Picospritzer 2

Recording of mEPC in the NMJ was performed as previously described with some modifications (47 (link)). Skinned larvae were pinned down to the recording chamber coated with Sylgard and immobilized by bathing in 10 μM nifedipine (Merck, Kenilworth, NJ). Patch-clamp recordings were made by the whole-cell ruptured technique of muscle cells. The pipette solution used for the voltage-clamp recording was 120 mM KCl, 5 mM BAPTA, and 5 mM Hepes (pH 7.2). The extracellular solution contained 112 mM NaCl, 2 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 3 mM glucose, and 5 mM Hepes (pH 7.4). Membrane currents were recorded with an EPC10 amplifier and PatchMaster. For mEPC recordings, muscle cells were voltage-clamped at −90 mV, and 1 μM Tetrodotoxin (TTX) was added to the recording solution. The currents were sampled at 50 kHz and filtered at 3 to 5 kHz before analysis. Capacitive transients were compensated manually.
For puff application, a glass electrode [opening, ∼30 μm; filled with bath solution containing 30 μM ACh and dextran–Alexa Fluor 488 (Thermo Fisher Scientific, Waltham, MA)] was placed near the voltage-clamped muscle cell and positive pressure was applied using Picospritzer II (Parker Hannifin; 30 ms, 1 psi). ACh-induced currents were recorded by the whole-cell ruptured technique.

All procedures were carried out in strict adherence to guidelines provided by The Guide for the Care and Use of Laboratory Animals and requirements of the institutional animal welfare committee. Animal subjects were C57 BL/6 mice aged 6 weeks or older, male and female unless otherwise stated. All surgeries were performed under deep anesthesia using isoflurane gas (Covetrus, 029405). CTB labeling was performed by exposing spinal level C8 and inserting a pulled glass pipette (World Precision Instruments, 1B150F-4) 0.5 mm lateral to midline, 0.6 mm deep. Using a picospritzer II (Parker Hannifin), 1 ul of 1% Cholera Toxin B (List Biological Labs, 104) was injected at a slow rate, injecting both left and right spinal cord. Dorsal column lesions were performed at spinal level C7 using a Kopf wire knife (David Kopf Instruments), 0.6 mm lateral to midline, 0.4 mm deep, and lifted to transect the dorsal column with coincident compression with a 28-ga blunt tip to ensure lesion completeness. Conditioning Lesion: dorsal column lesions were performed at spinal level C7 as described above. Cortices were isolated for culture at 1 hr, 6 hr, 24 hr, 72 h, and 7 days post lesion. Three animals were used per timepoint as individual biological replicates where each animal’s cortical neurons were isolated, cultured, plated, and imaged individually.