1. Neuroscience
Download icon

Fast and reversible neural inactivation in macaque cortex by optogenetic stimulation of GABAergic neurons

  1. Abhishek De
  2. Yasmine El-Shamayleh
  3. Gregory D Horwitz  Is a corresponding author
  1. Graduate Program in Neuroscience, University of Washington, United States
  2. Department of Physiology and Biophysics, Washington National Primate Research Center, University of Washington, United States
  3. Department of Neuroscience, Zuckerman Mind Brain Behavior Institute, Columbia University, United States
Research Article
Cite this article as: eLife 2020;9:e52658 doi: 10.7554/eLife.52658
9 figures, 1 table and 1 additional file

Figures

Figure 1 with 1 supplement
Immunohistochemical analysis of transduction by AAV1-mDlx5/6-ChR2-mCherry.

(A) A histological section of V1 from monkey 1 stained with DAPI (blue) and antibodies against parvalbumin (green) and mCherry (red). Scale bar is 250 μm. The pial surface is indicated by the dashed gray curve and the border between layers 1 and 2/3 is indicated by the solid gray curve. The laminar specificity is an idiosyncrasy of this particular injection; see Figure 1—figure supplement 1 for a histological section of the V1/V2 border. (B) Locations of cell bodies in (A) expressing mCherry (red), parvalbumin (green), or both (‘+').

Figure 1—figure supplement 1
Immunohistochemical analysis of transduction by AAV1-mDlx5/6-ChR2-mCherry.

(A) A histological section of V1/V2 from monkey 1 processed with antibodies against parvalbumin (green) and mCherry (red), imaged at 10X. Scale bar is 1 mm. (B) Locations of cell bodies in (A) expressing mCherry (red), parvalbumin (green) or both (‘+'). Sensitivity analysis of AAV–mDlx5/6–ChR2–mCherry transduction to PV+ neurons in two regions of efficient transduction.

Figure 2 with 3 supplements
Optogenetic activation and suppression of single- and multi-units.

(A,B) Responses (in impulses per second, ips) of two example single units, aligned to the onset of optical stimulation, which lasted 300 ms (blue rectangle). Rasters (tick marks) and peristimulus time histograms (blue traces) are shown for an activated single unit (A) and a suppressed single unit (B). Insets: Mean spike waveform (thick black curve) and noise waveform (thick gray curve) ± 1 standard deviation (thin curves). (C) Scatter plot of firing rate on laser trials against baseline firing rate of units from monkey 2 (squares) and monkey 3 (circles). Data from example activated and suppressed units are circled in red. Firing rates were computed during optical stimulation or the equivalent epoch on control trials.

Figure 2—figure supplement 1
Analysis of visually driven responses at activated and suppressed sites.

Visually driven firing rate was computed during the Gabor stimulus presentation period (200 ms) and plotted against the baseline firing rate. A total of 46 sites were driven by visual stimuli. 19 of those were significantly visually driven.

Figure 2—figure supplement 2
Analysis of latency at activated and suppressed sites.

(A) Analysis of latency to first spike at activated sites. Latency was defined as the time to first spike following optical stimulation on each trial. Black points represent medians across trials within a site, and the lower and the upper end of vertical black lines represent the 25th and 75th percentiles. (B) Histogram of average latencies to first spike following optogenetic activation. (C) Histogram of latencies at activated (black) and suppressed (gray) sites. For each site, firing rate was computed in a sliding 50-ms window from −50–150 ms after the laser was turned off. This firing rate was compared against the pre-laser firing rate (computed in a 50-ms window before optical stimulation). The time at which the firing rates in the two windows differed significantly was defined as the latency (p<0.05, Wilcoxon rank sum test).

Figure 2—figure supplement 3
Rasters from all of the 18 suppressed sites.

Responses are aligned to the onset of optical stimulation.

Analysis of activity rebound and recovery at suppressed sites.

(A) Responses of a single unit with a pre-laser firing rate that exceeded the post-laser firing rate. Recovery time to the baseline firing rate was 195 ms (vertical gray line). (B) Responses of another single unit with a post-laser firing rate that exceeded the pre-laser firing rate. Recovery time was 15 ms. (C) Scatter plot of pre-laser firing rates against post-laser firing rates. For each site, post-laser firing rate was computed in a sliding 50-ms window from 0 to 200 ms after the laser was switched off. The ranges of post-laser firing rates are plotted as black lines and averages are plotted as black points. Data from neurons in (A) and (B) are circled in red. (D) Histogram of recovery times following optogenetic suppression. Recovery times of example units are marked with red tick marks, and the median is marked with a triangle.

Figure 4 with 2 supplements
Effect of optogenetic inactivation of V1 on visually guided saccades.

(A) Task design. (B) Timing of events. The small overshoot in the laser trace accurately reflects the temporal profile of the light. (C,D) Eye position traces on control (gray) and laser (blue) trials are shown from 0 to 300 ms after the fixation point was extinguished for one block of trials. The RF location of the illuminated neurons (gray filled circle) and the target locations outside of the RF (gray open circles) are highlighted. (E,F) Eye positions on catch trials from the same blocks as (C,D).

