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An acetylcholine-activated microcircuit drives temporal dynamics of cortical activity

Abstract

Cholinergic modulation of cortex powerfully influences information processing and brain states, causing robust desynchronization of local field potentials and strong decorrelation of responses between neurons. We found that intracortical cholinergic inputs to mouse visual cortex specifically and differentially drive a defined cortical microcircuit: they facilitate somatostatin-expressing (SOM) inhibitory neurons that in turn inhibit parvalbumin-expressing inhibitory neurons and pyramidal neurons. Selective optogenetic inhibition of SOM responses blocked desynchronization and decorrelation, demonstrating that direct cholinergic activation of SOM neurons is necessary for this phenomenon. Optogenetic inhibition of vasoactive intestinal peptide-expressing neurons did not block desynchronization, despite these neurons being activated at high levels of cholinergic drive. Direct optogenetic SOM activation, independent of cholinergic modulation, was sufficient to induce desynchronization. Together, these findings demonstrate a mechanistic basis for temporal structure in cortical populations and the crucial role of neuromodulatory drive in specific inhibitory-excitatory circuits in actively shaping the dynamics of neuronal activity.

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Figure 1: Optogenetic stimulation of ChAT-ChR2–expressing axons induces LFP desynchronization and decorrelation in layer 2/3 V1 neurons.
Figure 2: ACh induces facilitation at different dynamic ranges in layer 2/3 SOM, VIP and L1 inhibitory neurons in V1 slices.
Figure 3: Direct cholinergic facilitation of SOM responses leads to indirect inhibitory responses in FS (putative PV) and PYR neurons.
Figure 4: Optogenetic stimulation of ChAT-ChR2–expressing axons evokes diverse responses in V1 layer 2/3 SOM, PV and putative PYR neurons.
Figure 5: ChAT-ChR2 stimulation–induced LFP desynchronization and decorrelation is mediated by SOM neurons.
Figure 6: VIP neurons do not contribute to ChAT-ChR2 stimulation–induced LFP desynchronization.
Figure 7: Direct ChR2 stimulation of SOM neurons is sufficient to induce LFP desynchronization.

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Acknowledgements

We thank G. Feng and H. Robertson (Massachusetts Institute of Technology) for providing ChAT-ChR2 mice, E. Boyden and A. Yang (Massachusetts Institute of Technology) for providing Arch virus, C. Le for technical assistance with viral injections and immunohistochemistry, S. El Boustani, R. Huda and M. Goard of the Sur laboratory for careful reading of manuscript and critical technical advice, T. Emery for technical assistance with the optogenetics laser setup, and J. Sharma for technical assistance. We especially thank B. Sabatini, A. Granger, W. Xu and Z. Fu for providing critical technical advice. This work was supported by an A*STAR (Singapore) Fellowship (N.C.), and grants from the US National Institutes of Health (R01EY007023, R01EY018648, U01NS090473), the National Science Foundation (EF1451125) and the Simons Foundation (M.S.).

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Authors and Affiliations

Authors

Contributions

N.C. and H.S. designed, conducted and analyzed the ex vivo experiments. H.S. and N.C. designed, conducted and analyzed the in vivo experiments. N.C., H.S. and M.S. wrote the manuscript. M.S. supervised the project.

Corresponding author

Correspondence to Mriganka Sur.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 ChAT-ChR2 induced desynchronization and decorrelation.

