Protocol to determine oligomerization states of proteins in living cells using photon counting histogram analysis.

Energy balance is coordinated by distributed neural circuits that regulate feeding, energy expenditure, and adaptive behavioral responses. It is now appreciated that widespread nuclei of the brain serve as entry points and relay stations for the integration of peripheral metabolic cues with central representations of internal state and environmental context to coordinate energy balance []. Recently, the locus coeruleus (LC), a nucleus classically implicated in arousal, vigilance, and stress responding [], has established itself as a contributor to feeding behavior. Optogenetic and chemogenetic studies in mice have shown that activation of LC norepinephrine neurons attenuates feeding behavior [,] and pharmacological manipulation of classical feeding receptors, including those for the hormones glucagon-like peptide-1 (GLP-1) and ghrelin, in the LC bidirectionally control appetite [,]. Together, these findings position the LC as an integrative node where metabolic signals, the extent of which is still unknown, shape feeding behavior. The calcitonin receptor (CTR) is abundantly expressed in the LC [,], suggesting that this nucleus is positioned to respond to multiple members of the calcitonin peptide family, including calcitonin, calcitonin gene-related peptide (CGRP), and amylin. Amylin is a well-established anorectic hormone that regulates energy balance and exerts its effects through amylin receptors, which are formed by heterodimerization of the CTR with one of three receptor activity-modifying proteins (RAMP1-3) [,]. Activation of these receptor complexes suppresses food intake, delays gastric emptying, and reduces postprandial glucagon secretion [,], making amylin an effective therapy for both diabetes and obesity [, , ]. More recently, Dual Amylin and Calcitonin Receptor Agonists (DACRAs), including cagrilintide, have demonstrated weight-loss efficacy comparable to current GLP-1 receptor agonist therapies [,]. Because DACRAs activate multiple CTR-containing receptor complexes, including the AMY1 receptor (CTR:RAMP1), understanding the anatomical distribution and functional roles of these receptors within the brain is of increasing therapeutic importance) [,,]. In addition to amylin, the AMY1 receptor is activated by CGRP) [,,]. CGRP signaling has an increasingly recognized role in feeding suppression and aversion [,, , , ], although these studies have implicated CGRP-expressing neurons rather than directly establishing CGRP as the critical neurotransmitter mediating these effects. CGRP also signals through the canonical CGRP receptor composed of the calcitonin receptor-like receptor (CLR) and RAMP1 (CLR:RAMP1) []. Because the LC expresses the molecular components required for both AMY and CGRP receptor complexes, it may represent an important yet understudied site through which calcitonin-family peptides influence ingestive behavior. Defining these central sites of action is critical for understanding the mechanisms through which current and next-generation CTR agonists produce their therapeutic effects [, , , , ]. Here, we use pharmacological and chemogenetic approaches to show that LC CTR activation suppresses feeding in the mouse and rat. We demonstrate that LC CTRs co-express the necessary RAMPs to permit amylin and CGRP signaling, and that pharmacological delivery of either CTR ligand to the LC suppresses food intake. Importantly, LC CTR manipulations do not recruit illness-like behaviors or autonomic impairments that are observed under other conditions of LC engagement [,,] or following CTR agonism in other areas of the brain , , , , , , , , . Together, these findings position the LC as a critical and dissociable node for calcitonin receptor signaling in the central control of feeding behavior.