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Towards optogenetic approaches for hearing restoration
Biochemical and Biophysical Research Communications xxx (xxxx) xxx
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Towards optogenetic approaches for hearing restoration Tobias Moser a, b, c, *, Alexander Dieter a, d €ttingen, 37075, Go €ttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Go €ttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, 37077, Go c €ttingen, Germany Auditory Neuroscience Group, Max Planck Institute of Experimental Medicine, 37075, Go d € €ttingen, 37075, Go €ttingen, Germany Gottingen Graduate School for Neurosciences and Molecular Biosciences, University of Go a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2019 Accepted 23 December 2019 Available online xxx
Hearing impairment (HI) is the most frequent sensory deficit in humans. As yet there is no causal therapy for sensorineural HI - the most common form e that results from cochlear dysfunction. Hearing aids and electrical cochlear implants (eCIs) remain the key options for hearing rehabilitation. The eCI, used by more than 0.7 Mio people with profound HI or deafness, is considered the most successful neuroprosthesis as it typically enables open speech comprehension in quiet. By electrically stimulating the auditory nerve, eCIs constitute a brain-machine interface re-connecting the patient with the auditory scene. Nonetheless, there are short-comings resulting from the wide spread of electric current inside the cochlea which limit the quality of artificial hearing. Since light can be better confined in space than electric current, optogenetic stimulation of the auditory nerve has been suggested as an alternative approach for hearing restoration, enabling higher resolution of artificial sound encoding. Future optical CIs (oCIS) promise increased spectral selectivity of artificial sound encoding, and hence might improve speech recognition in background noise as well as processing of music. © 2020 Elsevier Inc. All rights reserved.
Keywords: Neurotechnology Optics Cochlear implant Gene therapy Virus Channelrhodopsin
1. Current state of the art of hearing restoration and limitations Over 5% of the world’s population 466 million people e suffers from disabling HI (432 million adults and 34 million children) [1], with approximately one third of people being over the age of 65. Disabling hearing loss is defined as a hearing impairment greater than 40 dB (dB) in the better hearing ear in adults and 30 dB in children and impacts the individual’s ability to communicate with others if left untreated. HI reduces chances in the job market, causes social isolation, increases the risks of depression and cognitive decline, and poses an annual global cost of US$750 billion. WHO estimates that by 2050, over 900 million people e or one in every ten people e will suffer from disabling hearing loss. The majority of HI is due to disorders of the cochlea (see Fig. 1a for a primer of the function of the ear). Next to genetic causes, HI frequently commences throughout lifetime due to noise, ototoxic drugs, ischemia, trauma or infection damaging the cochlea. Degeneration of sensory cells and auditory neurons is a major
* Corresponding author. Institute for Auditory Neuroscience and InnerEarLab, €ttingen, 37075, Go €ttingen, Germany. University Medical Center Go E-mail address: [email protected] (T. Moser).
common outcome of these disorders. So far, a causal treatment of sensorineural hearing impairment is lacking. Regenerative approaches are pursued, and a first clinical gene therapy study of the ear aims to regenerate hair cells from supporting cells by forced expression of the transcription factor Atoh1. Several preclinical gene replacement therapy studies have been performed, demonstrating proof-of-principle typically in mouse models of human genetic deafness. Clinical translation will require appropriate timing of the intervention prior to cochlear degeneration and remain limited to subset of the more than 150 known deafness genes (for reviews on regenerative and gene therapeutic efforts refer to Refs. [2e4]). However, currently and likely also in the medium-term future, hearing aids and electrical cochlear implants (eCIs) provide the options of choice for hearing rehabilitation in the majority of hearing impaired, depending on the severity and site of lesion. Hearing aids analyze surrounding acoustic signals and provide the ear with an amplified and preprocessed version, via either an acoustic speaker or bone coupling. When sufficient hearing rehabilitation cannot be achieved by hearing aids in profound hearing impairment or deafness, eCIs are employed. eCIs directly stimulate cochlear spiral ganglion neurons (SGNs), thereby bypassing the dysfunctional or lacking hair cells
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Please cite this article as: T. Moser, A. Dieter, Towards optogenetic approaches for hearing restoration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.126
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Fig. 1. Natural vs. artificial activation of the auditory pathway. (a) Air pressure waves (left) are relayed into the cochlea via the ossicles. The resulting travelling wave is decomposed along the cochlear axis depending on its frequency and amplitude (center), activating mechanosensitive hair cells (red) which in turn pass the information to spiral ganglion neurons (yellow; right). (b/c) In case of deafness, e.g. due to loss of hair cells, cochlear implants extract predominant frequencies from the auditory scene. These frequencies are than mapped to electrodes (eCI, b) or optical emitters (oCI, c) close to the cochlear position that would naturally be activated by these frequencies and directly activate the auditory nerve at the respective location by electric current (b) or light (c). As optical stimulation can be better confined in space than electric current, oCIs might activate the auditory nerve with superior spatial (i.e. spectral) selectivity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
(Fig. 1B). While the eCI is considered the most successful neuroprosthesis, there is still room for improvement: Patients have trouble understanding speech in noisy backgrounds and often do not enjoy music. The largest bottleneck for hearing restoration by eCIs is the wide spread of electric current from each of the 12e24 electrode contacts, resulting in broad neural excitation of SGNs, which restricts the frequency resolution of artificial sound encoding. In order to improve the performance of eCIs current steering using multipolar stimulation [6] as well as intraneural stimulation [7] are being employed. As light can be better spatially confined, optogenetic stimulation of the auditory nerve might overcome this bottleneck of the eCI. Hence, future oCIs (Fig. 1C) promise increased spectral selectivity of artificial sound encoding, and hence might improve speech recognition in background noise as well as processing of music. 2. Current state of the biomedical and optoelectronic developments for future optical CIs General considerations: The development of optical cochlear
