Fast excitatory neurotransmission is mediated by activation of synaptic ionotropic glutamate receptors. In hippocampal slices, we report that stimulation of Schaffer collaterals evokes in CA1 neurons delayed inward currents with slow kinetics, in addition to fast excitatory postsynaptic currents. Similar slow events also occur spontaneously, can still be observed when neuronal activity and synaptic glutamate release are blocked, and are found to be mediated by glutamate released from astrocytes acting preferentially on extrasynaptic NMDA receptors. The slow currents can be triggered by stimuli that evoke Ca2+ oscillations in astrocytes, including photolysis of caged Ca2+ in single astrocytes. As revealed by paired recording and Ca2+ imaging, a striking feature of this NMDA receptor response is that it occurs synchronously in multiple CA1 neurons. Our results reveal a distinct mechanism for neuronal excitation and synchrony and highlight a functional link between astrocytic glutamate and extrasynaptic NMDA receptors.

Astrocytes modulate neuronal activity by releasing chemical transmitters via a process termed gliotransmission. The role of this process in the control of behavior is unknown. Since one outcome of SNARE-dependent gliotransmission is the regulation of extracellular adenosine and because adenosine promotes sleep, we genetically inhibited the release of gliotransmitters and asked if astrocytes play an unsuspected role in sleep regulation. Inhibiting gliotransmission attenuated the accumulation of sleep pressure, assessed by measuring the slow wave activity of the EEG during NREM sleep and prevented cognitive deficits associated with sleep loss. Since the sleep-suppressing effects of the A1 receptor antagonist CPT were prevented following inhibition of gliotransmission and because intracerebroventricular delivery of CPT to wildtype mice mimicked the transgenic phenotype we conclude that astrocytes modulate the accumulation of sleep pressure and its cognitive consequences through a pathway involving A1 receptors.

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In the mammalian brain, astrocytes modulate neuronal function, in part, by synchronizing neuronal firing and coordinating synaptic networks. Little, however, is known about how this is accomplished from a structural standpoint. To investigate the structural basis of astrocyte-mediated neuronal synchrony and synaptic coordination, the three-dimensional relationships between cortical astrocytes and neurons was investigated. Using a transgenic and viral approach to label astrocytes with enhanced green fluorescent protein, we performed a three-dimensional reconstruction of astrocytes from tissue sections or live animals in vivo. We found that cortical astrocytes occupy nonoverlapping territories similar to those described in the hippocampus. Using immunofluorescence labeling of neuronal somata, a single astrocyte enwraps on average four neuronal somata with an upper limit of eight. Single-neuron dye-fills allowed us to estimate that one astrocyte contacts 300 -600 neuronal dendrites. Together with the recent findings showing that glial Ca 2ϩ signaling is restricted to individual astrocytes in vivo, and that Ca 2ϩ signaling leads to gliotransmission, we propose the concept of functional islands of synapses in which groups of synapses confined within the boundaries of an individual astrocyte are modulated by the gliotransmitter environment controlled by that astrocyte. Our description offers a new structurally based conceptual framework to evaluate functional data involving interactions between neurons and astrocytes in the mammalian brain.

Status epilepticus (SE), an unremitting seizure, is known to cause a variety of traumatic responses including delayed neuronal death and later cognitive decline. Although excitotoxicity has been implicated in this delayed process, the cellular mechanisms are unclear. Because our previous brain slice studies have shown that chemically induced epileptiform activity can lead to elevated astrocytic Ca 2ϩ signaling and because these signals are able to induce the release of the excitotoxic transmitter glutamate from these glia, we asked whether astrocytes are activated during status epilepticus and whether they contribute to delayed neuronal death in vivo. Using two-photon microscopy in vivo, we show that status epilepticus enhances astrocytic Ca 2ϩ signals for 3 d and that the period of elevated glial Ca 2ϩ signaling is correlated with the period of delayed neuronal death. To ask whether astrocytes contribute to delayed neuronal death, we first administered antagonists which inhibit gliotransmission: MPEP [2-methyl-6-(phenylethynyl)pyridine], a metabotropic glutamate receptor 5 antagonist that blocks astrocytic Ca 2ϩ signals in vivo, and ifenprodil, an NMDA receptor antagonist that reduces the actions of glial-derived glutamate. Administration of these antagonists after SE provided significant neuronal protection raising the potential for a glial contribution to neuronal death. To test this glial hypothesis directly, we loaded Ca 2ϩ chelators selectively into astrocytes after status epilepticus. We demonstrate that the selective attenuation of glial Ca 2ϩ signals leads to neuronal protection. These observations support neurotoxic roles for astrocytic gliotransmission in pathological conditions and identify this process as a novel therapeutic target.

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...

Insights into the pathogenesis of migraine with aura may be gained from a study of human CaV2.1 channels containing mutations linked to familial hemiplegic migraine (FHM). Here, we extend the previous single-channel analysis to human CaV2.1 channels containing mutation V1457L. This mutation increased the channel open probability by shifting its activation to more negative voltages and reduced both the unitary conductance and the density of functional channels in the membrane. To investigate the possibility of changes in Ca V2.1 function common to all FHM mutations, we calculated the product of single-channel current and open probability as a measure of Ca 2؉ influx through single CaV2.1 channels. All five FHM mutants analyzed showed a single-channel Ca 2؉ influx larger than wild type in a broad voltage range around the threshold of activation. We also expressed the FHM mutants in cerebellar granule cells from CaV2.1␣1 ؊/؊ mice rather than HEK293 cells. The FHM mutations invariably led to a decrease of the maximal CaV2.1 current density in neurons. Current densities were similar to wild type at lower voltages because of the negatively shifted activation of FHM mutants. Our data show that mutational changes of functional channel densities can be different in different cell types, and they uncover two functional effects common to all FHM mutations analyzed: increase of single-channel Ca 2؉ influx and decrease of maximal CaV2.1 current density in neurons. We discuss the relevance of these findings for the pathogenesis of migraine with aura.

Summary The two basic processes underlying perceptual decisions – how neural responses encode stimuli, and how they inform behavioral choices – have mainly been studied separately. Thus, although many spatiotemporal features of neural population activity, or “neural codes,” have been shown to carry sensory information, it is often unknown whether the brain uses these features for perception. To address this issue, we propose a new framework centered on redefining the neural code as the neural features that carry sensory information used by the animal to drive appropriate behavior; that is, the features that have an intersection between sensory and choice information. We show how this framework leads to a new statistical analysis of neural activity recorded during behavior that can identify such neural codes, and we discuss how to combine intersection-based analysis of neural recordings with intervention on neural activity to determine definitively whether specific neural activity features are involved in a task.