Differential copper-induced death and regeneration of olfactory sensory neuron populations and neurobehavioral function in larval zebrafish

Abstract

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.

Larval zebrafish use olfactory detection of sodium and chloride to avoid salt-water

“…We next wanted to define the precise roles that the olfactory system and the lateral line each play in the generation of salt avoidance behaviors. To do this, we incubated larvae in various concentrations of copper sulfate, which kills cells in both the lateral line 27 and olfactory epithelia 28 . After treatment and recovery periods, we examined the larvae's behavioral responses to 50 mM NaCl pulses and found their reaction rate decreased with increasing copper concentration (4A).…”

The Olfactory System of Zebrafish as a Model for the Study of Neurotoxicity and Injury: Implications for Neuroplasticity and Disease

“…These olfactory alterations correlate with a long-term decrease in alarm and avoidance responses [88,114,115,116,117,118,119]. Exposure to Copper (Cu) causes marked OSN death, with a preferential loss of ciliated in comparison to microvillous OSNs; impairs the transcription of a wide variety of genes involved in olfactory processing, such as ORs and ionic channels; and reduces the recognition of bile salts while impairing odor-mediated alarm responses [120,121,122,123,124]. Cobalt (Co) is a naturally occurring and essential metal at low concentrations, but it is an environmental contaminant when found in high concentrations.…”

“…It has been proposed that microvillous OSNs, which respond to amino acids, may present protective mechanisms that render them more resistant to damage, in order to preferentially preserve food sensing and feeding behaviors. It has also been suggested that these neurons may be replaced faster or in greater numbers and, therefore, their numbers do not plunge following olfactory epithelium damage [19,22,124].…”

“…The olfactory rosettes show bimodal patterns of OSN recovery following Cu exposure. On one hand, acute Cu exposure targets ciliated OSNs, with an observed recovery of this cell population after 72 h, whereas chronic exposure produces both ciliated and microvillous OSN loss with a faster proliferation and regeneration of microvillous OSNs observed after 72 h. Olfactory-mediated behavior assessment showed a partial functional recovery of ciliated OSNs, suggesting that although ciliated OSNs are more susceptible to Cu exposure, they are able to recover faster, perhaps through repair mechanisms rather than neurogenesis, a mechanism that was observed for the replenishment of microvillous OSNs [124].…”

“…In control conditions, OSNs present a constant turnover rate and most proliferation occurs in discrete clusters located in the apex of the lamellae [13,38,40,152]. Following damage to the olfactory organ, cellular proliferation is increased and localized in broader regions on the olfactory epithelium, giving rise to a rapid recovery of olfactory organ size and function [18,123,124]. Newly generated OSNs can reinnervate the olfactory bulb in their corresponding glomeruli and make synaptic connections with mitral cells.…”