High-velocity stimulation evokes "dense" population response in layer 2/3 vibrissal cortex

Abstract

Supragranular layers of sensory cortex are known to exhibit sparse firing. In rodent vibrissal cortex, a small fraction of neurons in layer 2 and 3 (L2/3) respond to whisker stimulation. In this study, we combined whole cell recording and two-photon imaging in anesthetized mice and quantified the synaptic response and spiking profile of L2/3 neurons. Previous literature has shown that neurons across layers of vibrissal cortex are tuned to the velocity of whisker movement. We therefore used a broad range of stimuli that included the standard range of velocities (0-1.2 deg/ms) and extended to a "sharp" high-velocity deflection (3.8 deg/ms). Consistent with previous literature, whole cell recording revealed a sparse response to the standard range of velocities: although all recorded cells showed tuning to velocity in their postsynaptic potentials, only a small fraction produced stimulus-evoked spikes. In contrast, the sharp stimulus evoked reliable spiking in the majority of neurons. The action potential threshold of spikes evoked by the sharp stimulus was significantly lower than that of the spontaneous spikes. Juxtacellular recordings confirmed that application of sharp stimulus to single or multiple whiskers produced temporally precise spiking with minimal trial-to-trial spike count variability (Fano factors equal or close to the theoretical minimum). Two-photon imaging further confirmed that most neurons that were not responsive to the standard deflections responded to the sharp stimulus. Altogether, our results indicate that sparseness in L2/3 cortex depends on the choice of stimulus: strong single- or multiwhisker stimulation can induce the transition from sparse to "dense" population response.NEW & NOTEWORTHY In superficial layers of sensory cortex, only a small fraction of neurons fire most of the spontaneous and sensory evoked spikes. However, the functional relevance of such "sparse" activity remains unknown. We found that a "dense" population response is evoked by high-velocity micromotions applied to whiskers. Our results suggest that flashes of precisely timed population response on an almost silent background can provide a high capacity for coding of ecologically salient stimuli.

Keywords: AP threshold; Fano factor; postsynaptic potentials; somatosensory; sparse coding; two-photon imaging; whisker velocity.

Fig. 1.

Experimental setup and stimulus properties. A: schematic illustration of the experimental setup. B: average position profile of the piezo movement (100 trials per each stimulus). This color convention (grayscale for the standard and green for the sharp stimulus) is used in all figures. C: peak velocity of the piezo movement plotted against peak amplitude. Every dot represents 1 stimulus. D: example evoked LFPs averaged across 25 trials per stimulus.

Fig. 2.

Synaptic responses in L2/3 neurons. A: average PSPs of an example neuron plotted vs. time from the stimulus onset (25 trials per stimulus). Inset shows an expanded view of the first 50 ms. B: average PSPs across 20 neurons. C, top: peak of PSP is averaged over trials and then over neurons (mean ± SE over neurons). Bottom, delay of the peak PSP averaged over trials and then over neurons (means ± SE over neurons). Note that in both panels (and subsequent figures), x-axis is broken beyond 1.2. D: APs per trial vs. stimulus peak velocity for whole cell recordings (circles). Red circles represent significant response based on ROC analysis. Average AP per trial across 20 cells is slightly shifted rightward and plotted as black diamonds (means ± SE). Inset raster plot represents spikes in response to the sharp stimulus for an example neuron-stimulus pair (25 trials). The red circle corresponding to the example neuron-stimulus pair is filled in black. Pie charts at top represent fraction (in percent) of significantly responsive neurons for each stimulus. E: spontaneous (gray) and sharp stimulus evoked (green) APs of an example neuron. Individual AP thresholds are shifted to the right for better visibility and are plotted along with their mean and SD. Inset represents an expanded view of the initial segment of the AP waveforms (dashed box). The dots on each expanded waveform represent the position of the peak of the second derivative, representing the AP threshold. F: AP thresholds across 20 neurons for sharp stimulus evoked (green) and spontaneous (gray) spikes. Each line represents a neuron, and diamonds are means ± SE.

Fig. 3.

Spiking responses for juxtacellular recordings. A: spiking responses evoked by multiwhisker deflections (details as described in Fig. 2D). B: intrinsic signal optical imaging through intact skull. Top, vasculature pattern imaged using 527-nm light. Middle, intrinsic signal (dark spot) captured by whisker deflections under 626-nm light. Bottom, merged image of top (green) and middle (red) panels. C: spiking responses evoked by C2 whisker deflections (details as described in Fig. 2D).

Fig. 4.

A: box and whisker plot representing spike time variation about the median (black lines) in response to the sharp stimulus. B: Fano factor vs. APs per trial (75 ms). Color coding of the circles is retained from Fig. 1B. Solid brown curve represents the theoretical minimum Fano factor. C: color plot representing APs per trial (color coded) vs. stimulus intensity (x-axis) vs. depth (y-axis) across 84 neurons, including all previous neurons from Figs. 2 and 3 (71 neurons), 7 fast-spiking (FS) neurons, and 6 deep neurons (>400 µm). Significant responses are marked with cyan asterisks. Arrowheads at right indicate FS neurons. D: cumulative distribution of response across all 77 regular-spiking (RS) neurons color coded for different stimuli as in Fig. 1B. E: juxtacellular spike waveforms of RS (red; n = 19) and FS (blue; n = 7) neurons (means ± SD). F: average response of FS (blue; left y-axis) and RS (red; right y-axis) neurons vs. stimulus intensity.

Fig. 5.

Calcium imaging from L2/3 vS1. A: imaging field with green and red channels overlaid. Top image is the area outlined in white at bottom, recorded with higher magnification (scale bars are 10 µm). Neuron 1 is targeted for simultaneous juxtacellular recording. B: example calcium-dependent change in fluorescence (ΔF/F) of 4 neurons labeled in A. Black dots at top represent juxtacellular spikes of neuron 1. Subsequent spikes occurring in the same frame are shown above the previous ones. Vertical lines represent stimuli, color coded as per convention. C: an example session where the area under the ROC curve (AUROC) is plotted vs. stimulus velocity (n = 56). Red circles represent significant detection performance (P < 0.05). Pie charts at top represent fraction of neurons (in percent) with significant response. Inset shows average peristimulus (at zero) ΔF/F over trials, over neurons. D: average AUROC for each session (gray plots, 44 sessions). The average AUROC of the example session in C is plotted in blue. Pie charts at top show the fraction of all imaged neurons with significant response. E: cumulative distribution of AUROCs across 1,640 imaged cells color coded for different stimuli as in Fig. 1B. Cumm. Frac., cumulative fraction. F: average of the evoked ΔF/F (baseline subtracted) for each session (gray plots). Inset shows peristimulus ΔF/F averaged over all sessions. Black diamonds in all panels represent means ± SE.