Microstimulation of sensory cortex engages natural sensory representations

Abstract

Cortical activity patterns occupy a small subset of possible network states. If this is due to intrinsic network properties, microstimulation of sensory cortex should evoke activity patterns resembling those observed during natural sensory input. Here, we use optical microstimulation of virally transfected layer 2/3 pyramidal neurons in the mouse primary vibrissal somatosensory cortex to compare artificially evoked activity with natural activity evoked by whisker touch and movement ("whisking"). We find that photostimulation engages touch- but not whisking-responsive neurons more than expected by chance. Neurons that respond to photostimulation and touch or to touch alone exhibit higher spontaneous pairwise correlations than purely photoresponsive neurons. Exposure to several days of simultaneous touch and optogenetic stimulation raises both overlap and spontaneous activity correlations among touch and photoresponsive neurons. We thus find that cortical microstimulation engages existing cortical representations and that repeated co-presentation of natural and artificial stimulation enhances this effect.

Keywords: barrel cortex; cortex; cortical plasticity; microstimulation; pattern completion; recurrent networks.

Figure 1.. Model of vS1 L2/3 clustered subnetwork response to random stimulation

(A) Integrate-and-fire model of barrel cortex. Left, model includes 170 clustered excitatory neurons (blue), 1,530 unclustered excitatory neurons (grey), and 300 inhibitory neurons (black). Right, ‘random’ stimulation was simulated by assigning 20% of both clustered and unclustered neurons to the opsin-expressing group (Opsin⁺, orange) and stimulating these neurons with a simulated photostimulus (red, 5 ms current pulse). Both neurons that are directly stimulated and indirectly stimulated respond to simulated photostimulation (red outline, darker color). (B) Example response to simulated photostimulation. Left, raster plots showing response among the top 25 most responsive clustered neurons (blue) and top 100 most responsive unclustered neurons (grey). Right, mean firing rate response among directly (top) and indirectly stimulated (middle) pyramidal neurons (blue, clustered; grey, unclustered) and inhibitory neurons (bottom, black). (C) Mean pairwise Pearson’s correlation of firing rate during ‘spontaneous’ activity (Methods). Mean correlation among unclustered excitatory neurons (grey) and among excitatory clustered neurons (blue). Each line represents a single network (N=13). P-value across networks for paired Wilcoxon signed rank test. ***, P < 0.001. (D) Calculation of overlap between the clustered and photoresponsive populations relative to random. Left, composition of the example population (indirectly stimulated, opsin non-expressing population). Right, calculation of overlap relative to random in this simplified example (Methods). (E) Left, schematic showing propagation of activity from directly stimulated, opsin-expressing (Opsin⁺, orange) to indirectly stimulated, opsin non-expressing (Opsin⁻, green) neurons. Right, overlap relative to random for opsin-expressing and opsin non-expressing populations. P-value within a group is for Wilcoxon signed rank test evaluating whether the median differs from 0. P-value across groups is for paired Wilcoxon signed rank test. **, P < 0.01; ***, P < 0.001.

Figure 2.. Neurons in L2/3 of vS1 respond to photostimulation, touch, and whisking

(A) Opsin expression in barrel cortex. Left, widefield view of cranial window showing viral injection sites (numbers) and LED; center, widefield two-photon image of cranial window after onset of opsin expression (green, GCaMP6s fluorescence; red, mScarlet fluorescence); right, higher magnification two-photon image. (B) Left, trial structure during a rewarded photostimulation trial. Right, trial structure during a rewarded vibrissal touch trial. (C) Volumetric two-photon imaging. Subvolumes consist of 3 planes imaged simultaneously at 7 Hz. Left, six planes (800-by-800 μm, 20 μm inter-plane distance, 2 subvolumes); right, example plane with example opsin-expressing (orange) and opsin non-expressing (green) neurons. (D) Left, chematic showing the expected local propagation of activity from opsin-expressing (orange) to opsin non-expressing (green) neurons. Right, photostimulation-evoked $\text{ΔF}/\text{F}$ traces for 4 neurons. Light, individual trials; dark, mean. Orange, opsin- expressing; green, opsin non-expressing. A PMT shutter prevented acquisition during photostimulation (Methods). (E) Whisker videography in an example trial. Top, example frames; bottom, whisker angle ($\text{θ}$, black), and change in whisker curvature ($\text{Δκ}$, grey, see Methods), with touch periods indicated with colored circles. (F) $\text{ΔF}/\text{F}$ traces for 4 neurons aligned to whisker touch (left column) and to whisk onset (right column). Grey, individual trials; black, mean. Top two neurons, touch neurons; bottom two neurons, whisking neurons. See also Figure S1, Table S1.