(a) A raw example trace (b) Spectrogram and (c) Power-time graph of a 10 minutes continuous LFP recording, where power at a frequency of 1 Hz in the spectrogram was plotted (Clement E.A. et al., PLoS ONE, 2008). Red vertical lines indicate time of ChAT-ChR2 stimulation. The LFP state was stable throughout the recording for the ChAT-ChR2 stimulation induced changes to be discerned as shown by the clear decrease of power at 1 Hz upon ChAT-ChR2 stimulation (activated state). This finding agrees with previous work where urethane-anesthetized ChAT-ChR2 mice were used (Kalmbach A et al., Journal of Neurophysiology, 2012). Note that the LFP state described by the power at 1 Hz is not as stable in previous work using urethane-anesthetized rats (Clement E.A. et al., PLoS ONE, 2008). This may be due to use of different animal model or urethane dosing paradigm. (d) Coefficient of variances (CV) calculated from temporal change of power at 1 Hz from all LFP recordings (n = 111). The red line in the center of box indicates median value. The top and bottom edges of the box indicate 75 and 25 percentiles, respectively. The whiskers that extend from the box are the most extreme data points not considered outliers. Outliers are represented by the two crosses above the box. The single green circle indicates CV computed from a captured trace shown in previous publication using urethane anesthetized rats (Clement E.A. et al., PLoS ONE, 2008) where cyclic fluctuation of power was observed over time of EEG recording. The brain state in urethane anesthetized ChAT-ChR2 mice was more stable than that observed in this previous work. (e) Extent of desynchronization in V1 increased with the power of ChAT-ChR2 stimulation. (f) Scatter plot showing the between-cell correlation coefficients before and after ChAT-ChR2 stimulation when (Left) natural movies and (Right) random orientation gratings were shown. Each blue circle represents the averaged correlation coefficient between a single neuron and all other neurons in the same recording while the single red circle is the population averaged correlation coefficient. There is a significant decrease of between-cell correlation coefficient after ChAT-ChR2 stimulation in both natural movies (p <<< 0.0001, paired t-test) and random orientation grating experiments (p < 0.001, paired t-test). (g) Scatter plot showing the between-cell correlation coefficients before and after ChAT-ChR2 stimulation with responses binned at 200 ms (5 Hz) (p <<< 0.0001, paired t-test) and 50 ms (20 Hz) (p < 0. 001, paired t-test) respectively. A significant decrease of between-cell correlation coefficient after ChAT-ChR2 stimulation is observed in responses binned at 200 ms and 50 ms, similar to responses binned at 100 ms (10 Hz) (Fig. 1f). (h) Scatter plot showing the individual between-cell correlation coefficients before and after ChAT-ChR2 stimulation. Blue circles represent single Pearson correlation coefficients. Inset: Expanded scale; red circle represents averaged correlation coefficient. The correlation values for each cell are pooled and shown in Fig. 1f. (i) ChAT-ChR2 stimulation induced significant decrease in population averaged normalized correlation coefficient across experiments in both natural movies (p < 0.006, paired t-test) and random orientation grating experiments (p < 0.03, paired t-test). * p < 0.05, ** p<0.01, *** p<0.001, **** p<0.0001.

Supplementary Figure 2 Membrane properties of SOM neurons compared to VIP and L1 neurons.

(a) The firing responses in SOM, VIP and L1 neurons to 40 pA current injection. Black and red arrows indicate resting membrane potentials and action potential thresholds respectively. (b) Population averaged resting membrane potentials and action potential thresholds of SOM, VIP and L1 neurons. SOM neurons have significantly lower action potential thresholds compared to the other two neuron types. (c) Population averaged firing frequencies of SOM, VIP and L1 neurons as a function of stimulus current, measured during 750 ms constant-current steps. * p <0.05 *** p <0.001.

Supplementary Figure 3 ACh-induced IPSCs in SOM, VIP and L1 neurons.

