Astrocytes regulate the strength of synaptic networks and various aspects of neuronal activity (1). Neurotransmitters can activate astrocytic Gq-coupled receptors which results in accumulation of InsP3. InsP3 binds to its receptor located on endoplasmatic reticulum (ER) to release Ca2+. Ca2+ may also enter astrocytes from the extracellular space via various mechanisms. In order to use optogenetics to trigger [Ca2+]i elevations in astrocytes previously we expressed in these cells the channelrhodopsin-2 mutant, ChR2(H134R) (2),(3). However, Ca2+ permeability of ChR2(H134R) is relatively low. Therefore, a recently published Ca2+ translocating channelrhodopsin (CatCh) reported to have up to 6-fold higher Ca2+ permeability (4) is of interest as a tool for optogenetic control of astrocytes. Native ChR2 and similar opsins such as CatCh are normally targeted to the plasma membrane but many important signalling events in astrocytes are triggered by release of Ca2+ from the ER. As a step towards simulating this process, we have generated a fusion of CatCh, via a linker, to the transmembrane domains 1 and 2 of the InsP3 receptor 1 to achieve ER-specific targeting/retention. Enhanced yellow fluorescent protein (EYFP) was also fused into the construct to aid visualization. In HEK 293 cells, ER-CatCh-EYFP showed clear preference to localization within endomembranes, in contrast to native CatCh-EYFP, which is plasma membrane targeted. We evaluated CatCh-EYFP and its ER-CatCh-EYFP as optogenetic tools for control of astrocytic [Ca2+]i. Primary astrocytes from neonatal rats were transfected with 0.5 µg DNA plasmids to express the relevant constructs under control of cytomegalovirus (CMV) promoter using the TransIT-293 reagent (Mirus). After 48h, transfected astrocytes loaded with Rhod-2 AM were placed in a chamber mounted on a confocal microscope and perfused with Hank’s Balanced Solution (HBS) at 34oC. Light-stimulation of the ER-CatCh-EYFP astrocytes triggered increases in [Ca2+]i (130% ± 1; n= 80; p<0.001; Student’s paired t-test) and of the CatCh-EYFP (135% ± 2; n= 68; p<0.001; Student’s paired t-test). The latency of response was ≤ 2s for both ER-CatCh-EYFP and CatCh-EYFP and > 5s for ChR2(H134R). The [Ca2+]i peaked within ~30-35s with CatCh in contrast to >60s with ChR2(H134R) under comparable conditions. Thus, both constructs are superior to previously used ChR2(H134R) as tools for optogenetic control of astrocytic [Ca2+]i (5). Currently, we are working to establish exact contributions of extracellular and intracellular Ca2+ to the observed elevations of [Ca2+]i in astrocytes. Viral constructs to express these tools specifically in astrocytes are also being generated. This approach opens a wide range of opportunities for enquiries into the physiology and signalling properties of these cells.