8 ± 1.2 Hz; n = 4; p = 0.48; data not shown). Furthermore, coapplication of FAs with either 5 mM glucose (n = 4) or AA mix (n = 5) or both (n = 5) did not change the typical responses to these

nutrient mixtures (Figure 8C). This suggests that under our experimental conditions, FAs do not directly modulate the firing of orx/hcrt cells. Despite the importance CRM1 inhibitor of dietary timing and composition for healthy body weight and sleep-wake cycles (Flier, 2004 and Kohsaka et al., 2007), the effects of typical dietary nutrient mixtures on specific neurons regulating metabolic health are poorly understood. Our study uncovers several features of macronutrient interactions with cells that act as key regulators of energy balance. First, the orx/hcrt cells were directly stimulated by nutritionally relevant mixtures of dietary AA mixtures, both in vitro (Figure 1) and in vivo (Figures 2A and 2B). Peripheral administration of AAs produced locomotor effects consistent with orx/hcrt release (Figure 2C). Second, our data show that the stimulatory effects of AAs on orx/hcrt cell membrane involve an increase in the depolarizing activity of system-A AA transporters, and a concurrent reduction in the hyperpolarizing activity of KATP channels (Figure 4 and Figure 5).

Consistent with the involvement of the system-A transporters, which prefer nonessential AAs (Mackenzie 5 FU and Erickson, 2004), orx/hcrt cells were more potently stimulated by nonessential

AAs in vitro and in vivo (Figure 3). Third, the excitatory influence of AAs on orx/hcrt cells summed nonlinearly with the previously reported inhibitory effect of glucose, in favor of AA excitation (Figure 6). Ergoloid This is probably due to the suppression of the glucose response by AAs, and/or their metabolic derivatives (Figure 6). Because physiological AA fluctuations in the brain occur within a smaller concentration range than those of glucose (Choi et al., 1999, Choi et al., 2000 and Silver and Erecińska, 1994), it is possible that the suppression of glucose response by AAs may serve to amplify the relative influence of AAs on the orx/hcrt neurons. Recently, two hypotheses were proposed to explain how the AA composition of the extracellular space could be converted into appropriate changes in brain activity. One envisages sensing of essential AAs by deacylated tRNA in the piriform cortex, based on the observation that blocking tRNA synthetases of essential AAs in this region induces feeding behavior similar to that caused by essential AA deficiency (Hao et al., 2005). Another hypothesis involves sensing of leucine (an essential AA) by an mTOR-related pathway in the mediobasal hypothalamus (Blouet et al., 2009 and Cota et al., 2006). Both of these mechanisms are selective for essential AAs, unlike the mechanism described in our study, which is more sensitive to nonessential AAs.