brain's reward circuit, GABAergic neurons control the excitability of dopamine (DA) projection neurons and are the site of initial psychostimulant-dependent changes in signaling. Previous work established that cocaine/methamphetamine exposure increases protein phosphatase 2A (PP2A) activity, which dephosphorylates the GABABR2 subunit, promotes internalization of the GABAB receptor (GABABR) and leads to smaller GABABR-activated G-protein-gated inwardly rectifying potassium (GIRK) currents in VTA GABA neurons. How the actions of PP2A become selective for a particular signaling pathway is poorly understood. Here, we demonstrate that PP2A can associate directly with a short peptide sequence in the C terminal domain of the GABABR1 subunit, and that GABABRs and PP2A are in close proximity in rodent neurons (mouse/rat; mixed sexes). We show that this PP2A-GABABR interaction can be regulated by intracellular Ca2+ Finally, a peptide that potentially reduces recruitment of PP2A to GABABRs and thereby limits receptor dephosphorylation increases the magnitude of baclofen-induced GIRK currents. Thus, limiting PP2A-dependent dephosphorylation of GABABRs may be a useful strategy to increase receptor signaling for treating diseases.SIGNIFICANCE STATEMENT Dysregulation of GABAB receptors (GABABRs) underlies altered neurotransmission in many neurological disorders. Protein phosphatase 2A (PP2A) is involved in dephosphorylating and subsequent internalization of GABABRs in models of addiction and depression. Here, we provide new evidence that PP2A B55 regulatory subunit interacts directly with a small region of the C-terminal domain of the GABABR1 subunit, and that this interaction is sensitive to intracellular Ca2+ We demonstrate that a short peptide corresponding to the PP2A interaction site on GABABR1 competes for PP2A binding, enhances phosphorylation GABABR2 S783, and affects functional signaling through GIRK channels. Our study highlights how targeting PP2A dependent dephosphorylation of GABABRs may provide a specific strategy to modulate GABABR signaling in disease conditions.

Sustained laryngeal transient receptor potential vanilloid 1 activation inhibits mechanically induced swallowing in anesthetized rats.

A major component of gastric acid is hydrochloric acid (HCl), which can activate transient receptor potential vanilloid 1 (TRPV1). In the present study, we investigated how sustained laryngeal TRPV1 activation affects the frequency of the swallowing reflex. Experiments were carried out on 85 male Sprague-Dawley rats. The effects of short and sustained application of chemicals (3 µl of 0.1 N HCl or capsaicin) on the frequency of swallowing and on time-dependent changes in the occurrence of swallowing evoked by supralaryngeal nerve stimulation were determined. To evaluate vascular permeability of the larynx, Evans blue dye was intravenously injected after 5 or 60 min of sustained TRPV1 activation. SB366791 (a TRPV1 inhibitor) and Cap/QX-314 (a TRPV1-expressed neuronal inhibitor) significantly inhibited HCl/capsaicin-induced swallowing, but air flow-induced swallowing was not affected. Although the number of air flow-induced swallows followed by capsaicin stimulation was not affected within 5 min, it was significantly reduced by 60-min capsaicin or HCl application. The swallowing threshold associated with supralaryngeal nerve stimulation did not significantly change throughout the recording period. Evans blue dye concentrations in the larynx were significantly higher at 60 min in the 10-5 M capsaicin group than in the control group. Our results suggest that sustained TPRV1 activation not only desensitizes TRPV1 but also inactivates mechanoreceptors, which may be attributed to increases in vascular permeability and edema, as part of an inflammatory process.NEW & NOTEWORTHY Although a transient receptor potential vanilloid 1 (TRPV1) inhibitor or TRPV1-expressed neuronal inhibitor significantly inhibited HCl/capsaicin-evoked swallowing, air flow-induced swallowing was not affected. The number of air flow-induced swallows was significantly reduced within 60 min of TRPV1 activation. Evans blue dye concentration in the larynx increased 60 min after capsaicin application. TPRV1 activation not only desensitizes TRPV1 but also inactivates mechanoreceptors caused by increases in vascular permeability and edema.

The dynamic modulation of GABA(A) receptor trafficking and its role in regulating the plasticity of inhibitory synapses.

