Impact of opsin kinetics on high-rate stimulation of the auditory nerve in mice

doi:10.21203/rs.3.rs-3786291/v1

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Impact of opsin kinetics on high-rate stimulation of the auditory nerve in mice

https://doi.org/10.21203/rs.3.rs-3786291/v1

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published 09 Sep, 2024

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Optogenetic stimulation improves spectral resolution compared to electrical stimulation in preclinical cochlear implant studies but remains unreliable at the high stimulation rates needed for precise temporal resolution. Combined optogenetic-electrical stimulation has been shown to improve temporal resolution while maintaining good spectral resolution. However, the reliability of combined stimulation at clinically relevant stimulation rates (> 400 pulses per second (pps)) is yet to be tested, nor whether altering opsin channel kinetics impacts these findings. We investigated responses of the auditory nerve and inferior colliculus to electrical, optogenetic, and combined stimulation in mice virally transduced with one of three opsin variants with different opsin kinetics: ChR2-H134R, ChIEF, or ChR2-C128A. Robust optogenetic responses were elicited in ChR2-H134R and ChIEF mice but extended periods of stimulation led to severe and non-recoverable deterioration of optogenetic responses. Unlike previous studies, there was no consistent facilitation of electrical responses in combined stimulation trials. Although ChIEF responses outperformed ChR2-H134R at 100 pps, the temporal characteristics were similar at higher rates. Combined stimulation significantly improved response characteristics at 400 pps, shown here for the first time in mice virally transduced with the ChR2-H134R and ChIEF opsins. These results have significant implications for the translation of optogenetic-only and combined stimulation techniques for hearing loss.

Biological sciences/Neuroscience

Biological sciences/Neuroscience/Auditory system

Although cochlear implants have proven remarkably successful at restoring speech comprehension in quiet conditions, many users report difficulties understanding speech in noisy conditions, reduced music enjoyment, inability to recognise speakers, and trouble differentiating statements from questions. These limited outcomes are believed to be due to the broad spread of electrical charge throughout the perilymph-filled cochlear chambers, resulting in activation of the auditory nerve with low spectral resolution.

In contrast, optogenetics, which relies on light rather than electrical stimulation, has been demonstrated to activate the auditory nerve with high spectral resolution^1–3. This stimulation technique is enabled by genetic modification of tissues to express light-sensitive ion channels, known as opsins. Stimulation of these opsins by light permits the flow of ions across the cell membrane that, in turn, evokes a response from the tissue. However, optogenetics is unable to evoke responses with high fidelity (i.e., consistently evoke responses over time), stability (i.e., consistent response sizes) nor high temporal precision (i.e., consistent response latency) at clinically relevant rates (> 400 pulses per second, pps)^4,5. A combined optogenetic-electrical stimulation approach has been demonstrated to improve fidelity, stability, and temporal precision compared to optogenetic stimulation at rates up to 100 pps⁵. Additionally, near-threshold optogenetic stimulation reduces electrical activation thresholds, resulting in significantly higher spectral resolution than electrical stimulation alone⁶. These studies were conducted in transgenic mice with consistently high opsin expression in the entire auditory neuron population. Clinical translation of optogenetics requires introduction of opsins to the auditory nerve through techniques, such as viral transduction, which do not result in consistent expression throughout the auditory nerve^7,8. Therefore, it is important to validate these results using a clinically relevant method of genetic modification and at higher, clinically relevant stimulation rates. Furthermore, studies of combined stimulation in the cochlea have only considered the H134R variant of channelrhodopsin (ChR2) and it is yet to be determined if outcomes can be further improved using opsin variants with different characteristics.

