Optogenetics: Illuminating the Future of Hearing Restoration and Understanding Auditory Perception.

Hearing loss is a prevalent sensory impairment significantly affecting communication and quality of life. Traditional approaches for hearing restoration, such as cochlear implants, have limitations in frequency resolution and spatial selectivity. Optogenetics, an emerging field utilizing light-sensitive proteins, offers a promising avenue for addressing these limitations and revolutionizing hearing rehabilitation. This review explores the methods of introducing Channelrhodopsin- 2 (ChR2), a key light-sensitive protein, into cochlear cells to enable optogenetic stimulation. Viral- mediated gene delivery is a widely employed technique in optogenetics. Selecting a suitable viral vector, such as adeno-associated viruses (AAV), is crucial in efficient gene delivery to cochlear cells. The ChR2 gene is inserted into the viral vector through molecular cloning techniques, and the resulting viral vector is introduced into cochlear cells via direct injection or round window membrane delivery. This allows for the expression of ChR2 and subsequent light sensitivity in targeted cells. Alternatively, direct cell transfection offers a non-viral approach for ChR2 delivery. The ChR2 gene is cloned into a plasmid vector, which is then combined with transfection agents like liposomes or nanoparticles. This mixture is applied to cochlear cells, facilitating the entry of the plasmid DNA into the target cells and enabling ChR2 expression. Optogenetic stimulation using ChR2 allows for precise and selective activation of specific neurons in response to light, potentially overcoming the limitations of current auditory prostheses. Moreover, optogenetics has broader implications in understanding the neural circuits involved in auditory processing and behavior. The combination of optogenetics and gene delivery techniques provides a promising avenue for improving hearing restoration strategies, offering the potential for enhanced frequency resolution, spatial selectivity, and improved auditory perception.

Channel Gating in Kalium Channelrhodopsin Slow Mutants.

Kalium channelrhodopsin 1 from Hyphochytrium catenoides (HcKCR1) is the first discovered natural light-gated ion channel that shows higher selectivity to K+ than to Na+ and therefore is used to silence neurons with light (optogenetics). Replacement of the conserved cysteine residue in the transmembrane helix 3 (Cys110) with alanine or threonine results in a >1,000-fold decrease in the channel closing rate. The phenotype of the corresponding mutants in channelrhodopsin 2 is attributed to breaking of a specific interhelical hydrogen bond (the "DC gate"). Unlike CrChR2 and other ChRs with long distance "DC gates", the HcKCR1 structure does not reveal any hydrogen bonding partners to Cys110, indicating that the mutant phenotype is likely caused by disruption of direct interaction between this residue and the chromophore. In HcKCR1_C110A, fast photochemical conversions corresponding to channel gating were followed by dramatically slower absorption changes. Full recovery of the unphotolyzed state in HcKCR1_C110A was extremely slow with two time constants 5.2 and 70 min. Analysis of the light-minus-dark difference spectra during these slow processes revealed accumulation of at least four spectrally distinct blue light-absorbing photocycle intermediates, L, M1 and M2, and a UV light-absorbing form, typical of bacteriorhodopsin-like channelrhodopsins from cryptophytes. Our results contribute to better understanding of the mechanistic links between the chromophore photochemistry and channel conductance, and provide the basis for using HcKCR1_C110A as an optogenetic tool.

Impact of volume and expression time in an AAV-delivered channelrhodopsin.

Optogenetics has revolutionised neuroscience research, but at the same time has brought a plethora of new variables to consider when designing an experiment with AAV-based targeted gene delivery. Some concerns have been raised regarding the impact of AAV injection volume and expression time in relation to longitudinal experimental designs. In this study, we investigated the efficiency of optically evoked post-synaptic responses in connection to two variables: the volume of the injected virus and the expression time of the virus. For this purpose, we expressed the blue-shifted ChR2, oChIEF, employing a widely used AAV vector delivery strategy. We found that the volume of the injected virus has a minimal impact on the efficiency of optically-evoked postsynaptic population responses. The expression time, on the other hand, has a pronounced effect, with a gradual reduction in the population responses beyond 4 weeks of expression. We strongly advise to monitor time-dependent expression profiles when planning or conducting long-term experiments that depend on successful and stable channelrhodopsin expression.

Modulating cardiac physiology in engineered heart tissue with the bidirectional optogenetic tool BiPOLES.

