Neuronal excitotoxicity during stroke is caused by activation of unidentified large-conductance channels, leading to swelling and calcium dysregulation. We show that ischemic-like conditions [O(2)/glucose deprivation (OGD)] open hemichannels, or half gap junctions, in neurons. Hemichannel opening was indicated by a large linear current and flux across the membrane of small fluorescent molecules. Single-channel openings of hemichannels (530 picosiemens) were observed in OGD. Both the current and dye flux were blocked by inhibitors of hemichannels. Therefore, hemichannel opening contributes to the profound ionic dysregulation during stroke and may be a ubiquitous component of ischemic neuronal death.

SUMMARY Astrocytes are proposed to participate in brain energy metabolism by supplying substrates to neurons from their glycogen stores and from glycolysis. However, the molecules involved in metabolic sensing and the molecular pathways responsible for metabolic coupling between different cell types in the brain are not fully understood. Here we show that a recently cloned bicarbonate (HCO3−) sensor, soluble adenylyl cyclase (sAC), is highly expressed in astrocytes and becomes activated in response to HCO3− entry via the electrogenic NaHCO3 cotransporter (NBC). Activated sAC increases intracellular cAMP levels, causing glycogen breakdown, enhanced glycolysis, and the release of lactate into the extracellular space, which is subsequently taken up by neurons for use as an energy substrate. This process is recruited over a broad physiological range of [K+]ext and also during aglycemic episodes, helping to maintain synaptic function. These data reveal a molecular pathway in astrocytes that is responsible for brain metabolic coupling to neurons.

Cortical spreading depression (SD) is a propagating wave of neuronal and glial depolarization that manifests in several brain disorders. However, the relative contribution of neurons and astrocytes to SD genesis has remained controversial. This is in part due to a lack of utilizing sophisticated experimental methodologies simultaneously to quantify multiple cellular parameters. To address this, we used simultaneous two-photon imaging, intrinsic optical imaging, and electrophysiological recordings to ascertain the changes in cellular processes that are fundamental to both cell types including cell volume, pH, and metabolism during SD propagation. We found that SD was correlated in neurons with robust yet transient increased volume, intracellular acidification, and mitochondrial depolarization. Our data indicated that a propagating large conductance during SD generated neuronal depolarization, which led to both calcium influx triggering metabolic changes and H(+) entry. Notably, astrocytes did not exhibit changes in cell volume, pH, or mitochondrial membrane potentials associated with SD, but they did show alterations induced by changing external [K(+)]. This suggests that astrocytes are not the primary contributor to SD propagation but are instead activated passively by extracellular potassium accumulation. These data support the hypothesis that neurons are the crucial cell type contributing to the pathophysiological responses of SD.

Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K(+) concentrations ([K(+)]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K(+)]o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na(+)/Ca(2+) exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na(+) influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na(+)/Ca(2+) exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K(+)]o-triggered SD.

Two-photon laser scanning microscopy has revolutionized the ability to delineate cellular and physiological function in acutely isolated tissue and in vivo. However, there exist barriers for many laboratories to acquire two-photon microscopes. Additionally, if owned, typical systems are difficult to modify to rapidly evolving methodologies. A potential solution to these problems is to enable scientists to build their own high-performance and adaptable system by overcoming a resource insufficiency. Here we present a detailed hardware resource and protocol for building an upright, highly modular and adaptable two-photon laser scanning fluorescence microscope that can be used for in vitro or in vivo applications. The microscope is comprised of high-end componentry on a skeleton of off-the-shelf compatible opto-mechanical parts. The dedicated design enabled imaging depths close to 1 mm into mouse brain tissue and a signal-to-noise ratio that exceeded all commercial two-photon systems tested. In addition to a detailed parts list, instructions for assembly, testing and troubleshooting, our plan includes complete three dimensional computer models that greatly reduce the knowledge base required for the non-expert user. This open-source resource lowers barriers in order to equip more laboratories with high-performance two-photon imaging and to help progress our understanding of the cellular and physiological function of living systems.

According to the glutamate hypothesis of schizophrenia, the abnormality of glutamate transmission induced by hypofunction of NMDA receptors (NMDARs) is causally associated with the positive and negative symptoms of schizophrenia. However, the underlying mechanisms responsible for the changes in glutamate transmission in schizophrenia are not fully understood. Astrocytes, the major regulatory glia in the brain, modulate not only glutamate metabolism but also glutamate transmission. Here we review the recent progress in understanding the role of astrocytes in schizophrenia. We focus on the astrocytic mechanisms of (i) glutamate synthesis via the glutamate-glutamine cycle, (ii) glutamate clearance by excitatory amino acid transporters (EAATs), (iii) D-serine release to activate NMDARs, and (iv) glutamatergic target engagement biomarkers. Abnormality in these processes is highly correlated with schizophrenia phenotypes. These findings will shed light upon further investigation of pathogenesis as well as improvement of biomarkers and therapies for schizophrenia.

No abstract

Hyperekplexia is a neurological disorder associated primarily with mutations in the ␣1 subunit of glycine receptors (GlyRs) that lead to dysfunction of glycinergic inhibitory transmission. To date, most of the identified mutations result in disruption of surface expression or altered channel properties of ␣1-containing GlyRs. Little evidence has emerged to support an involvement of allosteric GlyR modulation in human hyperekplexia. Here, we report that recombinant human GlyRs containing ␣1 or ␣1␤ subunits with a missense mutation in the ␣1 subunit (W170S), previously identified from familial hyperekplexia, caused remarkably reduced potentiation and enhanced inhibition by Zn 2ϩ . Interestingly, mutant ␣1 W170S ␤ GlyRs displayed no significant changes in potency or maximum response to glycine, taurine, or ␤-alanine. By temporally separating the potentiating and the inhibitory effects of Zn 2ϩ , we found that the enhancement of Zn 2ϩ inhibition resulted from a loss of Zn 2ϩ -mediated potentiation. The W170S mutation on the background of H107N, which was previously reported to selectively disrupt Zn 2ϩ inhibition, showed remarkable attenuation of Zn 2ϩ -mediated potentiation and thus indicated that W170 is an important residue for the Zn 2ϩ -mediated GlyR potentiation. Moreover, overexpressing the ␣1 W170S subunit in cultured rat neurons confirmed the results from heterologous expression. Together, our results reveal a new zinc potentiation site on ␣1 GlyRs and a strong link between Zn 2ϩ modulation and human disease.