Patch clamp recordings in vitro

Whole-cell patch-clamp recordings were performed as described (39–42). Mice weighing 20 to 25 g were decapitated under 4% isoflurane anesthesia for induction, and slices containing the CeA and hippocampus from adult male and female mice were identified using the mouse brain atlas and then incubated in oxygenated artificial cerebrospinal fluid [(ACSF, composition in mM: 125 NaCl, 25 NaHCO₃, 2.5 KCl, 1.25 NaH₂PO₄, 2 CaCl₂, 1.5 MgCl₂, and 10 d-glucose (pH 7.4) 300 to 310 mosmol] at 35° ± 0.5°C for at least 1 hour. The slices were kept at room temperature for approximately 30 min before recording. Then, they were moved to a submerged chamber during recording sessions and continuously perfused with oxygenated ACSF at a rate of 2 ml/min maintained at 32 ± 0.5°C.

Recordings were conducted using patch electrodes made of borosilicate glass that were pulled using a Flaming-Brown puller (Sutter Instruments, CA) and polished with a polishing tool (Narishige, Tokyo, Japan) immediately before use. The pipette resistance typically ranged from 4 to 6 megohm. The internal solution comprised the following (in mM): 135 KMeSO₄, 10 Na2-phosphocreatine, 5 KCl, 0.5 CaCl₂, 5 Hepes, 5 EGTA, 2 Mg₂ATP, and 0.5 Na₃GTP, adjusted to pH 7.25 with KOH. CeA neurons were observed using an Olympus BX51WI microscope (Tokyo, Japan) equipped with an infrared differential interference contrast and fluorescence module. All images were captured using a Hamamatsu ORCA FLASH4.0 camera (Hamamatsu Photonics, Japan) and displayed on a laboratory computer. Patch clamp recordings were obtained using an Axopatch-700B amplifier (Axon Instruments, Sunnyvale, CA) and fed into a computer via a Digidata-1440 interface (Axon Instruments) for data acquisition and analysis (pClamp 10.0, Axon Instruments). Recordings of whole-cell currents were filtered at a low-pass of 2 kHz and were digitized at 10 kHz, while recordings of membrane potentials were filtered at a low-pass of 5 kHz and were digitized at 20 kHz. Neurons were kept at a membrane potential of −70 mV and were assessed by injecting a rectangular voltage pulse (5 mV, 50 ms) to measure the whole-cell membrane capacitance, series resistance, and membrane resistance. Any neurons with unstable series resistance or a resistance exceeding 30 megohm were excluded from the study. Neurons with seal resistance changes of more than 30% of the initial level were also abandoned and excluded from further analysis.

The neuronal resting membrane potential was recorded under a current clamp (I = 0 pA). Input resistance was measured through potential changes resulting from injected currents ranging from −30 to 150 pA. The action potential was assessed by injecting a series of current pulses with an intensity of −30 to 150 pA, incrementing by 30 pA, and lasting for 1 s. For chemogenetic experiments, DCZ (MedChem Express, NJ, 0.05 μM) was dissolved in DMSO and added to ACSF at a final concentration of 0.3% DMSO (43, 44).