All recordings were performed in voltage clamp mode with high chloride internal solution in the presence of NBQX. (a - c, Top) ACh-induced (black dot, 100 µM) inward currents recorded in SOM, VIP and L1 before and after bath application of gabazine and CGP55845 hydrochloride. (a - c, Bottom) ACh-induced changes in mean current amplitude (pA) of (a) SOM (b) VIP and (c) L1 neurons against range of pipette concentrations of ACh in the presence of NBQX before (solid line) and after (dotted line) application of gabazine and CGP55845 hydrochloride. Error bars indicate 0.5 SEM. (d) Difference in the ACh-induced changes in mean current amplitude (pA) of SOM, VIP and L1 neurons between responses recorded in the presence of NBQX (filled circles in a-c) and that of NBQX, gabazine and CGP55845 hydrochloride (open circles in a-c) against ACh concentrations. This difference represents the component of response that pertains to summated IPSCs that were sensitive to the GABAergic blockers. Error bars indicate 0.5 SEM. The inset in (d) shows responses demarcated by the dotted red box on an expanded scale. (e) (Left) High ACh concentration (black dot; 10 mM) evoked response in a putative VIP neuron (in GAD67-GFP-SOM-Cre-Arch slice) was partially reduced by green light exposure (green bar). (Right) Population average of ACh-induced changes in mean current amplitude of putative VIP neurons before, during and after Arch. See Fig. 2j-k. (f) Same as Supplementary Fig. 3e except for L1 neurons in a SOM-Cre-Arch slice. See Fig. 2m-n. * p <0.05, ** p <0.01.

Supplementary Figure 4 Proposed cholinergic mechanism, spike properties of specific neuron classes and green light controls.

(a) Proposed mechanism of cholinergic action on inhibitory neuron subtypes. Green ovals and red circles indicate cholinergic receptors and inhibitory synapses respectively. (b) Population average of the mean peak to valley ratio of spikes in GFP-expressing SOM neurons, non GFP-expressing PYR neurons and L1 neurons of SOM-Cre-Arch-GFP animals as well as FS (putative PV) and RS (putative VIP) inhibitory neurons in GAD-67-GFP-SOM-Cre-Arch animals studied with whole cell patch-clamp recordings ex vivo. (See Figs. 2 and 3). ** p <0.01, *** p <0.001, Wilcoxon rank-sum test. (c) ( Left) Merged fluorescence and DIC images of a SOM neuron patched in a SOM-TD slice. Relative positions of ACh pipette (1) and patch pipette (2) are as indicated. Scale bar, 10 µm. (Inset) Configuration of whole-cell patch-clamp recording of SOM neurons in slices during ACh application. (Right) Population average of ACh-induced changes in mean Vm of SOM neurons before and after green light exposure in control slices (n = 3, p > 0.4, paired t-test). Green light did not significantly change the ACh-induced response in SOM neurons in slices without Arch expression. N.S., not significant.

Supplementary Figure 5 Current and voltage clamp recordings of ACh-induced responses in SOM, PV and PYR neurons.

