Neovascular age-related macular degeneration (AMD) and retinitis pigmentosa (RP) affect more than 2 million people worldwide. A series of promising attempts for partial vision restoration exist, which comprise retinal implants, gene-replacement therapy, stem cell transplantation and optogenetics. Pre-clinical assessment of a successful restoration strategy may be obtained using a system identification approach. Here we investigate this hypothesis for stimulation of opsin-expressing retinal ganglion cells and simultaneous recording using a CMOS-based high-density microelectrode array (CMOS MEA).
Material and Methods:
1)Transgenic mice expressing a ChR2-EYFP fusion protein under control of the endogenous parvalbumine promoter were obtained by intercrossing of B6.129P2-Pvalbtm1(cre)Arbr/J and B6.Cg-Gt(ROSA)26Sortm32(CAG-COP4*H134R/EYFP)Hze/J mice (The Jackson Laboratory, stock No. 017320 and 024109). Genotyping by means of genomic PCR confirmed the expected genotype.
2)Customized spatial and temporal light patterns are created by using a combination of selectable LEDs and a digital mirror device (DMD). The patterned light stimuli allow to precisely stimulate retinal cells whose spiking response is recorded by the CMOS MEA beneath the retinal sample. The identification of spikes and spike sorting is performed based on a convolutive ICA approach [Leibig et al. 2016].
3)Based on a method pioneered by Chichilnisky and co-workers [Chichilnisky et al. 2001] the estimation of the mean effective visual stimulus for different RGC types can be derived by considering robust spikes in several repetitions of the same stimulus followed by the calculation of the spike triggered average stimulus (STA).
Results and Discussion:
Retinal ganglion cells (RGCs) were first stimulated by light flashes and by white noise stimuli (photopic regime). RGCs were broadly classified based on their temporal filters and the polarities of their response (ON vs. OFF). Temporal filters showed ON or OFF polarity with a mean time-to-peak of 50 ms.
The optogenetic response of that RGC subpopulation expressing ChR2 was identified upon blocking glutamate receptors. Brief (4 ms) light flashes revealed a reliable response of the Chr2 - positive cells.
White noise stimuli revealed that all optogenetically derived STAs are much shorter (~10ms) than optically derived STAs and show an ON-type polarity only. Localized stimulation was used to analyse the spatial selectivity, which could be obtained by optogenetic stimulation.
The presented method demonstrates that simultaneous optogenetic stimulation and identification of the induced signals is feasible without any artefact. Mean effective stimuli (temporal filters) can be recovered from optogenetic stimulation. Ongoing research investigates the spatial selectivity and to what degree the contrast coding is affected if all retinal output is converted into ON-type of stimulus.