Figure 4—figure supplement 1
Effect of optogenetic inactivation on visually guided saccades from monkey 3.

Data are shown from each individual block of trials. Gray traces show saccades on control trials, and the blue traces show saccades on laser trials. The position of the target inside the RF of the illuminated neurons (gray filled circle) and outside (gray unfilled circle) are shown.

Figure 4—figure supplement 2
Effects of optogenetic inactivation of V1 on visually guided saccade accuracy and latency.

(A,B) Saccade accuracy (the average distance between saccade end points and target location) on control and laser trials. Data from trials in which the target appeared inside the RFs of the stimulated neurons (filled symbols) were analyzed separately from interleaved trials in which the target appeared in other locations (open symbols). (C) Histograms of saccade latency on control (gray) and laser (blue) trials when the target was presented inside the RF. (E) Histograms of saccade latency on control (gray) and laser (blue) trials when the target was presented outside the RF. (B,D,F) Data from monkey 3 in the same format as (A,C,E).

Effect of V1 inactivation on visual contrast detection.

(A) Task design. (B) Timing of events. (C) Performance of monkey 2 over one block of trials. Hits (H) and misses (M) are proportions of Gabor-present trials that were answered correctly and incorrectly, respectively. Correct rejections (CR) and false alarms (FA) are proportions of Gabor-absent trials that were answered correctly and incorrectly, respectively. Insets show psychometric functions on control (gray) and laser (blue) trials. Symbol size in insets reflects the number of trials that contributed to each data point. (E) Contrasts selected by the staircase procedure on control (gray) and laser (blue) trials. (D,F) Performance of monkey 3 in the same format as (C,E). Luminance contrast could not exceed 0.66 because the gray background was close to the upper limit of the display range.

Figure 6 with 1 supplement
Retinotopic specificity of optogenetic effects on contrast detection.

Data are from a single session consisting of 4 blocks of trials from monkey 2. (A) On each trial, the Gabor stimulus appeared at one of three randomly interleaved locations (X, Y, or Z), all of which were 9.6° away from the fixation point (central black dot). Locations Y and Z were on the vertical and horizontal meridians, respectively. (B) The proportions of hits (H), misses (M), correct rejections (CR) and false alarms (FA) are plotted in the same format as in Figure 5C. The laser reduced the monkey’s contrast sensitivity when the Gabor stimulus appeared at the receptive fields of the transduced neurons (X, gray circle). No significant effect was observed at locations Y and Z (C,D).

Figure 6—figure supplement 1
Contrast detection thresholds were stable across seven blocks collected during a single session from monkey 3.

In the first block, the laser power was low (0.8 mW) and performance was statistically indistinguishable on laser and control trials. For this block only, data from laser and control trials were pooled. In the subsequent blocks, the laser power was >60 mW, and performance was significantly impaired on laser trials (p<0.05 in all cases, binomial test of proportions). For these blocks, data from control trials are only presented. (A) Proportion of hits in each block is plotted in chronological order. (B) d’ in each block is plotted in chronological order. (C) Psychometric functions in the first and the pooled subsequent blocks did not differ significantly (p=0.11, Likelihood ratio test of separate Weibull fits to the first and the subsequent blocks data versus the best single fit to the pooled data). (D) Luminance contrast values selected by the staircase procedure as a function of trial number in each block.

Figure 7 with 1 supplement
Effect of V1 inactivation on visual contrast detection across multiple sessions.

(A) Scatter plot showing proportion of hits on control trials against laser trials from each session in monkey 2. Sessions with significantly fewer hits on laser trials than control trials are shown in black (p<0.05, binomial test for equal proportions). Error bars represent the standard error of mean. (B) Data from monkey 3 in the same format as (A). (C) Scatterplot of d’ from control trials plotted against d’ from laser trials from each session performed by monkey 2. (D) Data from monkey 3 in the same format as (C).

Figure 7—figure supplement 1
Analysis of the relationship between d’ and c-criterion.

(A) Noise (gray, 𝒩 (0,1)) and signal (blue) distributions of decision variables with same variance are plotted. The effect of the laser is assumed to reduce the mean of the signal distribution. Shown are the monkey’s criterion (vertical dashed line) and the optimal criterion (blue triangle) for each signal distribution. The optimal criterion is the point of intersection between the signal and noise distributions. Even if the monkey’s criterion does not depend on laser power, c-criterion changes. This is because c-criterion is the difference between the optimal and the monkey’s criterion. (B) d’ is plotted against the c-criterion for different signal distributions. Under this model, changes in d’ are conflated with changes in c-criterion.

Figure 8 with 2 supplements
Effect of laser power and repeated optical stimulation on contrast detection.