All neurons in Supplementary Figs. 5c, g, k were recorded in a current clamp mode with low chloride internal solution. Neurons in Supplementary Fig. 5d, h, l were patched in voltage clamp mode with a low chloride internal solution at holding potential of – 70 mV. Neurons in Supplementary Fig. 5e, i, m were patched in voltage clamp mode with a high chloride internal solution in the presence of NBQX at holding potential of -70 mV. ACh (10 mM) was applied locally at 200 ms, 20 psi. (a) Population average of the mean peak to valley ratio of spikes of PV, SOM and PYR neurons studied in Supplementary Fig. 5 with whole cell patch-clamp recordings ex vivo in PV-TD, SOM-TD and GAD-67-GFP/WT animals respectively. ** p <0.01; N.S., not significant, Wilcoxon rank-sum test. (b) Merged fluorescence and DIC images of a tdTomato positive SOM neuron patched in a SOM-TD slice. Relative positions of ACh pipette (1) and patch pipette (2) were as indicated. Scale bar, 10 µm. (Inset Top) Configuration of whole-cell patch-clamp recording of SOM neurons in slices during ACh application. (Inset Bottom) A typical spike of a SOM neuron. (c) (Left) Local ACh application (black dot) induced transient action potentials in 2 example SOM neurons. (Right) Population temporal cholinergic response profile of SOM neurons. Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. (d) (Left) Local ACh application (black dot) induced inward currents in 2 example SOM neurons. (Right) Population temporal cholinergic response profile of inward current response in SOM neurons. See Supplementary Fig. 6c-e for the quantification of cholinergic inward currents in SOM neurons. (e) (Left) Local ACh application (black dot) induced NBQX-insensitive inward currents in 2 example SOM neurons. (Right) Population temporal cholinergic response profile of NBQX-insensitive inward current response in SOM neurons. (f) Similar to Supplementary Fig. 5b where fast-spiking, tdTomato positive PV neurons in PV-TD slices were recorded. Scale bar, 10 µm. (g) (Left) Local ACh application (black dot) did not evoke any facilitatory response in 2 example PV neurons. (Right) Population temporal cholinergic response profile of PV neurons shows no significant change in membrane potentials when recorded at resting membrane potential. ACh however can induce significant IPSCs as observed in voltage clamp recordings (Arroyo S et al., Journal of Neuroscience, 2012) (see Supplementary Fig. 5i). Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. (h) (Left) Local ACh application (black dot) did not induce inward currents in 2 example PV neurons. (Right) Population temporal cholinergic response profile in PV neurons. Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. (i) Similar to Supplementary Fig. 5e except in PV neurons. (j) Similar to Supplementary Fig. 5b where GFP negative, PYR neurons in GAD67-GFP slices were recorded. Scale bar, 10 µm. (k) (Left) Local ACh application (black dot) evoked hyperpolarization followed by slow depolarization in some PYR neurons (11/21) and depolarization only in others (10/21). The extent of hyperpolarization was dependent on membrane potential (data not shown) (McCormick DA & Prince DA, Journal of Physiol, 1987). (Right) Population temporal cholinergic response profile of PYR neurons shows no significant change in membrane potential. Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. (Right inset) Population temporal cholinergic response of PYR neurons demarcated by black dotted box in expanded scale. ACh (10 mM) was applied locally at 200 ms, 20 psi. (l) (Left) Local ACh application (black dot) induced inward currents in 2 example PYR neurons. (Right) Population temporal cholinergic response profile of inward current response in PYR neurons. (m) Similar to Supplementary Fig. 5e except in PYR neurons.

Supplementary Figure 6 ACh directly excites SOM neurons via nicotinic and muscarinic receptors.

(a) Local ACh application (black dot) evoked a transient train of action potentials in SOM neurons which were abolished by (left) mecamylamine and (right) atropine. This shows that the response is mediated by nAChRs and mAChRs. These receptors may convert transient ACh drive into the sustained facilitatory responses observed in SOM neurons via specific receptor subtypes with slower kinetics (Arroyo, S et al., J. Neurosci, 2012; Gulledge, A.T. et al., J. Neurosci., 2009) or modulation of potassium conductances (Carr, D.B et al., Journal of Neurophysiology, 2007; Zhang, H. et al., Neuron, 2003; Buchanan, K.A. et al., Neuron, 2010; Giessel, A.J. and Sabatini, B.L., Neuron, 2010) downstream of these cholinergic receptor types. An additional mechanism that prolongs these SOM responses may be via electrical coupling between them (Beierlein, M. et al., Nat. Neurosci., 2000; Skinner, F. et al., J. Neurophysiol.,1999; Gibson, J.R. et al., Nature, 1999; Ma, Y. et al., Cereb. Cortex, 2011). (b) Population average of ACh-induced changes in mean Vm of SOM neurons before and after bath application of mecamylamine and atropine. ACh was locally applied at 10 mM, 200 ms, 20 psi. Responses were recorded in current clamp mode with low chloride internal solution. Mecamylamine: n= 6 neurons in 6 slices from 3 animals, p < 0.0001, comparing ACh-induced depolarization before and after mecamylamine, paired t-test. Atropine: n = 5 neurons in 5 slices from 2 animals, p = 0.0050, paired t-test. (c) Merged fluorescence and DIC images of RFP positive SOM neuron patched in a ChAT-ChR2-SOM-Cre slice. Scale bar, 10 µm. (d) Voltage clamp recordings of the cholinergic currents in RFP-labeled SOM neurons of ChAT-ChR2-SOM-Cre slice during ChAT-ChR2 blue light stimulation. Green and red traces are the population averages of normalized response that occurred before and after application of mecamylamine or atropine. The response of each cell was normalized by the negative peak of its response before drug. Recordings were performed with low chloride internal solution in the voltage clamp mode at holding potential of – 70 mV, in the presence of NBQX. (e) Population averages of the latency, peak amplitude and charge of events that occurred during ChAT-ChR2 blue light stimulation before and after mecamylamine/atropine application. (See Online Methods - Slice physiology analysis). (f) (Left) Muscarinic (mAChR) and nicotinic (nAChR) receptors on tdTomato positive SOM neurons in V1 of SOM-TD mice. Immunohistochemistry (anti-mAChR M1/M2, anti-nAChR alpha4 and beta2, anti-nAChR alpha-7) showing co-localization of these receptors on SOM neurons. Scale bar, 10 µm. (Right) Percentage co-localization of each set of cholinergic receptors with tdTomato positive SOM neurons in fixed slices. * p <0.05, ** p <0.01, *** p <0.001, N.S., not significant.

