Borosilicate glass pipette

Borosilicate glass pipettes (Harvard Apparatus) were pulled with a Sutter puller, fired polished, and had a resistance between 1–2 MΩ. Series resistance was compensated up to 50% and was continually monitored during the experiment. The composition of the standard extracellular solution used to record APs was (mM): NaCl, 140; KCl, 4; CaCl2, 1.8; MgCl2, 1.1; HEPES, 10; glucose, 10; pH 7.4 (LiOH). When APs were recorded, the pipette solution contained (mM): KCl, 135; MgCl2, 4; Ethylene glycol-bis (2-aminoethyl ether)-N, N, N’, N’-tetraacetic acid (EGTA), 10; Glucose 10; HEPES, 10; Na2-ATP 5; Na2-CP 3, pH 7.2 (LiOH). In a current-clamp configuration, action potential was measured in response to brief depolarizing current (1–2 ms) injections at 1 Hz as described (18 (link)).

MVN neurons identified visually by location, size and fluorescence were recorded using borosilicate glass pipettes of external diameter 1.2 mm/internal diameter 0.69 mm (Harvard Apparatus) pulled from Flaming/Brown micropipette puller (Model P-97, Sutter Instrument) and filled with high chloride internal solution (in mM): 140 CsCl, 10 HEPES, 1 EGTA, 2 MgCl₂, 2 Na₂ATP, and 1 Na₂GTP (adjusted to pH 7.2, 290 mOsm). The advancement of the pipette was manually operated through the micromanipulator (Sutter Instrument).
Signals were amplified using MultiClamp700A (Axon Instruments) and acquired through a 16-bit data acquisition system (DIGIDATA 1322A; Axon Instruments). During whole-cell patch-clamp recording, membrane potentials were corrected for the liquid junction potential (10 mV), and the change of series resistance was sustained within 15%. Only recordings with series resistance smaller than 15 MΩ were included for subsequent analysis. Cell recording was discarded if the leaking currents went beyond 200 pA. The signals of the recording were digitized at 10 kHz and filtered at 3 kHz by the Multiclamp 700A amplifier, DIGIDATA 1322A analog/digital interface board and pCLAMP 10.2 software (Axon Instruments). Data were captured by Clampex 10.2/Multiclamp Commander 1 (Axon Instruments) package.

Borosilicate glass pipettes (Harvard Apparatus, Holliston, MA, USA) with resistance of 1.5–3 MΩ were fabricated using a PC-100 vertical Micropipette Puller (Narishige International Inc., East Meadow, NY, USA). Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Membrane capacitance and series resistance were estimated using the dial settings on the amplifier, and capacitive transients and series resistances were compensated by 70–80%. Data acquisition and filtering occurred at 20 and 5 kHz, respectively, before digitization and storage. Clampex 9 software (Molecular Devices) was used to set experimental parameters, and electrophysiological equipment was interfaced to this software using a Digidata 1200 analog–digital interface (Molecular Devices). Analysis of electrophysiological data was performed using Clampfit 11 software (Molecular Devices) and GraphPad Prism 8 software (La Jolla, CA, USA). Results were expressed as mean ± standard error of the mean (SEM). Except where otherwise noted, statistical significance was determined using a Student’s t-test comparing cells treated with vehicle (DMSO) or PW201, with p < 0.05 being considered statistically significant.

For hippocampal slice LTP recordings procedure, please refer to the method of our previous study [14 (link)].
Whole-cell patch clamp recordings were obtained from SF-1 neurons in the hypothalamus of ScKO mice and control mice using 4 to 8 MΩ borosilicate glass pipettes (Harvard Apparatus). The pipette recording solution contained (in mmol 1−1): 8.0 NaCl, nominally 0.0001 CaCl₂, 0.3 Na-GTP, 130 potassium gluconate, 10.0 Na-Hepes, 1.0 MgCl₂, and 2.0 Na-ATP (pH adjusted to 7.4 with methanesulfonic acid; 295 to 300 mosmol 1⁻¹). The pipettes with an Ag–AgCl electrode were connected to a CV-4 head stage and an Axopatch-1D amplifier with a Digi data 1200 interface (Axon Instruments). The pipettes are positioned within the tissue using a motorized patch-clamp micromanipulator. Seal resistance was typically 4 to 10 GΩ. Typical whole-cell access resistance (R_a) was 5 to 30 MΩ and whole-cell leak was below 20 pA [43 (link)]. Miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) were recorded in the presence of tetrodotoxin (500 nmol/L). We recorded mEPSCs and mIPSCs at holding potentials of –70 and 0 mV, respectively, in the same cell (3 min each; n > 20 cells/group).

Borosilicate glass pipettes (Harvard Apparatus, Holliston, MA, USA) with resistance of 3 – 5 MΩ were fabricated using a PC-100 vertical Micropipette Puller (Narishige International Inc., East Meadow, NY, USA). Recordings were obtained using an Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA). Membrane capacitance and series resistance were estimated using the dial settings on the amplifier, and capacitive transients and series resistance were compensated by 70–80%. Data acquisition and filtering occurred at 20 and 5 kHz, respectively, before digitization and storage. Clampex 9 software (Molecular Devices) was used to set experimental parameters, and electrophysiological equipment was interfaced to this software using a Digidata 1320A analog–digital interface (Molecular Devices). Analysis of electrophysiological data was performed using Clampfit 11 software (Molecular Devices) and GraphPad Prism 8 software (La Jolla, CA, USA). Results were expressed as individual replicates with SEM error bars. Statistical significance was determined using a one-way ANOVA with post hoc Tukey’s multiple comparisons test, with p < 0.05 being considered statistically significant.