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Targeted Expression of Channelrhodopsin-2 to the Axon Initial Segment Alters the Temporal Firing Properties of Retinal Ganglion Cells.

Zhang Z, Feng J, Wu C, Lu Q, Pan ZH - PLoS ONE (2015)

Bottom Line: Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important.We found that this targeted expression disrupted NaV channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient.Our results suggest that the clustering of membrane channels, including NaV channels, at the AIS is important for the ability of RGCs to generate sustained spike firing.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal excitability in axon-bearing neurons in the CNS. There is increasing interest in the targeting of optogenetic tools to subcellular compartments, including the AIS, to gain precise control of neuronal activity for basic research and clinical applications. In particular, targeted expression of optogenetic tools in retinal ganglion cells (RGCs) has been explored as an approach for restoring vision after photoreceptor degeneration. Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated targeted expression of channelrhodopsin-2 (ChR2)-GFP with a NaV channel motif in mouse RGCs. We found that this targeted expression disrupted NaV channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient. Our results suggest that the clustering of membrane channels, including NaV channels, at the AIS is important for the ability of RGCs to generate sustained spike firing. Additionally, the targeting of optogenetic tools to the AIS with the NaV channel motif may offer a way to create transient light responses in RGCs for vision restoration.

No MeSH data available.


Related in: MedlinePlus

Relationship between ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the NaVII-III targeting motif.(A) Co-staining of ChR2-GFP and anti-pan-NaV in motif-targeted RGCs. In the strongly ChR2-GFP-labeled AISs, co-staining with pan-NaV was absent or weak (arrows). In the unlabeled or weakly ChR2-GFP-labeled AISs, co-staining with pan-NaV was observed (arrowheads). Bottom panels are enlarged view of the boxed areas. (B) In non-motif-targeted RGCs, both ChR2-GFP expression and pan-NaV staining were observed (arrows). Bottom panels are enlarged view of the boxed areas. (C) Scatter plot with linear regression line showing the inverse relationship between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the targeting motif. (D) The scatter plot between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs in the control group.
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pone.0142052.g002: Relationship between ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the NaVII-III targeting motif.(A) Co-staining of ChR2-GFP and anti-pan-NaV in motif-targeted RGCs. In the strongly ChR2-GFP-labeled AISs, co-staining with pan-NaV was absent or weak (arrows). In the unlabeled or weakly ChR2-GFP-labeled AISs, co-staining with pan-NaV was observed (arrowheads). Bottom panels are enlarged view of the boxed areas. (B) In non-motif-targeted RGCs, both ChR2-GFP expression and pan-NaV staining were observed (arrows). Bottom panels are enlarged view of the boxed areas. (C) Scatter plot with linear regression line showing the inverse relationship between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the targeting motif. (D) The scatter plot between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs in the control group.

Mentions: To determine whether the targeted expression of ChR2-GFP with the NaVII-III motif could compete with the binding of NaV channels to AnkG [15,16], we used a pan-NaV antibody to examine the spatial distributions of NaV channels in the motif-targeted (Fig 2A) and control retinas (Fig 2B). NaV channel staining in the AISs of the motif-targeted RGCs was varied in a manner that appeared to be related to the GFP fluorescence intensity. The pan-NaV staining in areas of highly concentrated ChR2-GFP was minimal or absent (arrows in Fig 2A). In areas in which the expression of ChR2-GFP was absent, the pan-NaV staining (arrowheads in Fig 2A) was comparable to that in the controls (arrows in Fig 2B). We quantitatively examined this effect by measuring the FIs of the pan-NaV staining and GFP from each individual AISs of the motif-targeted RGCs (n = 42). We found an inverse relationship between the normalized pan-NaV-FI and the normalized GFP-FI (β = –0.91, F(1, 40) = 283.2, R2 = 0.88, p <0.001; Fig 2C). In contrast, no such relationship was shown for the non-motif target group (n = 33; β = –0.19, F(1, 31) = 113.2, R2 = 0.08, p = 0.267; Fig 2D). This result indicates that the expression of ChR2-GFP-NaVII-III disrupted NaV channel clustering at the AIS, which suggests that the NaVII-III targeting motif competed against the endogenous NaV channel for the AnkG binding site.


Targeted Expression of Channelrhodopsin-2 to the Axon Initial Segment Alters the Temporal Firing Properties of Retinal Ganglion Cells.

