Mechanisms underlying peptidergic modulation of feeding and feeding-associated behaviours

Abstract

Of the various neuromodulatory agents found in the central nervous system, the class of peptide modulators is of particular interest in the context of innate motivated behaviours, wherein they have been shown to modify the synaptic gain and alter intrinsic properties of neurons to initiate or extend stable behavioural states (e.g., sleep/arousal, roaming/dwelling). Feeding is an important behaviour where peptides are known to exert modulatory control. Although much is known about the neuropeptides and anatomical regions regulating feeding and associated behaviours, the underlying molecular mechanisms remain poorly understood. This thesis focuses on Cocaine-and-Amphetamine-Regulated-Transcript (CART) and Neuropeptide Y (NPY), two peptides strongly implicated in regulating feeding and associated modalities. Using a combinatorial approach, we explore the biochemical changes triggered due to CART and NPY signalling and their consequences on behaviour and neural activity in the Zebrafish (Danio rerio) nervous system. Previous data from our lab used a neural activity marker, phosphorylated extracellular signal-regulated kinase (p-ERK), to identify CART peptide responsive regions. In this study, we find a strong correlation between high levels of p-ERK in the dorsomedial telencephalon (Dm) and an associated anorexic behavioural output. We thus used pharmacological intervention coupled with behavioural monitoring and neuronal activity imaging in Dm to better understand the molecular mechanisms involved in establishing CART-mediated anorexia. We found that N-methyl-D-aspartate receptor (NMDAR) activity is necessary for CART signalling, as the CART-induced increase in activity of Dm neurons and reduction in feeding drive were both disrupted by blocking NMDAR signalling. We then tested if protein kinase A (PKA) is also involved in mediating this effect. Indeed, we found that PKA activity was required for CART-induced upregulation of neural activity in the Dm and suppression of feeding drive. PKA-mediated phosphorylation of NR1 subunits is known to alter NMDAR conductance and opening probability; we, therefore, tested if CART treatment alters phospho-NR1 (p-NR1) levels. Indeed, we found higher phospho- Ser 897 levels in Dm of CART injected animals which were suppressed by inhibiting PKA activity. Additionally, results of neuronal activity imaging in ex vivo brain preparations from transgenic animals expressing the calcium indicator GCaMP6f under a pan-neural promoter indicate that CART neuropeptide sensitises Dm neurons to excitatory inputs, which is correlated to CART-induced anorexia. Together these data suggest that CART action via PKA sensitises NMDA receptors via post-translational modification of receptor subunits, leading to heightened excitatory tone in Dm and a neural representation of the sated state. In the context of feeding behaviour, the sense of smell provides information about the exact location, palatability and reward value of food. It is thus a necessary input in the decision-making process when foraging and selecting between different food sources. Previous work done in the lab demonstrated that the levels of NPY, an orexigenic peptide, in the terminal nerve, olfactory epithelium, and olfactory bulb of Zebrafish reflect the animal's nutritional status. Furthermore, it was found that heightened NPY signalling under low energy conditions is necessary and sufficient to increase attraction towards amino acids, a major food cue. In the second study, we have investigated the mechanisms underlying this nutritional state-dependent increase in olfactory sensitivity. Results from activity-based p-ERK labelling revealed that amino acid detection requires Phospholipase C (PLC) activity. Additionally, we found that olfactory sensory neurons (OSNs) express NPY Y1 receptors and inhibiting its activity abrogates amino acid-induced OSN activation. Further testing confirmed crosstalk between odour-processing and peptide signalling pathways. We propose that this interaction of NPY signalling onto PLC-dependent odour processing allows for sensory gating at the level of OSNs. Altogether, our work has uncovered molecular pathways employed by neuropeptides to drive need-based alterations in neural activity and behaviour, which can be further explored to gain more insights into the neural computations underlying bistable behaviours.