The intracellular solution contained (in mM): 128 potassium gluconate, 4 MgCl2, 10 HEPES, 1 EGTA, 4 Na2ATP, 0.4 Na2GTP, 10 sodium phosphocreatine, 3 sodium L-ascorbate, and 0.02 Alexa-594 (Molecular Probes), and 3 mg/ml biocytin (pH 7.27; 287 mOsm). Cells were recorded at depths from 46 to 103 μm within the brain slice. Data were acquired using Ephus (www.ephus.org). Pyramidal neurons were selected based on MEK inhibitor their morphology confirmed under fluorescence microscopy (Alexa-594 in pipette solution) or post hoc by biocytin
staining. For sCRACM mapping, EPSC were recorded in voltage clamp while holding at −70 mV (L2/3 cells) or −75 mV (L5 cells). Access resistances ranged 10–40 MΩ. For every vM1 cell included in the data set, the site of viral infection was confirmed to lie within the barrel cortex by post hoc histological analysis. Most infections were roughly centered on the barrel field. The position of a blue laser beam (473 nm; Crystal Laser) was controlled with galvanometer scanners (Cambridge Scanning, learn more Inc.). The beam passed through an air objective (4×; 0.16 NA; UPlanApo, Olympus) and was nearly cylindrical (∼8–16 μm in diameter, full-width at half max at the specimen plane). The light pulses were controlled with
a Pockels cell (ConOptics). The power (0.7–1.8 mW) of the light pulses (duration, 1–2 ms) was adjusted so that the largest EPSCsCRACM had peak values in the range of 50–100 pA; in some Isotretinoin cases EPSCsCRACM were smaller even at the highest laser powers. Each trial consisted of approximately 100 ms baseline, the photostimulus, and 300 ms response period. Stimulation sites were on a 50 μm grid. Grid sizes (12 × 24, 12 × 26 or 12 × 28) were adjusted based on the size of the neuron; all
grids covered all potential sites of input within the dendritic arbor. Each map was repeated 2–4 times. The laser stimuli were given in a spatial sequence designed to maximize the intervals between stimuli arriving to neighboring spots (Shepherd et al., 2003). sCRACM pixel values corresponded to the mean EPSC amplitude in a 75 ms time window after the onset of the stimulus (given in picoamperes, pA, for consistency with previous studies). In some figure panels (Figures 3D and 4B), we display only pixels with significant responses (response amplitude >6× standard deviation of the baseline). For each cell, maps were averaged across repeats. To show the spatial distribution of input, maps were first peak-normalized (Figures 5, 7D, S9E, and S9F) and then averaged across cells within a class. Normalization was necessary because response amplitudes vary across experiments depending on the infection efficiency and the ChR2 expression level. To quantify the total input for pairs of neighboring neurons we summed all pixels that showed significant responses (>6× standard deviation of the baseline; Figures 3D, 4C–4F, 6, 7E, S8D, and S8E).