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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.

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