3.2. Dopaminergic Degeneration Minds the Duration of Exposure to 6-OHDA
The first week of zebrafish development is rapid and vital that every day counts for critical steps in forming a functional organism [46]. To assess the impact of age and treatment duration on the extent of developmental changes caused by 6-OHDA treatment, we repeated the experiments using 7dpf larvae. We exposed 2dpfpanx1a+/+ andpanx1a-/- larvae to 250µM 6-OHDA for either 72 or 120hrs and tested their performance at 7dpf (Fig. 3A and 3B). All p-values were presented in Table 2. A 72hrs treatment significantly reduced the RD and TBF of 7dpf panx1a+/+ larvae by 63% and 40%, respectively. The results were consistent with the data obtained for 5dpf larvae in section 3.1. The electric-induced RD and TBF of 7dpf panx1a-/- larvae were unchanged upon 72hrs treatment with 6-OHDA, like the behavioral response of 5dpf mutants.
Extending the treatment from 72 to 120hrs had no effect on 7dpfpanx1a+/+ larvae, suggesting that both exposure times led to the same and significant levels of motor deficiency and cell death of dopaminergic cells. The results suggested that panx1a+/+ larvae showed the greatest sensitivity to the neurotoxin within the first 5 days of development. In contrast to panx1a+/+ larvae, 7dpfpanx1a-/- larvae RD (24%) and TBF (52%) declined when the 6-OHDA exposure time was extended from 72 to 120 hour. This result showed that the duration of exposure of mutants to the neurotoxin dictated the behavioral outcomes. We concluded that the deregulation of the dopaminergic pathway inpanx1a-/- larvae shown previously[40] is the probable cause of the 6-OHDA susceptibility at 7dpf. This idea was tested using the rate-limiting enzyme TH as a surrogate for changes to dopaminergic signaling. A significant reduction in TH expression levels was observed after toxin treatments for both genotypes which was aligned with the behavioral outcomes (Fig. 3C). We concluded that the absence of functional Panx1a channels compromised dopaminergic signaling in 6-OHDA treated zebrafish larvae following a reduction in the expression of the critical rate-limiting enzyme TH.