Melatonin modulates habituation learning via the convergent action of two MT ₁ -type receptors

Melatonin is a hormone produced by the pineal gland and retina, with roles in sleep and circadian rhythms as well as other neuronal processes, including the regulation of learning and memory. Using larval zebrafish, we previously identified Melatonin as a potent modulator of habituation learning: the progressive suppression of responses to a repeated stimulus. Here we have extended these analyses and have found that Melatonin has strong effects on separate aspects of habituation learning: it potentiates habituation of response probability and latency, while simultaneously inhibiting habituation of movement amplitude and duration. We performed a systematic CRISPR mutagenesis of all six zebrafish Melatonin receptors, and found that only two MT ₁ -type receptors (Mtnr1aa and Mtnr1al) are required for Melatonin’s effects on habituation. These receptors showed a cooperative interaction, with each individual mutant showing reduced sensitivity to Melatonin, and mtnr1aa;mtnr1al double mutants showing complete insensitivity. To map where these receptors act, we used whole-brain activity mapping. Despite distinct footprints, both perturbed a shared set of brain areas, including the torus longitudinalis, cerebellum, and preoptic area, with the double mutant producing the most extensive phenotype by combining and expanding the effects seen in each single mutant. Thus, we propose that two MT ₁ -type receptors act at overlapping circuit nodes to reinforce Melatonin’s bidirectional control of learning.