In vivo recordings are obtained in the primary somatosensory cortex (S1) and primary motor cortex (M1) of P0–P7 Wistar rats using experimental protocols as described in detail before10 (Fig. 1). The animals are lightly anesthetized by intraperitoneal urethane (, Sigma-Aldrich, Taufkirchen, Germany) and the animal’s head is fixed with dental cement on the occipital bones via an aluminum holder into a stereotaxic apparatus. The skull above S1 and M1 is thinned on the left hemisphere using a miniature drill until the residual bone, but not the dura mater, can be carefully removed. The VSD RH1691 (Optical Imaging, Rehovot, Israel) is dissolved at in saline solution and topically applied to the cortical surface for 20 min [Fig. 1(b)]. In newborn rodents, all neocortical layers from the marginal zone (MZ)/layer (L) I to the subplate (SP) are stained [Fig. 1(c)]. The cortex is covered with 1% low-melting agarose and a cover slip is placed on top to stabilize the tissue. Excitation light from a red light-emitting diode (LED) (MRLED 625 nm, Thorlabs GmbH, Dachau, Germany) is band-pass filtered (), reflected by a 650-nm dichroic mirror, and focused onto the cortical surface with a 25-mm Navitar lens (Stemmer Imaging, Puchheim, Germany) [Fig. 1(a)]. The emitted fluorescence is collected via the same optical pathway, long-pass filtered (660 nm), and focused via another 25-mm Navitar lens onto the chip of a MiCam Ultima L high-speed camera (Scimedia, Costa Mesa, California). This tandem-type macroscope design34 results in a magnification, and every pixel of this camera collects light from a neocortical region of . Signals are recorded at a sampling frequency of 500 Hz, and the signal-to-noise ratio is improved using spatial binning followed by 60-Hz low-pass filtering. Bleaching of fluorescence is corrected by subtraction of a best-fit double-exponential or fifth-degree polynomial. The normalized change of fluorescence intensity () is calculated as the change of fluorescence intensity () in each pixel divided by the initial fluorescence intensity () from the same pixel. Fluorescent changes of are considered responses.