This will need to be tested as other antiobesity neurons are identified. Given

the above-mentioned findings, GABAergic output appears to be an important, direct target of leptin action. By using leptin-inducible P-STAT3 and GFP reporter expression (in Vgat-ires-Cre mice) to colocalize LEPRs and GABAergic neurons, we observed that LEPR-expressing GABAergic neurons are located in the arcuate, the DMH, and the lateral hypothalamus. Consequently, LEPR-expressing GABAergic neurons in one, two, or all three of these sites mediate leptin’s EX 527 clinical trial antiobesity effects. At present, our results do not allow us to rule in or out any one site or any combination of these sites. Nevertheless, for reasons listed below, we favor an important role for neurons in the arcuate. First, LEPR-expressing arcuate neurons have unparalleled access to circulating leptin ( Faouzi et al., 2007). Second, the arcuate has many GABAergic neurons, a small fraction of which are AgRP neurons ( Figure 3C and Acuna-Goycolea et al., 2005, Horvath et al., 1997 and Ovesjö et al., 2001). Third, POMC neurons, which are key targets of leptin-responsive GABAergic neurons ( Figure 5, Figure 6 and Figure 7

and BYL719 manufacturer Cowley et al., 2001), are located within the arcuate, surrounded by a dense population of GABAergic neurons ( Figure 3A). Fourth, neurons in the arcuate make many local connections ( Matsumoto and Arai, 1978 and van den Pol and Cassidy, 1982), providing the apparatus for local regulation of POMC neurons. One previously defined local circuit, likely to be physiologically important, is that between AgRP neuron collaterals and POMC neurons ( Cowley et al., 2001 and Horvath et al., 1992). As discussed below, we postulate that this is just one of many local leptin-responsive GABAergic inputs to POMC neurons. An earlier study (Cowley

et al., 2001) established that leptin reduces the frequency of IPSCs in POMC neurons (25% reduction in one-third of POMC neurons). The source of the reduced GABAergic input was attributed to AgRP neurons (which also express NPY). In the present study, we confirm leptin’s inhibitory effect on IPSC frequency, but, of interest, note a larger effect (40% inhibition in of all POMC neurons), perhaps because of our use of thicker brain slices (300 μm versus 200 μm). A key outcome of that prior study was the compelling proposal that leptin indirectly regulates POMC neurons via AgRP/NPY-GABAergic collaterals. The degree to which this accounts for leptin’s antiobesity effects, however, has been unclear, especially because deletion of LEPRs from AgRP neurons produces only a small disturbance in energy balance (van de Wall et al., 2008). In the present study, we show that the above-mentioned effect (i.e.