Cerebellar Shaping of Motor Cortical Firing Is Correlated with Timing of Motor Actions  Abdulraheem Nashef, Oren Cohen, Zvi Israel, Ran Harel, Yifat Prut  Cell Reports  Volume 23, Issue 5, Pages 1275-1285 (May 2018) DOI: 10.1016/j.celrep.2018.04.035 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2018 23, 1275-1285DOI: (10.1016/j.celrep.2018.04.035) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Cortical Response to Single-Pulse Activation of the CTC Pathway (A) Schematic illustration of the experimental setup. Primates were trained to perform an isometric wrist task with a pre-go delay period while neural activity was recorded from the motor cortex, somatosensory cortex, and motor thalamus. The sequence of events composing a single trial is shown (bottom left). An illustration of the CTC pathway is presented (right) and the part of the pathway in which the chronically stimulating electrode was implanted is highlighted. Four traces of recorded signals present the motor cortical and thalamic activity obtained simultaneously from different sites in a single recording session. In each recording site, after the monkey performed a sufficient number of trials, single-pulse stimulations were delivered via the stimulating electrode while the monkeys continued performing the task and data recording was maintained. (B) Recording maps of three different monkeys present the different areas from which recordings were obtained: motor cortex (red squares), premotor cortex (green circles), and somatosensory cortex (blue triangles). Yellow diamonds designate SCP-tested cortical sites that were unclassified. Gray symbols designate recordings sites that were not tested for an SCP response. (C) Example of a motor cortical response to single-pulse stimulations obtained in a single cortical site. The example contains an expanded view of a few traces (top chart), multiple repeated traces (middle chart), and mean rectified signals (bottom graph). All traces are aligned on stimulus onset time (t = 0). See also Figure S1. (D) Response map showing the relations between the recording depth (y axis) and the evoked multiunit response as a function of post-stimulation times (x axis). Each row of the color map corresponds to a cortical response from a single site. Single responses were normalized by subtracting the mean and dividing by the SD computed during the prestimulus baseline. Normalized responses were sorted according to their recording depth and aligned to stimulation onset-time (time 0). The left histogram shows the number of recording sites obtained at each depth. Depth measurements are relative to the first cells encountered in each track. The right plot shows the peak response times for each recording session as a function of recording depth. Top values show the correlation coefficient (r) and the significance level (p) computed between peak time and recording depth. (E) An example of MUA responses obtained from simultaneously recorded M1 (red) and premotor (green) responsive sites. The MUA data were first high-pass filtered (above 1 kHz) to extract spiking activity alone and then transformed into Z scores by subtracting the prestimulus baseline and dividing by the SD computed during this time. The amplitude (A), onset time (T), and duration (D) were computed for each response epoch (i.e., early excitation [exc] and subsequent inhibition [inh]). See also Video S1. (F) Statistical comparison of response timing of M1 and premotor computed for response latency (left), response duration (middle), and response amplitude (right). The top row shows analyses of early excitation, and the bottom row displays analyses of the subsequent inhibition. Mean and SEM were computed separately for different response polarities (exc. versus inh.) and recording sites (M1 versus premotor). For this analysis, we only considered cases where M1 and premotor activity were recorded simultaneously. A paired t test was used for statistical comparison, and n values are shown in the rightmost panels. Cell Reports 2018 23, 1275-1285DOI: (10.1016/j.celrep.2018.04.035) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Pattern of Single-Cell Response to SCP Stimulation (A) Example of a single motor cortical neuron that responded to SCP stimulation. All response sweeps are aligned on stimulus onset time (t = 0) and are further quantified using the peri-event time histogram (PETH) of the response (top histogram). The shape of the extracellularly recorded action potential of the unit is also shown by plotting 30 randomly selected waveforms. See also Figure S2. (B) Distribution of onset times computed for cortical cells that were excited by SCP stimulation. The response latency for each cell was only measured for the stimulation amplitude that triggered the most prominent excitatory responses. Note that for some neurons, the measured latency appeared earlier than the latency expected for this di-synaptic pathway. This reflects instances of poor performance of the algorithm used for determining response time due to the low signal-to-noise ratio of the cortical response compared to the pre-response baseline level. Mean response time is shown as well (dashed black line). The mean response time averaged across 22 thalamic cells is also shown (dashed gray line). (C–E) Mean response profiles computed by averaging single-cell response across neurons with similar response patterns. Single-cell response was first normalized by subtracting the baseline level and dividing by the baseline STD. Response shapes included excitation (C), excitation-inhibition (D), and inhibition (E). Cell Reports 2018 23, 1275-1285DOI: (10.1016/j.celrep.2018.04.035) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Inhibition Dominates the Response of Single Cortical Cells to SCP Stimulation (A) Single-cell example showing the impact of early post-response excitation on subsequent inhibition. Single stimulation sweeps were sorted into responsive (blue traces, n = 61) and nonresponsive (red traces, n = 146) sweeps, based on the existence of early response spikes (i.e., spikes in the 2–11 ms time interval after stimulus onset time). Mean response of the neuron across all sweeps (gray histogram) is shown above the raster plot. Mean responses obtained by summing responsive and nonresponsive traces separately (blue and red top histograms, respectively) are shown in the top panel. (B) A scatterplot depicting the relationships between the post-response depression obtained for responsive sweeps (x axis) and nonresponsive sweeps (y axis) measured individually for responsive cells. The scatter of points around the unity line (x = y) was unbiased (p > 0.47, paired t test). (C) Single-cell example of