Optogenetic actuators are rapidly advancing tools used to control physiology in excitable cells, such as neurons and cardiomyocytes. In neuroscience, these tools have been used to either excite or inhibit neuronal activity. Cell type-targeted actuators have allowed to study the function of distinct cell populations. Whereas the first described cation channelrhodopsins allowed to excite specific neuronal cell populations, anion channelrhodopsins were used to inhibit neuronal activity. To allow for simultaneous excitation and inhibition, opsin combinations with low spectral overlap were introduced. BiPOLES (Bidirectional Pair of Opsins for Light-induced Excitation and Silencing) is a bidirectional optogenetic tool consisting of the anion channel Guillardia theta anion-conducting channelrhodopsin 2 (GtACR2 with a blue excitation spectrum and the red-shifted cation channel Chrimson. Here, we studied the effects of BiPOLES activation in cardiomyocytes. For this, we knocked in BiPOLES into the adeno-associated virus integration site 1 (AAVS1) locus of human-induced pluripotent stem cells (hiPSC), subjected these to cardiac differentiation, and generated BiPOLES expressing engineered heart tissue (EHT) for physiological characterization. Continuous light application activating either GtACR2 or Chrimson resulted in cardiomyocyte depolarization and thus stopped EHT contractility. In contrast, short light pulses, with red as well as with blue light, triggered action potentials (AP) up to a rate of 240 bpm. In summary, we demonstrate that cation, as well as anion channelrhodopsins, can be used to activate stem cell-derived cardiomyocytes with pulsed photostimulation but also to silence cardiac contractility with prolonged photostimulation.

CaMKIIα Promoter-Controlled Circuit Manipulations Target Both Pyramidal Cells and Inhibitory Interneurons in Cortical Networks.

A key assumption in studies of cortical functions is that excitatory principal neurons, but not inhibitory cells express calcium/calmodulin-dependent protein kinase II subunit α (CaMKIIα) resulting in a widespread use of CaMKIIα promoter-driven protein expression for principal cell manipulation and monitoring their activities. Using neuroanatomical and electrophysiological methods we demonstrate that in addition to pyramidal neurons, multiple types of cortical GABAegic cells are targeted by adeno-associated viral vectors (AAV) driven by the CaMKIIα promoter in both male and female mice. We tested the AAV5 and AAV9 serotype of viruses with either Channelrhodopsin 2 (ChR2)-mCherry or Archaerhodopsin-T-green fluorescent protein (GFP) constructs, with different dilutions. We show that in all cases, the reporter proteins can visualize a large fraction of different interneuron types, including parvalbumin (PV), somatostatin (SST), neuronal nitric oxide synthase (nNOS), neuropeptide Y (NPY), and cholecystokinin (CCK)-containing GABAergic cells, which altogether cover around 60% of the whole inhibitory cell population in cortical structures. Importantly, the expression of the excitatory opsin Channelrhodopsin 2 in the interneurons effectively drive spiking of infected GABAergic cells even if the immunodetectability of reporter proteins is ambiguous. Thus, our results challenge the use of CaMKIIα promoter-driven protein expression as a selective tool in targeting cortical glutamatergic neurons using viral vectors.

Properties of a Single Amino Acid Residue in the Third Transmembrane Domain Determine the Kinetics of Ambient Light-Sensitive Channelrhodopsin.

Channelrhodopsins have been utilized in gene therapy to restore vision in patients with retinitis pigmentosa and their channel kinetics are an important factor to consider in such applications. We investigated the channel kinetics of ComV1 variants with different amino acid residues at the 172nd position. Patch clamp methods were used to record the photocurrents induced by stimuli from diodes in HEK293 cells transfected with plasmid vectors. The channel kinetics (τon and τoff) were considerably altered by the replacement of the 172nd amino acid and was dependent on the amino acid characteristics. The size of amino acids at this position correlated with τon and decay, whereas the solubility correlated with τon and τoff. Molecular dynamic simulation indicated that the ion tunnel constructed by H172, E121, and R306 widened due to H172A variant, whereas the interaction between A172 and the surrounding amino acids weakened compared with H172. The bottleneck radius of the ion gate constructed with the 172nd amino acid affected the photocurrent and channel kinetics. The 172nd amino acid in ComV1 is a key residue for determining channel kinetics as its properties alter the radius of the ion gate. Our findings can be used to improve the channel kinetics of channelrhodopsins.

