Starburst amacrine cells amplify optogenetic visual restoration through gap junctions

  1. Yusaku Katada
  2. Hiromitsu Kunimi
  3. Naho Serizawa
  4. Deokho Lee
  5. Kenta Kobayashi
  6. Kazuno Negishi
  7. Hideyuki Okano
  8. Kenji F. Tanaka
  9. Kazuo Tsubota
  10. Toshihide Kurihara

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Abstract

Ectopic induction of optogenetic actuators, such as channelrhodopsin, is a promising approach to restoring vision in the degenerating retina. However, the cell type-specific response of ectopic photoreception has not been well understood. It is limited to obtain efficient gene expression in a specifically targeted cell population by a transgenic approach. In the present study, we established a murine model with high efficiency of gene induction to retinal ganglion cells (RGCs)- and amacrine cells using an improved tetracycline transactivator-operator bipartite system (KENGE-tet system). To investigate the cell type-specific visual restorative effect, we expressed the channelrhodopsin gene into RGCs and amacrine cells using the KENGE-tet system. As a result, enhancement in the visual restorative effect was observed to RGCs and starburst amacrine cells. In conclusion, a photoresponse from amacrine cells may enhance the maintained response of RGCs and further increase/improve the visual restorative effect.

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  1. eLife

    Reviewer #3:

    This work by Katada and colleagues uses M4 and 5B transgenic lines to express ChR2 in starburst amacrine cells (SACs) and retinal ganglion cells (RGCs). It finds that ChR2 activation in SACs improves the ChR2 response in RGCs. Thus, in a gene therapy strategy that expresses optogenetic proteins in RGCs, SACs may be considered as a helpful additional target. The rationale of the manuscript basically regards RGCs as a uniform population and disregards all amacrine cells except SACs. If differences in RGC and amacrine subtypes are taken into consideration, some conclusions of this manuscript should be revised.

    Major comments:

    1. This manuscript makes one assumption: that the RGCs in M4-ChR2 and 5B-ChR2 have comparable ChR2 evoked response if activated alone, thus the difference between their ChR2 responses is entirely attributed to the activation of extra SACs in the M4 line. Yet there is no experimental evidence to support this assumption. Both M4-YC and 5B-YC label ~35% of the RGC consisting of multiple subtypes, the subtype compositions of the two populations are not shown. ChR2 response properties of a neuron may be influenced by its own ion channel composition that differ between cell types. The authors need to either a) show the 2 mouse lines label identical subsets of RGCs (unlikely, given FigS6E), or b) compare M4 line with or without coactivation of SACs to single out the effect of SACs.

    2. The experiment results using rAAV (Fig4) are hard to interpret:

    a) CAG promoter directs expression in most cell types. So other amacrine (Fig4D) and RGC cell types in addition to SACs and M4/5B RGCs are also infected. Comparison between rAAV/M4/5B retinas cannot provide clean insight into the effect of SAC.

    b) The manuscript makes comparisons within the rAAV experiments (Fig4I-K FigS8F-H), trying to link induction efficiency into SACs with visual restoration. However, it is a given that higher infection in RGCs also leads to better visual restoration. So SAC effect cannot be separated from RGCs (Fig4J-K FigS8G-H).

    c) The one exception shown in Fig4I and FigS8F, where SAC infection rate is linked to maintained/peak ratio, while RGC infection is not, has two caveat: First, the authors acknowledge that higher maintained response may not causally link to better restoration (line 235). Second, the same correlational analysis for other AC types is missing.

    d) At this stage, a simpler interpretation of the results is equally plausible: that higher infection in all retinal neurons (regardless of type) is correlated with better restoration.

    1. M4-ChR2 retina has very weak OFF response to regular light stimulus, but 5B has normal ON/OFF ratio. The authors speculate that SACs are responsible for this difference. But one observes that M4 labels mostly OFF RGCs while 5B labels equal amount of ON and OFF RGCs (S3 and S6E, lamination patterns of M4 and 5B), so there is a simpler explanation: RGCs that express tet-ON ChR2 are no longer very responsive to regular light stimuli. If that is true, that these cells are very unhealthy, then comparison of their ChR2 responses becomes less meaningful. The authors need to address the cell health problem caused by tet-ON ChR2 expression.

    2. Only a few RGC subtypes form synaptic connections with SACs in the rodent retina. Thus, the effect of SACs would be limited. In the case of primate retina, ChAT positive neurons are much fewer, so their effect in ChR2 gene therapy are likely even more limited.

    3. Lines 154-155: an equally likely explanation: M4 contains ON and ON-OFF DSGCs, which are known to be important for OKR, whereas 5B does not. This possibility needs to be considered.

