The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva

The role of the cannabinoid receptor 2 (CNR2) is still poorly described in sensory epithelia. We found strong cnr2 expression in hair cells (HCs) of the inner ear and the lateral line (LL), a superficial sensory structure in fish. Next, we demonstrated that sensory synapses in HCs were severely perturbed in larvae lacking cnr2. Appearance and distribution of presynaptic ribbons and calcium channels (Cav1.3) were profoundly altered in mutant animals. Clustering of membrane-associated guanylate kinase (MAGUK) in post-synaptic densities (PSDs) was also heavily affected, suggesting a role for cnr2 for maintaining the sensory synapse. Furthermore, vesicular trafficking in HCs was strongly perturbed suggesting a retrograde action of the endocannabinoid system (ECs) via cnr2 that was modulating HC mechanotransduction. We found similar perturbations in retinal ribbon synapses. Finally, we showed that larval swimming behaviors after sound and light stimulations were significantly different in mutant animals. Thus, we propose that cnr2 is critical for the processing of sensory information in the developing larva.

Irreversible hair cell (HC) loss in the inner ear is the leading cause for hearing and balance disorders. Discovery of therapeutic molecules preventing HC death and promoting regeneration, which does not occur in mammals like it does in lower vertebrates, is of major interest. In fish, HCs are also found in a superficial mechano-sensory organ called the lateral line (LL). LL-HCs are exposed to surrounding waters and are accessible to waterborne molecules providing a potent mean to study in vivo HC stability and regeneration. Commercial small molecule libraries were tested in screens for HC survival and regeneration in zebrafish, but ethnobotanical pharmacopeias remain totally unexplored because of the challenge that such complex mixtures represent. A rapid and cost-effective first-pass assay informing about the regenerative potential of an extract is therefore critical before embarking on cumbersome purification steps. We chose to test Valerian crude root extracts (Val), which are typically composed of more than 150 different components, amongst which is a main and abundant compound: valeric acid (VA). VA discovery and purification led to the commercialization of a synthetic analog: Valproic acid (VPA) which is a first-line drug for epilepsy and bipolar disorders that was also shown to significantly hamper LL-HC regeneration. We reasoned that if Val is not toxic, it would elicit effects like VPA. Thus, we synchronously ablated HCs in 5-day post-fertilization (dpf) larvae and monitored regeneration over the following 3 days in the presence of Val at the highest well-tolerated concentration (Val1 = 1mg/ml), or VPA (= 150M) as previously published. Both treatments significantly decreased HC regeneration without affecting HC-survival suggesting a similar mode of action. Furthermore, Val application as early as 3dpf and prolonged for up to 4 days did not affect larval survival, indicating that reduced HCregeneration was not due to overall toxicity. Taken together, Val and VPA-treatments displayed a comparable response in a simple and up-scalable HC-regeneration assay which is an in-first-pass potent approach for drug discovery.