Glia are non-excitable cells found in nervous tissue, and have an important role in synaptic plasticity and the maintenance of neuronal environment, as well as the activity, development, degeneration, and repair of neurons. Glial cells are interconnected via gap junctions to form a multicellular syncytium and utilize intercellular and intracellular Ca2+signals to regulate their functions. Glial Ca2+ signals regulate important cell functions that include gene expression, cell proliferation, metabolism, ion transport systems, release of cell products, and cell death. Consequently, significant alterations of glial Ca2+ signals are associated with pathological processes such as epilepsy, Alzheimer's disease and stroke. Two major forms of Ca2+ signals, intercellular Ca2+ waves and intracellular Ca2+ oscillations occur within glia. Intercellular Ca2+ waves consist of the propagation of elevations in intracellular calcium concentration ([Ca2+]i) between neighboring cells, while intracellular Ca2+ oscillations consist of repetitive elevations in [Ca2+]i that remain confined to single cells. Ca2+ signals are initiated by either a localized chemical, mechanical and electrical stimuli. However, the exact mechanism of their initiation, propagation and modulation is not fully understood. Previous studies have led to the hypothesis that mechanically-induced intercellular Ca2+ waves in glia are mediated by the diffusion of second messenger inositol (1,4,5)-trisphosphate (IP3) through the gap junctions (GJ). However, intracellular Ca2+ may also diffuse between cells during the spread of intercellular Ca2+ wave. Alternatively, Ca2+ waves may be mediated by the release of extracellular messengers, e.g. ATP, that act via phospholipase C (PLC) -linked receptors, e.g. P2y receptors. It is also unknown if the propagation of Ca2+waves requires the regeneration of the signaling message by each cell. An interesting consequence of the propagation of an intercellular Ca2+ wave in glia is that they induce intracellular Ca2+ oscillations in cells that participate in its propagation. These intracellular Ca2+ oscillations may serve to resolve information contained in the position and strength of a local stimulus that induces intercellular Ca2+ wave propagation. Although the mechanism by which Ca2+ waves initiate Ca2+ oscillations is unknown it would seem likely that the mechanism of wave propagation is linked to the mechanism of initiation of Ca2+ oscillations. Guided by previous findings, I hypothesized that intercellular Ca2+ waves propagate by the diffusion of IP3 via gap junctions between neighboring cells to establish an intercellular gradient of IP3 concentration ([IP3]i that within individual cells initiates distinct intracellular Ca2+ oscillations. Two specific aims were investigated to test this hypothesis. The First Specific Aim was to determine if intercellular Ca2+ waves in glia are initiated by the generation of IP3 within a stimulated cell, and propagated by diffusion of IP3 molecules between neighboring cells via gap junctions. The Second Specific Aim, was to determine if intercellular Ca2+ waves induce distinct intracellular Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The initiation and propagation of intercellular Ca2+ waves and intracellular Ca2+ oscillations were examined in primary cultures of rat neonatal cortical glia, utilizing the techniques of a) the intracellular measurement of [Ca2+]i by fluorescence videomicroscopy, b) the photorelease of second messengers IP3 and Ca2+from their photolabile carriers, c) the loading of specific drugs by electroporation into defined zones of glial cultures, and d) identification of cell types by immunocytochemistry. The results of the Specific Aim 1 demonstrated the following: Mechanically-induced intercellular Ca2+ waves reequired PLC activation, the subsequent production of IP3 within the stimulated cell, and release of Ca2+ from intracellular calcium stores. Propagation of Ca2+waves depended on the presence of gap junctions. The release of Ca2+ via IP3 receptor/channels (IP3Rs) was necessary for Ca2+ wave propagation. In contrast, release of Ca2+ from ryanodine receptor/channels (RyRs) occurred in the mechanically-stimulated cell as well as in cells propagating a Ca2+ wave, but was not required for Ca2+ wave initiation and propagation. The propagation of Ca2+ waves through