**3. The first human clinical trial of retinal prosthesis**

The first human clinical study has recently shown partial recovery of visual function in a blind patient with RP using optogenetics [25]. In the study, a serotype AAV encoding ChrimsonR fused to the red fluorescent protein tdTomoto was administered in one eye of the patient to target mainly foveal RGNs. After injecting the opsin into the ganglion cells, the retina was stimulated by an optoelectronic goggle after 7 months, as the expression takes two to six months to get stabilized in the cells [25, 39].

Recovery of visual function was tested under three conditions with three psychophysical tests. The conditions were as follows:

I.Both eyes were open in the absence of the goggles (Natural binoculars).


The conducted psychophysical tests were as follows:


The average response of the patient under the above conditions and psychological tests are summarized in **Table 1**.

The study has shown that wearing the goggle, which transforms the changes in intensity, pixel by pixel, into a monochromatic image and projects them in real time onto the retina, the patient could perceive, locate, count, and touch different objects (**Table 1**) [25].


**Table 1.**

*The success rate of visual perception under different conditions and psychological tests [25].*

#### **4. Challenges and limitations in optogenetic retinal prostheses**

Channelrhodopsin-2 (ChR2) from *Chlamydomonas reinhardtii* was the first lightsensitive opsin used to restore light responses in blind mice [40]. ChR2 was injected in thalamic projecting RGNs via adeno-associated virus (AAV) serotype 2 [40]. However, the required light intensity to evoke spiking with ChR2, having its maximum absorption at 460 nm, exceeds the standard safety threshold and might cause photochemical damage in the human retina and retinal pigment epithelium [41–43].

To overcome the limitation, new opsins with increased light sensitivity were developed through mutations and discovery that increased the dynamic range of neural activity and, thus, image contrast within regulatory limits. Since the safety limits of light intensity have a strong wavelength dependence, opsins with red-shifted wavelength activation peaks safely allow the use of higher irradiance than their blue-shifted counterparts. Shifting the wavelength from blue o red allows for safely increasing the photon flux by three orders of magnitude. Deeper penetration of longer wavelengths is an additional advantage with the red-shifted opsins [41].

To reduce irradiance requirement, opsin with Ca2+ selectivity named CatCh was expressed into RGNs [22, 44, 45]. Although the activation threshold of CatChexpressing RGNs is below the damage threshold and allows normal displays to evoke responses, CatCh suffers from inherent toxicity associated with Ca2+ influx [9]. Engineered mutants of CoChR, developed by optimizing the off-rate through sitedirected mutagenesis, have also been used to restore functional vision under normal light conditions. Although the mutants functions at safe irradiances, they do not provide sufficient temporal resolution [46].

At present, red-shifted opsins are being intensively investigated as red-shifted photons can reach deeper areas in the tissue, which is important for noninvasive light delivery in *in vivo* experiments [47–49]. Three orders of magnitude higher irradiance can be used at 590 nm in contrast to light at 470 nm for safe illumination of the retina [41]. The safety threshold shifts on changing wavelengths of light as 7.52 × 1013 photons mm−2 s−1 at 470 nm to 5.94 × 1015 photons mm−2 s−1 at 590 nm [41, 50]. Recently, a red-activable variant of ChR2, named ReaChR, has restored light sensitivity in blind rd1 mice, primate retina, and post-mortem human retinae when targeted to RGNs [41, 47]. However, ReaChR could not drive RGNs beyond 30 Hz [41]. Although ReaChR requires similar irradiance as ChR2, it is safe at the red-shifted wavelength [4, 41]. To get high-frequency along with red-shifted activation wavelength, ChrimsonR was used for optogenetic vision restoration [25, 39, 51–53]. Even though ChrimsonR uses a safe wavelength of light, it still requires extremely bright light [4]. ChrimsonR has been used for the first clinical study to restore vision in a human patient [25]. **Table 2** summarizes different opsins, their characteristic features, and their response in retinal neurons [9, 25, 39–41, 47, 54–59].

