Animals need to switch behavioral strategies to adapt to changing environmental conditions. The ability to choose the most advantageous response to a situation is crucial for survival. These decisions are influenced by environmental factors and internal signals such as physiological needs. During foraging behavior, larval zebrafish approach potential preys and avoid potential predators. The choice is influenced by external factors, such as the size of the object they are interacting with: small visual stimuli are perceived as preys while large objects are considered predators. Moreover, it was previously shown that behavioral choice during feeding behavior is modulated by metabolic state: food-deprived larvae are more likely to take risks during hunting and approach small, prey-like objects compared to fed fish. It was also demonstrated that this modulation is mediated by a differential processing of visual stimuli. In zebrafish the visual information is relayed from the retina to a midbrain structure, the optic tectum, where sensory inputs are integrated. Distinct downstream motor centers are subsequently activated to perform either an approach or an escape. This makes the optic tectum a major center for decision-making. In food-deprived larvae, tectal neurons respond preferentially to small visual stimuli compared to fed siblings, showing a shift of the tuning towards cues important for survival. These results suggest feeding induces a change in the excitability of tectal neurons to modulate behavioral choice, however the molecular mechanisms underlying this phenomenon are still unclear. To fill the gap by investigating the molecular pathways mediating the influence of metabolic state on behavioral choice, a proteomic study was performed to identify proteins differentially abundant in fed versus food-deprived larvae. Among all the hits, an especially interesting candidate was selected: a small peptide known to modulate neuronal excitability, Pcp4a, which was less abundant in fed larvae compared to food-deprived fish. pcp4a mRNA levels were also found to be lower in brain samples from fed larvae, suggesting that feeding modulates its expression through a transcriptional mechanism. PCP4, the mammalian ortholog of Pcp4a is known to bind to calmodulin, a molecule involved in several processes during neuronal activation, and to inhibit its target enzymes such as CaMKII. This results in modulation of neuron excitability in vivo. Since pcp4a is expressed in the optic tectum in zebrafish, we hypothesized that it may play a role in the regulation of tectal neurons excitability by feeding state. To test this hypothesis, I first looked at the role of pcp4a in the modulation of behavioral choice by feeding state using a loss-of-function model. Food-deprived larvae lacking Pcp4a show increased avoidance of small objects in a size discrimination assay, thus phenocopying behavioral choice of fed larvae. To understand if the effect was due to modulation of tectal neurons excitability, I investigated the response to visual stimuli of different size in tectal neurons expressing pcp4a (pcp4a+). Tectal pcp4a+ neurons in fed larvae responded preferentially to large objects compared to starved siblings; the same response profile was observed in larvae lacking Pcp4a, suggesting that Pcp4a mediates the effect of feeding on tectal neurons excitability. I then looked into the neuromodulatory mechanisms mediating the influence of metabolic signals on pcp4a expression. I found that feeding increased activity of dopaminergic neurons in the pretectum and hypothalamus, which could exert an effect on tectal pcp4a+ neurons through their direct and indirect projections to the optic tectum. Indeed, pharmacological activation of dopaminergic signaling through D2 receptors decreased pcp4a expression in food-deprived larvae, mimicking the effect of feeding. Dopamine controls pcp4a transcription through the D2 receptor - cAMP signaling cascade. Pharmacological activation of dopaminergic signaling induced a shift of the response profile of pcp4a+ tectal neurons towards large stimuli by altering the tuning properties of individual neurons. In this study we elucidate a novel molecular mechanism mediating the effect of metabolic state on behavioral choice in zebrafish. In our model, feeding activates dopaminergic neurons in the pretectum and hypothalamus, which project to the optic tectum. Dopaminergic signaling through D2 receptors induces a decrease of pcp4a expression through a transcriptional mechanism, which results in a shift of the response profile of pcp4a+ tectal neurons towards large visual stimuli through a cell-autonomous mechanism. This ultimately leads to increased avoidance of small stimuli during foraging behavior. This study advances our knowledge of the molecular mechanisms mediating neuromodulation of decision-making behavior.