Optogenetic restoration of high sensitivity vision with bReaChES, a red-shifted channelrhodopsin.

The common final pathway to blindness in many forms of retinal degeneration is the death of the light-sensitive primary retinal neurons. However, the normally light-insensitive second- and third-order neurons persist optogenetic gene therapy aims to restore sight by rendering such neurons light-sensitive. Here, we investigate whether bReaChES, a newly described high sensitivity Type I opsin with peak sensitivity to long-wavelength visible light, can restore vision in a murine model of severe early-onset retinal degeneration. Intravitreal injection of an adeno-associated viral vector carrying the sequence for bReaChES downstream of the calcium calmodulin kinase IIα promoter resulted in sustained retinal expression of bReaChES. Retinal ganglion cells (RGCs) expressing bReaChES generated action potentials at light levels consistent with bright indoor lighting (from 13.6 log photons cm-2 s-1). They could also detect flicker at up to 50 Hz, which approaches the upper temporal limit of human photopic vision. Topological response maps of bReaChES-expressing RGCs suggest that optogenetically activated RGCs may demonstrate similar topographical responses to RGCs stimulated by photoreceptor activation. Furthermore, treated dystrophic mice displayed restored cortical neuronal activity in response to light and rescued behavioral responses to a looming stimulus that simulated an aerial predator. Finally, human surgical retinal explants exposed to the bReaChES treatment vector demonstrated transduction. Together, these findings suggest that intravitreal gene therapy to deliver bReaChES to the retina may restore vision in human retinal degeneration in vivo at ecologically relevant light levels with spectral and temporal response characteristics approaching those of normal human photopic vision.

Hippocampal astrocytes modulate anxiety-like behavior.

Astrocytes can affect animal behavior by regulating tripartite synaptic transmission, yet their influence on affective behavior remains largely unclear. Here we showed that hippocampal astrocyte calcium activity reflects mouse affective state during virtual elevated plus maze test using two-photon calcium imaging in vivo. Furthermore, optogenetic hippocampal astrocyte activation elevating intracellular calcium induced anxiolytic behaviors in astrocyte-specific channelrhodopsin 2 (ChR2) transgenic mice (hGFAP-ChR2 mice). As underlying mechanisms, we found ATP released from the activated hippocampal astrocytes increased excitatory synaptic transmission in dentate gyrus (DG) granule cells, which exerted anxiolytic effects. Our data uncover a role of hippocampal astrocytes in modulating mice anxiety-like behaviors by regulating ATP-mediated synaptic homeostasis in hippocampal DG granule cells. Thus, manipulating hippocampal astrocytes activity can be a therapeutic strategy to treat anxiety.

Optogenetic activation of the diaphragm.

Impaired diaphragm activation is common in many neuromuscular diseases. We hypothesized that expressing photoreceptors in diaphragm myofibers would enable light stimulation to evoke functional diaphragm activity, similar to endogenous bursts. In a mouse model, adeno-associated virus (AAV) encoding channelrhodopsin-2 (AAV9-CAG-ChR2-mVenus, 6.12 × 1011 vg dose) was delivered to the diaphragm using a minimally invasive method of microinjection to the intrapleural space. At 8-18 weeks following AAV injection, mice were anesthetized and studied during spontaneous breathing. We first showed that diaphragm electromyographic (EMG) potentials could be evoked with brief presentations of light, using a 473 nm high intensity LED. Evoked potential amplitude increased with intensity or duration of the light pulse. We next showed that in a paralyzed diaphragm, trains of light pulses evoked diaphragm EMG activity which resembled endogenous bursting, and this was sufficient to generate respiratory airflow. Light-evoked diaphragm EMG bursts showed no diminution after up to one hour of stimulation. Histological evaluation confirmed transgene expression in diaphragm myofibers. We conclude that intrapleural delivery of AAV9 can drive expression of ChR2 in the diaphragm and subsequent photostimulation can evoke graded compound diaphragm EMG activity similar to endogenous inspiratory bursting.

Serotype-based evaluation of an optogenetic construct in rat cortical astrocytes.