implants (oCIs) requires multidisciplinary biomedical and optical engineering research as it builds on a combination of gene therapy and an optoelectronic medical device. Over recent years, major efforts have been made to establish gene therapeutic approaches for sensory restoration. Here, the retina has been at the forefront of gene therapy with recent approval by the Food and Drug Administration for an adeno-associated virus (AAV) based gene therapy treating juvenile Leber’s Congenital Amaurosis (Luxturna, Allergan). Retina and cochlea share advantages as gene therapy targets as both require tiny vector doses and are said to be immune privileged due to the blood-retinal/labyrinth-barrier and the local inhibition of immune responses by the unique intraocular/ intracochlear microenvironment. However, in contrast to optogenetic manipulation of retinal cells, optogenetic manipulation of SGNs that give rise to the auditory nerve bears specific challenges as the somata of SGNs hide in Rosenthal’s canal of the modiolus, the bony core of the cochlea. As the biomedical approaches can learn from optogenetic vision restoration, the development of the medical device oCI can, at least to some extent, build on the longstanding experience with
Please cite this article as: T. Moser, A. Dieter, Towards optogenetic approaches for hearing restoration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.126
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implementing and improving hearing rehabilitation with eCIs. Audio processing, transcranial signal and power transmission, as well as atraumatic insertion of a flexible and well encapsulated stimulating front-end of the implant into the fluid-filled natural cavity of scala tympani via the round window can be similarly implemented for the oCI. The switch to optical stimulation does, however, generate advantages and challenges that will be discussed below. Recent experiments employing optical fiber-based cochlear optogenetics in rodents whose SGNs have been optogenetically modified made considerable progress towards these goals [8e13]. 2.1. Biomedical development The first proof of principle study reported optogenetic activation of the auditory system [8] in transgenic mice and rats neurally expressing channelrhodopsin-2 (ChR2) under the Thy1.2 promoter [14]. Functional expression was demonstrated by ChR2-EYFP immunofluorescence in SGNs as well as optically evoked far-field potentials (interpreted as optical auditory brainstem responses, oABRs), single auditory nerve fiber firing and local field potentials in the auditory midbrain. The latter, when compared to acoustic and electric stimulation of the auditory nerve, indicated the feasibility of improved spectral selectivity at low light levels (threshold around 2 mJ). However, the temporal fidelity of optogenetic coding was relatively poor (<80 Hz) compared to the physiological firing rate of SNGs (a few hundreds of Hz). Optogenetic activation of the auditory nerve was also demonstrated in mice upon injections of adeno-associated virus (AAV2/6) carrying a calcium-translocating ChR2-variant (CatCh) [15] under the human synapsin promoter into the embryonic otocyst [8]. Follow-up studies used early postnatal intracochlear virus injections into the mouse inner ear. They corroborated optogenetic activation of the auditory pathway, employed fast channelrhodopsins (ChRs) and provided a more detailed characterization of optogenetic coding of temporal information with improved fidelity. Injections of AAV2/6 carrying the red-light shifted, fast-gating ChRvariant f-Chrimson under the human synapsin promoter enabled oABRs with low thresholds (0.5 mJ) up to 200 Hz and auditory nerve fiber responses up to a few hundred Hz [10]. In a second study, injections of the potent AAV-PHP.B carrying the fast ChR Chronos, containing ER-exiting and Golgi-trafficking sequences of an inwardly rectifying potassium channel, under the human synapsin promoter enabled SGN responses at the population and single neuron level up to a few hundreds of Hz [16]. Hence both fChrimson and Chronos support SGN firing at near physiological rates. Both studies achieved high transduction rates across all frequency (tonotopic) regions in the injected ear and did not cause obvious SGN-loss for the vast majority of injected mice [10,11]. In these studies, latency (~1 ms), waveform (3e5 waves) and amplitude (<1 mVe10 mV) of oABRs elicited by strong stimuli were similar to acoustic ABRs in mice, reflecting rather specific optogenetic activation of the auditory nerve in the injected ear. However, spread of virus was evident by ChR-expression in the non-injected contralateral ear. Another study performed in a different lab involved injection of AAV-Anc80 carrying the original Chronos under the control of the CAG promoter into the postnatal mouse ear and demonstrated optically evoked potentials that might have involved hair cell activation, as CAG promotes expression also beyond SGNs even with local AAV application to the inner ear [12]. Adult Mongolian gerbils, which have a frequency hearing range closer to humans, were targeted to achieve virus-mediated optogenetic SGN manipulation in an adult rodent preparation [9]. Direct injection of AAV2/6 carrying CatCh under the human synapsin promoter into the modiolus yielded CatCh expression in SGNs
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across all tonotopic regions with an average rate of 30% in approximately half of the injected animals. oABRs could be elicited with as little as 10% of the SGNs expressing CatCh. oABR amplitudes were smaller than in mice (typically up to 1 mV) but shared the short latency (~1 ms) and could follow stimulation up to approximately 200 Hz. A mild SGN-loss (20e30%) was found in the injected ear, which likely arose from intramodiolar pressure increase as it was also observed upon saline injection in control animals. Gerbils were then employed for demonstrating perception of optogenetic SGN stimulation [9]: A fiber-based single-channel oCI enabled gerbils to learn stimulus-cued avoidance behavior with laser thresholds below 2 mJ. Animals transferred the avoidance behavior from optogenetic to acoustic stimulation which indicates some generalization from perception of optogenetic stimuli to acoustic ones. The CatCh-expressing adult gerbil has recently also been used to comprehensively study the spectral selectivity of natural and artificial SGN stimulation [13]. Multi-channel recordings of neuronal cluster activity were performed in the auditory midbrain while stimulating the contralateral cochlea by acoustic, optical or electric means. Optical stimulation was performed with up to three lasercoupled optical fibers placed at distinct positions along the cochlear tonotopic axis and spatially selective neuronal activity was observed in tonotopically corresponding regions of the auditory midbrain [13]. Acoustic stimulation and stimulation via eCIs were done in naïve animals and an activity-based analysis enabled comparison of the spectral selectivity of acoustic, optogenetic, and electric SGN stimulation. Optogenetic stimulation was more selective than monopolar electrical stimulation at all activation strengths and outperformed bipolar electrical stimulation at medium and high activation strengths (as much as 2.04- and 1.94-fold, respectively). Optogenetic SGN stimulation was as selective as acoustic stimulation at low and modest activation levels, but caused broader activation at higher stimulus intensities (Fig. 2 [13]). The improved spectral selectivity demonstrated by physiological assessment was corroborated by Monte Carlo ray tracing simulations of light propagation in the cochlea [9,13]. The model further indicates that spectral selectivity of optical stimulation can be further improved by emitters with smaller aperture and narrower beam profile as well as close emitter proximity to the neural target tissue. Several of these rodent studies have demonstrated the feasibility of optogenetic hearing restoration by providing physiological or behavioral evidence for SGN stimulation in the deafened cochlea. In ChR-2 transgenic mice, oABRs could still be evoked after pharmacological or genetic interference with acoustic hearing [8]. Moreover, optogenetic SGN stimulation re-activated the auditory system on a physiological and on a behavioral level in gerbils following ototoxic deafening by aminoglycosides [9]. Stability and biosafety of opsin expression over time have been shown in mice: robust oABRs and stable expression of f-Chrimson without SGN loss have been observed 9 months after injection [10]. A different study found similar oABR appearance and expression levels of Chronos in the auditory nerve of mice 6e18 weeks after virus injection [12]. Finally, repeated oABR measurements in gerbils implanted with optical fibers showed stable responses to optogenetic SGN stimulation over more than 100 days after implantation [9]. Future experiments need to integrate these approaches and demonstrate the spectral specificity of cochlear optogenetics on a behavioral level, ideally in a longitudinal way and combined with biosafety studies. 2.2. Towards the development of multichannel optoelectronic cochlear implants The engineering of multi-channel oCIs is rapidly progressing
