Seizure-induced immediate early gene expression with, vs. without, isoflurane anesthesia

Description

A single electroconvulsive seizure (ECS), versus sham, was administered to anesthetized and non-anesthetized WT male mice. One hour after ECS, mice were sacrificed and perfused. Sectioned brain tissue was immunofluorescently labeled with an antibody against the IEG Arc. Complete methods are found in associated Data in Brief article (Data on serial electroconvulsive seizure in mice, effects of anesthesia and correlation between age and neurogenesis) and original associated article: Meyers, K.T., et al., Serial electroconvulsive Seizure alters dendritic complexity and promotes cellular proliferation in the mouse dentate gyrus; a role for Egr3. Brain Stimul, 2023. 16(3): p. 889-900. https://doi.org/10.1016/j.brs.2023.04.022

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Animals: Male C57Bl/6J mice, ages (1.5-2.5 mo.) were obtained from The Jackson Laboratory and housed on a 14:10 hour light:dark cycle with ad libitum access to food and water. Electroconvulsive Seizure: Following administration of local anesthetic eye drops, ECS was administered via ocular electrodes using a Ugo Basile instrument (Varese, Italy). Settings for ECS were: pulse frequency 260 Hz, pulse width 0.3 ms, duration 100 ms, and current 80 mA. For Arc immunofluorescent studies, half the cohort received a single ECS without the use of inhalational anesthesia while the other half received a single ECS following isoflurane (5%) anesthesia. For mice receiving isoflurane anesthesia, ECS was administered before mice fully recovered consciousness (2 mins, 30s). Under these conditions, following administration of electrical shock, anesthetized animals displayed behaviors such as lip smacking, arching, stiffening, and running but did not display tonic-clonic movements following delivery of current. Unanesthetized mice exhibited tonic-clonic seizure after current administration. Sham control animals underwent the identical procedure without current administration. Arc immunofluorescence: One hour after administration of ECS, mice were sacrificed via isoflurane overdose and perfused with 1x phosphate buffered saline (PBS) followed by 4% paraformaldehyde (PFA). Brains were post-fixed in PFA for 24 hours and then transferred to 30% sucrose at 4oC. Following sucrose saturation, brains were sectioned at 40μm on a sliding microtome (American Optical). Free-floating sections were washed in 1x tris-buffered saline (TBS), then blocked in 4% normal donkey serum, diluted in 1x TBS with 0.4% Triton-X 100, overnight at 4oC with constant agitation. Primary antibody, 1:1000 rabbit anti-ARC (Synaptic Systems, Cat. # 156 003), was applied to tissue sections and incubated overnight at 4oC with constant agitation. Following overnight incubation, sections were washed in 1x TBS and then incubated in secondary antibody (1:1000 donkey anti-rabbit, Alexa 568, Invitrogen) overnight at 4oC. Tissue sections were washed in 1x TBS and mounted with Vectashield containing DAPI (Vector Labs). Images were taken on the 10x objective of a Zeiss AXIOImager M2 epifluorescence microscope in the DsRed channel (300 ms).

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