Uneven balance of power between hypothalamic peptidergic neurons in the control of feeding Qiang Weia, David M. Krolewskia, Shannon Moorea, Vivek Kumara, Fei Lia, Brian Martina, Raju Tomerb, Geoffrey G. Murphya, Karl Deisserothc, Stanley J. Watson Jr.a, and Huda Akila,1 aMolecular and Behavioral Neuroscience Institute, University of Michigan, Ann Arbor, MI 48109; bDepartment of Biological Sciences, Columbia University, New York, NY 10027; and cDepartment of Bioengineering, Stanford University, Stanford, CA 94305 Contributed by Huda Akil, August 7, 2018 (sent for review February 6, 2018; reviewed by Olivier Civelli and Allen Stuart Levine) Two classes of peptide-producing neurons in the arcuate nucleus consumption, while anorexigenic neurons that express POMC in- (Arc) of the hypothalamus are known to exert opposing actions on hibit feeding behavior. AgRP/NPY neurons are active when the feeding: the anorexigenic neurons that express proopiomelano- energy stores are not sufficient to meet systemic demands, thereby cortin (POMC) and the orexigenic neurons that express agouti- releasing AgRP and promoting feeding (9, 10). POMC is a complex related protein (AgRP) and neuropeptide Y (NPY). These neurons polypeptide precursor cleaved into active peptides including mel- are thought to arise from a common embryonic progenitor, but anocortins and β-endorphin (11). While β-endorphin is a long- our anatomical and functional understanding of the interplay of acting opioid analgesic (12), the cosynthesized α-melanocyte stim- these two peptidergic systems that contribute to the control of ulating hormone (α-MSH) blocks appetite (13). These two sets of feeding remains incomplete. The present study uses a combination peptidergic neurons in the Arc respond to nutrient and hormonal of optogenetic stimulation with viral and transgenic approaches, signals from the periphery. Thus, leptin, a signal of positive energy coupled with neural activity mapping and brain transparency balance, stimulates POMC neurons, leading to decreased food i visualization to demonstrate the following: ( ) selective activation intake while inhibiting the release of the orexigenic peptides from of Arc POMC neurons inhibits food consumption rapidly in unsated neighboring AgRP/NPY neurons (10). Together, AgRP/NPY neu- ii animals; ( ) activation of Arc neurons arising from POMC- rons and POMC neurons work coordinately to integrate signals of expressing progenitors, including POMC and a subset of AgRP energy homeostasis and to regulate feeding behavior (8, 14). neurons, triggers robust feeding behavior, even in the face of iii Evidence suggests that these two peptidergic systems that satiety signals from POMC neurons; ( ) the opposing effects on control feeding emerge from a common cellular origin. A de- food intake are associated with distinct neuronal projection and velopmental study has demonstrated that embryonic POMC- activation patterns of adult hypothalamic POMC neurons versus expressing progenitors not only give rise to adult POMC cells, Arc neurons derived from POMC-expressing lineages; and (iv) the but also differentiate into non-POMC neurons, including NPY increased food intake following the activation of orexigenic neu- neurons (15). This means transgenic studies that target POMC rons derived from POMC-expressing progenitors engages an ex- tensive neural network that involves the endogenous opioid cells in early development can capture both adult populations, system. Together, these findings shed further light on the dynamic whereas adult delivery of viral vectors can target POMC cells balance between two peptidergic systems in the moment-to- selectively. Consequently, we used optogenetic stimulation re- moment regulation of feeding behavior. lying on both virally mediated and traditional transgenic ap- proaches to deliver blue light-activated channelrhodopsin 2 proopiomelanocortin | agouti-related protein | feeding | (ChR2) to POMC neurons in the Arc. We coupled this stimulation arcuate nucleus | satiety with behavioral assessments and anatomical analyses examining the Significance lobal epidemic obesity underscores the critical need for NEUROSCIENCE Gunderstanding how the brain regulates food intake (1). Feeding is essential for survival and relies on mechanisms that The interplay between the anorexigenic and orexigenic neurons monitor energy demands and metabolic status. But feeding be- in the arcuate nucleus that contributes to the control of feeding havior is more complex, as it has a strong rewarding component remains elusive. Using optogenetic stimulation, we show that (2) and can respond to environmental stimuli, including stress activation of POMC neurons rapidly inhibits feeding behavior in and social context (3). Therefore, it