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Fig. 2. Spectral selectivity in the auditory system. Neural activation (color-coded in d’ units) across the auditory midbrain (ordinate) upon (A) acoustic, (B) optogenetic, (C) monopolar and (D) bipolar electric activation of the auditory nerve with increasing stimulus intensity (abscissa). Data is reprinted from Ref. [13]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
and employs two basic strategies: active oCIs that bring optoelectronic emitters into the cochlea and passive oCIs that use waveguides to pipe the light of extracochlear emitters into the cochlea. The design requirements include power-efficient optical emitters for acceptable energy budget and heat liberation, narrow beam profile and appropriate positioning of emitters in scala tympani, suitable form-factor and low stiffness despite integration of tens to hundreds of stimulation channels. Finally, oCIs must be biocompatible, safe and long-term stable as oCIs need to work over decades. Active oCIs: Active oCIs offer the advantage of bringing the lightgenerating elements, such as light emitting diodes (LEDs), in close proximity to the SGNs and thereby avoid light loss during in- and outcoupling into and out of waveguides. Current implementations have considered LEDs as well as laser diodes. Commercially available LEDs (fabricated by Cree Europe GmbH; emission peak: 460 nm) were flip-chip bonded on a flexible polyimide substrate of 20 mm length and 0.24 mm width [17]. Ten LEDs of 220 270 mm with a pitch of either 350 or 500 mm were employed and could be individually addressed. Driven with a current of 5 mA, the optical power of LEDs amounted to ~0.3 mW (and could be as high as 1.5 mW when driven with 45 mA) [18]. Another study employed 15 LEDs (1 0.6 mm; max. 34 mW at 470 nm) embedded in biocompatible silicone and implanted this oCI into a human scala tympani model with insertion forces comparable to commercially available eCIs [19]. Custom-made thin film microscale (m)LEDs with dimensions as small as 50 50 mm have been developed [20,21] and parallelized wafer-level fabrication has been described [20]. Such an oCI with a total width of 230 mm and a length of 5 mm
housed four mLEDs of 50 50 15 mm and could be inserted into a mouse cochlea via the round window. Based on the established wafer-level processes, oCIs of 350 mm width and 15 mm length with a total of 144 individually addressable mLEDs of 50 50 mm have been engineered recently [21]. In this next generation mLED-based oCIs, the fully epoxy-based carrier minimizes the thermomechanical bending. Furthermore, the optical power is substantially increased: at 10 mA, the output power (at a wavelength of 462 nm) amounted to 0.82 mW. Finally, a maximum temperature increase of 1 C was measured when driving the mLEDs with DC pulses of ~20 ms duration and 10 mA intensity when the implant was placed on agarose, which makes these implants suitable for in vivo application [21]. In a subsequent study, the optical properties of these mLED-based oCIs have been improved by adding conical concentrators and spherical micro-lenses made from polydimethylsiloxane [22]. Long-time stable, transparent encapsulation that maintains sufficient flexibility of the oCI will be required for translation of active oCIs. The challenge is to achieve sufficient barrier function towards water vapor and ions without risking a brittle and eventually cracking oCI in order to protect the optoelectronic components and vice versa avoid diffusion of oCI particles into the cochlea. Passive oCIs: Passive oCIs use waveguides to deliver light from external sources to the cochlea. Hence, they separate optoelectronics from the cochlea and encompass its hermetic sealing as in state of the art eCIs and cardiac pacemakers. Further advantages are stability, biocompatibility, lower cochlear heat generation, and smaller size of the intracochlear light emitters. Disadvantages of passive oCIs are the light loss during in-coupling at the emitter-
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waveguide-interface and along the waveguides [23] as well as potential mechanical susceptibility of this in-coupling. Recently, a passive oCI composed of eight glass fibers of 25 mm diameter which were embedded in silicone has been reported [24]. Insertion forces for this device measured were comparable to those with a conventional eCI and insertion up to 20 mm depth was possible also in human temporal bones [24]. Polymer-based waveguides which have been manufactured with core thicknesses below 10 mm offer an alternative approach, especially when considering flexibility and the number of independent stimulation channels [19,23,25]. However, light propagation of polymer fibers is limited in a wavelength-dependent manner. 3. Outlook The gene therapy component: In order to pave the way for clinical application, efficiency, stability and biosafety of cochlear optogenetics are of utmost importance. Thus, the construct to render the auditory nerve light sensitive should be designed to enable robust optogenetic activation of SGNs at physiological firing rates and with low light requirements. Also, the vector needed for neural transduction should be optimized in order to safely and efficiently deliver its genetic load. AAVs will likely be the vectors of choice, since they enable foreign protein to be expressed at high levels and over long periods of time. Furthermore, AAVs have little risk for adverse reactions of the target cells [1], are used in various clinical trials for gene therapy (including retinal dysfunction) and have been successfully used to restore auditory function in animal models of deafness [2]. Most recently, two clinical trials involving AAV-mediated optogenetic vision restoration have been approved (NCT02556736 and NCT03326336), corroborating the potential of optogenetic therapies for neural restoration. The implementation of adequate promotors as well as local virus application in the ossified cochlea will contribute to selective transduction of SGNs while excluding opsin expression beyond the auditory nerve. Inspired by retinal optogenetics, tissue explants and inner ear organoids might help evaluating the construct of choice and optimize efficiency and specificity in human SNGs [21,25e27]. Upon successful implementation of the gene therapeutic part of optogenetic hearing restoration, also the effect of chronic illumination of intracochlear neural tissue needs to be evaluated. Safety limits for light intensity need to be defined in order to avoid tissue heating or phototoxic effects. Here, the implementation of opsins with a red-shifted action spectrum will reduce the risk of tissue damage, since radiation in the red spectrum is reportedly less phototoxic (safety limits for