Chemical flavorants differentially modulate appendage regenerative capacity in a juvenile vertebrate immersion model
July 20
Commercial aerosol flavorants constitute an expansive and understudied source of small molecules that may influence embryonic patterning and post-embryonic tissue repair. Prior work showed that menthol and green-apple derivatives can potentiate tail and fin regrowth by reshaping bioelectric gradients through α7-like cholinergic channel modulation in growth-control domains. Here, we examined the impact of vanilla (vanillin, ethyl-vanillin) and cherry (ethyl-vanillin, vanillin, ethyl acetate, ethyl maltol, maltol) mixtures on regenerative outcomes in the presence or absence of a low-dose cholinergic agonist. Juvenile C57BL/6J-equivalent zebrafish were placed in an electro-aerosol immersion regeneration (EAIR) assay following caudal-fin amputation. Four combinations elevated the regenerative index: agonist + menthol, agonist + cherry, agonist + vanilla, and vanilla alone. Immersion in cherry mixture without agonist did not enhance blastema growth relative to vehicle control. In contrast, vanilla mixture alone significantly accelerated outgrowth in both male and female juveniles; addition of the agonist did not further increase total regenerate length or cell-cycle entry but did improve patterning fidelity, as indicated by a higher dorsal/ventral ray ratio. Vanilla exposure alone produced measurable shifts in both tonic membrane potential and phasic bioelectric oscillations within the distal blastema, recorded by voltage-sensitive dye imaging. Ligand-binding simulations and fluorescence-polarization assays revealed direct interactions of green-apple and vanilla constituents with the extracellular loops of α7-like and α4β2-like channel subunits, suggesting a shared structural mechanism. These findings add to the evidence that specific aerosol flavor chemicals can act as standalone morphogenetic modulators, capable of promoting vertebrate appendage regeneration independently of exogenous cholinergic agonists.