*Recent Advances in Optogenetic Retinal Prostheses DOI: http://dx.doi.org/10.5772/intechopen.109205*


**Table 2.**

*Microbial rhodopsins in optogenetic retinal prostheses.*

Although recently discovered opsins could allow control at improved light levels, they have not yet been studied for that purpose. To drive opsin-expressing neurons at their firing frequencies, optogenetic approaches are combined with an extraocular device or optoelectronic goggles [26]. These devices consist of a camera and an image processing unit. These devices are crucial for optogenetic retinal prostheses for the following reasons [18]:


Optogenetic-based retinal prostheses involve the genetic expression of opsinconstruct into the retinal neurons. Neurotoxicity and immunogenicity are the key concerns for clinical trials. Attempting functional-level response in the system by enhancing opsin expression is problematic due to the increased risk of cell toxicity and immune response [27, 60]. Although AAVs have a favorable safety profile in gene therapy, their safety is dose-dependent [61]. This limits the expression of microbial opsin with a safe viral dose [9]. Another critical task in clinical trials is to achieve stable opsin expression in the retina. Earlier studies have shown that the virus-mediated expression of ChR2 remained stable beyond 3 months in nonhuman primates [62]. Similar safety and stability checks must be conducted while selecting new opsins for retinal prostheses. Therefore, long-term stability and neurotoxicity with new microbial rhodopsins will need to be carefully evaluated before they can be considered for clinical applications [18].

#### **5. New opsins in optogenetics**

In recent years, intense research efforts have been directed to design more efficient opsins with tailored properties, including unitary spectral conductance, retinal binding affinity, faster temporal kinetics, light actuation sensitivity, spectral tuning, and protein stability. Being a primary goal of optogenetics, new opsins are first investigated in neurons in the brain (**Table 3**) [29, 32, 63–68].

CsChrimson is a CsChR-Chrimson chimera, produced by replacing the Chrimson N terminus with the CsChR N terminus. It exhibits more sensitivity than Chrimson, while the spectral and kinetic properties are the same [51]. bReaChES was generated by introducing a Glu123Ser mutation and replacing the first 51 amino-terminal residues with the first 11 amino-terminal residues in the ReaChR construct [63]. It

*Recent Advances in Optogenetic Retinal Prostheses DOI: http://dx.doi.org/10.5772/intechopen.109205*


#### **Table 3.**

*Newly discovered or engineered microbial rhodopsins in optogenetics.*

has allowed simultaneous stimulation and recording using Ca2+ indicators [63]. Most recently, screening of residues forming the cation-conducting ChR pore guided by crystal structure-derived knowledge has resulted in ~1000 suitable new opsin sequences. A sequence of a marine opsin gene optimized for mammalian expression, named ChRmine from *Tiarina fusus*, exhibits a very large inward photocurrent ~4 nA with red-shifted excitable wavelength along with an order of magnitude faster recovery than for other red-shifted opsins [49, 68].

More recent efforts to understand the mechanism of ion transport by ChRmine has been enabled by its cryo-electron microscopy structure at 2.0 A° resolution. The structural knowledge has been used to design new variants with faster speed (hsChRmine) and greater red shift (rsChRmine), while retaining high current and light sensitivity [64]. Along with the development of red-shifted opsins, new efficient blue-activated opsins have also been discovered and engineered. *de novo* sequencing of opsins from over 100 algal species has resulted in Chronos, a blue light-sensitive opsin exhibiting high light sensitivity and unprecedented kinetics, with a turn-off of 3.6 ± 0.2 ms. It is the fastest blue-shifted opsin reported to date [51]. Recent efforts to engineer Chronos have resulted in a mutant, ST-Chronos-M140E, or ChroME. It exhibits rapid decay kinetics while exhibiting photocurrents more than 3-5 times larger than Chronos [65]. More recently, two new mutants have been engineered, namely ChroME2f and ChroME2s, with enhanced properties that can support largescale, temporally precise multiphoton excitation [66].

Although the newly discovered opsins can significantly improve optogenetic retinal prostheses, developing strategies for safe and sufficient expression of new opsins in retinal neurons and experimentation of each opsin pair with different targeted retinal neurons is a lengthy process and needs repetitive investigation and safety checks before clinical studies.