Supplementary Figure 7 ACh-induced IPSCs in PV and PYR neurons.

All recordings were performed in voltage clamp mode with high chloride internal solution in the presence of NBQX. (a - b, Top) ACh-induced (black dot, 10 mM) inward currents recorded in PV and PYR neurons in the presence of NBQX before and after bath application of gabazine and CGP55845 hydrochloride. (a - b, Bottom) ACh-induced changes in mean current amplitude (pA) of PV and PYR neurons against range of pipette concentrations of ACh in the presence of NBQX before (solid line) and after (dotted line) bath application of gabazine and CGP55845 hydrochloride. Error bars indicate 0.5 SEM. (c) (Left) Low ACh concentration (black dot; 100 µM) evoked response in a putative PV neuron (in GAD67-GFP-SOM-Cre-Arch slice) which was abolished by green light exposure (green bar). (Right) Population average of ACh-induced changes in mean current amplitude of putative PV neurons before, during and after Arch. See Fig. 3d-e. (d) Same as Supplementary Fig. 7c where PYR neurons in a SOM-Cre-Arch slice were recorded. See Fig. 3g-h. * p <0.05, ** p <0.01.

Supplementary Figure 8 ACh-induced excitatory responses in PYR neurons.

Recordings were performed with low chloride internal solution in the voltage clamp mode at holding potential of – 70 mV. (a, d, g) Proposed mechanism of cholinergic action on specific cortical cell types and connections. Red circles, blue circles and green ovals indicate inhibitory synapses, excitatory synapses and cholinergic receptors respectively. Possible mechanisms that mediate ACh-induced excitation in PYR neurons include (1) direct ACh action on PYR neurons and (2) Indirect ACh action on PYR-PYR recurrent connections via SOM-PV-PYR connection. Red crosses indicate the connections that were blocked either by pharmacology or optogenetics. (b) NBQX blocked the transient component but not the prolonged component of ACh-induced inward currents in PYR neurons. (Top) ACh-evoked (black dot) inward currents recorded in a PYR neuron before and after bath application of NBQX. Dotted red lines demarcate the time window analyzed in Supplementary Fig. 8c. Inset shows the trace segment demarcated by black box in expanded scales. (Bottom) Population response profile before and after bath application of NBQX. Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. Blue and black lines pertain to before and after NBQX respectively. (c) Population average of first ten seconds of ACh-induced changes in mean current amplitude (pA) of PYR neurons before and after NBQX. (e) Gabazine blocked the transient component but not the prolonged component of ACh-induced inward currents in PYR neurons. (Top) ACh-evoked (black dot) inward currents recorded in a PYR neuron before and after bath application of gabazine. Dotted red lines demarcate the time window analyzed in Supplementary Fig. 8f. Inset shows the trace segment demarcated by black box in expanded scales. (Bottom) Population response profile before and after bath application of gabazine. Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. Blue and black lines pertain to before and after gabazine respectively. (f) Population average of first ten seconds of ACh-induced changes in mean current amplitude (pA) of PYR neurons before and after gabazine. (h) Hyperpolarization of SOM neurons blocked the transient but not the prolonged components of ACh-induced inward currents. Alternative mechanisms may underlie the prolonged components of these currents (Buchanan K.A. et al., Neuron, 2010; Giessel A.J. et al., Neuron, 2010; Chen N, Sugihara. H et al., PNAS, 2012). (Top) ACh-evoked (black dot) inward currents recorded in a PYR neuron before and during green light stimulation (green bar) of Arch to hyperpolarize SOM neurons. Dotted red lines demarcate the time window analyzed in Supplementary Fig. 8i. Inset shows the trace segment demarcated by black box in expanded scales. (Bottom) Population response profile before and during Arch stimulation. Each open and filled dot pertains to time averaged responses of a single neuron and population of neurons respectively. Blue and green lines pertain to before and after Arch stimulation respectively. (i) Population average of ACh-induced changes in mean current amplitude (pA) of putative PYR neurons in the first ten seconds of response to ACh before, during and after Arch. n=6 neurons in 6 slices from 3 animals, p = 0.03, comparing ACh-induced current amplitudes before (-9.61 ± 3.79 pA) and after Arch (1.16 ± 0.479 pA), paired t-test. * p <0.05, *** p <0.001. ACh was applied at 10 mM, 200 ms, 20 psi.

