Ds3 constant current isolated stimulator

Following behavioural testing, rats implanted with recording or stimulating electrodes were anesthetised with gaseous isoflurane and intraperitoneal injection of pentobarbital (27.5 mg/kg) until hindpaw reflexes were absent. A current pulse of 100 µA for 2 s (DS3 isolated constant current stimulators, Digitimer Ltd.) was passed through the headstage to lesion electrode sites. Rats were then transcardially perfused with phosphate-buffered saline, followed by 4% paraformaldehyde (PFA). The brains were extracted and left in 4% PFA for 24 h. Brains were then cut into 80 µm sections on a vibratome or freezing microtome, and these sections mounted onto glass slides. Sections were then stained with cresyl violet acetate, covered with DPX mounting medium and coverslipped. A Leica DMR upright bright-field microscope was used to image the lesion site. Location of the lesion site was projected onto a schematic of the PAG.

Evoked local field potentials (eLFP) were recorded in the stratum radiatum of the CA1 hippocampal region using glass micropipette electrodes filled with ACSF (resistance 1–2 MΩ) and an HEKA EPC 10 Patch Clamp Amplifier (HEKA Elektronik, Lambrecht, Germany). The DS3 Constant Current Isolated Stimulator (Digitimer, Welwyn Garden, UK) and a bipolar stimulating electrode placed on the Schaffer’s collaterals at the hippocampal area CA2 were used for the induction of eLFPs. Current pulses 20–300 μA in amplitude and 200 μs in duration were applied to obtain reliable eLFPs. Single eLFPs were recorded continuously every 20 s. PatchMaster software (HEKA Electronik, Lambrecht, Germany) was used to record eLFPs, control the HEKA EPC 10 Patch Clamp Amplifier and the DS3 Constant Current Isolated Stimulator.

SCI rats were randomized into one of two treatment groups: repeated transcutaneous stimulation (SCI + tSCS group, n = 6) or no stimulation (SCI, n = 6). Starting 5 days post-injury, animals from the SCI + tSCS group were fitted with transcutaneous electrodes as described above (Section 2.1.1) and secured in a modified, custom-built apparatus to allow them to lie prone with hindlimbs hanging below at rest. The motor threshold (T) was evaluated visually and with light manual touch and was determined as the lowest intensity eliciting a twitch of the ankle. Stimulations were evoked using a DS3 constant current isolated stimulator (Digitimer Ltd., Hertfordshire, UK) and a customized script written in Signal (CED). The stimulation protocol consisted of single, monophasic pulses of 1 ms in duration delivered at 0.2 Hz. Stimulation intensity alternated in bouts of 3 min between suprathreshold (1.2 T) and subthreshold (0.8 T) for a total duration of 18 min per session. Untreated SCI animals were similarly secured to the apparatus for an equal amount of time but were not stimulated. Sessions were repeated 3 times a week for 4 weeks before the terminal experiment.

A rectangle of stacked copper plates was brought into contact with the fly’s abdomen and legs using a manually movable stage (DT12XYZ/M, Thorlabs). The design of the copper plates was based on Felsenberg et al., 2018 (link). Shocks were provided by a DS3 Constant Current Isolated Stimulator (1.2 s, 32 mA, Digitimer; maximum voltage 90 V) and triggered by the same software as the odor delivery. Successful shock delivery was confirmed by observing the fly’s physical reaction with a Genie Nano-M1280 camera (Teledyne Dalsa) coupled to a 1x lens with working distance 67 mm (SE-16SM1, CCS).