Inhibition in the adult mammalian central nervous system (CNS) is mediated by γ-aminobutyric acid (GABA). The fast inhibitory actions of GABA are mediated by GABA type A receptors (GABA(A)Rs); they mediate both phasic and tonic inhibition in the brain and are the principle sites of action for anticonvulsant, anxiolytic, and sedative-hypnotic agents that include benzodiazepines, barbiturates, neurosteroids, and some general anesthetics. GABA(A)Rs are heteropentameric ligand-gated ion channels that are found concentrated at inhibitory postsynaptic sites where they mediate phasic inhibition and at extrasynaptic sites where they mediate tonic inhibition. The efficacy of inhibition and thus neuronal excitability is critically dependent on the accumulation of specific GABA(A)R subtypes at inhibitory synapses. Here we evaluate how neurons control the number of GABA(A)Rs on the neuronal plasma membrane together with their selective stabilization at synaptic sites. We then go on to examine the impact that these processes have on the strength of synaptic inhibition and behavior.

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.

Diversity of structure and function of GABAB receptors: a complexity of GABAB-mediated signaling.

γ-aminobutyric acid type B (GABAB) receptors are broadly expressed in the nervous system and play an important role in neuronal excitability. GABAB receptors are G protein-coupled receptors that mediate slow and prolonged inhibitory action, via activation of Gαi/o-type proteins. GABAB receptors mediate their inhibitory action through activating inwardly rectifying K+ channels, inactivating voltage-gated Ca2+ channels, and inhibiting adenylate cyclase. Functional GABAB receptors are obligate heterodimers formed by the co-assembly of R1 and R2 subunits. It is well established that GABAB receptors interact not only with G proteins and effectors but also with various proteins. This review summarizes the structure, subunit isoforms, and function of GABAB receptors, and discusses the complexity of GABAB receptors, including how receptors are localized in specific subcellular compartments, the mechanism regulating cell surface expression and mobility of the receptors, and the diversity of receptor signaling through receptor crosstalk and interacting proteins.

A Role for the GIRK3 Subunit in Methamphetamine-Induced Attenuation of GABAB Receptor-Activated GIRK Currents in VTA Dopamine Neurons.

Repeated exposure to psychostimulants induces locomotor sensitization and leads to persistent changes in the circuitry of the mesocorticolimbic dopamine (DA) system. G-protein-gated inwardly rectifying potassium (GIRK; also known as Kir3) channels mediate a slow IPSC and control the excitability of DA neurons. Repeated 5 d exposure to psychostimulants decreases the size of the GABAB receptor (GABABR)-activated GIRK currents (IBaclofen) in ventral tegmental area (VTA) DA neurons of mice, but the mechanism underlying this plasticity is poorly understood. Here, we show that methamphetamine-dependent attenuation of GABABR-GIRK currents in VTA DA neurons required activation of both D1R-like and D2R-like receptors. The methamphetamine-dependent decrease in GABABR-GIRK currents in VTA DA neurons did not depend on a mechanism of dephosphorylation of the GABAB R2 subunit found previously for other neurons in the reward pathway. Rather, the presence of the GIRK3 subunit appeared critical for the methamphetamine-dependent decrease of GABABR-GIRK current in VTA DA neurons. Together, these results highlight different regulatory mechanisms in the learning-evoked changes that occur in the VTA with repeated exposure to psychostimulants. SIGNIFICANCE STATEMENT: Exposure to addictive drugs such as psychostimulants produces persistent adaptations in inhibitory circuits within the mesolimbic dopamine system, suggesting that addictive behaviors are encoded by changes in the reward neural circuitry. One form of neuroadaptation that occurs with repeated exposure to psychostimulants is a decrease in slow inhibition, mediated by a GABAB receptor and a potassium channel. Here, we examine the subcellular mechanism that links psychostimulant exposure with changes in slow inhibition and reveal that one type of potassium channel subunit is important for mediating the effect of repeated psychostimulant exposure. Dissecting out the components of drug-dependent plasticity and uncovering novel protein targets in the reward circuit may lead to the development of new therapeutics for treating addiction.

Neurosteroids promote phosphorylation and membrane insertion of extrasynaptic GABAA receptors.

Neurosteroids are synthesized within the brain and act as endogenous anxiolytic, anticonvulsant, hypnotic, and sedative agents, actions that are principally mediated via their ability to potentiate phasic and tonic inhibitory neurotransmission mediated by γ-aminobutyric acid type A receptors (GABAARs). Although neurosteroids are accepted allosteric modulators of GABAARs, here we reveal they exert sustained effects on GABAergic inhibition by selectively enhancing the trafficking of GABAARs that mediate tonic inhibition. We demonstrate that neurosteroids potentiate the protein kinase C-dependent phosphorylation of S443 within α4 subunits, a component of GABAAR subtypes that mediate tonic inhibition in many brain regions. This process enhances insertion of α4 subunit-containing GABAAR subtypes into the membrane, resulting in a selective and sustained elevation in the efficacy of tonic inhibition. Therefore, the ability of neurosteroids to modulate the phosphorylation and membrane insertion of α4 subunit-containing GABAARs may underlie the profound effects these endogenous signaling molecules have on neuronal excitability and behavior.