Figure 3.. Overlap between touch responsive and photoresponsive populations

(A) Photoresponsive (top, red) and touch responsive (bottom, blue) populations in an example mouse. Neurons are collapsed across six planes spaced 20 μm apart. Colored circles show the mean touch (blue) or photostimulation (red) evoked $\text{ΔF}/\text{F}$ across stimulus presentations. Black dots indicate neurons that did not belong to the top 10% of responders (Methods). (B) Top, overlay of the maps in (A), with non-responsive neurons removed. Neurons belonging to both representations are marked with a black dotted circle. Bottom, stimulus-aligned photostimulation (red, top) and touch (blue, bottom) $\text{ΔF}/\text{F}$ responses of four example neurons. Dark color, mean response; light color, individual responses. (C) Relationship between mean $\text{ΔF}/\text{F}$ touch and photostimulation responses for all responsive neurons in an example mouse (including both opsin-expressing and opsin non-expressing neurons). Blue, neurons that belong only to the top 10% of touch responders; red, neurons that belong only to the top 10% of photostimulation responders; magenta, neurons that belong to both groups (‘dual’). (D) Overlap between vibrissal and photostimulation responsive populations relative to chance for opsin-expressing (orange) and opsin non-expressing (green) neurons across mice (N=13). P-values are given for the Wilcoxon signed rank test assessing whether the median is different than 1 (chance); P-values for comparisons between opsin-expressing and opsin non-expressing populations and between touch-photoresponsive and whisking-photoresponsive populations are for the Wilcoxon signed rank test, paired by animal. Left, overlap between touch responsive and photoresponsive population; right, overlap between whisking responsive and photoresponsive population. See also Figures S2, S3, S4.

Figure 4.. Spontaneous activity correlations within different populations

(A) Pairwise correlations during the inter-stimulus (‘spontaneous’) period in an example mouse. Left, photoresponsive neurons. Middle, touch responsive neurons. Right, dual-representation neurons. Neurons are sorted by their mean correlation to the other neurons within the population. (B) Mean spontaneous pairwise correlations across mice (N=13) for photoresponsive (red), touch responsive (blue), and dual-representation (magenta) neurons. P-value provided Wilcoxon signed rank test comparing two populations, paired by animal. ***, P < 0.001; **, P < 0.01 ; *, P < 0.05. (C) As in (B), but for 95^th percentile of spontaneous pairwise correlations.

Figure 5.. Change in overlap between touch and photoresponsive populations following induction

(A) Timeline of the induction experiment. Top, breakdown of sessions in induction experiment. Ten session induction block is preceded and followed by touch-only sessions (Methods). Middle, dual-stimulus induction features photostimulation (9 pulses; Methods) during the period of pole accessibility. Bottom, photostimulation-only induction does not include pole presentation. (B) Maps showing the photoresponsive (red) and touch responsive (blue) populations in an example mouse before (left) and after (right) dual-stimulus induction. Colored circles show the mean touch (blue) or photostimulus (red) evoked $\text{ΔF}/\text{F}$ across stimulus presentations. Neurons belonging to both representations are circled with a dotted line. Bottom, response of two example neurons prior to (left) and after (right) induction. (C) Touch-photoresponsive overlap among opsin-expressing (orange) and opsin non-expressing (green) populations before and after induction. Mice were exposed to dual-stimulus induction (lighter color, N=7 mice) or photostimulation-only induction (darker color, N=6 mice). P-values provided for Wilcoxon signed rank test comparing response before and after induction. ***, P < 0.001; **, P < 0.01 ; *, P < 0.05. (D) As in (C), but for whisking-photoresponsive overlap. See also Figures S5, S6.

Figure 6.. Change in touch and photoresponsive populations following induction

(A) Photoresponsive (top, red) and touch responsive (bottom, blue) populations in an example mouse before (left) and after (right) dual-stimulus induction. Neurons are collapsed across six imaging planes spaced 20 μm apart. Colored circles show the mean touch (blue) or photostimulation (red) evoked $\text{ΔF}/\text{F}$ across stimulus presentations. (B) Change in mean stimulus-evoked $\text{ΔF}/\text{F}$ among the top 10% of most photoresponsive neurons following induction. Left, opsin-expressing; right, opsin non-expressing. In each case, we examined mice exposed to dual-stimulus induction (N=7; lighter color), and photostimulation-only induction (N=6; darker color). P-values provided for signed rank test comparing response before and after induction. ***, P < 0.001; **, P < 0.01 ; *, P < 0.05. (C) As in (B), but for touch responsiveness. (D) Stimulus response (mean evoked $\text{ΔF}/\text{F}$) before and after dual-stimulus induction. Left (red), photostimulation response among all responsive neurons before and after induction in an example mouse. Right (blue), touch response before and after induction. (E) Correlation of pre- and post-induction mean evoked $\text{ΔF}/\text{F}$ for photostimulation responsive (red) and touch responsive (blue) populations. Left, opsin-expressing; right, opsin non-expressing neurons. ***, P < 0.001; **, P < 0.01 ; *, P < 0.05; P-value for Wilcoxon signed rank test comparing touch and photoresponsive populations. See also Figure S7.

Figure 7.. Spontaneous activity correlations before and after induction

(A) Spontaneous pairwise correlation matrices for the photoresponsive population in an example mouse before (left) and after (right) dual-stimulus induction. Top, opsin-expressing population; bottom, opsin non-expressing. Neurons are sorted by their mean correlation to the other neurons within the population. (B) Mean spontaneous pairwise correlations before and after induction among photoresponsive neurons. Grey, mice that were exposed to dual-stimulus induction (N=7); black, mice that were exposed to photostimulus-only induction (N=6). P-values provided for Wilcoxon signed rank test comparing fraction before and after induction. ***, P < 0.001; **, P < 0.01 ; *, P < 0.05. Top, opsin-expressing neurons; bottom, opsin non-expressing neurons (C) As in (B), but for correlations between touch responsive and photoresponsive neurons. (D) As in (B), but for touch responsive neurons. (E) As in (B), but for dual-representation neurons.