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Experiments were performed on brain slices from 12- to 15-week-old genetically marked (with ZsGreen) Glp1r- and Lepr-expressing POMC neurons using POMCDre LeprCre ROSA26lSlrSrZsGreen+/− or POMCDre Glp1rCre ROSA26lSlrSrZsGreen+/− male and female mice. Animals were kept under standard laboratory conditions, with tap water and chow available ad libitum, on a 12 h light/dark cycle. The animals were lightly anesthetized with isoflurane (B506; AbbVie) and decapitated. Coronal slices (270–300 µm) containing the ARC were cut with a vibration microtome (HM-650 V; Thermo Scientific) under cold (4 °C), carbogenated (95% O2 and 5% CO2), glycerol-based modified artificial cerebrospinal fluid (GaCSF)50. GaCSF contained (in mM): 244 glycerol, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.2 NaH2PO4, 10 HEPES, 21 NaHCO3 and 5 glucose, adjusted to pH 7.2 with NaOH. If not mentioned otherwise, the brain slices were continuously superfused with carbogenated aCSF at a flow rate of ~2.5 ml min−1. aCSF contained (in mM): 125 NaCl, 2.5 KCl, 2 MgCl2, 2 CaCl2, 1.2 NaH2PO4, 21 NaHCO3, 10 HEPES and 5 glucose, adjusted to pH 7.2 with NaOH. To block GABAergic and glutamatergic synaptic input, in all recordings, the aCSF contained 10−4 M picrotoxin (P1675; Sigma-Aldrich), 5 × 10−6 M CGP (CGP-54626 hydrochloride; BN0597, Biotrend), 5 × 10−5 M DL-AP5 (DL-2-amino-5-phosphonopentanoic acid; BN0086, Biotrend) and 10−5 M CNQX (6-cyano-7-nitroquinoxaline-2,3-dione; C127, Sigma-Aldrich). To suppress action-potential-dependent synaptic release, blocked voltage-dependent Na+ channels were blocked by 10−6 M TTX (T-550, Alomone).

Current-clamp and voltage-clamp recordings of ZsGreen-expressing POMC neurons were performed at ~32 °C in the perforated patch-clamp configuration. Neurons were visualized with a fixed-stage upright microscope (BX51WI, Olympus) using ×40 and ×60 water-immersion objectives (LUMplan FL/N ×40, 0.8 numerical aperture, 2 mm working distance; LUMplan FL/N ×60, 1.0 numerical aperture, 2 mm working distance, Olympus) with infrared differential interference contrast optics51 and fluorescence optics. ZsGreen-expressing POMC neurons were identified by their anatomical location in the ARC and by their ZsGreen fluorescence that was visualized with an X-Cite 120 illumination system (EXFO Photonic Solutions) in combination with a Chroma 41001 filter set (ex: HQ480/×40; bs: Q505LP; em: HQ535/50m). Electrodes with tip resistances of between 4 and 6 MΩ were fashioned from borosilicate glass (0.86-mm inner diameter; 1.5-mm outer diameter; GB150-8P, Science Products) with a vertical pipette puller (PP-830, Narishige). All recordings were performed with an EPC10 patch-clamp amplifier (HEKA) controlled by the program PatchMaster (version 2.32; HEKA) running in Windows. In parallel, data were recorded using a micro1410 data acquisition interface and Spike 2 (version 7, both from CED). Current-clamp recordings were sampled at 25 kHz and low-pass filtered at 2 kHz with a four-pole Bessel filter. Voltage-clamp recordings were sampled at 5 kHz, smoothed (𝜏 = 0.2 s) and downsampled to 0.5 Hz. The calculated liquid junction potential of 14.6 mV between intracellular and extracellular solution was compensated or subtracted offline (calculated with Patcher’s Power Tools plug-in from for IGOR Pro 6; Wavemetrics).

Perforated patch experiments were conducted using protocols modified from previous studies52,53. Recordings were performed with pipette solution containing (in mM): 140 K-gluconate, 10 KCl, 10 HEPES, 0.1 EGTA and 2 MgCl2, adjusted to pH 7.2 with KOH. ATP and GTP were omitted from the intracellular solution to prevent uncontrolled permeabilization of the cell membrane54. The patch pipette tip was filled with internal solution and backfilled with internal solution, which contained the ionophore to achieve perforated patch recordings and 0.02% tetramethylrhodamine-dextran (3,000 MW, D3308, Invitrogen) to monitor the stability of the perforated membrane. Amphotericin B (A4888; Sigma) was dissolved in DMSO to a concentration of 40 µg µl−1 (D8418, Sigma) following the protocols of a previous study55. The used DMSO concentration (0.1–0.3%) had no obvious effect on the investigated neurons. The ionophore was added to the modified pipette solution shortly before use. The final concentration of amphotericin B was ~120–160 µg ml−1. Amphotericin solutions were prepared from undissolved weighted samples (stored at 4 °C protected from light) on every recording day. During the perforation process, access resistance (Ra) was monitored continuously and experiments started after Ra values reached steady state (~15–20 min) and the action potential amplitude was stable.