the changes in early excitation (blue) and subsequent inhibition (red) when the response of the same cell was tested at different stimulation intensities. (D) A scatterplot showing the intensity-dependent change in the area of the post-response trough (y axis) as a function of the change in area of the early response peak (x axis). The red-circled dot designates the example shown in (C), and the unity line (x = y) is presented for clarity. The deviation of the data from the unity line (which corresponds to a similar change in area for excitatory and inhibitor response components) was tested (paired t test), and the significance level is indicated (p < 0.0002). Note the different ranges of the x and y axes. (E) Relationship between the area (i.e., number of spikes per trigger) found for early excitation (y axis) and the area of subsequent inhibition. Each dot represents a single cell with an excitation-inhibition response pattern. The dominance of the inhibition is clearly shown from the significant deviation of the data points from the line of unity. (F) The distribution of the ratio between excitation and inhibition areas (taken from E) is shown in log scale for clarity. Mean ratio is shown (solid red line) as well the corresponding value obtained when calculating the response of M1 neurons to premotor ventral (PMv) stimulation (dashed red line, based on data published by Kraskov et al., 2011). Cell Reports 2018 23, 1275-1285DOI: (10.1016/j.celrep.2018.04.035) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Task-Related Synchrony Induced by the CTC System (A) An example of a raw-data trace obtained from the motor cortex from which two single units were isolated. Orange and green squares denote the units and the red asterisks denote the stimulation artifacts. (B) SCP-triggered response of the two units shown in (A). (C) Task-related activity of the two units shown in (A). All trials are aligned on movement onset (t = 0) and sorted according to the target the monkey had to acquire in the trial (1–8). PETHs were computed by averaging cell activity across all trials using a bin size of 25 ms). (D) Trial-to-trial co-modulation of normalized rate (noise correlation) was measured by the correlation coefficient (r) between the normalized rate of the reference unit (y axis) and the normalized rate of the trigger unit (x axis). (E) A color-coded plot of the mean noise correlation as a function of time relative to movement onset (time zero at x axis) and cell-to-cell time lag (y axis) averaged across all pairs of neurons in which both cells were excited by SCP stimulation (i) or both were inhibited (ii) and a mixed set of pairs (iii) including either nonresponsive cells or cells with a non-matching response pattern (e.g., one cell was excited and the other inhibited). (F) Bin-to-bin testing (using t test) between the leftmost matrix and the sum of the two remaining matrices revealed an area in which the correlation was significantly different between the two groups (white dots, taking into account the Bonferroni correction). (G) Task-related response shape computed for cells that were excited (blue), inhibited (red), or unaffected by SCP stimulation. PETHs were computed around torque onset using non-overlapping 30-ms bins. In cases of tuned neurons, PETHs were computed for the PD ± 1 target. PETH is also shown (cyan) for a subset of SCP-responsive cells with specifically high response gain (see Supplemental Experimental Procedures). (H) Mean PTI (phasic-tonic index) and SEM computed for the different cell groups. The mean PTI was higher for SCP-responsive neurons (specifically those with high response gain) than for cells that were inhibited by SCP stimulation. Nonetheless, the differences were not statistically significant. (I) For cells that were excited by SCP stimulation, we found a significantly positive correlation between response gain (i.e., the strength of single response to the stimulation) and the tendency to exhibit phasic response profile. This correlation may explain the high variability found for PTI among responsive neurons. See also Figure S3. Cell Reports 2018 23, 1275-1285DOI: (10.1016/j.celrep.2018.04.035) Copyright © 2018 The Authors Terms and Conditions

Figure 5 Neural Correlation with Temporal Properties of Motor Behavior (A) Task-related activity of a single cell that was excited by SCP stimulation. The raster plot was aligned on “go” signal (t = 0). Trials were sorted by target number (1–8), and the PETH (histogram above the raster) presents the average activity of the cell, computed across all trials. Red circles denote time of movement onset. The response of the cell to SCP stimulation reveals an excitation-inhibition response pattern. The shape of the action potential emitted by the cell is demonstrated by plotting 30 randomly selected action potentials (APs) (top right). (B) Relationships between spike counts (y axis) computed for a time window starting −280 to −130 ms before the go signal (highlighted by the blue background) and the single trial reaction times (x axis). Each dot represents data obtained from a single trial. In this specific time window, there was a significantly negative correlation between reaction times and spike counts. (C) The time-resolved rate-to-RT correlation for the single cell shown in (A). Correlation values were computed using 150 ms time windows that were advanced at 10 ms steps. Correlation was studied at a time window from −1,000 to +500 ms around the go signal. Red dot corresponds to the example shown in (B). (D) Population-based relationships between spike counts and reaction times were computed by averaging single-cell correlations across different groups of neurons as defined by their response pattern to SCP stimulation. The groups were made up of neurons that were excited by SCP stimulation (blue), inhibited by SCP stimulation (red), and nonresponsive neurons (black). The single-cell correlation function was first normalized using the Z-transform and then averaged across neurons. SEM is shown for each curve. See also Figure S5. (E) Time-resolved fraction of neurons that had a significant correlation with RT computed for each time bin for the three groups of neurons defined in (D). (F) Rate modulation of the three groups of cells relative to baseline level. Baseline rate level was computed during the pre-cue period. (G and H) Mean correlation (G) and per-bin fraction of cells with significant correlation (H) computed between spike-counts and movement times (MT). (I) Average activity of the three groups of neurons aligned on torque onset. Cell Reports 2018 23, 1275-1285DOI: (10.1016/j.celrep.2018.04.035) Copyright © 2018 The Authors Terms and Conditions