Low-Dimensional and Monotonic Preparatory Activity in Mouse Anterior Lateral Motor Cortex

Abstract

Neurons in multiple brain regions fire trains of action potentials anticipating specific movements, but this "preparatory activity" has not been systematically compared across behavioral tasks. We compared preparatory activity in auditory and tactile delayed-response tasks in male mice. Skilled, directional licking was the motor output. The anterior lateral motor cortex (ALM) is necessary for motor planning in both tasks. Multiple features of ALM preparatory activity during the delay epoch were similar across tasks. First, most neurons showed direction-selective activity and spatially intermingled neurons were selective for either movement direction. Second, many cells showed mixed coding of sensory stimulus and licking direction, with a bias toward licking direction. Third, delay activity was monotonic and low-dimensional. Fourth, pairs of neurons with similar direction selectivity showed high spike-count correlations. Our study forms the foundation to analyze the neural circuit mechanisms underlying preparatory activity in a genetically tractable model organism.SIGNIFICANCE STATEMENT Short-term memories link events separated in time. Neurons in the frontal cortex fire trains of action potentials anticipating specific movements, often seconds before the movement. This "preparatory activity" has been observed in multiple brain regions, but has rarely been compared systematically across behavioral tasks in the same brain region. To identify common features of preparatory activity, we developed and compared preparatory activity in auditory and tactile delayed-response tasks in mice. The same cortical area is necessary for both tasks. Multiple features of preparatory activity, measured with high-density silicon probes, were similar across tasks. We find that preparatory activity is low-dimensional and monotonic. Our study forms a foundation for analyzing the circuit mechanisms underlying preparatory activity in a genetically tractable model organism.

Keywords: motor planning; premotor cortex; preparatory activity; short-term memory.

Figure 1.

Behavioral tasks. a, Schematic, tactile task. During the delay epoch, an object is presented within reach of the whiskers in one of two locations. Mice report the location of the object by directional licking. b, Schematic, auditory task. Mice discriminate the frequency of tones presented during the sample epoch. c, Example learning curve, auditory task. The delay duration was gradually increased (top). Behavioral performance improved with training, while early lick rates decreased (middle and bottom).

Figure 2.

ALM is required for motor-planning. a, Schematic, photoinhibition of the ALM either during the sample epoch or the delay epoch. Cortical regions were photoinhibited for 1.2 s starting at the onset of the sample or delay epochs (relevant to b–d). The photostimulus was ramped down over the last 200 ms to avoid rebound activity (Guo et al., 2014a). b, Spatial maps of behavioral effects caused by photoinhibition during the sample epoch (left) and the delay epoch (right). Performance compared with the control condition (Δperformance) is shown in color code. Spot sizes indicate p values based on hierarchical bootstrap (see Materials and Methods). p values <0.001 were significant even after correction for multiple comparisons (Benjamini–Hochberg procedure). Data based on three animals, 104 sessions; 50,594 trials (75.1 ± 9.5 stimulation trials per spot, mean ± SD) for the sample photoinhibition; 50,585 trials (101.2 ± 10.8 stimulation trials per spot, mean ± SD) for the delay photoinhibition. Control performance was 83.0 ± 4.0% (mean ± SD) for lick right trials, 85.1 ± 0.9% (mean ± SD) for lick left trials. c, Behavioral effects of unilateral ALM photoinhibition. Top, Locations of photoinhibition. Bottom, Behavioral performance. Thick lines, Grand mean performance (n = 5 animals); thin lines, mean performance for each animal. Error bar, SEM based on hierarchical bootstrap (see Materials and Methods); Sample, Trials with photoinhibition in the sample epoch; Delay, trials with photoinhibition in the delay epoch. The whisker representation area of the primary somatosensory cortex (S1) was photoinhibited as a control (see Materials and Methods for coordinates). Data based on five animals, 47 sessions, 12,556 control trials, and 3385 stimulation trials in total. p values are based on hierarchical bootstrap. **p < 0.001; *p < 0.05 (both without correction for multiple comparisons. Only ** have p < 0.05 after Bonferroni correction). d follows the same format. See Table 4 for p values. d, Behavioral effects of bilateral ALM photoinhibition. Eight spots surrounding the ALM in both hemispheres were photoinhibited (see Materials and Methods). M1 was inhibited as a control (see Materials and Methods for coordinate). Data based on four animals, 40 sessions, 6096 control trials, and 1125 stimulation trials in total. From left to right, p values < 0.0001, 0.0001, and p = 0.0767, 0.0736 (hierarchical bootstrap, 10,000 iterations, without correction for multiple comparison).

Figure 3.

Classification of neural activity patterns. a, Parameters used to classify activity patterns. b, Schematics illustrating activity patterns. Blue, Spike rates in contra trials. Red, Spike rates in ipsi trials.

Figure 4.

