Next, we examined whether calcium transients could also be detected in individual somata of Thy1-GCaMP mice. We first tested whether we could detect GCaMP responses in the soma of neurons in layer II/III of the motor cortex in anesthetized animals. In Thy1-GCaMP2.2c mice, we observed GCaMP-expressing neurons but could not detect activated

cells within an imaging window of 250 × 250 μm during a 10 min recording period. In contrast, in Thy1-GCaMP3 mice, we observed 9.3 ± 0.5 cells (n = 4 areas from 3 mice) using the same imaging conditions ( Figures 6A and 6B). Differences between Thy1-GCaMP2.2c and Thy1-GCaMP3 mice are likely due to the fact that significantly fewer layer II/III neurons are labeled in Thy1-GCaMP2.2c selleck products mice ( Figure 1; Figure S2). We also imaged neuronal activity in the motor cortex of awake mice using a fixed-head imaging design (Dombeck Alectinib et al., 2007, 2009). In awake, behaving animals,

we were able to detect activated neurons both in Thy1-GCaMP2.2c and Thy1-GCaMP3 mice ( Figures 6A and 6B; Figure S6; Movie S7). In Thy1-GCaMP3 mice, we detected many more activated neurons (34.4 ± 1.7 cells in a 250 × 250 μm imaging window, n = 5 areas from 3 mice) over a 10 min period ( Figures 6A and 6B) compared to anesthetized animals (see above). The population activity in the primary motor cortex of both Thy1-GCaMP mice was correlated with locomotor activity ( Figure 6C; Figure S6).

Repeated imaging of the same brain area at 15 days after the first imaging showed that most of the same neurons were active in both views ( Figure 6D). From these observations, we conclude that both Thy1-GCaMP2.2c and Thy1-GCaMP3 mice can be used to monitor neuronal activity over extended periods of time in the motor cortex of living animals. In recent studies, viral expression of the ratiometric GECIs (YC3.60 and D3cpV) and GCaMP3 in pyramidal neurons of the mouse somatosensory (barrel) cortex allowed the detection of neuronal activity induced by whisker stimulation (Lütcke et al., 2010; Mittmann et al., 2011; O’Connor et al., 2010; Wallace et al., 2008). To determine whether Ca2+ transients could be detected in the barrel cortex in response to sensory out stimulation in our Thy1-GCaMP mice, we performed a similar test. We used Thy1-GCaMP3 mice because in vivo two-photon imaging in the barrel cortex revealed sparse labeling of layer II/III pyramidal neurons in Thy1-GCaMP2.2c mice and dense labeling in Thy1-GCaMP3 mice (data not shown). To induce sensory stimulation, we deflected multiple mystacial vibrissae ten times using 500 ms air puffs with 10 s interpuff intervals. In Thy1-GCaMP3 transgenic mice, we routinely detected calcium transients associated with whisker stimulation in both cell somata and the adjacent layer II/III neuropil ( Figures 7A–7C and Movie S8).