Developmental, molecular, and genetic dissection of INa in vivo in embryonic zebrafish sensory neurons.
The presence of multiple Nav1 isotypes within a neuron and the lack of specific blockers hamper identification of the in vivo roles of sodium current (INa) components, especially during embryonic stages. To identify the functional properties of INa 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: Nav1.1 and Nav1.6. To examine the properties of Nav1.1- and Nav1.6-encoded currents in RB cells at different developmental stages, we eliminated the contribution of Nav1.6 and Nav1.1 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 Nav1.1MO. Overall, the effect of the Nav1.1MO was a small 25% average reduction in current amplitude. Further, Nav1.1MO effects were most pronounced in RB cells of younger embryos. In contrast, the effects of the Nav1.6 MO were observed in all cells and increased as development proceeded. These results indicated that developmental upregulation of RB INa entailed an increase in the number of functional Nav1.6 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 INa 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.
Pineda
Ricardo H
RH
Department of Physiology and Biophysics, Mail Stop 8307, University of Colorado Health Sciences Center at Fitzsimons, P.O. Box 6511, Aurora, CO 80045, USA.
Heiser
Ryan A
RA
Ribera
Angeles B
AB
D000367:Age Factors; D000704:Analysis of Variance; D000818:Animals; D002467:Cell Nucleus / Q000187:drug effects / Q000502:physiology; D004625:Embryo, Nonmammalian; D015870:Gene Expression / Q000502:physiology; D018507:Gene Expression Regulation, Developmental / Q000187:drug effects / Q000502:physiology*; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology / Q000528:radiation effects; D008845:Microinjections; D008967:Molecular Biology / Q000379:methods; D009154:Mutation / Q000502:physiology; D009475:Neurons, Afferent / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods; D011930:Reaction Time / Q000502:physiology; D026941:Sodium Channel Blockers / Q000494:pharmacology; D015222:Sodium Channels / Q000235:genetics* / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology*; D013779:Tetrodotoxin / Q000494:pharmacology; D015027:Zebrafish / Q000235:genetics* / Q000254:growth & development
D020319:Oligodeoxyribonucleotides, Antisense; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D013779:Tetrodotoxin