Example neurons in the ALM. a–c, Nine example ALM neurons for each task type. Top, Spike rasters. Bottom, PSTH. Blue, correct lick right trials (Contra trials); red, correct lick left trials (Ipsi trials). Randomly selected 50 trials are shown per trial type. Dashed lines separate behavioral epochs. S, Sample epoch; D, delay epoch; R, response epoch. Time is relative to the go cue. Arrowheads indicate phasic activity at the beginning of the delay epoch. d–f, Grand mean PSTH for all pyramidal cells. Shadow, SEM (bootstrap).

Figure 5.

Selectivity in ALM. a–c, ALM population selectivity. Top, Cells with higher spike counts across all trial epochs in the contra trials. Bottom, Cells with higher spike counts in the ipsi trials. Each row represents contralateral selectivity normalized by the peak selectivity of individual neurons (blue, contralateral; red, ipsilateral). Vertical bars on the right. White, Neurons with premovement (sample or delay epoch) selectivity only; gray, both premovement and perimovement (response epoch) selectivity; black, perimovement selectivity. d–f, Proportion of neurons with selectivity in each epoch. g–i, Proportion of neurons that kept, lost, or switched selectivity between epochs.

Figure 6.

Selectivity and mean PSTH. a–c, Grand mean PSTH for cells with different types of delay selectivity. Shadow, SEM (bootstrap). d–f, Grand mean selectivity for cells with significant delay selectivity (see Materials and Methods). Shadow, SEM (bootstrap). g–i, Distribution of delay contralateral selectivity. Bin size, 2.5 spikes per second.

Figure 7.

Selectivity and ramping. a–c, Grand mean PSTH of contra-preferring and ipsi-preferring cells with different ramping directions. Ramping up/down was tested in the preferred direction (Mann–Whitney U test, p < 0.05). Shadow, SEM (bootstrap). d–f, Numbers of neurons with different ramping directions. g–i, Comparison of the absolute value of DA (see Materials and Methods) between the contra and ipsi trials. Mean ± SEM (bootstrap) is shown in the bar graph. All putative pyramidal cells were included. P value is based on bootstrap with 1000 iterations testing a null hypothesis that contralateral DA is lower than ipsilateral DA.

Figure 8.

Relationship of DA in contra and ipsi trials. a–c, Distribution of contralateral and ipsilateral DA of all putative pyramidal neurons. Each circle corresponds to one neuron. Contralateral ramping-up cells, ipsilateral ramping-up cells, and ramping-down cells (both contralateral and ipsilateral ramping-down cells) are shown as filled circles in different colors. Other cells are shown as open circles. Line represents linear regressions (blue, contralateral ramping-up cells; red, ipsilateral ramping-up cells). C.C., Correlation coefficient of contralateral and ipsilateral DA based on all cells. Diagonal dotted line, Line with slope 1. d–f, Normalized selectivity (see Materials and Methods) of ramping-up neurons (both contralateral and ipsilateral ramping-up neurons) and ramping-down neurons (both contralateral and ipsilateral ramping-down neurons). Central line in the box plot is the median. Top and bottom edges are the 75 and 25 percentage points, respectively. The whiskers show the lowest datum within 1.5 interquartile range (IQR) of the lower quartile, and the highest datum within 1.5 IQR of the upper quartile. P values, Mann–Whitney U test.

Figure 9.

Selectivity of putative FS neurons. a–c, Grand mean PSTH for all FS neurons. Shadow, SEM (bootstrap). d–f, Grand mean selectivity of FS neurons with significant delay selectivity. Shadow, SEM (bootstrap). g–i, Distribution of contralateral and ipsilateral DA for all FS neurons. Each circle corresponds to each cell. C.C., Correlation coefficient of contralateral and ipsilateral DA. Diagonal dashed line, Line with slope 1. j–l, Normalized selectivity of putative pyramidal neurons (pyr) and FS neurons. For these figures, normalized selectivity was measured for neurons with positive DA (DA_contra + DA_ipsi > 0). Central line in the box plot is the median. Top and bottom edges are 75 and 25 percentage points, respectively. The whiskers show the lowest datum within 1.5 interquartile range (IQR) of the lower quartile, and the highest datum within 1.5 IQR of the upper quartile. P values, Mann–Whitney U test.

Figure 10.

Coding during the delay epoch. a, b, Relationship between selectivity in correct and incorrect trials. We analyzed cells with >10 incorrect trials for each trial type (IR and IL trials). Each point corresponds to each cell. Dashed lines: horizontal, vertical, and lines with slope 1 and −1. c, d, Scatter plot and histogram of polar coordinates (r and θ) in a and b. Inset, The definition of r and θ. Bin size: 5° for θ, 1 spike per second for r. e, f, Distribution of θ across cortical depth. Bin size: 10° for θ, 100 μm for depth. g, h, Decoding of motor and sensory information using ROC (see Materials and Methods). θ And area under ROC curve for motor output (magenta) and sensory input (green) are shown for each cell. Dashed line, 95% confident interval of shuffled data (see Materials and Methods). Cells that have proportion correct higher than the dashed line were judged as cells decoding sensory or motor information.

Figure 11.