retinal exposure defined by the European commission (2006/25/EC) are three orders of magnitude higher for orange light as compared to red light [3]). Together these considerations will foster the conservation of cochlear structures and offtarget tissues and guarantee a maximum of safety for the patient. The medical device component: Unlike typical gene therapy, the optogenetic manipulation of the SGNs is not aimed to restore hearing by itself. It requires the combination with the oCI as a medical device e consisting of an external sound processor, an internal light emitting implant, as well as an appropriate coding strategy and communication between these devices. While communication and processor technology might readily be adapted from eCIs, implemented restrictions in the temporal domain (due to opsin kinetics) and the increase in stimulation channels most likely require mayor re-working of coding strategies. The minimal duration and intensity of light pulse sufficient for SGN activation will depend on channel kinetics of the chosen optogenetic tool. From this quantum, the sound intensity coding should utilize the full range of optogenetically evoked SGN activation at their natural firing rates until the upper limit is reached, where firing saturates
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or behavior signals discomfort. The energy of a single pulse will need to be balanced between its duration (which needs to be limited in order to allow high stimulation rates) and its intensity (which is restricted by biosafety limits) and should furthermore allow for reasonable battery lifetimes of oCIs. The resulting patterns of optical stimulation will then be mapped on an e compared to electrical CIs e increased set of independent stimulation channels in order to encode spectral information. While the final number of intracochlear optical emitters remains to be defined, the spectral selectivity of optogenetic SGN stimulation has been estimated two outperform electrical coding by a factor of at least two. As contemporary eCIs house between 12 and 24 electrodes, a range of 50e100 optical emitters seems like a reasonable number to aim for [4]. Acknowledgment The work was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 670759 e advanced grant “OptoHear”) to T.M. A.D. is a fellow of the German Academic Scholarship Foundation. We would like to thank Dr. Jakob Neef for help on the figures and manuscript. References [1] Deafness and hearing loss, World Health Organ.. (n.d.). http://www.who.int/ news-room/fact-sheets/detail/deafness-and-hearing-loss (accessed September 16, 2018). [2] H. Li, C.E. Corrales, A. Edge, S. Heller, Stem cells as therapy for hearing loss, Trends Mol. Med. 10 (2004) 309e315, https://doi.org/10.1016/ j.molmed.2004.05.008. [3] H. Ahmed, O. Shubina-Oleinik, J.R. Holt, Emerging gene therapies for genetic hearing loss, J. Assoc. Res. Otolaryngol. 18 (2017) 649e670, https://doi.org/ 10.1007/s10162-017-0634-8. [4] T. Moser, Gene therapy for deafness: how close are we? Sci. Transl. Med. 7 (2015) 295fs28, https://doi.org/10.1126/scitranslmed.aac7545. [6] C.K. Berenstein, L.H.M. Mens, J.J.S. Mulder, F.J. Vanpoucke, Current steering and current focusing in cochlear implants: comparison of monopolar, tripolar, and virtual channel electrode configurations: Ear Hear. 29 (2008) 250e260, https://doi.org/10.1097/AUD.0b013e3181645336. [7] J.C. Middlebrooks, R.L. Snyder, Auditory prosthesis with a penetrating nerve array, J. Assoc. Res. Otolaryngol. JARO. 8 (2007) 258e279, https://doi.org/ 10.1007/s10162-007-0070-2. [8] V.H. Hernandez, A. Gehrt, K. Reuter, Z. Jing, M. Jeschke, A. Mendoza Schulz, G. Hoch, M. Bartels, G. Vogt, C.W. Garnham, H. Yawo, Y. Fukazawa, G.J. Augustine, E. Bamberg, S. Kügler, T. Salditt, L. de Hoz, N. Strenzke, T. Moser, Optogenetic stimulation of the auditory pathway, J. Clin. Investig. 124 (2014) 1114e1129, https://doi.org/10.1172/JCI69050. [9] C. Wrobel, A. Dieter, A. Huet, D. Keppeler, C.J. Duque-Afonso, C. Vogl, G. Hoch, M. Jeschke, T. Moser, Optogenetic stimulation of cochlear neurons activates the auditory pathway and restores auditory-driven behavior in deaf adult gerbils, Sci. Transl. Med. 10 (2018), https://doi.org/10.1126/scitranslmed.aao0540 eaao0540. [10] T. Mager, D. Lopez de la Morena, V. Senn, J. Schlotte, A. D Errico, K. Feldbauer, C. Wrobel, S. Jung, K. Bodensiek, V. Rankovic, L. Browne, A. Huet, J. Jüttner, P.G. Wood, J.J. Letzkus, T. Moser, E. Bamberg, High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics, Nat. Commun. 9 (2018) 1750, https://doi.org/10.1038/s41467-018-04146-3. [11] D. Keppeler, R.M. Merino, D.L. de la Morena, B. Bali, A.T. Huet, A. Gehrt, C. Wrobel, S. Subramanian, T. Dombrowski, F. Wolf, V. Rankovic, A. Neef, T. Moser, Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos, EMBO J. 37 (2018), e99649, https://doi.org/ 10.15252/embj.201899649. [12] M.J. Duarte, V.V. Kanumuri, L.D. Landegger, O. Tarabichi, S. Sinha, X. Meng, A.E. Hight, E.D. Kozin, K.M. Stankovic, M.C. Brown, D.J. Lee, Ancestral adenoassociated virus vector delivery of opsins to spiral ganglion neurons: implications for optogenetic cochlear implants, Mol. Ther. 26 (2018) 1931e1939, https://doi.org/10.1016/j.ymthe.2018.05.023. [13] A. Dieter, C.J. Duque-Afonso, V. Rankovic, M. Jeschke, T. Moser, Near physiological spectral selectivity of cochlear optogenetics, Nat. Commun. 10 (2019) 1962, https://doi.org/10.1038/s41467-019-09980-7. [14] B.R. Arenkiel, J. Peca, I.G. Davison, C. Feliciano, K. Deisseroth, G.J. Augustine, M.D. Ehlers, G. Feng, In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2, Neuron 54 (2007) 205e218, https://doi.org/10.1016/j.neuron.2007.03.005. [15] S. Kleinlogel, K. Feldbauer, R.E. Dempski, H. Fotis, P.G. Wood, C. Bamann,
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Towards optogenetic approaches for hearing restoration Tobias Moser a, b, c, *, Alexander Dieter a, d €ttingen, 37075, Go €ttingen, Germany Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Go €ttingen, Germany Auditory Neuroscience and Optogenetics Laboratory, German Primate Center, 37077, Go c €ttingen, Germany Auditory Neuroscience Group, Max Planck Institute of Experimental Medicine, 37075, Go d € €ttingen, 37075, Go €ttingen, Germany Gottingen Graduate School for Neurosciences and Molecular Biosciences, University of Go a
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a r t i c l e i n f o
a b s t r a c t
Article history: Received 22 December 2019 Accepted 23 December 2019 Available online xxx
Hearing impairment (HI) is the most frequent sensory deficit in humans. As yet there is no causal therapy for sensorineural HI - the most common form e that results from cochlear dysfunction. Hearing aids and electrical cochlear implants (eCIs) remain the key options for hearing rehabilitation. The eCI, used by more than 0.7 Mio people with profound HI or deafness, is considered the most successful neuroprosthesis as it typically enables open speech comprehension in quiet. By electrically stimulating the auditory nerve, eCIs constitute a brain-machine interface re-connecting the patient with the auditory scene. Nonetheless, there are short-comings resulting from the wide spread of electric current inside the cochlea which limit the quality of artificial hearing. Since light can be better confined in space than electric current, optogenetic stimulation of the auditory nerve has been suggested as an alternative approach for hearing restoration, enabling higher resolution of artificial sound encoding. Future optical CIs (oCIS) promise increased spectral selectivity of artificial sound encoding, and hence might improve speech recognition in background noise as well as processing of music. © 2020 Elsevier Inc. All rights reserved.
