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Developmental, Molecular, and Genetic Dissection of I_Na 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_v1 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_v1.1 and Na_v1.6. To examine the properties of Na_v1.1- and Na_v1.6-encoded currents in RB cells at different developmental stages, we eliminated the contribution of Na_v1.6 and Na_v1.1 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_v1.1MO. Overall, the effect of the Na_v1.1MO was a small 25% average reduction in current amplitude. Further, Na_v1.1MO effects were most pronounced in RB cells of younger embryos. In contrast, the effects of the Na_v1.6 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_v1.6 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.

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