Coding of selectivity over time. a, b, Amplitude of selectivity (r) over time. Mean and SEM (1000 permutations). c, d, Histogram of θ over time. Bin size: 10° for θ and 250 ms for time. Mean of 1000 permutation. To calculate θ, we selected cells with r > 2 (Fig. 10a,b). Because of the ramping, the number of analyzed neurons increased during the delay epoch. e, f, Grand mean PSTH of cells coding motor output (−67.5° < θ < −22.5°). Shadow, SEM (bootstrap). g, h, Grand mean PSTH of cells with mixed-selectivity coding (−22.5° < θ < 22.5°). Shadow, SEM (bootstrap).

Figure 12.

Two possible models producing ramping activity on average. a, b, Mean PSTH of all modeled cells in c and d. c, d, PSTH of individual modeled cells with ramping (c) or with bumps at different timing (d; see Materials and Methods). Eleven of 50 modeled cells are shown. e, f, Rank correlation of neural activities over time (Fig. 14). g, h, Stability of CD over time (Fig. 15).

Figure 13.

Activity peaks are at the beginning or end of the delay epoch. a–c, Location of activity peaks in randomly selected half of trials. Neurons were sorted by the activity peak location. Each row, PSTH of each cell normalized by its peak (colormap). d–f, Location of activity peaks in the remaining half of trials. Neurons were sorted in the same order as in a–c. g–i, Heatmaps indicating the density of peaks in the randomly selected half of trials and in the rest of trials (see Materials and Methods). Bin size, 50 ms. j–l, Proportion of neurons with peak at each time point (magenta). This corresponds to proportion of neurons on the positive diagonal axis in g–i. Black, Chance level (Materials and Methods). Shadow, SEM (bootstrap).

Figure 14.

Rank correlation of DA. a–c, Change in spike rate (averaged over 100 ms) during the delay epoch in the contra trials (top) and ipsi trials (bottom). All cells with delay selectivity are shown. Each line, Individual neuron. Lines were color coded according to rank order of change in spike rate at the end of the delay epoch. d–f, Rank correlation of change in spike rate between different time points.

Figure 15.

Stability of the CD. a, Definition of CD as the direction that best differentiates trial-type-related activity. b–d, Projection to the CD. Shadow, SEM (bootstrap). e, The RM is the first SVD mode capturing the largest remaining variance not explained by CD (see Materials and Methods). It captures nonselective ramping as shown in f–h. f–h, Projection to the RM. Shadow, SEM (bootstrap). i–k, Stability of CD. CD was calculated at each time point and compared with CD calculated at a different time point using Pearson's correlation (see Materials and Methods). l–n, Relationship between normalized CD and reaction time (see Materials and Methods). Trials from all sessions were pooled and binned (see Materials and Methods). Lower values correspond to faster reaction times. Normalized projection to CD; 0, corresponds to median activity on ipsi trials; 1, same for contra trials. Note that the projection to the CD and the reaction time show negative correlations in contra trials but positive correlations in ipsi trials. p, Probability that correlation coefficient is significantly different from 0 (hierarchical bootstrap).

Figure 16.

Variance explained by each mode. a–c, Distribution of number of putative pyramidal neurons simultaneously recorded. Because we used probes with fewer dead sites in the auditory task with 2.0 s delay, the yield was better. d–f, Distribution of correlation coefficient between CD and RM (before orthogonalization to CD) weights. Bin size, 0.1. g–i, Selectivity explained by CD, RM, and remaining top two SVD modes (M3 and M4; see Materials and Methods). Sum of four modes are shown on top (mean across sessions). Central line in the box plot is the median. Top and bottom edges are the 75 and 25 percentage points, respectively. The whiskers show the lowest datum within 1.5 interquartile range (IQR) of the lower quartile, and the highest datum within 1.5 IQR of the upper quartile. j–o follow the same format. j–l, Trial-average variance explained by each mode (see Materials and Methods). m–o, Across-trial variance explained by each mode (see Materials and Methods).

Figure 17.

Example spike-count correlation. a, Top, Grand mean PSTH of ramping-up cells from the same session. Shank and the depth of each cell are shown. Bottom, Spike raster in contra trials. Contra trials in all cells were sorted based on the rank order of spike count during the delay epoch in Cell 1. b, Relationship of contralateral DA in two example cells. Each circle represents a trial. c, Spike-count correlation in contra trials calculated for all combinations of example cells. Grid color represents the value of spike-count correlation (color bar).

Figure 18.

Spike-count correlation. a–c, Distribution of spike-count correlation between different subtypes of neurons in contra trials (left). Distribution is overlaid on top of the distribution of nonselective cells (black) for comparison. As a control, trials were shuffled for each neuron. Spike-count correlation of the shuffled data is shown in the same format (right). C, Contra-preferring ramping-up cells; I, ipsi-preferring ramping-up cells; D, ipsi-preferring ramping-down cells; N, other cells. Numbers of pairs are shown on top of the histogram. d–f, Mean spike-count correlation between different subtypes of neurons in contra trials. Spike-count correlation is shown in pseudocolor (colormap: red, positive; blue, negative). Numbers of samples and p values (see Materials and Methods) are shown below the noise correlation in each grid. g–i, Mean noise correlation between different subtypes of neurons from different shanks.