is critical to unravel the fasted animals. However, simultaneous stimulation of both mechanisms that modulate both the initiation and the cessation POMC neurons and a subset of the orexigenic neurons that ex- of feeding. press AgRP is sufficient to reverse that inhibition and trigger It is well established that the hypothalamus plays an essential intense feeding behavior. We used 3D imaging and functional role in the regulation of feeding behavior and contributes to the studies to illuminate the anatomical underpinning of both the process of energy homeostasis (4, 5). Hypothalamic nuclei en- inhibitory and excitatory events. Our work suggests that trans- gaged in this regulation include the Arc, the paraventricular lational applications that aim to control appetite need to target nucleus (PVN), the lateral hypothalamic (LH) area, the dorso- the activation rather than the inhibition mechanisms. medial nucleus, and the ventromedial nucleus. Disruption of the Author contributions: Q.W., R.T., G.G.M., K.D., S.J.W., and H.A. designed research; Q.W., ventromedial hypothalamus induces hyperphagia and obesity, D.M.K., S.M., V.K., F.L., and B.M. performed research; Q.W., D.M.K., S.M., V.K., and B.M. while disruption of the lateral hypothalamus causes hypophagia analyzed data; and Q.W. and H.A. wrote the paper. and weight loss, suggesting the existence of ventromedial “satiety” Reviewers: O.C., University of California, Irvine; and A.S.L., University of Minnesota. and lateral “feeding” centers (6). Recent work has extended these The authors declare no conflict of interest. early observations and has focused on the Arc of the hypothala- This open access article is distributed under Creative Commons Attribution-NonCommercial- mus, a nucleus particularly important for the central regulation of NoDerivatives License 4.0 (CC BY-NC-ND). appetite, energy expenditure, and body weight (7, 8). 1To whom correspondence should be addressed. Email: [email protected]. Two classes of peptide-producing neuronal populations in the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. Arc play a central and antagonistic role in the control of appetite— 1073/pnas.1802237115/-/DCSupplemental. the orexigenic neurons that express AgRP and NPY promote food Published online September 17, 2018. www.pnas.org/cgi/doi/10.1073/pnas.1802237115 PNAS | vol. 115 | no. 40 | E9489–E9498 Downloaded by guest on September 30, 2021 extent of activation and its consequences on cFos expression DIO-ChR2(H134R)-eYFP (Fig. 1A) was microinjected into the throughout the brain. Thus, we selectively activated neurons Arc of adult POMC-Cre transgenic mice (16). Upon trans- derived from POMC-expressing progenitors in the Arc and duction, Cre-expressing POMC neurons invert the ChR2-eYFP ascertained the net impact on food intake in adult mice. We ORF in irreversible means and ChR2-eYFP proteins are expressed contrasted the effect of activating only the POMC neurons ver- under control of the elongation factor 1α promoter (referred to sus the impact of activating both POMC and AgRP/NPY neu- as viral POMC-ChR2). This approach allowed us to selectively rons simultaneously on food consumption in mice. Our findings stimulate Arc POMC neurons with blue light (473 nm). We indicate that selective activation of Arc POMC neurons inhibits validated the cell-type selectivity of this viral approach in coronal food consumption rapidly in fasted animals whereas activation of Arc sections of viral POMC-ChR2 mice. ChR2-eYFP specifically Arc neurons arising from POMC-expressing progenitors results colocalized with endogenous POMC neurons (Fig. 1B). On av- in a robust increase in food intake. We show that in the conflict erage, 80% of POMC-positive neurons were positive for ChR2- between the drive to consume food and the rapid satiety control eYFP in the virus injection sites (Fig. 1C). Of the ChR2-eYFP that inhibits feeding, the former dominates, resulting in an acute expressing cells, 93% were colocalized by POMC immunostain- increase in feeding. These opposing effects are associated with ing (Fig. 1C), demonstrating high specificity of ChR2 expression distinct projection and activation patterns. Thus, the regulation in the POMC neurons of the Arc of the viral POMC-ChR2 mice. of feeding behavior involves a dynamic balance between the two To confirm the functional impact of ChR2 activation in the viral peptidergic systems. POMC-ChR2 mice, the Arc was illuminated with blue light while the animal explored an open field chamber. Expression of the Results neuronal activity marker, immediate early gene cFos, was evalu- Characterization of Viral POMC-ChR2 Mice. To control hypothalamic ated in the Arc of the viral POMC-ChR2 mice with or without POMC neurons selectively, we used