2c and GCaMP2.0, whereas there was no significant difference between the basal fluorescence of GCaMP2.0

and GCaMP2.2c ( Figures S1B and S1C). In addition, we found that fluorescence intensity CH5424802 concentration changes elicited by 100 μM ATP are ∼1.9-fold (1.9 ± 0.1, n = 56) and ∼3.2-fold (3.2 ± 0.3, n = 61) higher in cells expressing GCaMP2.2c and GCaMP3 than in cells expressing GCaMP2.0, respectively ( Figure S1D). The studies above indicate that GCaMP2.2c has a low basal fluorescence with a modest fluorescence change in response to stimulation, whereas GCaMP3 shows higher basal fluorescence and a more robust change in fluorescence after stimulation. Because GCaMP (and any GECI) binds calcium, there is a risk of neuronal toxicity associated with calcium binding and expression level. To increase the chances of finding lines with both strong signal and low toxicity, we generated both GCaMP2.2c and GCaMP3 transgenic lines. To generate GCaMP transgenic mice, we used the RG7204 clinical trial well-characterized Thy1 promoter to express GCaMPs in neurons. We generated eight founder lines of GCaMP2.2c and six founder lines of GCaMP3. Our previous studies have shown that the Thy1 promoter predominantly drives transgene expression in projection neurons in the CNS. Due to strong transgenic position-effect variegation, a Thy1-driven

transgene is often stochastically and differentially expressed in subsets of neurons in different transgenic lines ( Feng et al., 2000; Young et al., 2008). Consistent with these findings, we found that all founder lines differed in levels and patterns of expression. For further characterization, we focused on Thy1-GCaMP2.2c SB-3CT line 8 and Thy1-GCaMP3 line 6 because these lines had the

highest levels of transgene expression. Both lines of mice are born at the expected Mendelian rate and are healthy with no apparent histological or behavioral abnormality. GCaMP2.2c and GCaMP3 expression in these lines was widespread in the CNS including cortex, hippocampus, thalamus, cerebellum, superior colliculus, amygdala, brain stem, retina, and spinal cord ( Figures 1A, 1B, and 2; Figure S2). However, some notable differences in expression between the two lines were observed. For example, although both lines have expression in layer V neurons of the cortex, expression in layer II/III is more widespread in the Thy1-GCaMP3 line ( Figures 1B, 1Bb1, and 1Bb2) compared to the Thy1-GCaMP2.2c line ( Figures S2B, S2Bb1, and S2Bb2). In addition, Thy1-GCaMP3 mice, but not Thy1-GCaMP2.2c mice, showed high expression in olfactory bulb ( Figures 1A and 2). At the single cell level, both GCaMP2.2c and GCaMP3 were homogeneously distributed in the cytoplasm without nuclear localization ( Figures 1B, 1Bb1, and 1Bb2; Figures S2B, S2Bb1, and S2Bb2). We further examined the effect of long-term GCaMP expression in both GCaMP transgenic lines.