Use and efficiency of morpholinos in Neotropical tadpole brains v1

Antisense morpholinos are a common tool used to knockdown protein abundance in target tissues in fish and amphibians. However, protocols are largely limited to common aquatic research organisms, such as zebrafish or Xenopus frogs. The goal of this protocol is to enable in vivo morpholino delivery, visualization, and knockdown evaluation in the Neotropical tadpoles. We tested the efficiency of standard and Vivo-morpholinos in the knockdown of tyrosine hydroxylase protein, the rate limiting enzyme in dopamine synthesis, in the Mimetic poison frog (Ramitomeya imitator). We compared the knockdown efficacy of each morpholino at two different time points and the effect of knockdown on tadpole behavior. To quantify tyrosine hydroxylase knockdown efficiency, we developed a quick and inexpensive dot blotting method to evaluate protein levels within a single tadpole brain. We compared this method to chemiluminescent imaging and found both methods are sufficient to assess protein levels and discuss important differences in blot visualization techniques. Finally, we found morpholino knockdown reduced tadpole swimming, consistent with other studies implicanting dopamine as important in motor behaviors. We complement this protocol with a discussion of common challenges, suggestions for troubleshooting, and ideas for future improvement. Extending morpholino delivery and protein knockdown assessment to diverse amphibian species and labs with low budgets will enable the field to move forward more rapidly in the study of tadpole physiology and behavior.