Keywords: Neurotechnology Optics Cochlear implant Gene therapy Virus Channelrhodopsin
1. Current state of the art of hearing restoration and limitations Over 5% of the world’s population 466 million people e suffers from disabling HI (432 million adults and 34 million children) [1], with approximately one third of people being over the age of 65. Disabling hearing loss is defined as a hearing impairment greater than 40 dB (dB) in the better hearing ear in adults and 30 dB in children and impacts the individual’s ability to communicate with others if left untreated. HI reduces chances in the job market, causes social isolation, increases the risks of depression and cognitive decline, and poses an annual global cost of US$750 billion. WHO estimates that by 2050, over 900 million people e or one in every ten people e will suffer from disabling hearing loss. The majority of HI is due to disorders of the cochlea (see Fig. 1a for a primer of the function of the ear). Next to genetic causes, HI frequently commences throughout lifetime due to noise, ototoxic drugs, ischemia, trauma or infection damaging the cochlea. Degeneration of sensory cells and auditory neurons is a major
* Corresponding author. Institute for Auditory Neuroscience and InnerEarLab, €ttingen, 37075, Go €ttingen, Germany. University Medical Center Go E-mail address: [email protected] (T. Moser).
common outcome of these disorders. So far, a causal treatment of sensorineural hearing impairment is lacking. Regenerative approaches are pursued, and a first clinical gene therapy study of the ear aims to regenerate hair cells from supporting cells by forced expression of the transcription factor Atoh1. Several preclinical gene replacement therapy studies have been performed, demonstrating proof-of-principle typically in mouse models of human genetic deafness. Clinical translation will require appropriate timing of the intervention prior to cochlear degeneration and remain limited to subset of the more than 150 known deafness genes (for reviews on regenerative and gene therapeutic efforts refer to Refs. [2e4]). However, currently and likely also in the medium-term future, hearing aids and electrical cochlear implants (eCIs) provide the options of choice for hearing rehabilitation in the majority of hearing impaired, depending on the severity and site of lesion. Hearing aids analyze surrounding acoustic signals and provide the ear with an amplified and preprocessed version, via either an acoustic speaker or bone coupling. When sufficient hearing rehabilitation cannot be achieved by hearing aids in profound hearing impairment or deafness, eCIs are employed. eCIs directly stimulate cochlear spiral ganglion neurons (SGNs), thereby bypassing the dysfunctional or lacking hair cells
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Please cite this article as: T. Moser, A. Dieter, Towards optogenetic approaches for hearing restoration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.126
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Fig. 1. Natural vs. artificial activation of the auditory pathway. (a) Air pressure waves (left) are relayed into the cochlea via the ossicles. The resulting travelling wave is decomposed along the cochlear axis depending on its frequency and amplitude (center), activating mechanosensitive hair cells (red) which in turn pass the information to spiral ganglion neurons (yellow; right). (b/c) In case of deafness, e.g. due to loss of hair cells, cochlear implants extract predominant frequencies from the auditory scene. These frequencies are than mapped to electrodes (eCI, b) or optical emitters (oCI, c) close to the cochlear position that would naturally be activated by these frequencies and directly activate the auditory nerve at the respective location by electric current (b) or light (c). As optical stimulation can be better confined in space than electric current, oCIs might activate the auditory nerve with superior spatial (i.e. spectral) selectivity. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
(Fig. 1B). While the eCI is considered the most successful neuroprosthesis, there is still room for improvement: Patients have trouble understanding speech in noisy backgrounds and often do not enjoy music. The largest bottleneck for hearing restoration by eCIs is the wide spread of electric current from each of the 12e24 electrode contacts, resulting in broad neural excitation of SGNs, which restricts the frequency resolution of artificial sound encoding. In order to improve the performance of eCIs current steering using multipolar stimulation [6] as well as intraneural stimulation [7] are being employed. As light can be better spatially confined, optogenetic stimulation of the auditory nerve might overcome this bottleneck of the eCI. Hence, future oCIs (Fig. 1C) promise increased spectral selectivity of artificial sound encoding, and hence might improve speech recognition in background noise as well as processing of music. 2. Current state of the biomedical and optoelectronic developments for future optical CIs General considerations: The development of optical cochlear
implants (oCIs) requires multidisciplinary biomedical and optical engineering research as it builds on a combination of gene therapy and an optoelectronic medical device. Over recent years, major efforts have been made to establish gene therapeutic approaches for sensory restoration. Here, the retina has been at the forefront of gene therapy with recent approval by the Food and Drug Administration for an adeno-associated virus (AAV) based gene therapy treating juvenile Leber’s Congenital Amaurosis (Luxturna, Allergan). Retina and cochlea share advantages as gene therapy targets as both require tiny vector doses and are said to be immune privileged due to the blood-retinal/labyrinth-barrier and the local inhibition of immune responses by the unique intraocular/ intracochlear microenvironment. However, in contrast to optogenetic manipulation of retinal cells, optogenetic manipulation of SGNs that give rise to the auditory nerve bears specific challenges as the somata of SGNs hide in Rosenthal’s canal of the modiolus, the bony core of the cochlea. As the biomedical approaches can learn from optogenetic vision restoration, the development of the medical device oCI can, at least to some extent, build on the longstanding experience with
