GABA A receptor-mediated inhibition depends on the maintenance of intracellular ClϪ concentration ([Cl Ϫ ] in ) at low levels. In neurons in the developing CNS, [Cl Ϫ ] in is elevated, E GABA 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 E GABA was positively shifted in granule cells. This shift in E GABA altered synaptic integration, increased granule cell excitability, and resulted in compromised "gate" function of the dentate gyrus. E GABA recovered to control values at longer latencies post-STEP (2-8 weeks), when animals had developed epilepsy. During this period of shifted E GABA , expression of the Cl Ϫ extruding K ϩ /Cl Ϫ cotransporter, KCC2 was decreased. Application of the KCC2 blocker, furosemide, to control neurons mimicked E GABA shifts evident in granule cells post-STEP. Furthermore, post-STEP and furosemide effects interacted occlusively, both on E GABA 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.

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 Ca 2؉ signaling in astrocytes and, as a consequence, the Ca 2؉ -dependent release of excitatory transmitters from these glia. In this study, we demonstrate that activation of mGluR5 induces Ca 2؉ oscillations in NAc astrocytes with the correlated appearance of NMDA receptor-dependent slow inward currents detected in medium spiny neurons (MSNs). Photolysis of caged Ca 2؉ loaded specifically into astrocytes evoked slow inward currents demonstrating that Ca 2؉ 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 Ca 2؉ 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.addiction ͉ astrocytes ͉ glutamate release ͉ NMDA receptors A lthough the dopaminergic system is known to play important roles in responses to drugs of abuse, there is an increasing awareness of the importance of glutamate in mediating effects of these drugs in the brain (1). Cocaine administration requires glutamatergic transmission in the nucleus accumbens (NAc) for drug-seeking behavior (2, 3) with accumulating evidence demonstrating the importance of metabotropic glutamate receptors (mGluRs) in mediating the actions of extracellular glutamate after cocaine use (4). mGluR5 knockout mice do not acquire cocaine self-administration behavior (4), and in wild-type mice the mGluR5 antagonist, 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP), decreases cocaine, morphine, and nicotine selfadministration and drug-seeking behavior (5-8).Although astrocytes are electrically inexcitable, emerging evidence demonstrates that these nonneuronal cells respond to neurotransmitters with elevations in their internal Ca 2ϩ and with the release of the chemical transmitters glutamate, ATP and D-serine (9, 10). Because astrocytes and neurons release common chemical transmitters, we use the term gliotransmitter and gliotransmission to refer to their release from astrocytes (11). Recent...

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 GABA A receptors (GABA A Rs), the major sites of fast synaptic inhibition in the brain. We demonstrate that SE selectively decreases the phosphorylation of GABA A Rs on serine residues 408/9 (S408/9) in the ␤3 subunit by intimately associated protein kinase C isoforms. Dephosphorylation of S408/9 unmasks a basic patchbinding motif for the clathrin adaptor AP2, enhancing the endocytosis of selected GABA A R subtypes from the plasma membrane during SE. In agreement with this, enhancing S408/9 phosphorylation or selectively blocking the binding of the ␤3 subunit to AP2 increased GABA A R cell surface expression levels and restored the efficacy of synaptic inhibition in SE. Thus, enhancing phosphorylation of GABA A Rs or selectively blocking their interaction with AP2 may provide novel therapeutic strategies to ameliorate SE.