#### **6. Computational modeling of optogenetic excitation of retinal neurons**

Computational modeling of optogenetic systems has made significant contributions in developing a better understanding of the reaction dynamics behind photocurrent generation in the opsin molecules and change in membrane potential in opsin-expressing neurons in response to light. It helps in correctly interpreting experiments and optimizing photostimulation conditions within a complex cell/tissue environment, which is often questionable and challenging [69–74]. Due to the naturally occurring structural and functional diversity in the cell types in living animals, a large data set is required for optogenetics to be used in different environments, which is a challenging task. The problem persists while selecting light-sensitive proteins to get desired control over cellular activity. Computational models can help quick (virtual) testing of newly developed light-sensitive proteins in different cell types and within realistic tissue and organ-level settings.

Initial efforts in the field of computational optogenetics include modeling of the photoresponse of ChR2 considering three, four and six-state models of its photocycle, light-mediated spiking in neurons under continuous and pulsed illumination, the effect of illumination of sub-cellular compartments, analysis of activation threshold of opsin-expressing cell within tissue while considering scattering and absorption [69–74]. Although there were many new opsins with improved kinetics, photocurrent, and light sensitivity, theoretical models of these opsins were not formulated until recently.

In the last few years, theoretical models of optogenetic systems consisting of new light-sensitive proteins have been formulated. Accurate theoretical models of optogenetic excitation of neurons with Chronos, CheRiff, ReaChR, Chrimson, ChrimsonR, CsChrimson, f-Chrimson, vf-Chrimson, bReaChES and ChRmine and inhibition with NpHR, eNpHR3.0, Jaws, and GtACR2 have been reported recently. Using these computational models, a detailed theoretical analysis of the effect of photostimulation and physiological parameters was also conducted, which has provided a better understanding of the mechanism, limitations, and advantages of different types of neurons [75, 76]. Further, accurate theoretical models of bidirectional optogenetic control with spectrally separated excitatory-inhibitory opsin pairs, namely ChR2(H134R)-eNpHR3.0, Chrimson-GtACR2, Chronos-eNpHR3.0, Chronos-Jaws, CheRiff-Jaws, and Vf-Chrimson-GtACR2 were also reported. A comprehensive theoretical study on high-frequency low-power bidirectional optogenetic control of neurons with single spike resolution with already tested opsin pairs and with new

#### **Figure 1.**

*Equivalent circuit diagram of biophysics model of opsin-expressing retinal ganglion neurons. Cm is the membrane capacitance. gNa, gK, gKA, gKCa, gCa and gL are maximal conductances of naturally occurring sodium, potassium, potassium (A-type), Ca2+ activated potassium, calcium, and leak channels. ENa, EK, ECa, and EL are the reversal potential for sodium-, potassium-, calcium-conducting, and leaky channels. gOpsin is the light-dependent conductance through opsin channels having reversal potential EOpsin. C1 and C2 are the closed states, and O1 and O2 are the open states. Ga1, Ga2, Gb, and Gf are the light-dependent rate functions, while Gd1, Gd2, and Gr are the light-independent rate constants. ɸ is the photon flux. (For details, see Ref. [38]).*

#### *Recent Advances in Optogenetic Retinal Prostheses DOI: http://dx.doi.org/10.5772/intechopen.109205*

opsin pairs not experimented till that time for better control was conducted using theoretical models [38]. Recently, computational simulations have proposed a novel method of co-expressing step-function opsins with fast channelrhodopsins to avoid spike failure from desensitization of photocurrent [77]. Integration of theoretical models of photoresponse of ChRmine into biophysical circuit models of cardiac cells has shown that ChRmine could be used for ultra-low power deep sustained optogenetic excitation/suppression of electrical activity in cardiomyocytes [78].

Theoretical simulations of optogenetic visual cortical prosthetic systems have provided a better understanding of the mechanism behind signal encoding by the visual cortex [79, 80]. Theoretical modeling of optogenetic excitation of retinal neurons would be a primary step to design ideal optogenetic prosthetic devices and circuits. Also, the newly discovered light-sensitive proteins have the potential to overcome current challenges. However, designing methods for the efficient and safe delivery of new opsins in the retina is a lengthy process. No mathematical model was reported until recently to study optogenetically evoked spiking in RGNs.