To analyze in detail the intrinsic electrophysiological properties of ZsGreen-expressing POMC neurons, a set of current-clamp protocols from a holding potential of −70 mV was applied. Cell input resistance was determined from a series of hyperpolarizing small current pulses (1 s, 2–10 pA increments) and the slope of the resulting I–V relations. Whole-cell capacitances were calculated from the membrane time constant (𝜏) and the input resistance (R): C = 𝜏/R. To analyze the IH-dependent sag potentials, the neurons were hyperpolarized with five consecutively incrementing current pulses. The increments were adjusted so that the last pulse hyperpolarized the membrane to −120 mV. The sag potential was defined as the difference between the lowest voltage reached at the beginning of the pulse and the membrane potential reached at the end of hyperpolarization. To analyze post-inhibitory rebound excitation, we used an ‘enhanced rebound protocol’, whereby the same current-step amplitudes were applied as those used for the sag-potential analysis, but this time as 2-s hyperpolarizing pre-pulses that were followed by a 1-s test pulse with the amplitude of a single increment. The maximum instantaneous frequencies during the rebound were determined and plotted over the membrane potentials of the pre-pulses. To analyze input–output relations, we applied a series of ascending and then descending current ramps (5 s each), where the ramp amplitudes were increased from 10 to 25 pA in 5-pA increments. Amplitudes were further increased if the 25-pA ramp did not elicit action potentials. Spike-number ratios were calculated by dividing the number of action potentials during the ascending ramp by the number of action potentials during the descending ramp. To further analyze excitability, that is, evoked action potential firing, a series of depolarizing current pulses (1 s; 5–50 pA in 5-pA increments) was applied. For each current pulse, the number of action potentials was determined, plotted over the current amplitude and linearly fit. Linear fits were performed for data points where action potentials were elicited. Only data points at which action potentials were triggered were considered for fit.

For SFA ratios, 10-s depolarizing stimuli were applied from a holding potential of −70 mV with initial instantaneous action potential frequencies between 30 and 40 Hz. Instantaneous frequencies were plotted (Y) over the 10-s time course, and fit to a mono-exponential decay equation with Y0 set to the initial instantaneous frequency: Y = (Y0 − plateau) × exp(−K × T) + plateau, where ‘plateau’ is the asymptotic frequency, K is the inverse time constant and T is the time. The SFA ratio is determined by dividing the maximum initial instantaneous frequency by the plateau frequency of the fit. Action potential waveform parameters were obtained from action potentials with instantaneous frequencies ≤ 5 Hz. If necessary, hyperpolarizing bias currents were used to decrease spontaneous firing.

Leptin (100 nM; L3772, Sigma-Aldrich), and Glp1 (300 nM; H-5956, Bachem AG) were bath applied for 15 or 30 min with a perfusion rate of 2.5 ml s−1. Npy (100 nM; N5071, Sigma-Aldrich) was bath applied for 10 min.

In line with previous studies, we found that the basic firing properties of POMC neurons and their responsiveness to leptin and Glp1 were not homogeneous. Therefore, we used the ‘three times standard deviation’ (3σ) criterion, and a neuron was considered responsive if the change in firing frequency or membrane potential induced by leptin or Glp1 was three times larger than the standard deviation. Means and respective standard deviations of spontaneous action potential firing or membrane potential were calculated from a period of 120 s, divided into 12 bins, each 10 s long. Data were taken immediately before and at the end of the peptide application.

The NPY action on POMC neurons was analyzed under voltage clamp. Based on previous studies5658 and on the peptide-induced action-potential frequency modulation, which we observed in current-clamp recordings, the holding potential was set to −55 mV to optimize the recording conditions to measure inward currents. NPY (100 nM) was bath applied for 10 min after a 5-min baseline recording. The recorded peptide-induced currents were baseline subtracted and the mean (±s.e.m.) was calculated. To quantify differences in the peptide-induced currents, the area under the curve (electrical charge in nC) during the 10-min application of NPY was calculated.

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