Spike2

The experimental protocol is the same as in Dilgen et al. (2013 (link)) and Oprisan et al. (2015 (link)). Briefly, male PV-Cre mice (B6; 129P2 – Pval^{btm1(Cre)Arbr/J} Jackson Laboratory (Bar Harbor, ME, USA) were infected with the viral vector (AAV2/5. EF1a. DIO. hChR2(H134R) – EYFP. WPRE. hGH, Penn Vector Core, University of Pennsylvania) delivered to the mPFC as described in detail in Dilgen et al. (2013 (link)). The extracellular signals were amplified using a Grass amplifier (Grass Technologies, West Warwick, RI, USA), digitized at 10 kHz by a 1401plus data acquisition system, visualized using Spike2 software (Cambridge Electronic Design, LTD., Cambridge, UK) and stored on a PC for offline analysis. A HumBug 50/60 Hz Noise Eliminator (Quest Scientific Inc., Canada) filter canceled out the line noise and the signal was band-pass filtered online between 0.1 and 130 kHz to obtain the LFPs. A 473 nm laser (DPSS Laser System, OEM Laser Systems Inc., East Lansing, MI, USA) delivered the light stimulation via a 1401plus digitizer and Spike2 software (Cambridge Electronic Design Ltd., Cambridge, UK).

After one week of recovery from surgery, animals were habituated in the recording cage (white plexiglass of 12′′ × 12′′ × 11′′ length, width, and height). The recording cage was placed in a well-ventilated, sound and light dampened (black color plexiglass) recording chamber (48′′ × 24′′ × 24′′) to minimize external disturbances during the experiments. The recording chamber was illuminated with 20 Lux light. Food and water bottle were placed in the food cup and bottle holder attached to the recording cage. Animals were tethered with the recording cable through a commutator and habituated to the recording chamber for 6 hrs (11:30 AM–5:30 PM) on two consecutive days. Also, during this period, the EEG and EMG signals were examined in a computer through Spike2 software (Cambridge Electronic Design, Cambridge, UK). EEGs were recorded in two channels and EMG was recorded in a single channel. Electrophysiological signals were amplified using 15LT Bipolar Portable Physiodata Amplifier System (Astro-Med, USA). EEG signals were processed with a high-pass filter of 0.1 Hz and a low-pass filter of 40 Hz, while EMG signal was processed with a high-pass filter of 10 Hz and a low-pass filter of 90 Hz digitized at 100 Hz sampling rate. Recordings were acquired in a personal computer using Spike2 software (Cambridge Electronic Design, Cambridge, UK) and were saved for offline analysis.

Surface electromyography (EMG) was performed using three Trigno Wireless electrode sensors (Delsys Inc. Ltd. Boston, United States), which had a predetermined bandwidth of 20–450 Hz, a gain of 1,000, a common-mode rejection ratio of >80 dB, and a sampling rate of 2,000 Hz. After standard skin preparation, the electrodes were placed over the MG, SOL and Tibialis anterior (TA) according to SENIAM guidelines (Hermens et al., 1999 (link)). The EMG signals were collected at a sampling rate of 2,100 Hz and stored for offline analysis. All EMG data were visually inspected prior to analysis. EMG data for each gait cycle were exported from Visual 3D and imported into Spike2 software (Cambridge Electronic Design, Cambridge, United Kingdom) for further analysis. EMG raw signals were notch filtered at 50 Hz (to remove ambient noise from power supply), rectified and smoothed using a 5-point moving average (Spike2, Cambridge Electronic Design, Cambridge, United Kingdom). The EMG amplitude for each muscle was calculated as root mean square (RMS) over five stance cycles (initial contact to toe-off).

In order to obtain a measure of nerve activity, the trace recorded in Spike 2 (Cambridge Electronic Design) was first duplicated to apply different filtering processes to the A and C fibre CAPs. A fibre CAP signals were filtered within Spike2 using DC removal at 20 ms time constant and smoothing at 100 μs time constant to remove DC offset and high-frequency noise, respectively. Conversely C fibre CAP signals were filtered using DC removal at 20 ms time constant and smoothing at 900 μs time constant respectively. Additionally, stimulation artifacts were digitally blanked in MATLAB (Mathworks) to produce the plots shown in Figure 3. For statistical analysis CAP signals were rectified and integrated in MATLAB to produce a value representing neural activity in response to stimulation. Measures of activity reported in Figures 4, 5 are reported as relative to the baseline activity of 1, comparing activity at start of trials with activity measured during the trial, such as in the block or recovery phases. Signal filtering was carried out in Spike2 (Cambridge Electronic Design) and rectification-integration analysis and plotting were carried out using MATLAB (Mathworks).

Data were digitized and stored onto a computer hard disk through an appropriate interface (Power1401) and software (Spike2) from Cambridge Electronic Design Ltd. (Cambridge, UK).
Data were analyzed using the Spike2 analysis software. Spikes recorded from the CBCO nerve were identified according to their waveform based on a template matching protocol (wavemark). Templates were built automatically and corresponded to the mean duration of sensory spikes (about 1.5 ms in duration). The sampling rate for CBCO nerve recording was set to 15 kHz, which resulted in templates containing 20–22 points. The procedure used two criteria to identify a spike: (1) more than 90% of the points should be in the confidence limits of the template and (2) the maximum amplitude change for a match was less than 5%. This procedure was applied off-line. After the completion of this protocol, each identified CBCO unit (spike shape) was assigned an arbitrary number. Subsequently, a spike triggered average was performed for each CBCO unit, allowing us to observe in a given MN the occurrence of any postsynaptic events related to this unit.

A smart device (iPod Touch 5th generation, iOS 12.11, Apple Inc., CA) and data logging App (Sensor Data, Wavefront Labs) recorded the outcomes for the motor battery tasks. Research assistants described and demonstrated each task briefly and provided reminders of technique between tasks. Each task was completed twice, with a rest period of 30s between trials. Data were transferred to a lab computer and imported into the Spike 2 software program (Spike 2, v. 7.14, Cambridge Electronic Design, Cambridge, UK) for visual inspection and analysis. Motor BatterySupplementary Material provide materials, setup, and processing details for all tasks.