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Calcium signalling in glial cells: a potential role for TRP channels

K.L. Richardson,1 C.A. Reid,2 K. Powell,3 M. Monif1 and D.A. Williams,1 1Department of Physiology, The University of Melbourne, Vic, Australia, 2Howard Florey Institute, Melbourne, Vic, Australia and 3Department of Medicine, The University of Melbourne, Vic, Australia.

Glia possess specific receptors which allow these cells to respond to changes in neuronal activity. This neuron-glia cross-talk suggests that glia are integral modulatory components of neuronal excitability and synaptic transmission. Intra-glial Ca2+ signals, including Ca2+ oscillations, are important arbiters of glial activity but are poorly understood. Our aims were threefold: i) to establish the role of the endoplasmic reticulum (ER) in oscillations, ii) to determine if store-operated channels (SOCs) subserve this role, and finally iii) to explore links between, and possible co-identity of, SOC and transient receptor potential (TRP) channels. Standard recombinant DNA procedures were used to detect TRPC1 mRNA expression in primary hippocampal cultures (0-3 day neurons and glia). A modified calcium phosphate-based technique was used to transfect cells with either TRPC1 siRNA and/or D1ER cameleon (ER Ca2+ sensor) DNA. siRNA TRPC1 down-regulation was assessed with semi-quantitative PCR. Confocal microscopy techniques determined biosensor localisation and measured Ca2+ dynamics (fluo-4 and D1ER). Glia (confirmed by GFAP staining) but not neurons, expressed fully functional D1-ER. Glutamate (1μM-1mM) induced complex Ca2+ oscillations (n=6) that were significantly reduced by removal of extracellular Ca2+ (n=3), and caused reversible release from ER Ca2+ stores (n=6) independent of extracellular Ca2+. Prior depletion of ER Ca2+ stores with thapsigargin (1μM) significantly reduced subsequent glutamate-induced Ca2+ responses (n=3) and triggered SOC function (n=3). TRPC1 mRNA was present in detectable levels in cell cultures (n=6). Low transfection efficiencies diluted overall siRNA TRPC1 down-regulation. ER function is clearly involved in glial calcium signaling and linked with activation of store-operated channels. Further assessment of the relationship between SOC and TRP channels requires improved siRNA delivery.