In summary, deficiency of leptin signaling in presynaptic, OSI 906 non-AgRP GABAergic neurons, but not postsynaptic POMC neurons, selectively increases inhibitory tone in POMC neurons. To determine if POMC neurons are affected by this increased GABAergic tone, we assessed their membrane potential and firing rate. In comparison with neurons from control mice, POMC neurons from Vgat-ires-Cre, Leprlox/lox

mice tended to be hyperpolarized (−62.1 ± 1.94 mV compared with −57.8 ± 2.8 mV in control mice; Figure 6B, left panel) and consistent with this, addition of the GABAA receptor blocker picrotoxin (PTX) in Vgat-ires-Cre, Leprlox/lox mice produced a greater degree of depolarization. PTX addition increased membrane potential by 6.4 ± 0.97 mV in Vgat-ires-Cre, Leprlox/lox mice compared with only 3.2 ± 1.01 mV in control mice (p < 0.05, t test). In agreement with this, their firing rate

was markedly reduced, 0.32 ± 0.11 Hz in Vgat-ires-Cre, Leprlox/lox mice compared with 1.81 Hz ± 0.37 U0126 cost in control mice (p = 0.01, t test; Figure 6B, right panel) and this reduction was markedly attenuated by PTX. PTX addition increased firing rate by 11.6 ± 6.2-fold in Vgat-ires-Cre, Leprlox/lox mice and by only 1.2 ± 0.1-fold in control mice (p = 0.01, Mann-Whitney test). These findings support the view that deficiency of leptin signaling in presynaptic GABAergic neurons inhibits the activity of POMC neurons. We next evaluated whether a physiologic reduction in circulating leptin, as occurs with fasting (Ahima et al., 1996), also increases inhibitory input to POMC neurons. This is a key question because the marked effects observed Carnitine dehydrogenase in Figure 6, while suggestive of important regulation, might be seen only with “unphysiologic,” total absence of leptin signaling. Our studies described below were motivated by a prior study in which fasting markedly increased the firing rate of AgRP neurons (which

are GABAergic), an effect that was prevented by leptin treatment 3 hr prior to sacrifice (Takahashi and Cone, 2005). Of interest, we found that fasting for 24 hr produced a marked increase in sIPSC frequency and amplitude in POMC neurons (Figure 7A). Importantly, these fasting-mediated effects were completely prevented by injection of leptin (4 mg/kg), but not by saline, 3 hr prior to sacrifice. Complete prevention of the fasting-stimulated increase in IPSCs by leptin treatment is consistent with the view that increased inhibitory tone caused by fasting is indeed due to the fasting-mediated fall in leptin. We then assessed the effects of fasting in mice lacking LEPRs on GABAergic neurons (Vgat-ires-Cre, Leprlox/lox mice). Of note, fasting in these mice failed to increase sIPSC frequency and amplitude ( Figure 7B), which as noted earlier are increased in the fed state compared with control mice ( Figure 6A).