Optogenetics is a modern technique which has been recently expanded to non-neuronal cell types, e.g., astrocytes, and involves targeted gene delivery of light-sensitive ion channels like Channelrhodopsin-2 (ChR2). Optogenetic regulation of astrocytic activity can be used for therapeutic intervention of several neurological disorders. Astrocytic gene delivery, viz adeno-associated viral (AAV) vectors, have proven to be robust, time-, and cost-efficient contrary to the generation of transgenic animal models. When transducing astrocytes with an AAV vector, it is imperative to perform a serotype evaluation of the AAV vector due to variability in serotype transduction efficiency depending on species, target region and construct length. Rats have been a very successful animal model for studying a variety of brain disorders, from which ChR2-based intervention of astrocytes will benefit. However, the most efficient AAV capsid serotype targeting astrocytes for ChR2 expression in the in vivo rat brain cortex has not been characterized. To address this, we have evaluated AAV serotypes 1, 5, and 8 of the vector AAV-GFAP-hChR2(H134)-mCherry targeting astrocytes in the rat brain neocortex. Results show that serotype 8 exhibits promising transduction patterns, as it has demonstrated the highest tangential and radial viral spread in the rat brain. Our research will facilitate translational research for future applications of optogenetics involving the transduction of rat brain cortical astrocytes.

Optical ventricular cardioversion by local optogenetic targeting and LED implantation in a cardiomyopathic rat model.

AIMS: Ventricular tachyarrhythmias (VTs) are common in the pathologically remodelled heart. These arrhythmias can be lethal, necessitating acute treatment like electrical cardioversion to restore normal rhythm. Recently, it has been proposed that cardioversion may also be realized via optically controlled generation of bioelectricity by the arrhythmic heart itself through optogenetics and therefore without the need of traumatizing high-voltage shocks. However, crucial mechanistic and translational aspects of this strategy have remained largely unaddressed. Therefore, we investigated optogenetic termination of VTs (i) in the pathologically remodelled heart using an (ii) implantable multi-LED device for (iii) in vivo closed-chest, local illumination. METHODS AND RESULTS: In order to mimic a clinically relevant sequence of events, transverse aortic constriction (TAC) was applied to adult male Wistar rats before optogenetic modification. This modification took place 3 weeks later by intravenous delivery of adeno-associated virus vectors encoding red-activatable channelrhodopsin or Citrine for control experiments. At 8-10 weeks after TAC, VTs were induced ex vivo and in vivo, followed by programmed local illumination of the ventricular apex by a custom-made implanted multi-LED device. This resulted in effective and repetitive VT termination in the remodelled adult rat heart after optogenetic modification, leading to sustained restoration of sinus rhythm in the intact animal. Mechanistically, studies on the single cell and tissue level revealed collectively that, despite the cardiac remodelling, there were no significant differences in bioelectricity generation and subsequent transmembrane voltage responses between diseased and control animals, thereby providing insight into the observed robustness of optogenetic VT termination. CONCLUSION: Our results show that implant-based optical cardioversion of VTs is feasible in the pathologically remodelled heart in vivo after local optogenetic targeting because of preserved optical control over bioelectricity generation. These findings add novel mechanistic and translational insight into optical ventricular cardioversion.

IL-23 Enhances C-Fiber-Mediated and Blue Light-Induced Spontaneous Pain in Female Mice.

The incidence of chronic pain is especially high in women, but the underlying mechanisms remain poorly understood. Interleukin-23 (IL-23) is a pro-inflammatory cytokine and contributes to inflammatory diseases (e.g., arthritis and psoriasis) through dendritic/T cell signaling. Here we examined the IL-23 involvement in sexual dimorphism of pain, using an optogenetic approach in transgenic mice expressing channelrhodopsin-2 (ChR2) in TRPV1-positive nociceptive neurons. In situ hybridization revealed that compared to males, females had a significantly larger portion of small-sized (100-200 µm2) Trpv1+ neurons in dorsal root ganglion (DRG). Blue light stimulation of a hindpaw of transgenic mice induced intensity-dependent spontaneous pain. At the highest intensity, females showed more intense spontaneous pain than males. Intraplantar injection of IL-23 (100 ng) induced mechanical allodynia in females only but had no effects on paw edema. Furthermore, intraplantar IL-23 only potentiated blue light-induced pain in females, and intrathecal injection of IL-23 also potentiated low-dose capsaicin (500 ng) induced spontaneous pain in females but not males. IL-23 expresses in DRG macrophages of both sexes. Intrathecal injection of IL-23 induced significantly greater p38 phosphorylation (p-p38), a marker of nociceptor activation, in DRGs of female mice than male mice. In THP-1 human macrophages estrogen and chemotherapy co-application increased IL-23 secretion, and furthermore, estrogen and IL-23 co-application, but not estrogen and IL-23 alone, significantly increased IL-17A release. These findings suggest a novel role of IL-23 in macrophage signaling and female-dominant pain, including C-fiber-mediated spontaneous pain. Our study has also provided new insight into cytokine-mediated macrophage-nociceptor interactions, in a sex-dependent manner.