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implementing and improving hearing rehabilitation with eCIs. Audio processing, transcranial signal and power transmission, as well as atraumatic insertion of a flexible and well encapsulated stimulating front-end of the implant into the fluid-filled natural cavity of scala tympani via the round window can be similarly implemented for the oCI. The switch to optical stimulation does, however, generate advantages and challenges that will be discussed below. Recent experiments employing optical fiber-based cochlear optogenetics in rodents whose SGNs have been optogenetically modified made considerable progress towards these goals [8e13]. 2.1. Biomedical development The first proof of principle study reported optogenetic activation of the auditory system [8] in transgenic mice and rats neurally expressing channelrhodopsin-2 (ChR2) under the Thy1.2 promoter [14]. Functional expression was demonstrated by ChR2-EYFP immunofluorescence in SGNs as well as optically evoked far-field potentials (interpreted as optical auditory brainstem responses, oABRs), single auditory nerve fiber firing and local field potentials in the auditory midbrain. The latter, when compared to acoustic and electric stimulation of the auditory nerve, indicated the feasibility of improved spectral selectivity at low light levels (threshold around 2 mJ). However, the temporal fidelity of optogenetic coding was relatively poor (<80 Hz) compared to the physiological firing rate of SNGs (a few hundreds of Hz). Optogenetic activation of the auditory nerve was also demonstrated in mice upon injections of adeno-associated virus (AAV2/6) carrying a calcium-translocating ChR2-variant (CatCh) [15] under the human synapsin promoter into the embryonic otocyst [8]. Follow-up studies used early postnatal intracochlear virus injections into the mouse inner ear. They corroborated optogenetic activation of the auditory pathway, employed fast channelrhodopsins (ChRs) and provided a more detailed characterization of optogenetic coding of temporal information with improved fidelity. Injections of AAV2/6 carrying the red-light shifted, fast-gating ChRvariant f-Chrimson under the human synapsin promoter enabled oABRs with low thresholds (0.5 mJ) up to 200 Hz and auditory nerve fiber responses up to a few hundred Hz [10]. In a second study, injections of the potent AAV-PHP.B carrying the fast ChR Chronos, containing ER-exiting and Golgi-trafficking sequences of an inwardly rectifying potassium channel, under the human synapsin promoter enabled SGN responses at the population and single neuron level up to a few hundreds of Hz [16]. Hence both fChrimson and Chronos support SGN firing at near physiological rates. Both studies achieved high transduction rates across all frequency (tonotopic) regions in the injected ear and did not cause obvious SGN-loss for the vast majority of injected mice [10,11]. In these studies, latency (~1 ms), waveform (3e5 waves) and amplitude (<1 mVe10 mV) of oABRs elicited by strong stimuli were similar to acoustic ABRs in mice, reflecting rather specific optogenetic activation of the auditory nerve in the injected ear. However, spread of virus was evident by ChR-expression in the non-injected contralateral ear. Another study performed in a different lab involved injection of AAV-Anc80 carrying the original Chronos under the control of the CAG promoter into the postnatal mouse ear and demonstrated optically evoked potentials that might have involved hair cell activation, as CAG promotes expression also beyond SGNs even with local AAV application to the inner ear [12]. Adult Mongolian gerbils, which have a frequency hearing range closer to humans, were targeted to achieve virus-mediated optogenetic SGN manipulation in an adult rodent preparation [9]. Direct injection of AAV2/6 carrying CatCh under the human synapsin promoter into the modiolus yielded CatCh expression in SGNs
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across all tonotopic regions with an average rate of 30% in approximately half of the injected animals. oABRs could be elicited with as little as 10% of the SGNs expressing CatCh. oABR amplitudes were smaller than in mice (typically up to 1 mV) but shared the short latency (~1 ms) and could follow stimulation up to approximately 200 Hz. A mild SGN-loss (20e30%) was found in the injected ear, which likely arose from intramodiolar pressure increase as it was also observed upon saline injection in control animals. Gerbils were then employed for demonstrating perception of optogenetic SGN stimulation [9]: A fiber-based single-channel oCI enabled gerbils to learn stimulus-cued avoidance behavior with laser thresholds below 2 mJ. Animals transferred the avoidance behavior from optogenetic to acoustic stimulation which indicates some generalization from perception of optogenetic stimuli to acoustic ones. The CatCh-expressing adult gerbil has recently also been used to comprehensively study the spectral selectivity of natural and artificial SGN stimulation [13]. Multi-channel recordings of neuronal cluster activity were performed in the auditory midbrain while stimulating the contralateral cochlea by acoustic, optical or electric means. Optical stimulation was performed with up to three lasercoupled optical fibers placed at distinct positions along the cochlear tonotopic axis and spatially selective neuronal activity was observed in tonotopically corresponding regions of the auditory midbrain [13]. Acoustic stimulation and stimulation via eCIs were done in naïve animals and an activity-based analysis enabled comparison of the spectral selectivity of acoustic, optogenetic, and electric SGN stimulation. Optogenetic stimulation was more selective than monopolar electrical stimulation at all activation strengths and outperformed bipolar electrical stimulation at medium and high activation strengths (as much as 2.04- and 1.94-fold, respectively). Optogenetic SGN stimulation was as selective as acoustic stimulation at low and modest activation levels, but caused broader activation at higher stimulus intensities (Fig. 2 [13]). The improved spectral selectivity demonstrated by physiological assessment was corroborated by Monte Carlo ray tracing simulations of light propagation in the cochlea [9,13]. The model further indicates that spectral selectivity of optical stimulation can be further improved by emitters with smaller aperture and narrower beam profile as well as close emitter proximity to the neural target tissue. Several of these rodent studies have demonstrated the feasibility of optogenetic hearing restoration by providing physiological or behavioral evidence for SGN stimulation in the deafened cochlea. In ChR-2 transgenic mice, oABRs could still be evoked after pharmacological or genetic interference with acoustic hearing [8]. Moreover, optogenetic SGN stimulation re-activated the auditory system on a physiological and on a behavioral level in gerbils following ototoxic deafening by aminoglycosides [9]. Stability and biosafety of opsin expression over time have been shown in mice: robust oABRs and stable expression of f-Chrimson without SGN loss have been observed 9 months after injection [10]. A different study found similar oABR appearance and expression levels of Chronos in the auditory nerve of mice 6e18 weeks after virus injection [12]. Finally, repeated oABR measurements in gerbils implanted with optical fibers showed stable responses to optogenetic SGN stimulation over more than 100 days after implantation [9]. Future experiments need to integrate these approaches and demonstrate the spectral specificity of cochlear optogenetics on a behavioral level, ideally in a longitudinal way and combined with biosafety studies. 2.2. Towards the development of multichannel optoelectronic cochlear implants The engineering of multi-channel oCIs is rapidly progressing