Postsynaptic GABAB receptor activity regulates excitatory neuronal architecture and spatial memory.

Cognitive dysfunction is a common symptom in many neuropsychiatric disorders and directly correlates with poor patient outcomes. The majority of prolonged inhibitory signaling in the brain is mediated via GABAB receptors (GABABRs), but the molecular function of these receptors in cognition is ill defined. To explore the significance of GABABRs in neuronal activity and cognition, we created mice with enhanced postsynaptic GABABR signaling by mutating the serine 783 in receptor R2 subunit (S783A), which decreased GABABR degradation. Enhanced GABABR activity reduced the expression of immediate-early gene-encoded protein Arc/Arg3.1, effectors that are critical for long-lasting memory. Intriguingly, S783A mice exhibited increased numbers of excitatory synapses and surface AMPA receptors, effects that are consistent with decreased Arc/Arg3.1 expression. These deficits in Arc/Arg3.1 and neuronal morphology lead to a deficit in spatial memory consolidation. Collectively our results suggest a novel and unappreciated role for GABABR activity in determining excitatory neuronal architecture and spatial memory via their ability to regulate Arc/Arg3.1.

Glutamine synthetase stability and subcellular distribution in astrocytes are regulated by γ-aminobutyric type B receptors.

Emerging evidence suggests that functional γ-aminobutyric acid B receptors (GABABRs) are expressed by astrocytes within the mammalian brain. GABABRs are heterodimeric G-protein-coupled receptors that are composed of R1/R2 subunits. To date, they have been characterized in neurons as the principal mediators of sustained inhibitory signaling; however their roles in astrocytic physiology have been ill defined. Here we reveal that the cytoplasmic tail of the GABABR2 subunit binds directly to the astrocytic protein glutamine synthetase (GS) and that this interaction determines the subcellular localization of GS. We further demonstrate that the binding of GS to GABABR2 increases the steady state expression levels of GS in heterologous cells and in mouse primary astrocyte culture. Mechanistically this increased stability of GS in the presence of GABABR2 occurs via reduced proteasomal degradation. Collectively, our results suggest a novel role for GABABRs as regulators of GS stability. Given the critical role that GS plays in the glutamine-glutamate cycle, astrocytic GABABRs may play a critical role in supporting both inhibitory and excitatory neurotransmission.

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.

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.

Behavioral analysis of kainate receptor KO mice and the role of GluK3 subunit in anxiety.

Kainate receptors (KARs) are one of the ionotropic glutamate receptors in the central nervous system (CNS) comprised of five subunits, GluK1-GluK5. There is a growing interest in the association between KARs and psychiatric disorders, and there have been several studies investigating the behavioral phenotypes of KAR deficient mice, however, the difference in the genetic background has been found to affect phenotype in multiple mouse models of human diseases. Here, we examined GluK1-5 single KO mice in a pure C57BL/6N background and identified that GluK3 KO mice specifically express anxiolytic-like behavior with an alteration in dopamine D2 receptor (D2R)-induced anxiety, and reduced D2R expression in the striatum. Biochemical studies in the mouse cortex confirmed that GluK3 subunits do not assemble with GluK4 and GluK5 subunits, that can be activated by lower concentration of agonists. Overall, we found that GluK3-containing KARs function to express anxiety, which may represent promising anti-anxiety medication targets.

Prolonged activation of NMDA receptors promotes dephosphorylation and alters postendocytic sorting of GABAB receptors.

Slow and persistent synaptic inhibition is mediated by metabotropic GABAB receptors (GABABRs). GABABRs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABABRs are predominantly found on dendritic spines, adjacent to excitatory synapses, but the control of their plasma membrane availability is still controversial. Here, we explore the role of glutamate receptor activation in regulating the function and surface availability of GABABRs in central neurons. We demonstrate that prolonged activation of NMDA receptors (NMDA-Rs) leads to endocytosis, a diversion from a recycling route, and subsequent lysosomal degradation of GABABRs. These sorting events are paralleled by a reduction in GABABR-dependent activation of inwardly rectifying K+ channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABABR2 subunit, an established substrate of AMP-dependent protein kinase (AMPK). NMDA-R activation leads to a rapid increase in phosphorylation of S783, followed by a slower dephosphorylation, which results from the activity of AMPK and protein phosphatase 2A, respectively. Agonist activation of GABABRs counters the effects of NMDA. Thus, NMDA-R activation alters the phosphorylation state of S783 and acts as a molecular switch to decrease the abundance of GABABRs at the neuronal plasma membrane. Such a mechanism may be of significance during synaptic plasticity or pathological conditions, such as ischemia or epilepsy, which lead to prolonged activation of glutamate receptors.

Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo.

Gliotransmission, the release of molecules from astrocytes, regulates neuronal excitability and synaptic transmission in situ. Whether this process affects neuronal network activity in vivo is not known. Using a combination of astrocyte-specific molecular genetics, with in vivo electrophysiology and pharmacology, we determined that gliotransmission modulates cortical slow oscillations, a rhythm characterizing nonrapid eye movement sleep. Inhibition of gliotransmission by the expression of a dominant negative SNARE domain in astrocytes affected cortical slow oscillations, reducing the duration of neuronal depolarizations and causing prolonged hyperpolarizations. These network effects result from the astrocytic modulation of intracortical synaptic transmission at two sites: a hypofunction of postsynaptic NMDA receptors, and by reducing extracellular adenosine, a loss of tonic A1 receptor-mediated inhibition. These results demonstrate that rhythmic brain activity is generated by the coordinated action of the neuronal and glial networks.

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.

The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells.

Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.

Phospho-dependent functional modulation of GABA(B) receptors by the metabolic sensor AMP-dependent protein kinase.

GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels (GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.

Astrocytic activation of A1 receptors regulates the surface expression of NMDA receptors through a Src kinase dependent pathway.

Chemical transmitters released from astrocytes, termed gliotransmitters, modulate synaptic transmission and neuronal function. Using astrocyte-specific inducible transgenicmice (dnSNARE mice), we have demonstrated that inhibiting gliotransmission leads to reduced activation of adenosine A1 receptors (A1R) and impaired sleep homeostasis (Halassa et al. (2009) Neuron 61:213-219); Pascual et al. (2005) Science 310:113-116). Additionally, synaptic N-methyl-D-aspartate receptor (NMDAR) currents are reduced in these astrocyte-specific transgenic animals (Fellin et al. (2009) Proc Natl Acad Sci USA 106:15037-15042). Because of the importance of adenosine and NMDA receptors to sleep processes we asked whether there is a causal linkage between changes in A1R activation and synaptic NMDA receptors. We show that astrocytic dnSNARE expression leads to reduced tyrosine phosphorylation of Srckinase and NR2 subunits concomitant with the decreased surface expression of the NR2 subunits. To test the role of A1R signaling in mediating these actions, we show that incubation of wildtype (WT) slices with an A1R antagonist reduces tyrosine phosphorylation of Src kinase and NR2B, decreases the surface expression of the NR2B subunits and leads to smaller NMDA component of miniature EPSCs. In dnSNARE mice we could rescue WT phenotype by incubation in an A1R agonist:activation of A1 receptor led to increased tyrosine phosphorylation of Src kinase and NR2B subunits as well as increased the surface expression of the NR2B subunit and increased NMDA component of the synaptic mEPSC. These results provide the first demonstration that astrocytes can affect neuronal excitability on a long time scale by regulating the surface expression of NMDA receptors through the activation of specific intracellular signaling pathways.

A comparative analysis of kainate receptor GluK2 and GluK5 knockout mice in a pure genetic background.

Kainate receptors (KARs) are members of the glutamate receptor family that regulate synaptic function in the brain. Although they are known to be associated with psychiatric disorders, how they are involved in these disorders remains unclear. KARs are tetrameric channels assembled from a combination of GluK1-5 subunits. Among these, GluK2 and GluK5 subunits are the major heteromeric subunits in the brain. To determine the functional similarities and differences between GluK2 and GluK5 subunits, we generated GluK2 KO and GluK5 KO mice on a C57BL/6N background, a well-characterized inbred strain, and compared their behavioral phenotypes. We found that GluK2 KO and GluK5 KO mice exhibited the same phenotypes in many tests, such as reduced locomotor activity, impaired motor function, and enhanced depressive-like behavior. No change was observed in motor learning, anxiety-like behavior, or sociability. Additionally, we identified subunit-specific phenotypes, such as reduced motivation toward their environment in GluK2 KO mice and an enhancement in the contextual memory in GluK5 KO mice. These results revealed that GluK2 and GluK5 subunits not only function in a coordinated manner but also have a subunit-specific role in regulating behavior. To summarize, we demonstrated subunit-specific and common behavioral effects of GluK2 and GluK5 subunits for the first time. Moreover, to the best of our knowledge, this is the first evidence of the involvement of the GluK5 subunit in the expression of depressive-like behavior and contextual memory, which strongly indicates its role in psychiatric disorders.