Background and Purpose-We investigated the contribution of gap junctions to brain damage and delayed neuronal death produced by oxygen-glucose deprivation (OGD). Methods-Histopathology, molecular biology, and electrophysiological and fluorescence cell death assays in slice cultures after OGD and in developing rats after intrauterine hypoxia-ischemia (HI). Results-OGD persistently increased gap junction coupling and strongly activated the apoptosis marker caspase-3 in slice cultures. The gap junction blocker carbenoxolone applied to hippocampal slice cultures before, during, or 60 minutes after OGD markedly reduced delayed neuronal death. Administration of carbenoxolone to ischemic pups immediately after intrauterine HI prevented caspase-3 activation and dramatically reduced long-term neuronal damage. Conclusions-Gap

Microglia, the tissue macrophages of the central nervous system (CNS), intimately interact with neurons physically and through soluble factors that can affect microglial activation state and neuronal survival and physiology. We report here a new mechanism of interaction between these cells, provided by the formation of gap junctions composed of connexin (Cx) 36. Among eight Cxs tested, expression of Cx36 mRNA and protein was found in microglial cultures prepared from human and mouse, and Cx45 mRNA was found in mouse microglial cultures. Electrophysiological measurements found coupling between one-third of human or mouse microglial pairs that averaged below 30 pico-Siemens and displayed electrical properties consistent with Cx36 gap junctions. Importantly, similar frequency of low-strength electrical coupling was also obtained between microglia and neurons in cocultures prepared from neocortical or hippocampal rodent tissue. Lucifer yellow dye coupling between neurons and microglia was observed in 4% of pairs tested, consistent with the low strength and incidence of electrical coupling. Cx36 expression level and/or the degree of coupling between microglia did not significantly change in the presence of activating agents, including lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, interferon-gamma, and tumor necrosis factor-alpha, except for some reduction of Cx36 protein when exposed to the latter two agents. Our findings that intercellular coupling occurs between neuronal and microglial populations through Cx36 gap junctions have potentially important implications for normal neural physiology and microglial responses in neuronopathology in the mammalian CNS.

Astrocytes are coupled to one another by gap junction channels that allow the diffusion of ions and small molecules throughout the interconnected syncytium. In astrocytes, gap junctions are believed to participate in spatial buffering removing the focal excess of potassium resultant from intense neuronal activity by current loops through the syncytium and are also implicated in the propagation of astrocytic calcium waves, a form of extraneuronal signaling. Gap junctions can be modulated by several factors, including elevation of extracellular potassium concentration. Because K ϩ elevation is a component of spinal cord injury, we evaluated the extent to which cultured spinal cord astrocytes is affected by K ϩ levels and obtained evidence suggesting that a Ca 2ϩ-calmodulin (CaM) protein kinase is involved in the K ϩ-induced increased coupling. Exposure of astrocytes to high K ϩ solutions induced a dose-dependent increase in dye coupling; such increased coupling was greatly attenuated by reducing extracellular Ca 2ϩ concentration, prevented by nifedipine, and potentiated by Bay-K-8644. These results indicate that K ϩ-induced increased coupling is mediated by a signaling pathway that is dependent on the influx of Ca 2ϩ through L-type Ca 2ϩ channels. Evidence supporting the participation of the CaM kinase pathway on K ϩ-induced increased coupling was obtained in experiments showing that calmidazolium and KN-93 totally prevented the increase in dye and electrical coupling induced by high K ϩ solutions. Because no changes in connexin43 expression levels or distribution were observed in astrocytes exposed to high K ϩ solutions, we propose that the increased junctional communication is related to an increased number of active channels within gap junction plaques.