Viral Transduction of Human Rod Opsin or Channelrhodopsin Variants to Mouse ON Bipolar Cells Does Not Impact Retinal Anatomy or Cause Measurable Death in the Targeted Cells.

The viral gene delivery of optogenetic actuators to the surviving inner retina has been proposed as a strategy for restoring vision in advanced retinal degeneration. We investigated the safety of ectopic expression of human rod opsin (hRHO), and two channelrhodopsins (enhanced sensitivity CoChR-3M and red-shifted ReaChR) by viral gene delivery in ON bipolar cells of the mouse retina. Adult Grm6Cre mice were bred to be retinally degenerate or non-retinally degenerate (homozygous and heterozygous for the rd1Pde6b mutation, respectively) and intravitreally injected with recombinant adeno-associated virus AAV2/2(quad Y-F) serotype containing a double-floxed inverted transgene comprising one of the opsins of interest under a CMV promoter. None of the opsins investigated caused changes in retinal thickness; induced apoptosis in the retina or in transgene expressing cells; or reduced expression of PKCα (a specific bipolar cell marker). No increase in retinal inflammation at the level of gene expression (IBA1/AIF1) was found within the treated mice compared to controls. The expression of hRHO, CoChR or ReaChR under a strong constitutive promoter in retinal ON bipolar cells following intravitreal delivery via AAV2 does not cause either gross changes in retinal health, or have a measurable impact on the survival of targeted cells.

Optogenetic control of cancer cell survival in ChR2-transfected HeLa cells.

Optogenetics is a molecular biological technique involving transfection of cells with photosensitive proteins and the subsequent study of their biological effects. The aim of this study was to evaluate the effect of blue light on the survival of HeLa cells, transfected with channelrhodopsin-2 (ChR2). HeLa wild-type cells were transfected with a plasmid that contained the gene for ChR2. Transfection and channel function were evaluated by real-time polymerase chain reaction (RT-PCR), fluorescence imaging using green fluorescent protein (GFP) and flow cytometry for intracellular calcium changes using a Fura Red probe. We developed a platform for optogenetic stimulation for use within the cell culture incubator. Different stimulation procedures using blue light (467 nm) were applied for up to 24 h. Cell survival was determined by flow cytometry using propidium iodide and rhodamine probes. Change in cell survival showed a statistically significant (p < 0.05) inverse association with the frequency and time of application of the light stimulus. This change seemed to be associated with the ChR2 cis-trans-isomerization cycle. Cell death was associated with high concentrations of calcium in the cytoplasm and stimulation intervals less than the period of isomerization. It is possible to transfect HeLa cells with ChR2 and control their survival under blue light stimulation. We suggest that this practice should be considered in the future development of optogenetic systems in biological or biomedical research.

Corticotropin-Releasing Hormone from the Pontine Micturition Center Plays an Inhibitory Role in Micturition.

