, 2004, Dhillon et al., 2006 and van de Wall et al., 2008) and possibly serotonin neurons in the raphe (Yadav et al., 2009). An important question is which, if any, of these neurons are GABAergic as determined by Cre activity in Vgat-ires-Cre mice and consequently contribute to the obesity phenotype of Vgat-ires-Cre, Leprlox/lox mice. As previously

mentioned, SF1 neurons are glutamatergic (Figure 1 and Tong et al., 2007). In support of this, no GABAergic neurons were found in the VMH (Figure 1). To determine if POMC neurons are GABAergic or glutamatergic and to confirm that AgRP neurons are GABAergic (Cowley et al., 2001 and Tong et al., 2008), we used immunodetectable hrGFP expressed from POMC-hrGFP (Parton et al., 2007) and NPY-hrGFP (van MK-8776 chemical structure den Pol et al., 2009) BAC transgenes to identify POMC and AgRP neurons and colocalized this with Cre activity (tdTomato, as described below). BIBW2992 supplier Note that NPY and AgRP are coexpressed in the arcuate nucleus (van den Pol et al., 2009). GABAergic (VGAT+) and glutamatergic (VGLUT2+) neurons were identified by immunodetectable tdTomato in Vgat-ires-Cre, lox-tdTomato mice and Vglut2-ires-Cre, lox-tdTomato mice, respectively (lox-tdTomato, Ai9;

Madisen et al., 2010). Of note, essentially no POMC neurons (<1%) were VGAT+ ( Figure 3A) and ∼10% of POMC neurons were VGLUT2+ ( Figure 3B). AgRP neurons, as expected, were GABAergic ( Figure 3C). It is important to note, however, that AgRP neurons represent only a subset of all GABAergic neurons in the arcuate ( Figure 3C). Also of note, POMC neurons, which are not GABAergic, are situated in a dense background of GABAergic neurons ( Figure 3A). Finally, serotonin neurons in the raphe PDK4 (as identified by immunohistochemistry for TpH), do not express Cre activity in Vgat-ires-Cre mice ( Figure S3). Thus, AgRP neurons, but not POMC or SF1 neurons, are GABAergic and therefore are the only previously established first-order neurons that contribute directly to the obesity seen

in Vgat-ires-Cre, Leprlox/lox mice. However, as previously discussed, the contribution of LEPRs on AgRP neurons to regulation of energy balance is small ( van de Wall et al., 2008). Thus, the majority of leptin’s antiobesity must be mediated by previously uncharacterized first-order leptin-responsive GABAergic neurons. Given the above, a key question becomes the location of leptin-responsive GABAergic neurons. To address this, we colocalized LEPR activity, as assessed by leptin-inducible STAT3 phosphorylation, with Cre activity in Vgat-ires-Cre and Vglut2-ires-Cre, lox-GFP reporter mice, with or without neuron-specific deletion of LEPRs. Note that leptin-inducible P-STAT3 is a robust means of detecting LEPR activity ( Münzberg et al., 2004). Leptin was injected (4 mg/kg body weight i.p.) into mice that were fasted overnight and 1 hr later brains were removed and assessed for P-STAT3 and GFP expression.