PMC

Intrinsic excitability varies with L4 activation threshold. A and B: spike threshold and resting potential (V_rest) for all neurons (n = 160 cells). n.s., Not significant. C: I_h measured as membrane potential (V_m) sag ratio in response to a 500-ms, −200-pA current step from V_rest. n = 124 cells. D: input resistance (R_input) for all cells (n = 158 cells). For all panels, red symbols are low-threshold cells and black symbols are high-threshold cells. Black P values show 2-group comparison between low- and high-threshold cells. Gray P values are for linear regressions for all cells, including cells with intermediate activation thresholds (gray lines). Bars show means ± SE.

Comparison of activation thresholds on 2 stimulation pathways. A: stimulation electrodes and imaging field for a 2-pathway experiment. The field potential recording electrode (for stimulus normalization) is shown in the center of the imaging field. B: L4-evoked spiking recruitment for 6 cells, all imaged simultaneously in the same field. Dark gray, spike probability in response to home-column stimulation; light gray, adjacent-column stimulation. Dashed lines connect activation thresholds on both pathways. Cells are ordered by home-column activation threshold. C: correlation between activation thresholds (ranked within each imaging field) for home- and neighboring-barrel stimulation (54 cells, 4 slices). Filled symbols show all cells in 1 example experiment (triangles are the 6 cells in B).

Dendritic morphology and spine density in low- and high-threshold cells. A: Neurolucida reconstructions of a low (red)- and a high (black)-threshold cell from the same slice. B: Sholl analysis showing mean dendritic length in radial 10-μm bins around the soma. Cell numbers are indicated. Shading indicates mean ± SE across neurons. C: example of Alexa Fluor 594-filled high-threshold pyramidal cell. Arrowhead indicates apical trunk. A tertiary basal dendrite is boxed and enlarged at bottom to show spines. D: spine density for each analyzed basal branch, plotted vs. the activation threshold for each cell (89 branches from 20 cells). Red and black points are branches on low- and high-threshold cells. Bars indicate means ± SE.

Excitatory (G_e) and inhibitory (G_i) synaptic conductances in low- and high-threshold pyramidal cells. A, top: 3 cocolumnar pyramidal cells filled with Alexa Fluor 594. Red and black dots indicate low- and high-threshold cells. Arrowheads show apical dendritic trunks. Bottom: example currents from a synaptic conductance recording of a low-threshold cell, averaged across 5 sweeps. Cell was stimulated at its activation threshold of 37.6% maximum L4 stimulation. Arrowhead indicates L4 stimulation. B: average G_e and G_i waveforms measured in low-threshold (red) and high-threshold (black) cells. Shading indicates SE. L4 was stimulated at 0 ms. C, left: postsynaptic potential (PSP) predicted for each cell with a single-compartment model, based on the actual G_e and G_i waveforms measured in each cell. Right: predicted PSP peak for each low-and high-threshold cell (open symbols). Filled symbols are means ± SE.

Correlation between L4-evoked and whisker-evoked responsiveness in vivo. A: experimental setup for 2-photon calcium imaging of L2/3 pyramidal cells in the anesthetized mouse. Calcium transients were measured in response to both whisker stimulation and direct electrical stimulation of L4. B: example field of view after bolus loading with OGB-1 AM in a P36 mouse. C: ΔF/F traces from 4 cells (labeled 1–4 in B) during repeated L4 stimulation (left) and deflection of the principal whisker (PW; right). Vertical red lines indicate stimulation times. D: comparison of responses to electrical and whisker stimulation for each neuron (n = 132 cells, n = 6 fields, 4 animals). Filled points indicate cells from a single case.

Initial doublet spiking is inversely related to L4-evoked responsiveness. A: example firing patterns in response to 500-ms current injection from V_rest. The high-threshold cell generates a higher-frequency initial doublet at 140–200 pA. ADP, afterdepolarization. B: rheobase for all cells (n = 160). C: firing rate of first 2 spikes [inverse of first interspike interval (ISI)] as a function of current injection amplitude, for all cells (n = 160). Cells are divided into quartiles of L4-evoked activation threshold (n = 34–50 cells/group). Dashed lines show comparisons plotted in D and E. D: firing rate for first 2 spikes for current injection of 240 pA above rheobase, for all cells (n = 160). E: current injection (above rheobase) required to elicit a first ISI of ≤10 ms, termed the “burst step,” for all cells (n = 160). Conventions for colors and P values are as in . Bars indicate means ± SE.

L4-evoked activation of L2/3 neurons measured by population calcium imaging. A: the S1 slice preparation. White box shows imaging L2/3 field after Oregon Green BAPTA-1 AM (OGB-1 AM) loading. Letters indicate whisker identity of visualized barrel columns. Inset: schematic of the feedforward circuit. B: example ΔF/F traces (black) and detected spikes (green dots) from 7 neurons in a L2/3 imaging field. L4 was stimulated periodically (blue diamonds) at 17 μA intensity (corresponding to 40% of max stimulation for this column). A simultaneous loose-seal recording of spiking was made in 1 neuron (top right). Only a subset of cells spiked to this stimulation intensity. C: pharmacological test for antidromic activation of L2/3 cells. Spike probability was measured for 191 cells (14 slices), of which 148 spiked in response to strong L4 stimulation (mean 89.3 ± 0.9% normalized stimulus intensity). All but 6 cells (red) ceased spiking when kynurenic acid (kyn) and picrotoxin (picro) were added, indicating a 4% antidromic activation rate at high stimulation intensity.

Distribution of L4-evoked activation thresholds among L2/3 pyramidal cells. A: L4-evoked activation curves for all neurons in a single column, imaged simultaneously. Dots show L4 stimulation intensities that were tested. For the 1 neuron highlighted in black, the red line marks the activation threshold. Inset: method for normalizing stimulation intensity. Stimulation intensity was measured as the L2/3 field potential amplitude, normalized to the maximal (saturating) local field potential amplitude recorded in that column. B: distribution of activation thresholds across all cells in calcium imaging experiments. Red and black bars indicate the bottom and top quartiles of activation thresholds, which define low- and high-threshold cell populations. NR, nonresponsive. Black curve, cumulative fraction of cells responding. C: distribution of ΔF/F peak amplitudes for all detected calcium transients in low- and high-threshold cells. N = 193 low-threshold events; N = 189 high-threshold events; P < 0.0001 rank sum test; 618 cells from 69 slices. Bin size = 0.5. D: L4-evoked activation threshold as a function of absolute subpial depth, for all neurons. Line, linear regression. E: distribution of activation thresholds for all cells measured with loose-seal recordings of L4-evoked spikes. Black and gray bars, activation thresholds for physiologically and/or morphologically identified neurons (targeted as low- and high-threshold cells in the overall population). Black, confirmed pyramidal cells; gray, confirmed interneurons.