Article Figures & Data
Figures
- Figure 1.
Optogenetic tagging and functional identification of genetically defined L6 CT neurons in wS1 of the Ntsr1-Cre driver mouse line. A, Left, Confocal image of a fixed brain section (40 µm), centered on wS1, from an Ntsr1-Cre x floxed-tdTomato reporter (Ai14) mouse. White arrows indicate Ntsr1 expressing CT somata in L6. Right, Confocal image from the same fixed section, centered on the somatosensory thalamus, showing tdTomato-labeled CT axons terminating densely. VPm, Ventral posterior medial nucleus; POm, posterior medial nucleus; TRN, thalamic reticular nucleus. The tissue was stained immunohistochemically for NeuN (blue). B, Schematic of the experimental setup. Head-fixed Ntsr1-Cre x floxed-ChR2-EYFP reporter (Ai32) mice stood or walked/ran on a flexible circular treadmill during juxtacellular recordings from neurons in wS1 while we simultaneously monitored mystacial pad EMG, pupil diameter, and locomotion. We used an optical fiber located inside the glass recording pipette to deliver blue light locally through the tip for stimulation and identification of ChR2-expressing L6 CT cells in vivo (optogenetic tagging; 465 nm LED). C, Top, Raster plot of light-evoked spiking (blue dots) order by trial number for a ChR2-expressing L6 CT neuron. Brief LED flashes (5 ms) from the pipette tip evoked reliable spikes with short onset latencies (blue voltage trace) across all trials (gray traces). Bottom, Peristimulus time histogram of spike probability for 64 trials aligned on LED onset (blue line; bin size, 1 ms). We observed reliable light-evoked spikes within 2 ms of LED onset for this neuron. D, Post hoc confocal images of the same neuron (shown in C) labeled with neurobiotin by in vivo juxtacellular methods during the recording. Membrane ChR2-EYFP expression histologically confirmed that this recorded cell was a ChR2-expressing L6 CT neuron in the Ntsr1 x Ai32 mouse. E, Top, The normalized spike probability of all optogenetically tagged L6 CT neurons during brief photostimulation (n = 115 cells from 27 mice), sorted by mean spike probability in the 5 ms after LED onset. The white line indicates LED onset. Middle and bottom, The normalized spike probability of all RS (n = 53; peak-trough duration ≥ 0.5 ms) and FS cells (n = 60; peak-trough duration ≤ 0.4 ms, peak-trough ratio ≤ 2.5), respectively. F, Same as E but with a different scale (0–0.1 normalized spike probability) for better visualization of spike latency and suppressed units. G, Top left, Waveforms of spikes (recorded juxtacellularly) of a photoactivated L6 CT neuron (blue), putative FS interneuron (red), and RS cell (gray). Top right, Example waveform of a spike recorded extracellularly (n = 6 of 115 cells) from a photoactivated L6 CT cell (blue). Insets, Parameters measured. Bottom left, Plot showing the separation of putative FS interneurons (red) and RS cells (gray) from optically identified L6 CT (blue) based on the peak-to-trough duration and the peak-to-trough ratio of their average recorded spike waveform. Right, Plot showing the magnitude and timing of peak spike probability during photostimulation. Light-evoked response of L6 CT cells exhibited greater spike probability and shorter latency to peak spiking probability than putative FS interneurons and RS cells. The squares indicate cells confirmed based on neurobiotin staining and EYFP expression (6 CT cells and 6 non-CT cells), whereas the triangles represent the six extracellular CT units included in the dataset. The trough-to-peak ratio and interval were plotted for all extracellularly recorded CT cells. The absolute peak-to-trough ratio was plotted for all recorded cells.
- Figure 2.
L6 CT neuron spiking as a function of behavioral state in awake, head-fixed mice. A, Representative data from two types of Whisking-unresponsive L6 CT neurons recorded during quiet awake periods and self-initiated whisking-related behaviors (two examples highlighted by gray shading), Sparse-type (left) and Silent-type (right). The mean firing rate for the Sparse-type CT cell was 0.012 spikes/s, whereas the Silent-type never fired during the entire recording session. Instantaneous firing rate is shown, together with rectified EMG of the contralateral mystacial pad musculature, pupil diameter, and walking patterns on the same time base. The instantaneous firing rate and pupil diameter were smoothed with a low-pass Butterworth filter in both directions (100 ms window). B, Representative data from two types of Whisking-responsive L6 CT neurons recorded during quiet awake periods and self-initiated whisking-related behaviors (two examples highlighted by gray shading), Activated-type (left) and Suppressed-type (right). The Activated-type cell increased spiking rates during self-initiated whisking-related behavior, whereas the Suppressed-type cell decreased spiking rates. C, Overview of all recorded activity from L6 CT cells, sorted by firing rate difference between baseline (−4 to −1 s) and whisking (0–3 s) during both whisking/locomotion (left) and whisking only (right). The white line indicates EMG onset, and the color coding corresponds to the mean firing rate. D, Pie chart displaying the proportions of CT cell types identified based on their activity patterns. E, Left, Fluorescent image of a fixed slice (100 µm) showing a post hoc histologically verified ChR2-expressing CT cell within L6. Note the robust EYFP expression in L6 and a weaker fluorescence band near the L4–5 border. Right, Depth distribution of all histologically confirmed CT neurons.