Supplementary Figure 9 Electrophysiological properties of SOM and PV neurons studied with cell attached recordings in vivo in ChAT-ChR2-SOM-Cre-RFP and ChAT-ChR2-PV-Cre-RFP animals.

Population average of the (Left) mean spike half-width and (Right) mean peak to valley ratio of spikes in the PV (n = 23) and SOM (n = 11) neurons recorded. ** p <0.007, + p = 0.0926, t-test. See Fig. 4 (Ma W et al., J. Neurosci, 2010).

Supplementary Figure 10 Relationship between cortical state before ChAT-ChR2 stimulation and extent of ACh-induced desynchronization.

(Top) Mean power relative to control of low frequency events (<10 Hz) during ChAT-ChR2 stimulation (blue)/simultaneous ChAT-ChR2 and SOM-Arch stimulation (green) in ChAT-SOM-Arch animals against the cortical state before stimulation. The x-axis shows the pre-stimulation cortical state (degree of activation/inactivation) defined by the power at a frequency of 1 Hz in the LFP power spectrum (see Supplementary Fig. 1b for spectrogram and Supplementary Fig. 1c for power-time graph at 1 Hz). The higher the power at 1 Hz, the more deactivated the cortical state. Low blue laser ChAT-ChR2 stimulation (10 mW/mm2): n = 13 measurements in 6 animals. Filled dots on the right indicate mean power relative to control (p<0.01 between blue and green) across measurements. Blue and green lines are regression lines (p = 0.165 and p = 0.455, respectively). High blue laser ChAT-ChR2 stimulation (30-60 mW/mm2): n = 19 measurements in 6 animals. p<0.001 comparing mean power relative to control between blue and green across measurements. Blue and green lines are regression lines (p = 0.00871 and p = 0.0256, respectively). (Bottom) Same as (Top) except in ChAT-VIP-Arch animals. Low blue laser: n = 16 measurements in 5 animals. p > 0.07 comparing mean power relative to control between blue and green. Blue and green lines are regression lines (Blue: p = 0.770, Green: p = 0.612). High blue laser: n = 16 measurements in 5 animals. p > 0.3 comparing mean power relative to control between blue and green. Blue and green lines are regression lines (p = 0.0175 and p = 0.000905 respectively).

Supplementary Figure 11 Green light stimulation does not abolish the in vivo ChAT-ChR2 stimulation induced desynchronization of LFP and neuronal decorrelation in ChAT-ChR2 animals without Arch expression.

