Neuromodulatory mechanisms underlying the regulation of feeding drive in zebrafish

Abstract

Feeding, one of the innate, survival-oriented behaviours, is intricately controlled by conserved neural mechanisms within vertebrates. The feeding behaviour is composed of two alternating states-hunger and satiety, which are predominantly regulated by the internal energy needs of the body. Interoceptory neurons in the brain sense the internal energy conditions and regulate the release of orexic (e.g., NPY) and anorexic (e.g., CART) neuropeptides. These neuropeptides reconfigure the activities of feeding circuit elements to tune food intake with the body's metabolic needs. In mammals, the role of these neuropeptides in the establishment of and transition between hunger and satiety states is well documented. However, the complexity of neural mechanisms in higher vertebrates limits the understanding of circuit-level modulatory mechanisms regulating feeding behaviour. Using a simple vertebrate system, zebrafish, this study elucidates molecular level modulatory mechanisms regulating food intake and characterizes a novel neuroanatomical region that is correlated with the regulation of hunger-satiety bistable states. We demonstrate that antagonistic actions of orexic neuropeptide NPY and anorexic neuropeptide CART regulate the activity of dorsomedial telencephalon (Dm) to establish hunger-satiety states. CART facilitates the activation of Dm neurons to induce anorexia. In contrast, NPY leads to decreased activation of Dm neurons to increase food intake. To identify the plausible molecular candidates involved in feeding regulatory actions of CART and NPY, we performed pharmacological interventions and monitored their effect on feeding drive and Dm activity. Our data suggest that antagonistic activities of CART and NPY activate opposing biochemical signaling pathways that converge on NMDA receptor (NMDAR) function in Dm. Opposing actions of CART and NPY differentially phosphorylate serine 897 on the NR1 subunit of NMDARs. Phosphorylation of this residue is known to increase the calcium permeability of the receptor and thereby enhance NMDAR function. We find CART treatment leads to increased phosphorylation NR1 subunit by Protein kinase A (PKA) and Protein kinase C (PKC) activation to increase NMDAR activity and thereby increase the excitability of Dm neurons. On the other hand, NPY activates the protein phosphatase calcineurin and downregulates PKA activity to reduce NR1 phosphorylation, consequently de-potentiates NMDAR activity and reduces Dm excitability neurons. The modulatory configuration of biochemical signaling allows Dm to exhibit bi-stable excitability, which forms a neural representation of the energy state of the body and can be correlated with the modulation of feeding drive in zebrafish. Based on these results, we propose that antagonistic actions of CART and NPY tune the excitability of Dm neurons by modulating the NMDA receptor activity to regulate hunger-satiety behavioural outputs in zebrafish. Parallelly, we have undertaken the work to identify novel receptor/s for CART peptide. This study has identified a candidate CART responsive cell line- GH3, which shows receptor-like activity and produces a robust response to the CART treatment by upregulating intracellular calcium levels. We have optimized the conditions for the Ligand-based Receptor Capture method (LRC), which will be further used in the future to isolate and identify CART receptor/s from GH3 cells.