The theoretical framework of the biophysical mechanism of optogenetic excitation of already tested opsins, namely, ChR2, ReaChR, and ChrimsonR, and newly discovered potential opsins, *i.e.,* CsChrimson, bReaChES, and ChRmine has contributed significantly to our knowledge and provided insights into the design of optimized optogenetic retinal prosthetic circuits [38] (**Figure 1**). In the first step, the photocurrent characteristics of different opsins were compared over a wide range of irradiances (**Figure 2**). The variation of photocurrent with time is shown in **Figure 2A**. Peak and plateau photocurrent of these opsins were compared over a wide range of irradiance **Figure 2B** and **C**. Theoretical simulations have helped in determining the minimum pulse width to achieve maximum photocurrent at each irradiance, also called saturating pulse width (SPW), which is important to minimize delivered light power while getting maximum output (**Figure 2D**). SPW decreases on increasing irradiance and saturates at higher irradiances for each opsin [38].

Further, the variation of photocurrent with time on illuminating with repetitive light pulses at different light stimulation frequencies was compared (**Figure 2E**). At higher frequencies, the photocurrent in all the opsins does not reach the baseline before the arrival of the subsequent light pulse, which results in a non-zero photocurrent plateau indicated by an arrow in **Figure 2F**. The percentage of return to baseline (RTB %) plays a crucial role in temporal resolution at high frequencies. In all the opsins, RTB % decreases with stimulation frequency. The potential interactions between pulse width, irradiance, and stimulation frequency can have a profound impact on temporal fidelity due to the inverse relationship between kinetics and photosensitivity of opsins. The study also reported that RTB % does not change with irradiance, while it is lower at longer pulse [38].

To determine the irradiance range at which each opsin-expressing RGN exhibit spiking, the variation of membrane potential with time were simulated on illuminating with long (500 ms) and short (10 ms) light pulses at different irradiances (**Figure 3**). The variation of membrane potential with time on illuminating 500 ms light pulses at increasing irradiances has been shown in **Figure 3A**. From the variation of the number of spikes with pulse irradiance, the study has revealed that ChRmine is most sensitive among the studied opsins and the maximum firing frequency within the safety limit of irradiance is two-three orders of magnitude higher than the opsins tested for retinal prostheses (**Figure 3A**). Further, the response of these RGNs was simulated under randomized photon fluxes of increasing contrast (**Figure 4B**). Interestingly, ChRmine can respond to irradiances changes from one order to four orders (**Figure 3B**).

#### **Figure 2.**

*Photocurrent characteristics of ChR2, ReaChR, ChrimsonR, CsChrimson, bReaChES and ChRmine on illuminating with their peak absorption wavelength i.e., 460 nm for ChR2 and 590 nm for other opsins. (A) Variation of photocurrent with time on illuminating with single light pulse (1 s, 1 mW/mm2 ), and (B–D) corresponding variation of (B) peak, (C) plateau photocurrent and (D) saturating pulse width (SPW) with irradiance. (E) Variation of photocurrent with time on illuminating with multiple light pulses at indicated frequencies and different pulse widths (50 ms for 10 Hz, 25 ms for 20 Hz, 12.5 ms for 40 Hz, and 8.5 ms for 60 Hz) at 1 mW/mm<sup>2</sup> , and (F) corresponding variation of return to baseline (%) with stimulation frequency, calculated using the formula shown above the box. © IOP Publishing. Reproduced with permission. All rights reserved [38].*

In moving picture frames, time-dependent change in irradiances of each pixel is the key photostimulation parameter to encode changes in visual scenes. In optogenetics, irradiances below certain threshold results in spike failure, while pulses longer than a certain limit result in spurious spiking patterns. Thus, it would be necessary to determine how larger fluctuations in irradiance and pulse-width can be tolerated by retinal neural circuits. The theoretical study determined a region in the irradiancepulse-width plot in which each point is a combination of allowed irradiance and pulse-width, while maintaining single-spike resolution (**Figure 4**).

*Recent Advances in Optogenetic Retinal Prostheses DOI: http://dx.doi.org/10.5772/intechopen.109205*

#### **Figure 3.**

*Frequency response of opsin expressing retinal ganglion neurons in response to long- and short-duration light pulses at different irradiances. (A) Variation of membrane potential with time in response to light pulses (500 ms) at increasing irradiances, and (B) corresponding variation of the number of spikes during illumination at different irradiances. (C) Light-evoked spiking in different opsins-expressing RGNs on illuminating with multiple light pulses each of 10 ms at 50 Hz for 1 s and at randomized photon fluxes of increasing contrast from 1011 to 1012 photons mm−2 s−1 to 1011 to 1015 photons mm−2 s−1. © IOP Publishing. Reproduced with permission. All rights reserved [38].*