(A) Contrast values selected by the staircase procedure on laser trials (solid lines) and interleaved control trials (dashed lines) across seven blocks. (B) The difference in d’ between control and laser trials as a function of laser power calculated from the data in (A). A Naka-Rushton fit to the data is shown in black. (C) Differences in d’ between control and laser trials as a function of trial number in each session. Each session consisted of at least five blocks of 120 trials. The duration of an individual trial was 2.80 ± 0.51 s (mean ± SD), and the number of trials per session was 813 ± 253. Points are means and error bars are standard error of the mean (SEM). SEM was not plotted for the final two points, each of which represent data from a single session. (D) Scatter plot of the differences in d’ for early trials (1–480) vs. late trials (480–beyond) within each session.

Figure 8—figure supplement 1
Correlation between optogenetic effects on neural activity and behavior.

(A) Effect of laser power on firing rate across seven blocks from a single session. The difference in laser-evoked firing rate and baseline firing rate is plotted as a function of laser power. Behavioral effects for these blocks of trials are shown in Figure 8B. (B) Relationship between neurophysiological and behavioral effects at the activated (black) and suppressed (gray) sites during the Gabor contrast detection task. Neurophysiological effects were computed as the absolute value of the difference between laser-evoked and baseline firing rate, divided by the sum of two.

Figure 8—figure supplement 2
Analysis of visual sensitivity in monkey 2.

The data in panels (B,C and D) were collected 840 days after the vector injections, and 663 days after the termination of optogenetic silencing experiments that contributed to the manuscript. (A) Electrophysiologically mapped receptive fields (RFs) before (unfilled circles) and after (filled circles) AAV injections into area V1. The polygon (black outline) enclosing all the RFs represents the region of interest where monkey’s visual sensitivity could be affected. (B) Saccade accuracy data from a visually guided saccade task. On each trial, a target appeared, the fixation point disappeared, and the monkey was rewarded for making a saccade to the target within ~300 ms. Targets were randomly drawn from two 7 × 7° grids (98 locations), one in the upper visual field and one in the lower visual field. (10 repetitions at each location). The size of each disk represents the proportion of saccades made to the corresponding target (landing within a 5 × 5° window). Each target location tested is plotted in a unique color which is preserved across panels. The monkey’s performance was ≥60% at all the tested locations. (C) Average saccade latencies are plotted as a function of target location. (D) Saccade end points are plotted as a function of target location in the unique color assigned to each location. Relative to saccades up and left, saccades down and right were less likely to be correct, had longer latencies, and were less accurate. The ‘shearing’ of the saccade end point distributions relative to the target positions is due to a small tilt in the infrared camera (SMI Inc, Hi-Speed Primate) relative to the eye.

Responses of a putative GABAergic, direction-selective single unit to optical (450 nm laser) and visual (drifting achromatic 3 Hz sinusoidal grating) stimulation (A).

Peristimulus time histograms (black) in response to sinusoidal laser modulation from 2 Hz to 254 Hz (blue). Inset: Mean spike waveforms (thick black curve) and noise waveforms (thick gray curve) ± 1 standard deviation (thin curves). (B) Direction tuning curve showing individual (black points) and mean responses (black line) across repeated presentations of a drifting sinusoidal grating. (C) Spatial frequency tuning curve with symbols identical to (B).

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
AntibodymCherry
monoclonal antibody
ClontechCat. No. 632543 RRID:AB_2307319(1:250)
AntibodyRabbit anti-parvalbuminSwantCode: 27 RRID:AB_2631173(1:5000)
AntibodyDonkey anti-
Mouse IgG (H+L) highly cross-adsorbed secondary antibody, Alexa
Fluor 568
Molecular ProbesCat. No. A10037 RRID:AB_2534013(1:200)
AntibodyDonkey anti-Rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 488Molecular ProbesCat. No. A21206 RRID:AB_2535792(1:200)
AntibodyDonkey anti-Rabbit IgG (H+L) highly cross-adsorbed secondary antibody, Alexa Fluor 350Molecular ProbesCat. No. A10039 RRID:AB_2534015(1:200)
OtherDAPIInvitrogenCat. No. D21490(1:5000)
Recombinant DNA reagentpAAV-mDlx-ChR2-mCherry-Fishell-3AddgeneCat. No. 83898 RRID:Addgene_83898
Cell line (Homo-sapiens)HEK293TAmerican Type Culture CollectionCRL-3216 RRID:CVCL_0063
Biological sample (Macaca Mulatta)Rhesus monkeyWashington National Primate Research CenterNA
Software/AlgorithmMATLABMathworkshttps://www.mathworks.com/products/matlab.html RRID:SCR_001622
Software/AlgorithmFijiNIH (ImageJ)https://imagej.net/Fiji RRID:SCR_002285
Software/AlgorithmPlexon Sort ClientPlexonhttp://www.plexon.com RRID:SCR_003170
Software/AlgorithmPlexon Offline SorterPlexonhttp://www.plexon.com RRID:SCR_000012

Additional files

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)