Zhang Z, Feng J, Wu C, Lu Q, Pan ZH - PLoS ONE (2015)

Relationship between ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the NaVII-III targeting motif.(A) Co-staining of ChR2-GFP and anti-pan-NaV in motif-targeted RGCs. In the strongly ChR2-GFP-labeled AISs, co-staining with pan-NaV was absent or weak (arrows). In the unlabeled or weakly ChR2-GFP-labeled AISs, co-staining with pan-NaV was observed (arrowheads). Bottom panels are enlarged view of the boxed areas. (B) In non-motif-targeted RGCs, both ChR2-GFP expression and pan-NaV staining were observed (arrows). Bottom panels are enlarged view of the boxed areas. (C) Scatter plot with linear regression line showing the inverse relationship between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the targeting motif. (D) The scatter plot between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs in the control group.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4633179&req=5

pone.0142052.g002: Relationship between ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the NaVII-III targeting motif.(A) Co-staining of ChR2-GFP and anti-pan-NaV in motif-targeted RGCs. In the strongly ChR2-GFP-labeled AISs, co-staining with pan-NaV was absent or weak (arrows). In the unlabeled or weakly ChR2-GFP-labeled AISs, co-staining with pan-NaV was observed (arrowheads). Bottom panels are enlarged view of the boxed areas. (B) In non-motif-targeted RGCs, both ChR2-GFP expression and pan-NaV staining were observed (arrows). Bottom panels are enlarged view of the boxed areas. (C) Scatter plot with linear regression line showing the inverse relationship between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs with the targeting motif. (D) The scatter plot between the normalized FIs of ChR2-GFP expression and pan-NaV immunostaining in the AISs of RGCs in the control group.
Mentions: To determine whether the targeted expression of ChR2-GFP with the NaVII-III motif could compete with the binding of NaV channels to AnkG [15,16], we used a pan-NaV antibody to examine the spatial distributions of NaV channels in the motif-targeted (Fig 2A) and control retinas (Fig 2B). NaV channel staining in the AISs of the motif-targeted RGCs was varied in a manner that appeared to be related to the GFP fluorescence intensity. The pan-NaV staining in areas of highly concentrated ChR2-GFP was minimal or absent (arrows in Fig 2A). In areas in which the expression of ChR2-GFP was absent, the pan-NaV staining (arrowheads in Fig 2A) was comparable to that in the controls (arrows in Fig 2B). We quantitatively examined this effect by measuring the FIs of the pan-NaV staining and GFP from each individual AISs of the motif-targeted RGCs (n = 42). We found an inverse relationship between the normalized pan-NaV-FI and the normalized GFP-FI (β = –0.91, F(1, 40) = 283.2, R2 = 0.88, p <0.001; Fig 2C). In contrast, no such relationship was shown for the non-motif target group (n = 33; β = –0.19, F(1, 31) = 113.2, R2 = 0.08, p = 0.267; Fig 2D). This result indicates that the expression of ChR2-GFP-NaVII-III disrupted NaV channel clustering at the AIS, which suggests that the NaVII-III targeting motif competed against the endogenous NaV channel for the AnkG binding site.

Bottom Line: Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important.We found that this targeted expression disrupted NaV channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient.Our results suggest that the clustering of membrane channels, including NaV channels, at the AIS is important for the ability of RGCs to generate sustained spike firing.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan, United States of America.

ABSTRACT
The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal excitability in axon-bearing neurons in the CNS. There is increasing interest in the targeting of optogenetic tools to subcellular compartments, including the AIS, to gain precise control of neuronal activity for basic research and clinical applications. In particular, targeted expression of optogenetic tools in retinal ganglion cells (RGCs) has been explored as an approach for restoring vision after photoreceptor degeneration. Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated targeted expression of channelrhodopsin-2 (ChR2)-GFP with a NaV channel motif in mouse RGCs. We found that this targeted expression disrupted NaV channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient. Our results suggest that the clustering of membrane channels, including NaV channels, at the AIS is important for the ability of RGCs to generate sustained spike firing. Additionally, the targeting of optogenetic tools to the AIS with the NaV channel motif may offer a way to create transient light responses in RGCs for vision restoration.

No MeSH data available.


Related in: MedlinePlus