Previous reports suggested that TRPC channels are selectively permeable to both Na+ and Ca2+ (Clapham et al., 2001); thus, we replaced extracellular NaCl with equimolar choline chloride. Extracellular CaCl2 was also replaced with equimolar MgCl2 and 0.1 mM EGTA, which has previously been shown to greatly decrease the permeable cations through the TRPC channel (Qiu et al., 2010). Ion replacement of buy PCI-32765 extracellular Na+ and Ca2+ resulted in a failure of mCPP to depolarize all POMC neurons tested (0.1 ± 0.1 mV, n = 12; Figures 1H and 4E). Similarly, no change in input resistance was observed in the presence of mCPP in all three conditions (Figure 4C).

These pharmacological and ion substitution experiments suggest the involvement of TRPC channels in the mCPP-induced POMC neuronal activation. TRPC channels may be activated by PLC and Gq protein-coupled receptors (GqPCRs) (Strübing et al., 2001). Since 5-HT2CRs are coupled to Gq proteins, we predicted that mCPP may activate the TRPC channel via the Gq-phospholipase C (PLC) signaling pathway. We tested this hypothesis using the PLC inhibitor, U73122. Preapplication of U73122 (5 μM) prevented the depolarization of POMC neurons by mCPP Alectinib supplier in all neurons examined (−0.2 ± 0.2 mV, n = 12; Figures 1H and 4C). Thus, mCPP-induced POMC neuronal depolarization involves PLC-dependent activation of TRPC channels. The distribution of

mCPP-treated POMC-hrGFP neurons for these experiments is illustrated in Figure S4. Serotonin and leptin both inhibit food intake and regulate energy balance and both activate TRPC channels to excite POMC neurons. We recently reported that there is a functional segregation below of the acute effects of leptin and insulin in POMC neurons (Williams et al., 2010). Our current data suggest

that serotonin and leptin share common signaling mechanisms (TRPC channels) in order to modify POMC neuronal activity. Thus, it is formally possible that 5-HT and leptin target the same POMC neurons. To further delineate whether POMC neurons could respond to both serotonin and leptin, identified POMC cells were next assessed for effects of leptin and serotonin on membrane potential following successive application of both compounds. Application of mCPP depolarized 25% of arcuate POMC neurons and was readily reversed upon washout (Figure 1). Subsequent application of mCPP resulted in a depolarization that was 51.0% ± 9.9% (n = 6) of the first depolarization and suggests that although the response is smaller and maybe subject to desensitization, TRPC channels can be activated during subsequent applications. Following washout of mCPP, neurons were examined for the effects of leptin on membrane potential in 32 cells. Perfusion of mCPP depolarized 4 of 32 POMC neurons (5.8 ± 0.9 mV, n = 4). The remaining 28 neurons were unresponsive to mCPP (0.1 ± 0.1 mV; n = 28).