Developmental, Molecular, and Genetic Dissection of INa In Vivo in Embryonic Zebrafish Sensory Neurons

Department of Physiology and Biophysics, University of Colorado Health Sciences Center at Fitzsimons, Aurora, Colorado Submitted 12 October 2004; accepted in final form 25 January 2005 The presence of multiple Na v 1 isotypes within a neuron and the lack of specific blockers hamper identification of the in vivo roles of sodium current ( I Na ) components, especially during embryonic stages. To identify the functional properties of I Na components in vivo in developing neurons, we took a molecular genetic approach. Embryonic zebrafish Rohon–Beard (RB) mechanosensory neurons express two different sodium channel isotypes: Na v 1.1 and Na v 1.6. To examine the properties of Na v 1.1- and Na v 1.6-encoded currents in RB cells at different developmental stages, we eliminated the contribution of Na v 1.6 and Na v 1.1 channels, respectively, using an antisense morpholino (MO) approach. MOs were injected into one-cell stage embryos, and RB sodium currents were recorded using patch-clamp techniques in both conventional whole cell mode as well from nucleated patches. Only a subset of RB cells appeared to be affected by the Na v 1.1MO. Overall, the effect of the Na v 1.1MO was a small 25% average reduction in current amplitude. Further, Na v 1.1MO effects were most pronounced in RB cells of younger embryos. In contrast, the effects of the Na v 1.6 MO were observed in all cells and increased as development proceeded. These results indicated that developmental upregulation of RB I Na entailed an increase in the number of functional Na v 1.6 channels. In addition, analysis of voltage-dependent steady-state activation and inactivation parameters revealed that specific functional properties of channels were also developmentally regulated. Finally, analysis of macho mutants indicated that developmental upregulation of I Na was absent in RB cells. These results indicate that MOs are a useful tool for the molecular dissection and analysis of ion channel function in vivo. Address for reprint requests and other correspondence: A. B. Ribera, Department of Physiology and Biophysics, Mail Stop 8307, University of Colorado Health Sciences Center at Fitzsimons, P.O. Box 6511, Aurora, CO 80045 (E-mail: angie.ribera{at}uchsc.edu )