Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements

Size: px

Start display at page:

Download "Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements"

Norma Hamilton
5 years ago
Views:

1 Y. Isomura et al. 1 Microcircuitry coordination of cortical motor information in self-initiation of voluntary movements Yoshikazu Isomura, Rie Harukuni, Takashi Takekawa, Hidenori Aizawa & Tomoki Fukai Supplementary Figures Supplementary Figure 1. Regular experiment schedule and primary skull surgery. (a) Weekly experiment schedule for two Long-Evans rats (A, B). The schedule includes primary skull surgery (S; 1st week), operant task training (1-8; 2nd and 3rd weeks), room transfer (T; 4th week), secondary dura-opening surgery (D), and a recording experiment (R). Two task-trained rats were available for final recording experiments every week using this schedule. (b) Handling to habituate rats to the experimenters (15 min). (c) Adaptation to the body-supporting cylinder (dummy) in their home cage (2-3 days before surgery). (d) Lightweight, reusable, sliding head-attachment (13 g, aluminum). (e) Primary surgery, under isoflurane anesthesia, to mount the head-attachment on the animal s skull. Exposed skull surface (1) was covered with silicon sealant (2). FL is showing the skull surface above the forelimb (FL) area of motor cortex. (f) Full recovery from skull surgery. The treated animals behaved normally in their home cage (e.g., eating, drinking, running, sleeping). See the Methods for details.

Y. Isomura et al. 2 Supplementary Figure 2. Efficient multi-rat task-training system. (a) Six task-training boxes (1-6) were controlled by one computer (inset).

2 Y. Isomura et al. 2 Supplementary Figure 2. Efficient multi-rat task-training system. (a) Six task-training boxes (1-6) were controlled by one computer (inset). Note that all doors are shown open with the stereotaxic frames slid out. (b) The same training boxes are shown with the doors closed. Syringe pumps (red objects) were placed on the top of training boxes. (c) Spatial layout of the lever (1), water spout (2), sliding head-receptacles (3), body-supporting cylinder (4), side handrail (5), and optional armrest (6). The layout was properly adjusted for each animal. (d) Basement layout (1, infrared video camera; 2, horn tweeter; 3, arrow, lever-lock bar) below the stereotaxic frame with the head-receptacle and cylinder, which was removed for demonstration purposes. (e) Water spout for reward delivery was carefully placed into the left side of the mouth (circle). (f) Optimal angle of the right forelimb (shown by black lines) for pulling and pushing the lever. Note that the animal is stepping on the edge of the cylinder with both hindlimbs in the best sitting posture (circle; see non-slip edge in panel c, 4). (g) Typical posture of task-learning rat holding the side handrail with his left forelimb in the first training week. (h) Relaxed posture with the left forelimb on the optional armrest (circle) in the second training week.

3 Y. Isomura et al. 3 Supplementary Figure 3. Operant learning of voluntary forelimb movement task. (a) Scheduled criteria required for reward acquisition: start (hold) and goal (release) lever positions (left; final 0-20% and %, respectively) and lever hold time (right; final 1 s). (b) Body weight was maintained at more than 80% of pre-surgery weight. (c) Lever trajectories in a trainee (1 min trace; upward trace, pull; downward trace, push; bottom, holding) on the 1st, 2nd, 4th, 6th, and 8th training days. (d) Typical task performance (upper, histograms showing the trial number with actual lever-holding times; lower, temporal changes in trial number during 2-h task-training sessions; filled columns represent rewarded trials) during the two-week training period (1st, 2nd, 8th days), the transfer day, and the recording day. A lever lock (waiting) period was imposed for an hour before the start of each daily task session in the second week.

(b) Stereotaxic frame installed with all behavioral and electrophysiological devices.

4 Y. Isomura et al. 4 Supplementary Figure 4. Task performance in a novel environment for recording. (a) Recording room where task-trained animals performed the same forelimb movement task during the final electrophysiological experiment. (b) Stereotaxic frame installed with all behavioral and electrophysiological devices. (c) Task performance by a head-restraint rat during the simultaneous recording of neural activity in the left FL area through juxtacellular and multiunit electrodes. (d) Evoked electromyography (EMG) activity was observed selectively in the right upper forelimb (FL), but not in the right upper hindlimb (HL), in response to intracortical microstimulation (ICMS) at the left FL area of the motor cortex under urethane anesthesia (preliminary experiments). (e) Consecutive video pictures before and after the onset (0.000 s) of lever movement (pull). Note that the forelimb begins to move one or two images (about ms) prior to the onset of the lever movement. Supplementary Figure 5. Stable and reliable juxtacellular spike recordings. (a) Round-shape tip of a juxtacellular glass electrode (< 1 µm diameter) observed by scanning electron microscopy (SEM). (b) Development (traces 1 and 2) and maintenance (3 and 4) of positive spikes (light green) recorded juxtacellularly from a single neuron. (c) Temporal changes in the 1st, 2nd, and 3rd principle components (PC1-3) of isolated spikes from the same recorded neuron (light green dots) and the noise detected in the recorded trace (white dots). Juxtacellular spikes of single neurons were isolated off-line using KlustaKwik software with a principle component analysis 25 and refined using Klusters and NeuroScope 37.

