3 ± 0.9 mV, n = 7, p > 0.05 versus wild-type). These results suggest that GIRK channels, which contain GIRK1 subunits,

are constitutively active at rest in POMC neurons and contribute to the resting membrane potential of POMC neurons. In support of this, POMC neurons from GIRK1 knockout mice had a significantly higher input resistance as determined by hyperpolarizing current steps (1,514 ± 118 MΩ in GIRK1 knockout versus 1,142 ± 76 MΩ in wild-type mice) (Figure S2B). POMC neurons from GIRK2 knockout mice had a slightly higher input resistance (1,382 ± 112 MΩ), but the difference was not significant. We next examined the requirement of GIRK1 or GIRK2 subunits in the baclofen-induced hyperpolarization of the membrane potential of POMC neurons. Baclofen hyperpolarized 11 of 14 (78.6%) POMC-hrGFP neurons from wild-type mice by −15.1 ± 2.1 mV (from −54.3 ± Selleckchem IBET151 1.7 mV in control to −69.4 ± 2.4 mV in baclofen, n = 11; Figure S2C). The hyperpolarization was accompanied by a 40.8% ± 6.2% decreased input resistance with a reversal potential of −91.2 ± 1.6 mV, supportive of K+ as the major cation responsible for the membrane hyperpolarization (Figures S2D and S2E). In GIRK1 knockout mice, baclofen hyperpolarized 2 of 16 (12.5%) POMC-hrGFP neurons (hyperpolarized by −8 mV and −9 mV), while

the remaining neurons were unchanged in response to baclofen (Figure S2F). In GIRK2 knockout mice, baclofen hyperpolarized 4 of 7 (57.1%) POMC-hrGFP neurons by −4.0 ± 0.7 mV (from −51.8 ± learn more 1.1 mV in control to −55.8 ± 1.8 mV in baclofen, n = 4) (Figure S2G). These results support a key role of GIRK1 subunits, but not GIRK2, in both constitutively active and GABAB-activated GIRK currents in POMC neurons (Figures S2H and S2I). We next determined the requirement of GIRK1 subunits in the mCPP induced depolarization of POMC-hrGFP neurons in GIRK1 knockout mice (Figure 3E). Perfusion of mCPP depolarized the membrane potential of 6 of 18 (33.3%) POMC-hrGFP neurons from GIRK1 knockout

mice by 5.2 ± 0.3 mV (n = 6), Linifanib (ABT-869) which was similar to the effect of mCPP observed in POMC neurons from wild-type mice. Together, these data suggest that inhibition of constitutively active GIRK channels (Chen and Johnston, 2005) is not responsible for the mCPP-induced excitation of POMC neurons. In order to further determine the conductance involved in the mCPP-induced depolarization, POMC neurons from wild-type mice were monitored for changes in input resistance and neuronal excitability. In current clamp configuration, continuous recordings of membrane potential were interrupted by hyperpolarizing rectangular current steps (500 ms; −10 to −50 pA; arrows in Figures 1F and 1G). In control ACSF, the whole-cell input resistance of POMC neurons was 1,323 ± 60 MΩ (n = 59), similar to previous reports (Hill et al., 2008 and Hill et al., 2010). The mCPP-induced depolarization of POMC neurons was accompanied by a reversible 17.1% ± 1.