- Figure 3.
Changes in activity during whisking with concomitant locomotion align better with EMG onset than locomotion. A, Juxtacellular unit responses recorded from an example Activated-type CT neurons displayed as raster plots (top) and peristimulus time histograms (PSTHs; bottom) aligned on locomotion onset (blue raster and line) and EMG onset (red raster; time = 0) during bouts of whisking/locomotion. Note the better temporal alignment between the change in firing rates and EMG onset than locomotion. B, Left, The normalized spiking patterns of all Whisking-responsive wS1 L6 CT neurons aligned on locomotion onset (left) or EMG onset (right) during whisking/locomotion. Each row is an individual neuron normalized to maximum firing rate and sorted by the mean baseline activity (3 s preceding EMG onset; white line; time = 0). C, Population PSTHs for both Activated-type (n = 19 cells) and Suppressed-type CT cells (n = 13 cells) aligned on locomotion onset (blue line) and EMG onset (red line; time = 0). Neural activity is plotted as mean ± SEM (gray shadow indicating SEM). D, Summary graphs of spiking activity for all Activated-type CT cells. Top, Mean firing rate during whisking/locomotion (red circles) and solitary whisking (blue circles), plotted as a function of mean baseline activity preceding EMG onset (n = 19 cells; p = 2.44e-04 and 4.88e-04, respectively; Wilcoxon signed-rank test). Bottom, Peak firing rate for each neuron during whisking/locomotion plotted as a function of peak activity during whisking (p = 2.44e-04; Wilcoxon signed-rank test). E, Summary graphs of spiking activity for all Suppressed-type CT cells. Top, Mean firing rate during whisking/locomotion (red circles) and whisking (blue circles), plotted as a function of mean baseline activity preceding EMG onset (n = 13 cells; p = 0.03 and 4.88e-04, respectively; Wilcoxon signed-rank test). Bottom, Trough (lowest) firing rate for each neuron during whisking/locomotion plotted against that for whisking (p = 2.44e-04, Wilcoxon sign-rank test). F, G, Summary graphs of spiking activity for all Sparse- and Silent-type CT cells. Top, Mean firing rate during whisking/locomotion (red circles) and whisking (blue circles), plotted as a function of mean baseline activity preceding EMG onset (Sparse-type, n = 50, p = 0.1 and 0.09, respectively; Wilcoxon signed-rank test). Bottom, Peak firing rate for each neuron during whisking/locomotion plotted as a function of peak activity during whisking (Sparse-type, n = 50, p = 0.3; Wilcoxon signed-rank test). The filled circles represent means in D–G. Values indicate mean ± SEM.
- Figure 4.
Diverse response profiles exhibited by whisker-related L6 CT neurons in wS1. A, Top, Examples of juxtacellular unit responses recorded from individual L6 CT neurons displayed as raster plots aligned on locomotion onset (top row, blue) and EMG onset (bottom row, red dashed line; time = 0) during whisking/locomotion. Bottom, Peristimulus time histograms (PSTHs) from the same cell aligned to EMG onset. The first four examples (left to right) show Activated-type CT neurons, whereas the last two are Suppressed-type CT cells. Activated-type cells had either tonic firing at EMG onset (left), phasic increases in activity at EMG onset (second from left), a slow buildup of activity preceding EMG onset (third from left), or delayed increases in activity from EMG onset (fourth from left). Suppressed-type neurons showed less diversity and tended to have decreased spiking rates before EMG onset. For the PSTHs, neural activity (black line) and EMG activity (red line) are plotted as mean ± SEM (gray shadow indicating SEM). B, Juxtacellular unit responses for the same CT neurons (shown in A) displayed as raster plots and PSTHs aligned on EMG onset (red dashed line; time = 0) during whisking and no locomotion. We found that most CT cells were modulated during solitary whisking similarly to whisking/locomotion. The only exception were cells that had delayed increases in activity during locomotion (i.e., the fourth cell from left), which did not respond during whisking only. Values indicate mean ± SEM.
- Figure 5.