5 Y. Isomura et al. 5 Supplementary Figure 6. Several examples of juxtacellularlly recorded neurons. (a) Layer 6 pyramidal cell with steep Pre-movement activity before the onset of pull movement and antagonistic inactivation in the non-preferred (push) direction. Left, neuron morphology (upper) and axons extending into the white matter (lower). Top, task-related firing activity aligned to the onset of pull or push movement (bin, 20 ms). Bottom; spike waveforms (left), traces of spontaneous and evoked (I) spiking (middle), and auto-correlation histogram (right; bin, 1 ms; gray, ongoing firing rate). (b) Layer 3 pyramidal cell with intrinsically bursting activity related to local field potential during slow-wave sleep. This neuron was not involved in task behavior (data not shown). (c) Putative pyramidal cell in the superficial layer (395 µm in depth) showing Movement activity with very few spikes. The neuron fired only one or two spikes every two to four trials, but most spikes were associated with lever movement. (d) Medium spiny neuron in the striatum, which displayed task-related activation in the juxtacellular recording (a preliminary experiment). The neuron expressed mrna for dopamine D1 receptor, as indicated by in situ hybridization (arrowhead).

6 Y. Isomura et al. 6 Supplementary Figure 7. Physiological properties of functionally different groups of pyramidal cells and FS interneurons. (a) Correlation between the physiological depth of juxtacellular recording site and the histological depth of the visualized neuron in a counter-stained section. Thus, physiological depth is a good measure of the cortical position of identified and unidentified neurons. (b) Cortical position (i.e., physiological depth) plotted against ongoing firing rate for identified and putative pyramidal cells (PC) and interneurons (FS) from juxtacellular recordings. Note that some pyramidal cells in superficial layers (above 800 µm) discharged at a very low firing rate. (c) Average firing rate at a baseline level (in lever-holding period) plotted for functionally different groups of RS and FS neurons in multiunit recordings (left) and pyramidal cells and FS interneurons in juxtacellular recordings (right). See Fig. 4 for Symbol legend.

7 Y. Isomura et al. 7 Supplementary Figure 8. Verification for excitatory synaptic connectivity in multiunit data analysis. (a) Original analysis using the entire spike dataset of the same RS (green) and FS (gray) neurons shown in Fig. 6b. Left, spike distribution in a two-dimensional plane out of 17 spike feature parameters 25,37. Middle, auto-correlation histograms (a.c.; bin, 1 ms). Right, cross-correlation histogram (c.c.; bin, 1 ms) between these neurons showing a single, asymmetric peak around +1.5 ms from spiking of a triggering (RS) neuron. The gap at 0 ms is due to the lack of spike-detection within ±0.5 ms from each spike 30. (b) Strict analysis by excluding all overlapping outlier spikes from the same neurons in the two-dimensional plane. The single peaks are almost identical in a and b, suggesting true short-latency interaction between two neurons with completely distinct spike features. (c) Representative raw traces of their spike pairs in each channel (1-4ch; 6 sets). Note that the first spikes (green) are largest in channel 1, while the following spikes (gray) are largest in channel 3, supporting evidence that they are really different neurons. (d) Excitatory synaptic interaction during task performance in the same RS and FS neurons shown in Fig. 6c (upper). A single peak was consistently observed throughout lever-hold and pull periods (analyzed for a duration of 500 ms, starting 1000, 750, 500, and 250 ms before the onset of pull movement). The single peak was completely abolished after shuffling task-trials in a triggering RS neuron (lower).

respectively. Effective excitatory response during task performance ( 1 to + 0.25 s) was also confirmed in 12 neuron pairs (bold numbers). Supplementary Videos (Legends) Supplementary Video 1.

8 Y. Isomura et al. 8 (e) A summary diagram of putative excitatory connections between functionally different neurons. Arrows and numbers indicate the delay direction and strength (peak amplitude normalized by baseline activity in the cross-correlation histogram) of individual excitatory synaptic interactions, respectively. Effective excitatory response during task performance ( 1 to s) was also confirmed in 12 neuron pairs (bold numbers). Supplementary Videos (Legends) Supplementary Video 1. Initial behavior of an untrained rat in the training box. This beginner rat entered the body-supporting cylinder by himself, because he had become accustomed to a dummy cylinder (see Supplementary Figure 1c). After his head-attachment was fastened to the stereotaxic frame, the rat grasped the lever naturally and tried to move it with his right forelimb without struggling. Supplementary Video 2. Efficient task-training of six rats in separate training boxes. Up to six rats were simultaneously and separately trained in our multi-rat task-training system to perform the voluntary forelimb movement task in the head-restraint condition. The rat in the lower right box (no. 6) was a beginner in the forelimb movement task. Supplementary Video 3. Pre-movement spiking activity during task performance (with sound). Pre-movement activity was recorded juxtacellularly from the layer 6 pyramidal cell shown in Supplementary Figure 6a while the rat was moving the lever. The sound represents the spiking of this neuron.

Nature Methods: doi: /nmeth Supplementary Figure 1. Activity in turtle dorsal cortex is sparse.

Nature Methods: doi: /nmeth Supplementary Figure 1. Activity in turtle dorsal cortex is sparse. Supplementary Figure 1 Activity in turtle dorsal cortex is sparse. a. Probability distribution of firing rates across the population (notice log scale) in our data. The range of firing rates is wide but