(a) Experimental setup for in vivo LFP recording using a glass pipette during blue and blue/green stimulation of ChAT-ChR2 through objective. (b) Desynchronization of LFP during ChAT-ChR2 stimulation at t = 0 s (arrow) (Left) is not blocked by green light stimulation (green bar) (Right). (Top) Raw trace (Bottom) Low-pass filtered (<5 Hz). (c) ChAT-ChR2 blue light stimulation induces a decrease in power of low frequency events (<10 Hz, p <0.05, paired t-test) and increase in high frequency events (10 – 100 Hz, p <0.02, paired t-test) respectively. This is not blocked during simultaneous green and blue light stimulation in the ChAT-ChR2 control animals without Arch expression (low frequency: p <0.04; high frequency: p <0.005). (d) Experimental setup for in vivo single unit recording using a tungsten electrode array during blue and blue/green stimulation of ChAT-ChR2 through objective. (e) Scatter plot showing the between-cell correlation coefficients (Left) before and after ChAT-ChR2 blue light stimulation as well as (Right) before and after simultaneous ChAT-ChR2 blue and green light stimulation. Each blue circle represents the averaged correlation coefficient between a single neuron and all other neurons in the same recording while the single red circle is the population averaged correlation coefficient. There is a significant decrease of between-cell correlation coefficient during ChAT-ChR2 blue light stimulation (p < 0.01, paired t-test) and also during simultaneous blue and green light stimulation (p < 0.005, paired t-test). (f) There is a significant decrease in population averaged normalized correlation coefficient across experiments during ChAT-ChR2 blue light stimulation (p <0.02, paired t-test) as well as during simultaneous blue and green light stimulation (p < 0.02, paired t-test). * p <0.05, ** p <0.01, paired t-test.

Supplementary Figure 12 ChAT-ChR2 stimulation induced decorrelation is accompanied by improved coding to visual stimulation.

(a) (Top) Schematic illustration of the experimental setup for in vivo single unit recording using a tungsten electrode array during ChAT-ChR2 blue light stimulation and SOM-Arch green light stimulation through objective. (Bottom) Image of a tungsten electrode array used for single-unit recording in the Arch virus injected region in V1 in ChAT-ChR2-SOM-Cre-Arch-GFP animals. Scale bar, 200 µm. (b) (Top) An example experiment showing neuronal decorrelation (i) before and (ii) after ChAT-ChR2 stimulation as well as (iii) before and (iv) after simultaneous ChAT-ChR2 and SOM-Arch stimulation. Each panel shows the responses of multiple single units recorded simultaneously during presentation of natural movies. Each unit is indicated by a different color. (Bottom) Scatter plot showing the between-cell correlation coefficients (Left) before and after ChAT-ChR2 stimulation, and (Right) before and after simultaneous SOM-Arch and ChAT-ChR2 stimulation. Blue circles represent single Pearson correlation coefficients; Inset: Expanded scale; red circle represents averaged correlation coefficient. The correlation values for each cell are pooled and shown in Fig. 5f. (c) Percentage of cells that have responses which are significantly suppressed (p <0.05), facilitated (p <0.05) and unchanged during blue light stimulation of ChAT-ChR2 animals as well as blue and blue+green light stimulation of ChAT-SOM-Arch animals. (d) ( Left) In vivo ChAT-ChR2 stimulation enhances discrimination performance via SOM neurons. Normalized discrimination performance with (blue curve) and without (black curve) blue light stimulation. Discrimination performance was plotted as a function of number of units assessed for computation of population performance and normalized by the control performance at 9 units. Only data sets with more than 9 units were included. Performance improved with increasing number of units assessed as well as with blue light stimulation (Two-way ANOVA: effect of number of units: p <0.03, effect of light stimulation: p <0.04, no significant interaction). (Right) Normalized discrimination performance with (green curve) and without (black curve) blue + green light stimulation. The effect of light stimulation is absent (Two-way ANOVA: effect of number of units: p <0.003, effect of light stimulation: p > 0.1, no significant interaction). * p <0.05, *** p <0.001, N.S., not significant. See also Online Methods: In vivo single unit recording and data analysis.

Supplementary Figure 13 Blockade of ACh-induced facilitatory responses in VIP and 5HT3aR-expressing L1 neurons does not block ACh-induced IPSCs in PYR neurons, and VIP hyperpolarization induces IPSCs in PYR neurons.