A variety of opsins have been discovered and developed to date, displaying a wide range of characteristics suited for different applications⁹. Many opsins are engineered variants with specific mutations to alter their characteristics, such as wavelength sensitivity, kinetics, and/or light sensitivity^9,10. For the high-rate stimulation needed for cochlear implants, the primary characteristic typically considered is the closing kinetics of the opsin ion channel. However, mutations that result in faster channel kinetics often negatively affect light sensitivity, demanding higher power for neural activation⁹. The opposite is also true: slower channel kinetics translating to lower power requirements⁹. ChR2-H134R (hereafter referred to as H134R), one of the earliest opsins developed, demonstrates good light sensitivity but relatively poor kinetics for high-rate stimulation applications such as cochlear implants¹¹. In contrast, the ChIEF opsin has closing kinetics nearly twice as fast as H134R (9.8 ms versus ~ 19 ms^10,12), but significantly reduced light sensitivity (EC50 0.9 mW/mm² versus 0.7 mW/mm²)¹². Conversely, the closing rate of ChR2-C128A (hereon referred to as C128A) is one of the slowest (52 ms) but C128A has extremely high light sensitivity (EC50 0.1 mW/mm²)^12,13. C128A is a member of the step-function opsin family, which have been shown to sensitize neurons to native synaptic inputs for extended periods^14,15. As such, C128A may be useful for a combined stimulation technique, whereby auditory neurons are primed with low light levels, allowing high rate, subthreshold electrical stimulation to be used. In contrast to the other two opsins mentioned, the closing kinetics of C128A can be accelerated by applying amber light, permitting greater control over its activity.

In this study, we investigate three channelrhodopsin variants – H134R, ChIEF, and C128A – in virally transduced mice for optogenetic and combined stimulation at rates up to 400 pps. The temporal resolution of responses (i.e., fidelity, temporal precision, and stability) and facilitation between stimuli were assessed via auditory nerve and inferior colliculus recordings.

Optical responses

We first compared the response characteristics of auditory neurons expressing one of H134R, ChIEF, or C128A opsin to 452 nm wavelength optical stimulation in vivo through compound action potential or tungsten electrode auditory nerve recordings and 32-channel shank probe recordings of the inferior colliculus.

Auditory nerve or inferior colliculus responses to 1–4 pps optical stimulation could be elicited in H134R and ChIEF mice, but not C128A expressing mice (n = 7 of 7 H134R mice; n = 8 of 9 ChIEF mice; n = 0 of 9 C128A mice) (Fig. 1A,B). Optical activation thresholds were not significantly different between H134R (0.66 ± 0.4 mW) and ChIEF (0.18 ± 0.1 mW) (p = 0.3, Student’s t-test) and, for both opsins, the optical activation thresholds reduced with increasing phase at both the auditory nerve and inferior colliculus (Fig. 1C,D).

To compare the dynamic ranges of the two opsins, we fitted sigmoids to the response curves (at the best electrode for inferior colliculus recordings) and calculated the slope at 50% (i.e., L₅₀). Dynamic range of the auditory nerve reduced with increasing stimulation phase and was significantly (p < 0.05, Student’s t-test) elevated for ChIEF (1.77 ± 0.2 dB/dB) compared to H134R (0.91 ± 0.3 dB/dB) for a 1 ms stimulation phase (Fig. 1D). Both were significantly reduced compared to electrical stimulation (3.9 ± 0.4 dB/dB and 4.4 ± 0.5 dB/dB for H134R and ChIEF, respectively; p < 0.05, paired t-test, n = 6 for each group, data not shown). In contrast, the dynamic range of inferior colliculus responses at 1 ms were not significantly different between the two opsins (1.56 ± 0.8 dB/dB for H134R, 1.50 ± 0.5 dB/dB for ChIEF; p > 0.9, Student’s t-test) and, neither were significantly different from the dynamic ranges of electrical responses (2.29 ± 0.2 dB/dB, p > 0.4, paired t-test, n = 7 for H134R; 2.78 ± 0.4 dB/dB, p > 0.08, paired t-test, n = 8 for ChIEF, data not shown).

