Studies have shown that olfactory-mediated behaviors that are critical to survival can be disrupted by exposure to certain metals. Aquatic Superfund sites often contain elevated levels of various metals, yet few have been characterized for their potential to cause olfactory toxicity. A larval zebrafish behavioral assay was developed to characterize concentration-response curves for zinc (Zn), hexavalent chromium (Cr), and arsenate (As) olfaction inhibition. Cadmium (Cd), an established olfactory toxicant, was used as a positive control. As expected, following a 24-hour exposure to Cd, we observed a reduced response to taurocholic acid (TCA), a behaviourally-active odorant, thus validating the behavioral assay. Zn exposure also decreased TCA response (IC50: 34.9 µg/L and 69.0 µg/L respectively). Behavioral responses to another odorant, L-cysteine, revealed that Zn was potentially of specific toxicity to ciliated olfactory sensory neurons.No significant changes in olfactory responses were observed from Cr and As exposures, even at exposures far greater than observed environmental concentrations.Exposures to binary mixtures of Cd and Zn indicated that Zn had a protective effect against Cd toxicity at low concentrations, but this effect disappeared at higher Zn concentrations. Quantitative analysis of whole larvae gene expression of 8 genes 4 important in mitigating metal induced oxidative stress, confirmed an antioxidant response was evoked by Cd, but not for the other metals, suggesting that oxidative stress was not a primary mechanism of Zn induced olfactory dysfunction. In summary, our study identified that Zn inhibits olfaction at environmentally-relevant concentrations, and that it may reduce Cd when present in mixtures. Our study extends the utility of zebrafish as a model species to assess olfactory toxicity in fish.5 Abbreviations: Nrf2: nuclear factor, erythroid 2-like 2, omp: olfactory marker protein b, dpf: days post fertilization, EM: E3 embryo medium, SRP: superfund research project, Zn: zinc, As: arsenic, Cr: chromium, Cd: cadmium, TCA: taurocholic acid, OE: olfactory epithelium, OSN: olfactory sensory neuron, OR: olfactory receptor, MOR: major olfactory receptor, TRPC2: Transient Receptor Potential Cation Channel Subfamily C Member 2, OB: olfactory bulb, hmox1a: heme oxygenase 1a, gstp: glutathione s-transferase pi, gclc:glutamate-cysteine ligase, catalytic subunit, nqo1: NAD(P)H dehydrogenase, quinone 1, prdx1: peroxiredoxin 1, gpx1a: glutathione peroxidase 1a, sod1: superoxide dismutase 1, soluble, sod2: superoxide dismutase 2, mitochondrial, hsp70: heat shock cognate 70-kd protein, tandem duplicate 3, actb1: actin, beta 1, gapdh: glyceraldehyde-3-phosphate dehydrogenase, hprt1: hypoxanthine phosphoribosyltransferase 1, gadd45bb: growth arrest and DNA-damage-inducible, beta b

Fish rely heavily on their sense of smell to maintain behaviors essential for survival, such as predator detection and avoidance, prey selection, social behavior, imprinting, and homing to natal streams and spawning sites. Due to its direct contact with the outside environment, the peripheral olfactory system of fish is particularly susceptible to dissolved contaminants. In particular, environmental exposures to copper (Cu) can cause a rapid loss of olfactory function. In this study, confocal imaging of double-transgenic zebrafish larvae with differentially labeled ciliated and microvillous olfactory sensory neurons (OSNs) were used to examine cell death and regeneration following Cu exposure. Changes in cell morphologies were observed at varying degrees within both ciliated and microvillous OSNs, including the presence of round dense cell bodies, cell loss and fragmentation, retraction or loss of axons, disorganized cell arrangements, and loss of cells and fluorescence signal intensity, which are all indicators of cell death after Cu exposure. A marked loss of ciliated OSNs relative to microvillous OSNs occurred after exposure to low Cu concentrations for 3 hours, with some regeneration observed after 72 hours. At higher Cu concentrations and 24-h exposures, ciliated and microvillous OSNs were damaged with increased severity of injury with longer Cu exposures. Interestingly, microvillous, but not ciliated OSNs, regenerated rapidly within the 72-h time period of recovery after death from Cu exposure, suggesting that microvillous OSNs may be replaced in lieu of ciliated OSNs. An increase in bromodeoxyuridine labeling was observed 24 h after Cu-induced OSN death, suggesting that increased proliferation of the olfactory stem cells replaced the damaged OSNs. Olfactory behavioral analyses supported our imaging studies and revealed both initial loss and restoration of olfactory function after Cu exposures. In summary, our studies indicate that following zebrafish OSN damage by Cu, regeneration of microvillous OSNs may occur exceeding ciliated OSNs, likely via increased proliferation of the cellular reservoir of neuronal OSC precursors. Transgenic zebrafish are a valuable tool to study metal olfactory injury and recovery and to characterize sensitive olfactory neuron populations in fish exposed to environmental pollutants.