Lower urinary tract or voiding disorders are prevalent across all ages and affect >40% of adults over 40 years old, leading to decreased quality of life and high health care costs. The pontine micturition center (PMC; i.e., Barrington's nucleus) contains a large population of neurons that localize the stress-related neuropeptide, corticotropin-releasing hormone (CRH) and project to neurons in the spinal cord to regulate micturition. How the PMC and CRH-expressing neurons in the PMC control volitional micturition is of critical importance for human voiding disorders. To investigate the specific role of CRH in the PMC, neurons in the PMC-expressing CRH were optogenetically activated during in vivo cystometry in unanesthetized mice of either sex. Optogenetic activation of CRH-PMC neurons led to increased intermicturition interval and voided volume, similar to the altered voiding phenotype produced by social stress. Female mice showed a significantly more pronounced phenotype change compared with male mice. These effects were eliminated by CRH-receptor 1 antagonist pretreatment. Optogenetic inhibition of CRH-PMC neurons led to an altered voiding phenotype characterized by more frequent voids and smaller voided volumes. Last, in a cyclophosphamide cystitis model of bladder overactivity, optogenetic activation of CRH-PMC neurons returned the voiding pattern to normal. Collectively, our findings demonstrate that CRH from PMC spinal-projecting neurons has an inhibitory function on micturition and is a potential therapeutic target for human disease states, such as voiding postponement, urinary retention, and underactive or overactive bladder.SIGNIFICANCE STATEMENT The pontine micturition center (PMC), which is a major regulator of volitional micturition, is neurochemically heterogeneous, and excitatory neurotransmission derived from PMC neurons is thought to mediate the micturition reflex. In the present study, using optogenetic manipulation of CRH-containing neurons in double-transgenic mice, we demonstrate that CRH, which is prominent in PMC-spinal projections, has an inhibitory function on volitional micturition. Moreover, engaging this inhibitory function of CRH can ameliorate bladder hyperexcitability induced by cyclophosphamide in a model of cystitis. The data underscore CRH as a novel target for the treatment of voiding dysfunctions, which are highly prevalent disease processes in children and adults.

Current Topics of Optogenetics for Medical Applications Toward Therapy.

The optogenetics approach uses a combination of genetic and optical methods to initiate and control functions in specific cells of biological tissues. Since the high-speed control of neuronal activity by irradiating channelrhodopsin-2 with blue light was reported in 2005, tremendous advancement and application of optogenetics in the field of neuroscience, such as in studies that associate neuronal activity with behaviors, have been initiated. Optogenetics is not only used as a research tool, but is also started to apply in the diagnosis of a disease or as therapy in various studies. Here, I summarize current reports on therapy using a typical photopigment used in optogenetics, channelrhodopsin-2.

The functional characteristics of optogenetic gene therapy for vision restoration.

Optogenetic strategies to restore vision in patients blind from end-stage retinal degenerations aim to render remaining retinal neurons light-sensitive. We present an innovative combination of multi-electrode array recordings together with a complex pattern-generating light source as a toolset to determine the extent to which neural retinal responses to complex light stimuli can be restored following viral delivery of red-shifted channelrhodopsin in the retinally degenerated mouse. Our data indicate that retinal output level spatiotemporal response characteristics achieved by optogenetic gene therapy closely parallel those observed for normal mice but equally reveal important limitations, some of which could be mitigated using bipolar-cell targeted gene-delivery approaches. As clinical trials are commencing, these data provide important new information on the capacity and limitations of channelrhodopsin-based gene therapies. The toolset we established enables comparing optogenetic constructs and stem-cell-based techniques, thereby providing an efficient and sensitive starting point to identify future approaches for vision restoration.

Selecting Channelrhodopsin Constructs for Optimal Visual Restoration in Differing Light Conditions.

Channelrhodopsin (ChR)-based optogenetics is one promising approach to restore vision in photoreceptor degenerative diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD). Currently, a large number of ChRs from different alga species as well as engineered variants are available. They vary with their light response properties like peak sensitive wavelength (λmax), current amplitude, and kinetics. Therefore, it is important to choose an appropriate ChR for practical applications, such as vision restoration. Here we describe a standard laboratory protocol for characterizing properties of ChRs in in vitro in human embryonic kidney (HEK) cells. Based on such characterization, we also discuss the criteria for selecting optimal ChRs for optogenetic vision restoration.

Application of Targeting-Optimized Chronos for Stimulation of the Auditory Pathway.

In the last 15 years, optogenetics has revolutionized the life sciences and enabled studies of complex biological systems such as the brain. Applying optogenetics also has great potential for restorative medicine, such as hearing restoration, by stimulating genetically modified spiral ganglion neurons of the cochlea with light. To this end, opsins with short closing kinetics are required, given the high firing rates and utmost temporal precision of spiking in these neurons. Chronos is the fastest native blue channelrhodopsin (ChR) reported so far with a closing kinetics bellow 1 ms at body temperature and an interesting candidate for the development of the future optogenetic cochlear implants. This book chapter explains in more details the development and application of Chronos with optimized membrane targeting for temporally precise optical stimulation of spiral ganglion neurons. In addition, the generation of adeno-associated virus (AAV) and AAV delivery to the cochlea of postnatal mice and the procedure to record optically evoked auditory brainstem responses are described.