  2. eLife

    Reviewer #2:

    This paper presents the results of a study of optogenetic visual restoration. ChR2 was targeted either to a subset of ganglion cells (GCs) or to a subset of ganglion cells-not necessarily the same ones-plus starburst amacrine cells (SACs) using an intersectional genetic strategy. Photoreceptors were ablated using MNU in animals expressing ChR2, and then retinal and whole animal responses to visual stimuli were assessed. Interestingly, co-expression of ChR2 in SACs and GCs resulted in different, potentially more "naturalistic" responses than expression in GCs alone. This is an interesting result, but given the number of possible explanations for it, the lack of any rigorous investigation of the underlying mechanism is problematic. Results presented by the authors indicate that ACh release from stimulated SACs acts upon some network(s) containing electrical synapses and presynaptic to the GCs to alter GC responses, but the identities of these network(s) remain unknown. Given that ACh is considered to act in a paracrine manner within the retina, the affected cells could be any number of amacrine or bipolar cells.

    There are a number of lines of investigation that the authors could pursue to identify-or at least, rule out-specific presynaptic networks. While too numerous to discuss individually, potential lines of investigation could differentiate nicotinic from muscarinic effects and effects on inhibitory and excitatory inputs to ganglion cells. As well, it would be important to express ChR2 in SACs alone to see if this drives changes in GC spiking.

    In all, the authors here have the opportunity to examine the effects of paracrine signaling by SACs on inner retinal network excitability and function using a nice model system, and they should take advantage of it.

  3. eLife

    Reviewer #1:

    The authors use a tetracycline controlled gene expression system to compare the effectiveness of two difference promoters to express channelrhodopsin in different populations of retinal neurons with the goal of rescuing visual function in mouse models of photoreceptor degeneration. The expression patterns of two promoters were compared - the first a muscarinic AChR (referred to as M4 in the manuscript) led to expression in a subset of RGCs and a subset of amacrine cells, while the second a 5-HT receptor (5B) led to expression in a subset of RGCs only. In the M4 line, the amacrine cells that were labeled were a subset of starburst amacrine cells located in the INL and did not label the SACs displaced in the GCL. Also, it was a subset of the INL-SACs. To assess the impact of these different expression patterns on vision restoration, mice expression ChR under these two different promoters were treated with MNU to induce PR degeneration. The light responses restored by ChR were assessed with a MEA recordings cortical VEPs and behavior. The M4 promoter had stronger light evoked responses. The authors used pharmacology to assess how the M4 retinal circuit might explain the enhanced light response.

    There were several fundamental problems with the manuscript that need to be addressed. These problems range from experimental design, interpretation of findings, some mistakes in description of retina circuits. Moreover, there is no context given comparing these results to the multiple other studies on vision restoration impact on visual-guided behaviors. These problems are listed here:

    1. The choice of promoters and expression patterns need to be further explored. The motivation for a particular subtype of mAChRs and 5-HT is not given. Though M4 and 5b drives expression in roughly the same percentage of total RGCs, there is no way to know whether they drive expression in the same subtypes of RGCs. Hence differences in firing patterns are not likely to be fully explained by the fact that M4 promoter also drives expression I a subset of INL-SACs.

    2. The observation that M4 drives expression in a subset of OFF SACs was quite intriguing. Though there are ways to distinguish ON from OFF SACs, this is the first example of which I am aware that a subset of OFF-SACs is labeled. Does this mean only a subset of OFF-SACs have mAChRs? Or was this reflective of the partial express induced by Tet? It is worth the authors quantifying the percent of OFF-SACs labeled in the M4 mouse line.

    3. The observation that they are able to rescue the OKR result in MNU treated mice using the M4-promoter is impressive. Again, the authors conclude that this is due to presence of ChR in INL-SACs but it could be they also have ChR expression in direction selective ganglion cells themselves. Hence the rescue is impressive, it is difficult to interpret. Also, this important behavior is confined to a supplemental figure.

    4. The authors conclude that M4-driven expression of ChR rescues the OKR in MNU-treated mice and not rd-mice because the rd mice have a "thinner INL" and therefore may have a depletion of INL-SACs. This appears to be an easy test for the authors using immunofluorescence.

    5. The authors do some pharmacology to test whether SACs are the basis of the larger sustained response observed in M4 vs 5B . However, the assumptions/interpretations for these experiments are based on some mistakes regarding retinal circuits. SACs release GABA and acetylcholine. However, the pharmacology they do is quite limited. Namely they use TPMPA, which blocks GABA-C receptors which are found on a subset of bipolar cell terminal and by no means represent the major source of GABAergic signaling in retina which is via GABA-A and GABA-B receptors. Similarly, the authors assess impact of ACh release by using atropine, which blocks muscarinic receptors but not nicotinic ACh receptors. Finally, the authors use MFA, a blocker of gap junctions, which does have clear impact on sustained responses. However, SACs are not thought to be gap junction coupled to anything. So, it is more likely MFA is acting via RGC-RGC gap junction coupling or having an off-target effect. Much more needs to be done to have a complete understanding of the circuits that mediate the ChR-mediated light responses.

    6. 226-227 - what is the conclusion - results suggest not due entirely to gene transfer? This needs further explanation.

    7. Comparison of light induced responses in MNU vs non MNU treated rather confusing. Authors should consider revising this point.

  4. Version published to 10.1101/2020.08.11.246686 on bioRxiv