Recordings in Supplementary Fig. 13a-b, 13e-f were performed in current clamp mode with low chloride internal solution. IPSCs in Supplementary Fig. 13c-d, 13g-h, 13i-j were recorded in voltage clamp mode (holding potential – 70 mV) with a high chloride internal solution in the presence of NBQX. (a) ( Top Left) Configuration of whole-cell patch-clamp recording of neurons during ACh pressure application, in VIP-Cre slices at a location where AAV-flex-Arch-GFP virus was injected (green shaded region), with green light stimulation of Arch to hyperpolarize VIP neurons. (Top Right) Merged fluorescence and DIC images of a GFP-positive, VIP neuron patched in a VIP-Cre slice at a location where AAV-flex-Arch-GFP virus was injected. Scale bar, 20 µm. (Bottom) Local ACh application (black dot) evoked depolarization in an Arch-expressing VIP neuron which was abolished by green light exposure (green bar). (b) Population average of mean Vm of VIP neurons when ACh was applied before, during and after Arch. n = 9 neurons in 8 slices from 3 animals, p = 0.0429, comparing ACh-induced depolarization in VIP neurons before and after Arch activation, paired t-test. (c) (Top) Similar to (a, Top) where GFP negative, putative PYR neuron in a VIP-Cre slice was recorded. Scale bar, 10 µm. (Bottom) Local ACh application (black dot) evoked IPSCs in a putative PYR neuron which increased upon green light exposure (green bar). (d) Population average of ACh-induced changes in mean current amplitude (pA) of putative PYR neurons before, during and after Arch. n=7 neurons in 7 slices from 3 animals, p = 0.0008, comparing ACh-induced current amplitudes before and after Arch, paired t-test. (e) ( Top Left) Configuration of whole-cell patch-clamp recording of neurons during ACh pressure application, in 5HT3aR-Cre slices at a location where AAV-flex-Arch-GFP virus was injected, with green light stimulation of Arch to hyperpolarize 5HT3aR-expressing L1 neurons. Green light stimulation was localized to layer 1 only (green shaded region). (Top right) Merged fluorescence and DIC images of a GFP-positive, 5HT3aR-expressing L1 neuron patched in a 5HT3aR-Cre slice at a location where AAV-flex-Arch-GFP virus was injected. Scale bar, 10 µm. (Bottom) Local ACh application (black dot) evoked depolarization in an Arch-expressing 5HT3aR neuron which was abolished by green light exposure (green bar). (f) Population average of mean Vm of 5HT3aR-expressing neurons when ACh was applied before, during and after Arch (the Arch and after Arch values were normalized by before Arch values for statistical comparisons). n=5 neurons in 4 slices from 3 animals, p = 0.01277, comparing ACh-induced depolarization in 5HT3aR neurons before and after Arch activation, paired t-test. (g) (Top) Similar to (e, Top) where GFP negative, putative PYR neuron in a 5HT3aR-Cre slice was recorded. Scale bar, 10 µm. (Bottom) Local ACh application (black dot) evoked IPSCs in a putative PYR neuron which is not affected by green light exposure (green bar). (h) Population average of ACh-induced changes in mean current amplitude (pA) of putative PYR neurons before, during and after Arch. n=8 neurons in 7 slices from 3 animals, p = 0.5357, comparing ACh-induced current amplitudes before and after Arch, paired t-test. (i) Green light induced hyperpolarization of VIP neurons in Arch injected VIP-cre animals evoked IPSCs in putative PYR neurons. (j) Population average of VIP hyperpolarization induced changes in mean current amplitude of putative PYR neurons in comparison to the ACh-induced and co-ACh and VIP hyperpolarization induced changes in the same neurons. * p <0.05, ** p <0.01, *** p <0.001, N.S., not significant. ACh (10 mM) was applied at 200 ms, 20 psi.

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Chen, N., Sugihara, H. & Sur, M. An acetylcholine-activated microcircuit drives temporal dynamics of cortical activity. Nat Neurosci 18, 892–902 (2015). https://doi.org/10.1038/nn.4002

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