They can be targeted to specific cell types or subcellular compartments when used

in combination with cell type-specific promoters or cellular targeting sequences. In addition, GECIs can be delivered and expressed in brain tissues via viral vectors, in utero electroporation, or through transgenic techniques (Hasan et al., 2004; Mao et al., 2008; Wallace et al., 2008; Yamada et al., 2011). Importantly, recently developed GECIs are capable of detecting calcium dynamics at the sensitivity level close to that of synthetic calcium dyes (Hendel et al., 2008; Pologruto et al., 2004). At least one class of Doxorubicin green fluorescent protein (GFP)-based GECIs, the GCaMP family, has been effective for detecting calcium dynamics induced by neuronal activity in multiple

model organisms (Muto et al., 2011; Reiff et al., 2005; Tian et al., 2009; Warp et al., 2012). Recently, a new generation of GCaMPs (e.g., GCaMP3) has been successfully used to monitor neuronal activity in rodents using viral approaches (Borghuis et al., 2011; Dombeck et al., 2010; Mittmann et al., 2011; Osakada et al., 2011; Tian et al., 2009). Here we report the generation and characterization of new transgenic mouse lines that express the improved GCaMP2.2c and GCaMP3 indicators (Tian et al., 2009) in subsets of excitatory neurons in the mouse brain using the Thy1 promoter. We demonstrate long-term, stable expression of GCaMPs Depsipeptide solubility dmso in subpopulations of neurons with no apparent toxicity. Both GCaMP2.2c and GCaMP3 show strong and sensitive changes in fluorescence upon neuronal stimulation. We further demonstrate the broad utility of Thy1-GCaMP2.2c and Thy1-GCaMP3 transgenic mice in reporting neuronal activity in vitro and in vivo. To generate GCaMP transgenic mice, we utilized the previously described GCaMP3 and a further modified over GCaMP2.2b (Tian et al., 2009). Previous studies suggested that the N-terminal arginine located immediately after the initiator methionine of GCaMP2.0 destabilizes the protein, and changing the serine

at 118 to cysteine could improve brightness and sensor response (Tian et al., 2009). Thus, we changed the second arginine in GCaMP2.0 to valine to increase its stability according to the N-terminal rule of protein degradation (Varshavsky, 2011) and changed the serine at 118 to cysteine as in GCaMP2.2b to create GCaMP2.2c. The domain structure and specific mutations of GCaMP2.2c and GCaMP3 are summarized in Figure S1A, available online. Two important properties to consider when evaluating GECIs are basal levels of fluorescence and stimulation-induced changes in fluorescence (ΔF/F). To assess these properties for GCaMP2.2c and GCaMP3, we coexpressed GCaMPs and the red fluorescence protein tdTomato in the same construct using the 2A peptide (P2A) sequence ( Szymczak et al., 2004) in HEK293 cells. To normalize for transfection efficiency, we used the fluorescence intensity ratio of GCaMPs/tdTomato.