Abstract
Purpose: :
AII amacrine cells have been shown to display tetrodotoxin (TTX)-sensitive action potentials. Voltage-gated sodium channel 1.1 (Nav1.1) has been reported in AII amacrine cells; however, its subcellular location has yet to be determined. In this study, we examined the localization of Nav1.1 in AII amacrine cells through the use of viral-mediated expression of the membrane anchored protein Chop2-GFP linked to the conserved ankyrin binding domain (ABD) responsible for sodium channel clustering in the axon initial segment of neurons.
Methods: :
C57BL/6J mice were divided into three groups: the non-injected control group did not receive intravitreal injections, the injected control group received intravitreal injections of AAV2 carrying Chop2-GFP, and the experimental group received intravitreal injections of AAV2 carrying Chop2-GFP-ABD. The expression of GFP was assessed in retinal whole-mounts and vertical sections. Immunohistochemical staining was done with antibodies against ankyrin-G, pan-neurofascin (Pan-NF), Nav1.1, PKC, connexin-36 (Cx36), vesicular glutamate transporter 1 (VGluT1), synaptoporin (SPo), and glycine receptor alpha 1 subunit (GlyRa1). Sodium channel-mediated spike activities were assessed by whole-cell patch-clamp recordings in retinal slices.
Results: :
Unlike the uniform membrane expression of GFP with the viral vector carrying Chop2-GFP, the expression of GFP with viral vector carrying the ABD motif showed a consistent and remarkable polarization to a single process emerging from one lobular appendage of each AII amacrine cell. This process co-localized with antibodies staining against ankyrin-G, pan-NF, Nav1.1. It is also positive for SPo and appears to terminate adjacent to GlyRa1 puncta. Targeted Chop2-GFP-ABD expression appears to disrupt native Nav1.1 clustering. Patch-clamp studies showed disrupted Nav1.1 clustering consequently abolished spontaneous spiking in the AII amacrine cell, suggesting the axon-like process as the site of spike initiation.
Conclusions: :
Our studies revealed a sodium channel clustering process in AII amacrine cells. The clustering of sodium channels in this axon-like process may be important in the generation of sodium spiking in AII amacrine cells.
Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • ion channels