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. 2023 Sep 13:31:101107.
doi: 10.1016/j.omtm.2023.09.003. eCollection 2023 Dec 14.

Optogenetic targeting of AII amacrine cells restores retinal computations performed by the inner retina

Hanen Khabou  1 , Elaine Orendorff  1 , Francesco Trapani  1 , Marco Rucli  1 , Melissa Desrosiers  1 , Pierre Yger  1 , Deniz Dalkara  1 , Olivier Marre  1
Affiliations

Optogenetic targeting of AII amacrine cells restores retinal computations performed by the inner retina

Hanen Khabou et al. Mol Ther Methods Clin Dev. .

Abstract

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of inner retinal neurons is a promising avenue to restore vision in retinas having lost their photoreceptors. Expression of optogenetic proteins in surviving ganglion cells, the retinal output, allows them to take on the lost photoreceptive function. Nonetheless, this creates an exclusively ON retina by expression of depolarizing optogenetic proteins in all classes of ganglion cells, whereas a normal retina extracts several features from the visual scene, with different ganglion cells detecting light increase (ON) and light decrease (OFF). Refinement of this therapeutic strategy should thus aim at restoring these computations. Here we used a vector that targets gene expression to a specific interneuron of the retina called the AII amacrine cell. AII amacrine cells simultaneously activate the ON pathway and inhibit the OFF pathway. We show that the optogenetic stimulation of AII amacrine cells allows restoration of both ON and OFF responses in the retina, but also mediates other types of retinal processing such as sustained and transient responses. Targeting amacrine cells with optogenetics is thus a promising avenue to restore better retinal function and visual perception in patients suffering from retinal degeneration.

Keywords: gene therapy; optogenetics; retina; vision restoration.

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Conflict of interest statement

D.D. reports grants from Foundation Fighting Blindness, USA and European Research Council, during the conduct of the study; D.D. is a co-inventor on patent #9193956 (Adeno-associated virus virions with variant capsid and methods of use thereof), with royalties paid to Adverum Biotechnologies. D.D. also has personal financial interests in Tenpoint Tx. and SparingVision, outside the submitted work. D.D., H.K., O.M., F.T., and E.O. are inventors on pending patent applications on methods to target A2 amacrine cells related to this work.

