Ect unit

Eleven to 15-week old mice were arbitrarily assigned to ECS and sham groups, with the exception that male and female mice were equally distributed across groups. Bilateral auricular ECS was administered via padded ear clip electrodes connected to a pulse generator (ECT Unit; Ugo Basile, Gemonio, Italy). Ear clip electrodes were soaked in saline solution and placed on the base of the pinna. Stimulation parameters were adapted from Yanpellewar et al. [41 (link)] and set at 0.5 ms pulse width, 50 Hz frequency, 0.5 s total shock duration, and 20–24 mA current. ECS stimulation generated a seizure scoring 5 on the Racine seizure scale for rodents [42 (link)] for all stimulated animals. Animals displayed a loss of posture, rearing, and tonic–clonic limb movements, with full recovery. Sham animals were handled and ear clipped in the same fashion (for 1 min) but did not receive any stimulation. Stimulated animals were removed and placed back to the home cage once they regained posture and motor function.

Young adult and middle-aged rats received a series of 1 ECS or sham treatment per day for 7 consecutive days. Bilateral ECS treatment was administered via spring-loaded ear clip electrodes (ECT unit, UGO Basile, Comerio, Italy), and sham treatment involved the application of ear clip electrodes without electrical stimulation. Chronic ECS parameters for each ECS treatment were as follows: current strength: 70 mA; duration: 0.5 seconds; frequency: 100 pulses/s; pulse width: 0.9 milliseconds. Behavioral assays or brain tissue harvesting for further processing was performed 2 hours post the last ECS treatment on the seventh day (Dias et al., 2003 (link)). The choice of the older age cohort for chronic ECS was based on preliminary experiments that determined minimal mortality following chronic ECS in the middle-aged (12 months) Sprague-Dawley rats.

Beginning on the day after 15-day CSS/control, half of the CSS and control mice received one daily electroconvulsive seizure (ECS) over a period of 10 days (study days 16–25). Between 3 and 5 pm, mice were anesthetized with isoflurane (3% in O₂ at 800 ml/min) and the ears were cleaned with 70% alcohol. An electrical current of 80 mC (80 mA, 50 Hz, 1s duration and 0.5 ms pulse width) generated by an ECT unit (Ugo Basile, Italy) was applied via ear clip electrodes. This current induced a tonic-clonic seizure lasting 5–20 seconds, with an overall average seizure duration (± SD) of 13.0 ± 2.2 seconds (12.9 ± 2.2 for the control group and 13.1 ± 2.2 for the CSS group) Averages of individual mice over all 10 ECS sessions ranged from 11.1 to 14.5 seconds, which was comparable to seizure duration induced by ECS in other studies [42 (link),44 (link),45 (link)]. The other half of the mice underwent a sham procedure comprising anaesthesia, ear cleaning and electrode attachment in the absence of ECS.