Miho Terunuma

mGluR5 stimulates gliotransmission in the nucleus accumbens

Although metabotropic glutamate receptor 5 (mGluR5) is essential for cocaine self-administration and drug-seeking behavior, there is limited knowledge of the cellular actions of this receptor in the nucleus accumbens (NAc). Although mGluR5 has the potential to regulate neurons directly, recent studies have shown the importance of mGluR5 in regulating Ca2+ signaling in astrocytes and, as a consequence, the Ca2+-dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca2+ oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca2+ loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca2+ elevations in astrocytes are responsible for these excitatory events. Pharmacological evaluation of these glial-evoked NMDA currents shows that they are mediated by NR2B-containing NMDA receptors, whereas synaptic NMDA receptors rely on NR2A-containing receptors. Stimulation of glutamatergic afferents activates mGluR5-dependent astrocytic Ca2+ oscillations and gliotransmission that is sustained for minutes beyond the initial stimulus. Because gliotransmission is mediated by NMDA receptors, depolarized membrane potentials exhibited during up-states augment excitation provided by gliotransmission, which drives bursts of MSN action potentials. Because the predominant mGluR5-dependent action of glutamatergic afferents is to cause the sustained activation of astrocytes, which in turn excite MSNs through extrasynaptic NMDA receptors, our results raise the potential for gliotransmission being involved in prolonged mGluR5-dependent adaptation in the NAc.

Disrupted Dentate Granule Cell Chloride Regulation Enhances Synaptic Excitability during Development of Temporal Lobe Epilepsy

GABAA receptor-mediated inhibition depends on the maintenance of intracellular Cl− concentration ([Cl−]in) at low levels. In neurons in the developing CNS, [Cl−]in is elevated, EGABA is depolarizing, and GABA consequently is excitatory. Depolarizing GABAergic synaptic responses may be recapitulated in various neuropathological conditions, including epilepsy. In the present study, rat hippocampal dentate granule cells were recorded using gramicidin perforated patch techniques at varying times (1–60 d) after an epileptogenic injury, pilocarpine-induced status epilepticus (STEP). In normal, non-epileptic animals, these strongly inhibited dentate granule cells act as a gate, regulating hippocampal excitation, controlling seizure initiation and/or propagation. For 2 weeks after STEP, we found that EGABA was positively shifted in granule cells. This shift in EGABA altered synaptic integration, increased granule cell excitability, and resulted in compromised “gate” function of the dentate gyrus. EGABA recovered to control values at longer latencies post-STEP (2–8 weeks), when animals had developed epilepsy. During this period of shifted EGABA, expression of the Cl− extruding K+/Cl− cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked EGABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on EGABA in granule cells, and on gatekeeper function of the dentate gyrus. This suggests a shared mechanism, reduced KCC2 function. These findings demonstrate that decreased expression of KCC2 persists for weeks after an epileptogenic injury, reducing inhibitory efficacy and enhancing dentate granule cell excitability. This pathophysiological process may constitute a significant mechanism linking injury to the subsequent development of epilepsy.

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 Phosphorylation of GABAA Receptors by Intimately Associated Protein Kinase C Activity Underlie Compromised Synaptic Inhibition during Status Epilepticus

Status epilepticus (SE) is a progressive and often lethal human disorder characterized by continuous or rapidly repeating seizures. Of major significance in the pathology of SE are deficits in the functional expression of GABAA receptors (GABAARs), the major sites of fast synaptic inhibition in the brain. We demonstrate that SE selectively decreases the phosphorylation of GABAARs on serine residues 408/9 (S408/9) in the β3 subunit by intimately associated protein kinase C isoforms. Dephosphorylation of S408/9 unmasks a basic patch-binding motif for the clathrin adaptor AP2, enhancing the endocytosis of selected GABAAR subtypes from the plasma membrane during SE. In agreement with this, enhancing S408/9 phosphorylation or selectively blocking the binding of the β3 subunit to AP2 increased GABAAR cell surface expression levels and restored the efficacy of synaptic inhibition in SE. Thus, enhancing phosphorylation of GABAARs or selectively blocking their interaction with AP2 may provide novel therapeutic strategies to ameliorate SE.