In visual systems, internal delays in signal processing may lead to mislocalization while capturing things changing with time [81]. Thus, the latency of a system is a critical feature for information processing which is determined by the turn-on rate of the opsin photocurrent [48, 81]. Furthermore, due to desensitization of photocurrent in opsins, sustained excitation of opsin-expressing neurons results in spike failure [27]. In the study, they have shown the variation of first spike latency with irradiance

#### **Figure 4.**

*Tolerance for maintaining single spike resolution by different opsin-expressing neurons against fluctuations in irradiance and pulse width. Variation of minimum (lower boundary of the shaded region) and maximum (lower boundary of the shaded region) irradiance thresholds with pulse-width to evoke 100% spiking at 10 Hz for 2 s in (A) ChR2, (B) ReaChR, (C) ChrimsonR, (D) CsChrimson, (E) bReaChES, and (F) ChRmine-expressing retinal ganglion neurons. © IOP Publishing. Reproduced with permission. All rights reserved [38].*

#### **Figure 5.**

*Latency of spikes in opsin-expressing retinal ganglion neurons in response to light stimulus. (A) Variation of first spike latency with pulse irradiance, and (B) variation of normalized spike latency with sequential position of light pulse each of 5 ms for ChR2, 4 ms for ChrimsonR, 1.8 ms for CsChrimson, 6 ms for ReaChR, 0.4 ms for bReaChES, and 0.13 ms for ChRmine in the stimulus train at 10 Hz and 0.6 mW/mm2 . © IOP Publishing. Reproduced with permission. All rights reserved [38].*

*Recent Advances in Optogenetic Retinal Prostheses DOI: http://dx.doi.org/10.5772/intechopen.109205*

in different opsin-expressing RGNs (**Figure 5A**). Further, latencies of subsequent spikes were also determined to check consistency on illuminating with a light pulse train (**Figure 5B**). Theoretical simulations revealed that under sustained excitation of RGNs, ChRmine exhibits the most stable and shortest latency during the entire stimulus train (**Figure 5B**).

For accurate information processing to higher visual centers of the brain, it is essential to drive RGNs up to their natural firing rates. Thus, it was important to determine high-frequency limits of each opsin-expressing RGNs. From MITirradiance graph, it is clear that there is a trade-off between pulse-width and required irradiances to evoke spike. However, the light pulse cannot be increased beyond a certain limit at a particular frequency (*e.g.,* 5 ms for 100 Hz). On the other hand, higher irradiance above its saturating value is totally waste of energy. Thus, a systematic study of the effect of photostimulation conditions was carried out to determine the high-frequency limit with each opsin under pulsed stimulation.

Further, the high-frequency limit was determined with each opsin. The variation of spiking frequency with stimulation frequency at optimized photostimulation conditions is shown in **Figure 6A**. Each opsin maintains 100% spike fidelity up to a certain frequency limit. The spiking patterns at the high-frequency limit are shown in **Figure 6B**. Among these opsins, ChrimsonR is the only opsin that allows 100% spiking fidelity up to 100 Hz. However, the required photon flux density with ChrimsonR ~8.3 × 1015 photons. mm−2 s−1 at 590 nm is beyond the safety threshold for the human retina [38, 41]. On the other hand, ChRmine maintains single-spike resolution up to 40 Hz, which is sufficient for RGNs, it needs pulses of the lowest light power (**Figure 6**) [38].

#### **Figure 6.**

*Frequency response of different opsin-expressing retinal ganglion neurons. (A) Variation of spiking frequency with stimulation frequency on illuminating with 20 light pulses at different frequencies and at different irradiances and pulse widths as follows: 5 mW/mm2 and 2.5 ms for ChR2, 2.8 mW/mm2 and 1.2 ms for ChrimsonR, 0.13 mW/ mm2 and 5 ms for CsChrimson, 5 mW/mm2 and 1 ms for ReaChR, 0.02 mW/mm2 and 6 ms for bReaChES, and 0.013 mW/mm2 and 0.93 ms for ChRmine, and (B) corresponding spiking patterns at the high-frequency limit with 100% spike fidelity. ©IOP Publishing. Reproduced with permission. All rights reserved [38].*
