Grass s88

Electrical stimulation was delivered to the IL or PrL through the implanted concentric bipolar electrode (Grass S88, Grass Instruments). Stimulation consisted of single pulses (0.2 Hz, 0.1–0.9 mA, 0.2 ms duration) or trains (10–40 Hz, 10 pulses, 0.1–0.9 mA, 0.1–0.2 ms duration).

Fish larvae were subjected electrical stimulation at room temperature using a custom-built electric stimulation set-up (ESS) (Fig. 1A). To minimize damage to the fish, the ESS consists of two silver electrodes (0.5 mm ∅, 99.9%, Arcos Organics, New Jersey, U.S.A.) arrayed parallel and 10 mm apart passing through the drilled holes of a 6-well tissue culture plate or a 60 mm ∅ petri dish. Chambers with electrodes were filled with 2% agarose gel, such that the silver electrodes are completely within the gel. A circular well of volume 0.5 mL was created by removing the agarose in the centre of the chamber between the two electrodes. Fish are placed carefully into the well with desired orientation. Care was taken not to expose the electrodes to the fish swimming in the well. The electrodes are connected to a stimulator (Grass S-88, Grass Instruments, U.S.A) that generated square wave pulse trains of indicated lengths. Polarity of electrodes was reversed between pulse trains to avoid electrolysis. A range of stimulation regimes was tested, with the aim of attaining a brief twitch response, and all experiments shown used a train of 200 20 V pulses, with 0.5 ms pulse duration (100 ms total train duration) once every 5 seconds (Fig. 1A).

Mice were anesthetized by isoflurane. EMG recordings were made using procedures modified from Arnold et al, 2015 [98 ]. The sciatic nerve was stimulated at the proximal hind limb above the site of transection using two 28 G monopolar electrodes (DTM Series, Electrode Store). Stimulation was provided by a Grass S88 (Quincy, MA) using square-wave pulses of 0.1 ms duration and intensity ranging from 1–10 mA. Recordings were made using one fine ring electrode (reference) placed around the ankle and a second 28 G monopolar electrode (active) placed mid-belly in the TA. A ground electrode was placed around the tail to allow for differential amplification of the CMAP waveform (BDA-H-4, WPI, Inc). Signals were acquired digitally and analyzed using Spike2 software (CED, Inc, Cambridge, UK). Stimulation intensity was increased until there was no more increase in CMAP amplitude. To ensure supramaximal stimulation, stimulation intensity was increase to ~120% of maximal response. EMG recording were made from both the left control non-denervated TA and on the side of transection (Right). EMG CMAP results are presented as ratio of the recordings from left and right sides.

Bipolar self-adhesive neuromuscular stimulation electrodes (7 × 10 cm) were placed over the distal-medial and proximal-lateral portion of the quadriceps muscle group, as reported previously [17 (link)]. Stimulation pulses were delivered using a Grass S88 stimulator with a Grass Model SIU8T stimulus isolation unit (Grass Technologies, West Warwick, RI). Stimulation pulses were 450 μs in duration and delivered at 100 Hz for 100 ms (10 pulses); voltage was set at 125 V. Participants were familiarized with maximum voluntary isometric contraction (MVIC). During MVIC testing, the burst of stimulation was applied. CAR was calculated: MVIC torque/(MVIC torque + Stimulation). The 30-repetition fatigue test (CAR30) was considered central fatigue. CAR was measured on only 40 of the subjects because data was collected on only the last 50 subjects entered in the study and because of inability of 10 subjects to complete protocol.

The MgN and PFC were stimulated via concentric bipolar electrodes controlled by a pulse stimulator (Grass S88, Grass Instruments, Warwick, RI, USA). LFPs as a result of single stimulation (0.2 Hz, 0.3–1.0 mA, 0.2 ms duration) of the PFC or MgN were recorded in the BLA, and a stimulation intensity of approximately half of the amplitude measured at 1.0 mA was used for subsequent recording protocols. When specified, train stimulations of the PFC occurred at 20 Hz (10 pulses/train, 50 ms inter-stimulus interval, 0.1–0.7 mA). To understand the degree of PFC influence on BLA activity, the stimulation intensity of 0.1 mA for the PFC train was also used to assess minimal BLA responses to PFC stimulation. MgN single stimulation occurred 100 ms following PFC train. The duration of the stimulation protocol was five seconds. Each stimulation protocol was run a minimum of 20 sweeps (trials). Each stimulation intensity was then averaged and analyzed. The order of stimulation protocol (0 mA PFC train, 0.1 mA PFC train, 50% PFC train) was counterbalanced between rats in all groups.

All intracellular recordings were performed on wandering third instar larvae as described previously (Choudhury et al., 2016 (link)). Briefly, HL3 buffer containing 1.5 mm Ca²⁺ was used for larval dissection. Recordings from muscle 6 of A2 hemisegment were performed using sharp glass electrodes having a resistance of 20–25 MΩ. Miniature excitatory junction potentials (mEJPs) were recorded for 60 s, followed by recordings of EJPs at 1 Hz stimulation. For High-frequency recording, nerves were stimulated at 10 Hz, and EJPs were recorded for 5 min. For recording EJPs, stimulation pulse was delivered using Grass S88 stimulator (Grass Instruments, Astro-Med, Inc). The signals were amplified using Axoclamp 900A, digitized using Digidata 1440A, and acquired using pClamp10 software (Molecular Devices). Muscles with resting membrane potential between −60 and −75 mV were used for analysis. The data were analyzed using the Mini Analysis program (Synaptosoft, Decatur).