CB1 CONTROL OF OREXINERGIC NEURONS IN THELATERAL HYPOTHALAMUS SHIFTS FROM INHIBITIONTO DISINHIBITION IN OBESE MICE

The lateral hypothalamus (LH) is the region of the brain previously denoted as the “hunger center”. Orexin A and B (OX-A and OX-B) are both orexigenic neuropeptides synthesized by perifornical neurons of the LH. OX-A is a short-term modulator of appetite since it increases food intake in pre-fed rats. Pretreatment with subeffective doses of Rimonabant blocks this effect, suggesting that hypothalamic orexinergic circuits cross-talk with cannabinoid CB1 receptors in the regulation of appetite (Crespo et al., Neuropharmacology 2008). Both orexins interact with other modulators of feeding behaviour and leptin, since orexinergic neurons express the leptin receptor. Endocannabinoids exert a bimodal control of stimulated-food intake, as shown by studies with CB1 conditional mutant mice (Bellocchio et al., Nat Neurosci. 2010), and can inhibit orexinergic neurons in lean rodents (Huang et al., J. Neurosci. 2007). However, it remains to be clarified if this latter phenomenon also occurs in obesity, during which high hypothalamic neural plasticity is required for both adequate regulation of energy balance (Horvart & Gao, Cell Metab. 2005) and leptin mophoregulatory effects (Valerio et al., JBC 2006). With this purpose, we have investigated endocannabinoid/orexin-A interactions in ob/ob, leptin-knockout, mice. Immunohistochemical studies were performed in the LH for OX-A, CB1, DAGL-α, NAPEPLD, MAGL, synaptophisin and vescicular GABA (VGAT) or glutamate (VGluT) transporters. Hypothalamic endocannabinoid levels were measured by LC-MS. Whole cellpatch clamp recordings was performed to measure the effect of 5 μM of WIN55,212 on the frequency of spontaneous IPSC (cell clamped at -70mV; 10 μM of CPP and NBQX in the bath) in orexinergic neurons of acute hypothalamic brain slices from p27-33 ob/ob vs. littermate mice. DAGL-α colocalized on the membrane of OX-A-ir neurons post-synaptically to CB1-expressing axon terminals and, unlike NAPE-PLD-ir, was up-regulated both in the LH and arcuate nucleus of ob/ob mice vs. littermates. Optical and electronic microscopy data showed a prevalent colocalization of CB1 and MAGL with GABAergic rather than glutamatergic afferents onto OX-A-ir neurons in ob/ob vs. littermates mice. WIN55,212 more strongly reduced the sIPSC frequency in ob/ob (70% of reduction, n=6) than wild-type mice (40%, n=8). These findings support the existence of a shift of CB1 from excitatory to inhibitory inputs onto orexinergic neurons in ob/ob mice and, hence, suggest that inhibitory inputs on these neurons may be functionally depressed by endocannabinoids more in obese than in normal mice, thus potentially contributing to hyperphagia