cells that contained heparin to block IP3Rs, or additional [Ca2+]i buffers, demonstrated that the regeneration of IP3 in the non-stimulated cells was not necessary for the propagation of the Ca2+ wave. Ca2+ waves were not mediated by extracellular signals, since Ca2+ waves were not affected by the extracellular perfusion or the inhibition of G proteins. Ca2+ was found to be a poor propagating signal of Ca2+ waves, since intercellular Ca2+ diffusion was not detected during Ca2+ wave propagation. These results are consistent with the hypothesis that Ca2+ waves propagate by diffusion of IP3molecules between neighboring cells via GIs. The [Ca2+]i increase in the stimulated cell occurred due to a Ca2+ influx from extracellular environment, and a release of Ca2+ from intracellular Ca2+ stores, and appeared to contribute to the activation of PLC and the generation of IP3. Ca2+ influx however, was not a necessary event in Ca2+ wave initiation or propagation, because Ca2+ waves occurred in the absence of extracellular Ca2+. By contrast, a [Ca2+]i increase in the absence of [IP3]i increase did not generate intercellular Ca2+waves. The results of the Specific Aim 2 demonstrated the following: An intercellular Ca2+ wave induced intracellular Ca2+ oscillations in a zone of cells at a specific distance from the stimulated cell. The initiation, frequency and duration of Ca2+ oscillations depended on the cells' distance from the Ca2+ wave origin, and not on the cell type or the magnitude of the Ca2+ wave. Modulation of the [IP3]i achieved by acetylcholine (ACh), a neurotransmitter that initiates IP3 production, or by intracellular photorelease of IP3 altered the oscillatory activity of individual cells and shifted the zone of oscillating cells away from the stimulated cell. Ca2+ oscillations spread through individual cells as an intracellular Ca2+ wave that was initiated from a specific site within the cell, independent of the orientation of the initial intercellular Ca2+ wave. These results are consistent with the hypothesis that an intercellular Ca2+ wave initiates Ca2+ oscillations by establishing a specific gradient of oscillation-promoting [IP3]i within the glial syncytium. The findings of this study support the hypothesis that intercellular diffusion of IP3 is the dominant mechanism of Ca2+ wave propagation and initiation of Ca2+ wave-induced Ca2+ oscillations. The significance of these results is that the glial syncytium may utilize specific intracellular Ca2+ oscillations to decode the position and strength of stimuli that induce intercellular Ca2+ waves, and thus integrate and coordinate multicellular functions of glia in the CNS.

Interactions between astrocytes and endothelial cells are believed to play an important role in the control of blood-brain barrier permeability and transport. Astrocytes and endothelial cells respond to a variety of stimuli with an increase of intracellular free calcium ([Ca2+]i) that is propagated to adjacent cells as an intercellular Ca2+ wave. We hypothesized that intercellular Ca2+ signaling also occurs between astrocytes and endothelial cells, and we investigated this possibility in co-cultures of primary astrocytes and an endothelial cell line using caged messengers. Intercellular Ca2+ waves, induced by mechanical stimulation of a single cell, propagated from astrocytes to endothelial cells and vice versa. Intercellular Ca2+ waves could also be induced by flash photolysis of pressure-injected caged inositol trisphosphate (IP3) and also by applying the flash to remote noninjected cells. Ca2+ waves induced by flash photolysis propagated from endothelial cells to astrocytes but not from astrocytes to endothelial cells even though caged IP3 diffused between the two cell types. Flash photolysis of caged Ca2+ (NP-EGTA) resulted in an increase of [Ca2+]i but did not initiate an intercellular Ca2+ wave. We conclude that an increase of IP3 in a single cell is sufficient to initiate an intercellular Ca2+ wave that is propagated by the diffusion of IP3 to neighboring cells and that can be communicated between astrocytes and endothelial cells in co-culture. By contrast, Ca2+ diffusion via gap junctions does not appear to be sufficient to propagate an intercellular Ca2+ wave. We suggest that intercellular Ca2+ waves may play a role in astrocyte-endothelial interactions at the blood-brain barrier.