GABAA Receptor Phospho-Dependent Modulation Is Regulated by Phospholipase C-Related Inactive Protein Type 1, a Novel Protein Phosphatase 1 Anchoring Protein

GABAA receptors are critical in controlling neuronal activity. Here, we examined the role for phospholipase C-related inactive protein type 1 (PRIP-1), which binds and inactivates protein phosphatase 1α (PP1α) in facilitating GABAA receptor phospho-dependent regulation using PRIP-1-/- mice. In wild-type animals, robust phosphorylation and functional modulation of GABAA receptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished in PRIP-1-/- mice. PRIP-1-/- mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABAA receptor β subunits, and moreover, these proteins were found to be PP1α substrates. Finally, phosphorylation of PRIP-1 on threonine 94 facilitated the dissociation of PP1α-PRIP-1 complexes, providing a local mechanism for the activation of PP1α. Together, these results suggest an essential role for PRIP-1 in controlling GABAA receptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

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.

Methamphetamine-evoked depression of GABA(B) receptor signaling in GABA neurons of the VTA.

Psychostimulants induce neuroadaptations in excitatory and fast inhibitory transmission in the ventral tegmental area (VTA). Mechanisms underlying drug-evoked synaptic plasticity of slow inhibitory transmission mediated by GABA(B) receptors and G protein-gated inwardly rectifying potassium (GIRK/Kir(3)) channels, however, are poorly understood. Here, we show that 1 day after methamphetamine (METH) or cocaine exposure both synaptically evoked and baclofen-activated GABA(B)R-GIRK currents were significantly depressed in VTA GABA neurons and remained depressed for 7 days. Presynaptic inhibition mediated by GABA(B)Rs on GABA terminals was also weakened. Quantitative immunoelectron microscopy revealed internalization of GABA(B1) and GIRK2, which occurred coincident with dephosphorylation of serine 783 (S783) in GABA(B2), a site implicated in regulating GABA(B)R surface expression. Inhibition of protein phosphatases recovered GABA(B)R-GIRK currents in VTA GABA neurons of METH-injected mice. This psychostimulant-evoked impairment in GABA(B)R signaling removes an intrinsic brake on GABA neuron spiking, which may augment GABA transmission in the mesocorticolimbic system.

The Availability of Surface GABAB Receptors Is Independent of γ-Aminobutyric Acid but Controlled by Glutamate in Central Neurons

The efficacy of synaptic transmission depends on the availability of ionotropic and metabotropic neurotransmitter receptors at the plasma membrane, but the contribution of the endocytic and recycling pathways in the regulation of γ-aminobutyric acid type B (GABAB) receptors remains controversial. To understand the mechanisms that regulate the abundance of GABAB receptors, we have studied their turnover combining surface biotin labeling and a microscopic immunoendocytosis assay in hippocampal and cortical neurons. We report that internalization of GABAB receptors is agonist-independent. We also demonstrate that receptors endocytose in the cell body and dendrites but not in axons. Additionally, we show that GABAB receptors endocytose as heterodimers via clathrin- and dynamin-1-dependent mechanisms and that they recycle to the plasma membrane after endocytosis. More importantly, we show that glutamate decreases the levels of cell surface receptors in a manner dependent on an intact proteasome pathway. These observations indicate that glutamate and not GABA controls the abundance of surface GABAB receptors in central neurons, consistent with their enrichment at glutamatergic synapses.

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 transgenic mice (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 Src kinase 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.

Direct interaction of N-ethylmaleimide-sensitive factor with GABAA receptor β subunits

GABAA receptors mediate most of the fast inhibitory neurotransmission in the brain, and are believed to be composed mainly of α, β, and γ subunits. It has been shown that GABAA receptors interact with a number of binding partners that act to regulate both receptor function and cell surface stability. Here, we reveal that GABAA receptors interact directly with N-ethylmaleimide-sensitive factor (NSF), a critical regulator of vesicular dependent protein trafficking, as measured by in vitro protein binding and co-immunoprecipitation assays. In addition, we established that NSF interacts with residues 395–415 of the receptor β subunits and co-localizes with GABAA receptors in hippocampal neurons. We also established that NSF can regulate GABAA receptor cell surface expression depending upon residues 395–415 in the β3 subunit. Together, our results suggest an important role for NSF activity in regulating the cell surface stability of GABAA receptors.