Auditory nerve response size diminished substantially over day-long optical stimulation for both opsins (Fig. 1E) until, in many cases (6 of 6 ChIEF mice, 2 of 7 H134R mice), the response was lost and could not be recovered, even after 1–2 hours of darkness. Loss of responses occurred more often and more rapidly in ChIEF mice compared to H134R mice. Similarly, inferior colliculus activation thresholds increased during the course of extended optical stimulation for both opsins, again being more severe for ChIEF mice (Fig. 1F). Irreversible loss of inferior colliculus responses occurred in 5 of 6 ChIEF mice following extended stimulation, whereas responses were elicited in all 7 H134R mice for the duration of the experiment. In contrast, electrical stimulation was reliable and stable, with minimal change in auditory nerve response size and inferior colliculus activation threshold (Fig. 1E,F).

Facilitation of auditory nerve responses

Following characterisation of the optogenetic response, we investigated the facilitation of electrical activation by simultaneous stimulation with light (combined stimulation). Electrical-only and optogenetic-only trials were interleaved with combined stimulation trials, and responses from the auditory nerve and inferior colliculus recorded. The optogenetic-only response was subtracted from the combined response to calculate changes in the electrical threshold and changes in the auditory nerve response size. A range of electrical levels were combined with subthreshold light (0.7–19 dB below threshold), parathreshold light (0 dB), or suprathreshold light (0.5–25 dB above threshold).

Addition of subthreshold and parathreshold optogenetic stimulation to electrical stimulation had no significant effect on electrical thresholds in H134R and ChIEF mice (p > 0.4, paired t-test) (Fig. 2A). In C128A mice, however, subthreshold optogenetic stimulation significantly reduced electrical thresholds albeit not substantially by 0.39 ± 0.1 dB (p < 0.05, paired t-test). A similarly modest reduction was seen in ChIEF mice when using suprathreshold optogenetic stimulation (-0.39 ± 0.2 dB; p = 0.05, paired t-test). Thresholds in C128A and H134R mice with suprathreshold optogenetic stimulation were not substantially nor significantly different to electrical stimulation alone.

We quantified the change in response size at the electrical threshold and at 0.7 dB above electrical threshold (i.e., at the electrical stimulation level that results in a 3 dB increase in response size, calculated using the L₅₀ for electrical stimulation). At electrical threshold, we observed no significant difference in the response size with the addition of optogenetic stimulation at any intensity for all opsins (p > 0.05, Student’s t-test) (Fig. 2B). Above electrical threshold (Fig. 2C), auditory nerve responses sizes were slightly but significantly larger (p < 0.05, Student’s t-test) with the addition of para- and suprathreshold optogenetic stimulation to electrical stimulation for H134R mice (0.36 ± 0.1 dB and 0.42 ± 0.1 dB, respectively). Both ChIEF and C128A mice showed no significant effect at any optogenetic stimulation power.

Facilitation of inferior colliculus activity

Similar to the auditory nerve, we looked at the inferior colliculus responses for evidence of facilitation during combined stimulation by quantifying changes in the electrical activation threshold (Fig. 3A) and the activation width (recording electrode distance between crossings of the spatial tuning curve) at the stimulation level midway between threshold and the maximum current threshold tested (Fig. 3B). Both H134R and ChIEF mice demonstrated a reduction in electrical activation thresholds around the optogenetic activation threshold (p < 0.001, Student’s t-test), and an increase in activation width above threshold (p < 0.05, Student's t-test). C128A mice did not show any significant effects (p = 0.4 for thresholds and p = 0.06 for activation width, Student’s t-test).

To understand how much of this facilitation was due to the interaction between the two stimuli rather than the two stimuli acting separately, we quantified the increase in spikes at the best recording electrode for combined stimulation compared to electrical stimulation (Fig. 3C). A median smoothing was applied to the difference. Consistent with the results of the auditory nerve, we observed no substantial effect of optogenetic stimulation in the inferior colliculus activity at subthreshold electrical levels for all opsins (Fig. 3D).

