Figure 2 .

In vivo whisker stimulation, two-photon imaging and neuronal tagging. (A) Intrinsic imaging was performed prior to each two-photon imaging session to identify barrel location (white circle). Vasculature under green light illumination (left) and change in reflected red light RD/R0 during single whisker stimulation (right). (B) Mice (n = 35) were kept under light isoflurane anesthesia during two-photon imaging in barrel cortex L2/3 (green square; left) and piezo-electric stimulation of single whiskers at 90 Hz (right). (C) Co-expression of red calcium indicator for two-photon imaging (here jRGECO1a) and photo-activatable green fluorescent protein (H2B-PAGFP) for photo-tagging cells. Overlay of green and red channels before (top) and after photo-activation of H2B-PAGFP (bottom). (D) Single-neuron calcium transients Δf/f0 (black) in response to whisker stimulation (blue). HRs #1–7 showed large average responses (red curves), while LRs #8–14 showed small average responses (blue curves). (E-G) Criteria for defining HRs (red) and LRs (blue): Peak calcium signals (Δf/f0) averaged across all trials (E), fraction of trials with significant activation (F), and response latency (G). In (G), only neurons for which the response latency could be calculated were included (100% of all HRs, 99% of MR and 65% of LRs). Response latencies take discrete values from the sampling time of calcium imaging at our sampling rate (11.4 Hz). Neurons that did not match these criteria were categorized as medium responders.