Subpopulations of Whisking-responsive L6 CT cells are modulated before whisker movement. A, Top, Two Activated-type CT neurons exhibiting pre-EMG spike enhancement. Bottom, Two Suppressed-type CT neurons exhibiting pre-EMG spike suppression. The red and black vertical dashed lines indicate EMG onset and the detected whisker-related spike modulation, respectively. B, Shown are the onset latencies of the pre-EMG spiking modulation for five Activated- and 10 Suppressed-type CT neurons with a mean baseline firing rate >2 spikes/s before whisking/locomotion. Most Activated-type CT cells (8 of 13) had little or no baseline firing rate, rendering them unsuitable for analysis (see text), whereas most Suppressed-type CT neurons (9 of 10) exhibited pre-EMG spiking suppression. C, Shown are the onset latencies of the pre-EMG spiking modulation for seven Activated- and 10 Suppressed-type CT neurons with a mean baseline firing rate >2 spikes/s before solitary whisking episodes. Like B, most Suppressed- and some Activated-type CT neurons exhibited pre-EMG spiking modulation. D, Averages of mean normalized pupil diameter and mean locomotion activity of all locomotion bouts for each neuron. Data are aligned on the onset of locomotion across all sessions when a Whisking-responsive neuron was recorded (n = 32 sessions). Pupil diameter was normalized to maximum dilation during the session. Values indicate mean ± SEM. E, Averages of mean EMG and mean pupil diameter during whisking/locomotion (same data shown in D) but now aligned on EMG onset. Values indicate mean ± SEM. F, Averages of mean EMG and mean normalized pupil diameter during whisking aligned on EMG onset across all sessions (n = 32 sessions). Pupil modulation occurs after EMG onset. Values indicate mean ± SEM.
- Figure 6.
Contributions of whisking and arousal to L6 CT neuron in wS1 of awake mice. A, B, Cross-correlograms of spiking activity and EMG signals (top, red) or pupil dynamics (bottom, blue) from Activated-type (A) and Suppressed-type CT neurons (B) during whisking/locomotion (left) and whisking (right). Light-colored lines (red and blue) represent the average trial-by-trial cross-correlations for each Whisking-responsive CT neuron. The dark-colored lines (red and blue) represent the mean cross-correlation across all neurons (n = 13/13 Activated-type cells; n = 11/12 Suppressed-type cells for whisking/locomotion and whisking, respectively). C, D, Left, Summary graphs showing the absolute peak cross-correlation of pupil dynamics to firing activity as a function of the peak cross-correlation of EMG and firing activity for each Activated- (C, left) and Suppressed-type CT cell (D, left) during whisking/locomotion (black circles) and whisking (gray circles). Right, Summary graphs showing the comparison of lags at which peak absolute correlation coefficients were obtained for Activated- (C, right) and Suppressed-type CT neurons (D, right) during whisking/locomotion and whisking (Activated-type mean cross-correlation values, EMG during whisking/locomotion, 0.56 ± 0.20; pupil during whisking/locomotion, 0.51 ± 0.21; EMG during whisking, 0.31 ± 0.16; pupil during whisking, 0.22 ± 0.12; Suppressed-type mean cross-correlation values, EMG during whisking/locomotion, 0.56 ± 0.17; pupil during whisking/locomotion, 0.50 ± 0.16; EMG during whisk only, 0.27 ± 0.16; pupil during whisk only, 0.23 ± 0.17). Data in C (right) and D (right) are plotted as median, interquartile distance, and range. Individual data points are represented as open circles.
- Figure 7.
Whisking-unresponsive L6 CT cells with sparse firing preferentially fired during pupil constrictions. A, Pupil diameter (black trace), rectified EMG (red trace), and spike times (gray ticks) are shown as a function of recording time for a Sparse-type CT cell. Insets, Expansion in time showing the relationship between spiking, EMG, and pupil diameter (1, 2, and 3; gray shading). Note small increases in pupil diameter are seen even in the absence of whisker movement (i.e., EMG changes). Note the increased incidence of spikes during pupil constriction. B, Spike-triggered pupil average for the same pupil cell pair (shown in A). Left, Raw pupil traces aligned on spike onset (n = 362 spikes; time = 0, dotted vertical line). It is hard to appreciate any trends in pupillary responses as they spanned a broad range of pupil diameters. For better visualization, we normalized the extracted pupillary responses to the peak of each extracted pupillary response. Middle, The same spike-aligned pupil traces normalized to their peak value. Note the clear tendency of preceding pupil constriction in most instances just before spiking. Right, Mean and SEM of the normalized spike-triggered pupil (black line and gray shadow). C, The normalized spike-triggered pupil average for each Sparse-type CT neuron (n = 43/50 cells; similar to B, right). Only Sparse-type cells with more than five spikes recorded in the entire session were used for analysis (7 cells excluded with <5 spikes). D, A summary graph of all normalized spike-triggered pupil traces (n = 5243 traces) across all the silent CT cells (n = 50 cells). E, A summary graph showing the spike-triggered pupil traces shown in D after separating them based on EMG activity at the time of the spike. Inset, The mean spike-triggered EMG signal across the two behavioral states (Whisking/high EMG activity, n = 1347 spikes; No whisking/low EMG activity, n = 3896 spikes). Whisking/high EMG activity was defined as an average EMG exceeding 20 µV in the 1.5 s preceding the spike. Values indicate mean ± SEM.