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Fig. 2. Spectral selectivity in the auditory system. Neural activation (color-coded in d’ units) across the auditory midbrain (ordinate) upon (A) acoustic, (B) optogenetic, (C) monopolar and (D) bipolar electric activation of the auditory nerve with increasing stimulus intensity (abscissa). Data is reprinted from Ref. [13]. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
and employs two basic strategies: active oCIs that bring optoelectronic emitters into the cochlea and passive oCIs that use waveguides to pipe the light of extracochlear emitters into the cochlea. The design requirements include power-efficient optical emitters for acceptable energy budget and heat liberation, narrow beam profile and appropriate positioning of emitters in scala tympani, suitable form-factor and low stiffness despite integration of tens to hundreds of stimulation channels. Finally, oCIs must be biocompatible, safe and long-term stable as oCIs need to work over decades. Active oCIs: Active oCIs offer the advantage of bringing the lightgenerating elements, such as light emitting diodes (LEDs), in close proximity to the SGNs and thereby avoid light loss during in- and outcoupling into and out of waveguides. Current implementations have considered LEDs as well as laser diodes. Commercially available LEDs (fabricated by Cree Europe GmbH; emission peak: 460 nm) were flip-chip bonded on a flexible polyimide substrate of 20 mm length and 0.24 mm width [17]. Ten LEDs of 220 270 mm with a pitch of either 350 or 500 mm were employed and could be individually addressed. Driven with a current of 5 mA, the optical power of LEDs amounted to ~0.3 mW (and could be as high as 1.5 mW when driven with 45 mA) [18]. Another study employed 15 LEDs (1 0.6 mm; max. 34 mW at 470 nm) embedded in biocompatible silicone and implanted this oCI into a human scala tympani model with insertion forces comparable to commercially available eCIs [19]. Custom-made thin film microscale (m)LEDs with dimensions as small as 50 50 mm have been developed [20,21] and parallelized wafer-level fabrication has been described [20]. Such an oCI with a total width of 230 mm and a length of 5 mm
housed four mLEDs of 50 50 15 mm and could be inserted into a mouse cochlea via the round window. Based on the established wafer-level processes, oCIs of 350 mm width and 15 mm length with a total of 144 individually addressable mLEDs of 50 50 mm have been engineered recently [21]. In this next generation mLED-based oCIs, the fully epoxy-based carrier minimizes the thermomechanical bending. Furthermore, the optical power is substantially increased: at 10 mA, the output power (at a wavelength of 462 nm) amounted to 0.82 mW. Finally, a maximum temperature increase of 1 C was measured when driving the mLEDs with DC pulses of ~20 ms duration and 10 mA intensity when the implant was placed on agarose, which makes these implants suitable for in vivo application [21]. In a subsequent study, the optical properties of these mLED-based oCIs have been improved by adding conical concentrators and spherical micro-lenses made from polydimethylsiloxane [22]. Long-time stable, transparent encapsulation that maintains sufficient flexibility of the oCI will be required for translation of active oCIs. The challenge is to achieve sufficient barrier function towards water vapor and ions without risking a brittle and eventually cracking oCI in order to protect the optoelectronic components and vice versa avoid diffusion of oCI particles into the cochlea. Passive oCIs: Passive oCIs use waveguides to deliver light from external sources to the cochlea. Hence, they separate optoelectronics from the cochlea and encompass its hermetic sealing as in state of the art eCIs and cardiac pacemakers. Further advantages are stability, biocompatibility, lower cochlear heat generation, and smaller size of the intracochlear light emitters. Disadvantages of passive oCIs are the light loss during in-coupling at the emitter-
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waveguide-interface and along the waveguides [23] as well as potential mechanical susceptibility of this in-coupling. Recently, a passive oCI composed of eight glass fibers of 25 mm diameter which were embedded in silicone has been reported [24]. Insertion forces for this device measured were comparable to those with a conventional eCI and insertion up to 20 mm depth was possible also in human temporal bones [24]. Polymer-based waveguides which have been manufactured with core thicknesses below 10 mm offer an alternative approach, especially when considering flexibility and the number of independent stimulation channels [19,23,25]. However, light propagation of polymer fibers is limited in a wavelength-dependent manner. 3. Outlook The gene therapy component: In order to pave the way for clinical application, efficiency, stability and biosafety of cochlear optogenetics are of utmost importance. Thus, the construct to render the auditory nerve light sensitive should be designed to enable robust optogenetic activation of SGNs at physiological firing rates and with low light requirements. Also, the vector needed for neural transduction should be optimized in order to safely and efficiently deliver its genetic load. AAVs will likely be the vectors of choice, since they enable foreign protein to be expressed at high levels and over long periods of time. Furthermore, AAVs have little risk for adverse reactions of the target cells [1], are used in various clinical trials for gene therapy (including retinal dysfunction) and have been successfully used to restore auditory function in animal models of deafness [2]. Most recently, two clinical trials involving AAV-mediated optogenetic vision restoration have been approved (NCT02556736 and NCT03326336), corroborating the potential of optogenetic therapies for neural restoration. The implementation of adequate promotors as well as local virus application in the ossified cochlea will contribute to selective transduction of SGNs while excluding opsin expression beyond the auditory