Acute brain slice electrophysiology

Mice were anesthetized with a mixture of ketamine (90 mg/kg) and xylazine (4.5 mg/kg) and transcardially perfused with ice-cold, oxygenated (95% O₂, 5% CO₂) artificial cerebrospinal fluid (ACSF) containing (in mM) 118 NaCl, 2.5 KCl, 1.5 MgSO₄, 1 CaCl₂, 1.25 NaH₂PO₄, 10 d-glucose, 30 NaHCO₃, (pH = 7.4). The brain was rapidly excised, and coronal brain slices of 250 µm thickness were prepared with a Dosaka Linear-Pro7. For SNX-482 experiments in rostral IPN, brain slices were prepared at a 54° angle to allow for electrical stimulation of FR. For angled slice recordings in lateral IPN of WT and Cav2.3 KO mice (Figure 1H–K), slices were cut at 1 mm thickness to assure that FR remained intact all the way to the lateral IPN. Slices were recovered at 35°C for 20 min and thereafter slowly cooled down to RT over the course of 1 hr. After recovery, one slice was transferred to the recording chamber (RC-26GLP, Warner Instruments, Holliston, MA) and superfused with ACSF containing 2.5 mM CaCl₂, 20 µM bicuculline methiodide, 50 µM hexamethonium bromide, and 5 µM mecamylamine hydrochloride at a rate of 3–4 ml/min at 32.0 ± 2.0°C. Rostral or lateral IPN nuclei were visually identified using an infrared differential interference contrast video system in a BX51 microscope (Olympus, Tokyo, Japan). Electrical signals were acquired at 10–50 kHz and filtered at 2 kHz using an EPC 10 (HEKA, Lambrecht/Pfalz, Germany) amplifier. Glass pipettes (BF150-86-10, Sutter Instrument, Novato, CA) with resistances of 3–4 MΩ were crafted using a P97 horizontal pipette puller (Sutter Instrument) and filled with internal solution containing (in mM) 130 K-Gluconate, 10 KCl, 5 MgCl₂, 5 MgATP, 0.2 NaGTP, 0.5 EGTA, 5 HEPES; pH 7.4 adjusted with KOH. Whole-cell patch-clamp recordings were performed in voltage-clamp mode at a holding potential of –60 mV, and access resistance was constantly monitored via a –10 mV voltage step at the end of each sweep. Recordings with access resistances exceeding 20 MΩ or with changes in access resistance or holding current by more than 20% were discarded. To evoke glutamatergic excitatory postsynaptic currents (EPSCs) in rostral IPN neurons, voltage pulses (0.5–3.5 V, 0.2 ms duration) were applied with an ISO-Flex stimulus isolator (AMPI, Jerusalem, Israel) to a concentric bipolar stimulating electrode (CBBPC75, FHC, Bowdoin, ME) located inside the IPN, ~200–300 µm distal to the recorded neuron. For optogenetic stimulation in Tac1-ChR2 mice, blue light (λ = 465 nm) was emitted directly onto the lateral IPN through a 5 mm long mono fiber-optic cannula (fiber diameter 200 µm, total diameter 230 µm, Doric lenses, Quebec, Canada) connected to a PlexBright LED (Plexon, Dallas, TX), with an optical patch cable (fiber diameter 200 µm, total diameter 230 µm, 0.48 NA). The LED was triggered via 200–290 mA current pulse (2 ms duration) from a LED Driver (LD-1, Plexon), which was controlled directly via the HEKA EPC10 amplifier. The sweep interval of all stimulation protocols (electrical and optogenetic) was 10 s. For the application of SNX-482 (1 µM, hellobio, Bristol, UK) and ω-Conotoxin GVIA (1 µM, Alomone Labs, Jerusalem, Israel), 0.1% bovine serum albumin was added to the ACSF. For variance-mean analysis, ACSF with four different Ca²⁺ concentrations (1.5–8 mM) was applied to measure variance and mean EPSC amplitude at different release probabilities. Each Ca²⁺ concentration was washed in for 5–10 min. Once EPSC amplitudes stabilized, 15–20 consecutive EPSC responses were used for the calculation of mean EPSC amplitude and variance, followed by the wash-in of the next Ca²⁺ concentration. The order of application was 2.5 mM Ca²⁺, followed by 1.5 mM Ca²⁺, followed by 6–8 mM Ca²⁺, followed by 4–5 mM Ca²⁺. Baseline noise was much smaller than synaptic noise. The values for release probability and quantal size were calculated according to the equations Var = Iq – I²/N and I = Nqp, with I being the average EPSC amplitude, N the number of release sites, and q the quantal size. To measure the paired-pulse ratio (PPR) of two consecutively evoked EPSCs at 20 Hz, the PPRs of 20–30 EPSC pairs evoked at 10 s intervals were averaged. To study the effect of GBR activation on EPSC amplitude, R(+)-Baclofen hydrochloride (1 µM) was bath applied for 3 min and washed for 10 min. For PPR and variance-mean experiments, data from wild-type C57BL/6J and BALB/c mice were found to not differ significantly and thus were pooled. To measure action potential properties in ventral MHb neurons, a 50 pA current was injected for 100 ms, which reliably evoked a train of action potentials. Action potential peak times were measured as the time from the threshold potential to the peak. Action potential full-width at half maximum was measured as the time between rising and the falling phase of the action potential at half of the maximal action potential amplitude. Only the first action potential in the train was analyzed.