Cellular mechanisms for response heterogeneity among L2/3 pyramidal cells in whisker somatosensory cortex

Abstract

Whisker deflection evokes sparse, low-probability spiking among L2/3 pyramidal cells in rodent somatosensory cortex (S1), with spiking distributed nonuniformly between more and less responsive cells. The cellular and local circuit factors that determine whisker responsiveness across neurons are unclear. To identify these factors, we used two-photon calcium imaging and loose-seal recording to identify more and less responsive L2/3 neurons in S1 slices in vitro, during feedforward recruitment of the L2/3 network by L4 stimulation. We observed a broad gradient of spike recruitment thresholds within local L2/3 populations, with low- and high-threshold cells intermixed. This recruitment gradient was significantly correlated across different L4 stimulation sites, and between L4-evoked and whisker-evoked responses in vivo, indicating that a substantial component of responsiveness is independent of tuning to specific feedforward inputs. Low- and high-threshold L2/3 pyramidal cells differed in L4-evoked excitatory synaptic conductance and intrinsic excitability, including spike threshold and the likelihood of doublet spike bursts. A gradient of intrinsic excitability was observed across neurons. Cells that spiked most readily to L4 stimulation received the most synaptic excitation but had the lowest intrinsic excitability. Low- and high-threshold cells did not differ in dendritic morphology, passive membrane properties, or L4-evoked inhibitory conductance. Thus multiple gradients of physiological properties exist across L2/3 pyramidal cells, with excitatory synaptic input strength best predicting overall spiking responsiveness during network recruitment.

Keywords: cortex; map plasticity; somatosensory; synaptic mechanisms; vibrissa.

Fig. 1.

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.

Fig. 2.

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.

Fig. 3.

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).

Fig. 4.

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.

Fig. 5.

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 Fig. 4. Bars indicate means ± SE.

Fig. 6.

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.

Fig. 7.

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.

Fig. 8.

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.