Figures

None
Graphical abstract
Figure 1
Figure 1
AAV-mediated gene delivery to AII amacrine cells from the vitreous (A) Whole-mount view showing eGFP expression (green) in the plane of AII somas (INL). (B) Retinal cryosections showing eGFP expression (green) and labeling of starburst amacrine cells with a ChAT antibody (red). (C, C′, C″) Retinal cryosections showing eGFP expression under the control of our HKamac sequence (in green) and Prox1 immunostaining (in red) (and DAPI in blue). (D, D′, D″) Retinal cryosections showing ReaChR-eYFP expression under the control of our HKamac sequence (in green), Prox1 immunostaining (in red) and DAPI in blue). For all panels, retinal layers are shown on the left: inner nuclear layer (INL), ganglion cell layer (GCL). DAPI labels all nuclei, while Prox1 labels bipolar and AII amacrine cells. Co-labeled AII amacrine cells are indicated with white arrows. Co-labeled RGCs are indicated by white arrowheads. Scale bars, 50 μm.
Figure 2
Figure 2
ON and OFF RGC responses elicited upon light stimulation of ReaChR-expressing AII amacrine cells (A) AII amacrine cells connect to the ON pathway (in red) through gap junctions, and to the OFF pathway (in blue) through glycinergic inhibitory connections. Stimulation of AIIs hence produces responses of opposite polarity on ON and OFF RGCs. We target AIIs through optogenetic stimulation consisting of a series of full-field flashes, and record the responses of the RGCs with a multielectrode array. Pharmacology (ACET and L-AP4) blocks synaptic transmission from photoreceptors. (B) Responses of representative ON (left column, red) and OFF (right column, blue) RGCs to photoreceptor stimulation with white full-field flashes. Top: spiking activity across different trials. Bottom: mean response. The time intervals of the flashes are shown in gray. (C) Responses of the same ON (left column, red) and OFF (right column, blue) RGCs shown in (B) to optogenetic stimulation, after blocking photoreceptor synaptic transmission. Top: spiking activity across different trials. Bottom: mean response. (D) Percentage of RGCs responding to optogenetic stimulation. Left: percentage of ON RGCs responding to the flash respectively at onset (red), offset (blue), or never (black), for both low and high luminance. Center and Right: same plot for OFF and ON-OFF RGCs, respectively. Error bars represent the standard error of the sample mean. (E) Percentage of RGCs responding to optogenetic stimulation for a control population with no opsin expressed. Same plots as in (D). Error bars represent the standard error of the sample mean.
Figure 3
Figure 3
Off RGC responses of inverted polarity are due to off-target opsin expression (A) Control protocol showing direct ganglion cell activation due to off-target expression of ReaChr: the application of CNQX and CPP disrupts all the excitatory synaptic connections. Responses induced by visual stimulations under this condition are only due to direct activation of the RGCs expressing the opsin. (B) Mean responses of three representative RGCs (one ON and two OFF) to simple photoreceptor stimulation. The stimulation time interval is depicted in gray. (C) Mean responses of the same three RGCs to optogenetic stimulation, after blocking the photoreceptor transmission. (D) Mean responses of the same RGCs after blocking all the excitatory synaptic connections. Responses of RGC 3 can only be explained by off-target expression of the opsin in ganglion cells. (E) Percentage of ganglion cells responding to direct optogenetic stimulation, due to off-target expression. Left: percentage of ON RGCs responding respectively to the stimulus onset or offset (or not responding), at both low and high luminance. Center and Right: same plot for OFF and ON-OFF RGCs, respectively. Error bars represent the standard error of the sample mean. (F) Percentage of ganglion cells responding to direct optogenetic stimulation for a control population with no opsin expressed. Same plots as in (E). Error bars represent the standard error of the sample mean.
Figure 4
Figure 4
The diversity of RGC responses is really due to AII activation, and not to photoreceptor transmission or off-target expression (A) Examples of responses to photoreceptor stimulation for representative ON (left column, red) and OFF (right column, blue) RGCs. Top: Raster plot of RGC responses across trials. Bottom: mean responses. The stimulation period is represented by the gray regions. (B) same as (A) for optogenetic stimulation, after blocking photoreceptor transmission: this time AII amacrine cells express an inhibitory opsin, gtACR1. (C) Percentage of RGCs responding to optogenetic stimulation (with the inhibitory opsin gtACR1). Top: percentage of ON RGCs responding respectively at stimulus onset (red), stimulus offset (blue), or not responding (black), for both low and high luminance. Bottom and Right: same plot for OFF and ON-OFF RGCs, respectively. Error bars represent the standard error of the sample mean.
Figure 5
Figure 5
AII activation generates diverse RGC responses (A) On top in black: the temporal profile of the full-field chirp stimulus (luminance over time). Below: mean responses of four representative RGCs to the chirp stimulus. In orange: responses mediated by photoreceptors (no optogenetics involved). In green: responses mediated by the optogenetic activation of AIIs (with blocked photoreceptor transmission). (B) Comparison between the sustained-transient index computed on photoreceptor and optogenetic responses for 48 selected RGCs. Each dot shows the index for a different RGC, for both photoreceptor responses (x axis) and optogenetic responses (y axis). Values close to 1 mean the response is predominantly sustained; values close to zero mean the response is predominantly transient. (C) Distribution of the comparative sustained-transient index ΔST for the ON (red) and OFF (blue) RGC populations (same 48 RGCs shown in B). An index close to 1 means that the cell responds more transiently when activated optogenetically with respect to its normal photoreceptor responses. Conversely, an index close to −1 means that the optogenetic responses are more sustained than the normal photoreceptor responses. (D) Response correlations across RGC pairs from a population of 40 RGCs, for both photoreceptor and optogenetic stimulations. Pearson correlation coefficient computed on the responses of pairs of RGCs to photoreceptor stimulation (x axis) and to optogenetic stimulation (y axis). Red dots represent ON to ON response correlations: blue dots represent OFF to OFF response correlations. (E) Principal-component analysis of the mean RGC responses to the chirp stimulus for a selected population of 40 cells (same shown in D). Each curve shows the number of principal components needed (x axis) to explain a given percentage of variance in the ganglion cell responses (y axis). We show the curves for both photoreceptor responses (orange) and optogenetic responses (green). Light dashed curves represent analysis conducted on the individual experiments. Dark, continuous lines represent the average across all experiments.
Figure 6
Figure 6
Optogenetic activation of AIIs produces ON and OFF RGC responses also in dystrophic retinas (A, B, and C) Cross section of a dystrophic mouse retina (rd1) showing ReaChR-eYFP expression (green, panels A and C) under HKamac sequence; co-localization with Prox1 antibody (red, panels B and C) indicates ReaChR expression in AII amacrine cells. Expression under control of DAPI is shown in blue (C). AII amacrine cells are indicated with white arrows. Retinal layers are shown on the left: outer plexiform layer (OPL), inner nuclear layer (INL), inner plexiform layer (IPL), ganglion cell layer (GCL). (D) Optogenetic responses of 10 representative RGCs from dystrophic retinas to a series of flashes. Left: raster plots of the responses; each row (and color) represents a different cell; the stimulation period is highlighted in gray. Right: mean responses over trials. (E) Activation of RGCs in dystrophic retina due to optogenetic stimulation. For each of five luminance levels, we plot the percentage of RGCs that responded to the optogenetic stimulation with pure ON (red), pure OFF (blue), ON-OFF (magenta), or no responses (black). Error bars represent the standard error of the sample mean.
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