nerve. Inspired by retinal optogenetics, tissue explants and inner ear organoids might help evaluating the construct of choice and optimize efficiency and specificity in human SNGs [21,25e27]. Upon successful implementation of the gene therapeutic part of optogenetic hearing restoration, also the effect of chronic illumination of intracochlear neural tissue needs to be evaluated. Safety limits for light intensity need to be defined in order to avoid tissue heating or phototoxic effects. Here, the implementation of opsins with a red-shifted action spectrum will reduce the risk of tissue damage, since radiation in the red spectrum is reportedly less phototoxic (safety limits for retinal exposure defined by the European commission (2006/25/EC) are three orders of magnitude higher for orange light as compared to red light [3]). Together these considerations will foster the conservation of cochlear structures and offtarget tissues and guarantee a maximum of safety for the patient. The medical device component: Unlike typical gene therapy, the optogenetic manipulation of the SGNs is not aimed to restore hearing by itself. It requires the combination with the oCI as a medical device e consisting of an external sound processor, an internal light emitting implant, as well as an appropriate coding strategy and communication between these devices. While communication and processor technology might readily be adapted from eCIs, implemented restrictions in the temporal domain (due to opsin kinetics) and the increase in stimulation channels most likely require mayor re-working of coding strategies. The minimal duration and intensity of light pulse sufficient for SGN activation will depend on channel kinetics of the chosen optogenetic tool. From this quantum, the sound intensity coding should utilize the full range of optogenetically evoked SGN activation at their natural firing rates until the upper limit is reached, where firing saturates
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or behavior signals discomfort. The energy of a single pulse will need to be balanced between its duration (which needs to be limited in order to allow high stimulation rates) and its intensity (which is restricted by biosafety limits) and should furthermore allow for reasonable battery lifetimes of oCIs. The resulting patterns of optical stimulation will then be mapped on an e compared to electrical CIs e increased set of independent stimulation channels in order to encode spectral information. While the final number of intracochlear optical emitters remains to be defined, the spectral selectivity of optogenetic SGN stimulation has been estimated two outperform electrical coding by a factor of at least two. As contemporary eCIs house between 12 and 24 electrodes, a range of 50e100 optical emitters seems like a reasonable number to aim for [4]. Acknowledgment The work was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 670759 e advanced grant “OptoHear”) to T.M. A.D. is a fellow of the German Academic Scholarship Foundation. We would like to thank Dr. Jakob Neef for help on the figures and manuscript. References [1] Deafness and hearing loss, World Health Organ.. (n.d.). http://www.who.int/ news-room/fact-sheets/detail/deafness-and-hearing-loss (accessed September 16, 2018). [2] H. Li, C.E. Corrales, A. Edge, S. Heller, Stem cells as therapy for hearing loss, Trends Mol. Med. 10 (2004) 309e315, https://doi.org/10.1016/ j.molmed.2004.05.008. [3] H. Ahmed, O. Shubina-Oleinik, J.R. Holt, Emerging gene therapies for genetic hearing loss, J. Assoc. Res. Otolaryngol. 18 (2017) 649e670, https://doi.org/ 10.1007/s10162-017-0634-8. [4] T. Moser, Gene therapy for deafness: how close are we? Sci. Transl. Med. 7 (2015) 295fs28, https://doi.org/10.1126/scitranslmed.aac7545. [6] C.K. Berenstein, L.H.M. Mens, J.J.S. Mulder, F.J. Vanpoucke, Current steering and current focusing in cochlear implants: comparison of monopolar, tripolar, and virtual channel electrode configurations: Ear Hear. 29 (2008) 250e260, https://doi.org/10.1097/AUD.0b013e3181645336. [7] J.C. Middlebrooks, R.L. Snyder, Auditory prosthesis with a penetrating nerve array, J. Assoc. Res. Otolaryngol. JARO. 8 (2007) 258e279, https://doi.org/ 10.1007/s10162-007-0070-2. [8] V.H. Hernandez, A. Gehrt, K. Reuter, Z. Jing, M. Jeschke, A. Mendoza Schulz, G. Hoch, M. Bartels, G. Vogt, C.W. Garnham, H. Yawo, Y. Fukazawa, G.J. Augustine, E. Bamberg, S. Kügler, T. Salditt, L. de Hoz, N. Strenzke, T. Moser, Optogenetic stimulation of the auditory pathway, J. Clin. Investig. 124 (2014) 1114e1129, https://doi.org/10.1172/JCI69050. [9] C. Wrobel, A. Dieter, A. Huet, D. Keppeler, C.J. Duque-Afonso, C. Vogl, G. Hoch, M. Jeschke, T. Moser, Optogenetic stimulation of cochlear neurons activates the auditory pathway and restores auditory-driven behavior in deaf adult gerbils, Sci. Transl. Med. 10 (2018), https://doi.org/10.1126/scitranslmed.aao0540 eaao0540. [10] T. Mager, D. Lopez de la Morena, V. Senn, J. Schlotte, A. D Errico, K. Feldbauer, C. Wrobel, S. Jung, K. Bodensiek, V. Rankovic, L. Browne, A. Huet, J. Jüttner, P.G. Wood, J.J. Letzkus, T. Moser, E. Bamberg, High frequency neural spiking and auditory signaling by ultrafast red-shifted optogenetics, Nat. Commun. 9 (2018) 1750, https://doi.org/10.1038/s41467-018-04146-3. [11] D. Keppeler, R.M. Merino, D.L. de la Morena, B. Bali, A.T. Huet, A. Gehrt, C. Wrobel, S. Subramanian, T. Dombrowski, F. Wolf, V. Rankovic, A. Neef, T. Moser, Ultrafast optogenetic stimulation of the auditory pathway by targeting-optimized Chronos, EMBO J. 37 (2018), e99649, https://doi.org/ 10.15252/embj.201899649. [12] M.J. Duarte, V.V. Kanumuri, L.D. Landegger, O. Tarabichi, S. Sinha, X. Meng, A.E. Hight, E.D. Kozin, K.M. Stankovic, M.C. Brown, D.J. Lee, Ancestral adenoassociated virus vector delivery of opsins to spiral ganglion neurons: implications for optogenetic cochlear implants, Mol. Ther. 26 (2018) 1931e1939, https://doi.org/10.1016/j.ymthe.2018.05.023. [13] A. Dieter, C.J. Duque-Afonso, V. Rankovic, M. Jeschke, T. Moser, Near physiological spectral selectivity of cochlear optogenetics, Nat. Commun. 10 (2019) 1962, https://doi.org/10.1038/s41467-019-09980-7. [14] B.R. Arenkiel, J. Peca, I.G. Davison, C. Feliciano, K. Deisseroth, G.J. Augustine, M.D. Ehlers, G. Feng, In vivo light-induced activation of neural circuitry in transgenic mice expressing channelrhodopsin-2, Neuron 54 (2007) 205e218, https://doi.org/10.1016/j.neuron.2007.03.005. [15] S. Kleinlogel, K. Feldbauer, R.E. Dempski, H. Fotis, P.G. Wood, C. Bamann,
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Please cite this article as: T. Moser, A. Dieter, Towards optogenetic approaches for hearing restoration, Biochemical and Biophysical Research Communications, https://doi.org/10.1016/j.bbrc.2019.12.126