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.adenosine ͉ astrocytes ͉ gliotransmission ͉ NMDA receptor N onrapid eye movement (NREM) sleep is characterized by global cortical oscillations of synchronized neuronal activity (1, 2). Major components of this activity are slow oscillations (Ͻ1 Hz), observed also under some forms of anesthesia (2-5). Slow oscillations are a fundamental network phenomenon that organizes other sleep rhythms (1), and have been suggested to have a role in sleep-dependent memory consolidation (6, 7).Different mechanisms have been proposed to control slow oscillations, including excitatory-inhibitory interactions (8), neuronal intrinsic properties (9, 10), and the modulation of synaptic transmission (4, 11). Although pharmacological (4, 11) studies have shown the importance of synaptic modulation on slow oscillations, it is not known whether endogenous cellular systems capable of modulating synaptic transmission have a role in shaping this cortical rhythm.Recently, brain slice experiments showed that a glial cell subtype, the astrocyte, modulates synaptic transmission through the release of different molecules (gliotransmission) including D-serine and ATP (12, 13). D-serine acts as a coagonist of NMDA receptors (14) increasing postsynaptic NMDA currents (15), whereas ATP is rapidly hydrolyzed to adenosine, which acts on A1 receptors to suppress synaptic transmission (16,17). Because astrocytes are intimately associated with pre and postsynaptic terminals (18, 19), on which they exert modulatory actions, they have the potential to act as endogenous regulators of slow oscillations.Support of this hypothesis comes from the observation that gliotransmission is essential for the normal accumulation of the homeostatic sleep pressure (20). This fundamental process is assessed by measuring the dynamic change in slow wave activity (SWA; 0.5-4 Hz) of the EEG during NREM sleep. Because slow oscillations underlie low frequency (Ͻ1 Hz) components of SWA, we hypothesized that gliotransmission may directly impact brain d...

γ-aminobutyric acid type-A receptors (GABAARs) mediate the majority of fast synaptic inhibition and are the principle sites of action for anxiolytic, sedative, hypnotic and anti-convulsant agents that include benzodiazepines, barbiturates, neurosteroids and some general anesthetics. GABAARs are hetero-pentameric ligand-gated ion channels that are found concentrated at inhibitory postsynaptic sites where they mediate phasic inhibition. Specialized populations of extrasynaptic GABAARs that mediate tonic inhibition are also expressed by neurons. The efficacy of phasic inhibition and thus neuronal excitability is critically dependent on the accumulation of specific GABAAR subtypes at inhibitory synapses. Here we evaluate how neurons control the number of GABAARs 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 their possible impact on behavior and the etiology of neuropsychiatric disorders.

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.

GABAAreceptors 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 GABAAreceptor phospho-dependent regulation usingPRIP-1-/-mice. In wild-type animals, robust phosphorylation and functional modulation of GABAAreceptors containing β3 subunits by cAMP-dependent protein kinase was evident, which was diminished inPRIP-1-/-mice.PRIP-1-/-mice exhibited enhanced PP1α activity compared with controls. Furthermore, PRIP-1 was able to interact directly with GABAAreceptor 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 GABAAreceptor activity via regulating subunit phosphorylation and thereby the efficacy of neuronal inhibition mediated by these receptors.

Slow and persistent synaptic inhibition is mediated by metabotropic GABA B receptors (GABA B Rs). GABA B Rs are responsible for the modulation of neurotransmitter release from presynaptic terminals and for hyperpolarization at postsynaptic sites. Postsynaptic GABA B Rs 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 GABA B Rs 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 GABA B Rs. These sorting events are paralleled by a reduction in GABA B R-dependent activation of inwardly rectifying K + channel currents. Postendocytic sorting is critically dependent on phosphorylation of serine 783 (S783) within the GABA B R2 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 GABA B Rs 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 GABA B Rs 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.he availability of neurotransmitter receptors, a major determinant of synaptic efficacy, is regulated by coordinated mechanisms of intracellular trafficking that deliver newly synthesized receptors to the plasma membrane and remove them for storage, recycling, or degradation (1). The molecular mechanisms controlling the availability of GABA B receptors (GABA B Rs), which are central players in the modulation of excitatory and inhibitory synaptic activity, are unclear.GABA B Rs mediate slow and prolonged inhibitory synaptic signals (2, 3). Consistent with these roles, modifications in the function of GABA B Rs are implicated in epilepsy, anxiety, stress, sleep disorders, nociception, depression, cognition, and addictive mechanisms to drugs of abuse (3-7). GABA B Rs are members of the G protein-coupled receptor (GPCR) superfamily and are obligatory heteromers composed of two related subunits, namely GABA B R1 and GABA B R2 (3, 8). GABA B R1 binds agonist with high affinity, whereas GABA B R2 mediates coupling to Gαi (9, 10). GABA B Rs are located in GABA-ergic and glutamatergic pre-and postsynaptic terminals, but their distribution does not coincide with the active zone, postsynaptic density, or inhibitory postsynaptic specializations. Rather, they are perisynaptic receptors activated by GABA spillover (3, 11). Stimulation of GABA B Rs decreases the levels of cAMP, inhibits neurotr...