Temporal fidelity, precision and stability of high-rate stimulation

Next, we compared the temporal characteristics of auditory nerve responses to optical, electrical, or combined stimuli in H134R and ChIEF mice. Stimuli were presented as a 300 ms burst at 100, 200, and 400 pps at a 1 Hz burst presentation rate. Electrical-only, optogenetic-only, and combined stimulation trials were randomly interleaved. The combined stimulation response was isolated by subtracting the electrical-only and optogenetic-only responses from the combined stimulation responses.

Three characteristics were extracted from the responses: fidelity (measured as the percentage of stimuli that elicited responses), temporal spread (measured as the change in response latency between the earliest and latest response) and stability (measured as the change in response size between the first and last response) (Fig. 4A). To best compare temporal spread and stability, we consider trials which achieve fidelity equal to or above 50% in our analyses.

In mice transduced with the H134R opsin, auditory nerve responses to 100 pps optical stimulation showed significantly worse fidelity (median ± interquartile range: 20 ± 22%) compared to electrical only stimulation (100 ± 0%) (p < 0.001, Wilcoxon-Mann-Whitney test) (Fig. 4B). Over the duration of the 300 ms burst, the responses to optical stimulation increased in latency by 491 ± 167 µs and decreased in amplitude by -5.6 ± 0.9 dB, while there was minimal change to the responses to electrical stimulation (20 ± 26 µs change in latency and − 0.3 ± 0.4 dB change in amplitude) (Fig. 4C,D). Critically, optogenetic responses consistently demonstrated fidelity less than 50% for 200 and 400 pps, and were thus excluded from response size stability and temporal spread analyses (Fig. 4B).

Compared to H134R mice, the auditory nerve responses of ChIEF mice to 100 pps optical stimulation demonstrated significantly greater fidelity (80 ± 47%; p < 0.05, Wilcoxon-Mann-Whitney test), similar temporal spread (376 ± 347 µs; p > 0.4, Wilcoxon-Mann-Whitney test), and significantly poorer stability (–8.3 ± 2.7 dB; p < 0.05, Wilcoxon-Mann-Whitney test) (Fig. 4B-D). Although the fidelity of auditory nerve responses to optical stimulation at 200 and 400 pps were not significantly different between H134R and ChIEF mice (p > 0.2, Wilcoxon-Mann-Whitney test), a subset of 200 pps trials in ChIEF mice achieved fidelity above 50% and were included in temporal spread and stability analyses.

Compared to electrical stimulation, optical auditory nerve responses of ChIEF mice demonstrated significantly worse fidelity at all rates (p < 0.001, Wilcoxon-Mann-Whitney test) (Fig. 4B). Temporal spread was also significantly worse at 100 and 200 pps (p < 0.001, Wilcoxon-Mann-Whitney test) (Fig. 4C). Stability was significantly worse at 100 pps (p < 0.001, Wilcoxon-Mann-Whitney test) but not at 200 pps (p > 0.8, Wilcoxon-Mann-Whitney test) (Fig. 4D).

Application of combined stimulation using suprathreshold optical stimulation significantly improved the fidelity compared to optogenetic-only stimulation with either opsin at all rates (p < 0.05, Wilcoxon-Mann-Whitney test), except for ChIEF at 100 pps, where optogenetic stimulation already demonstrated high fidelity (p > 0.4, Wilcoxon-Mann-Whitney test) (Fig. 4B). Compared to electrical stimulation, however, the fidelity of combined stimulation responses using suprathreshold optical stimulation was significantly worse (p < 0.05 at all rates and for both opsins, Wilcoxon-Mann-Whitney test).

At 100 pps, temporal spread and stability were significantly improved for combined stimulation using suprathreshold optical stimulation compared to optical stimulation for both opsins (p < 0.001, Wilcoxon-Mann-Whitney test) (Fig. 4C,D). The responses of ChIEF mice to 200 pps combined stimulation also demonstrated significantly better temporal spread (p < 0.01, Wilcoxon-Mann-Whitney test) but not stability (p > 0.8, Wilcoxon-Mann-Whitney test) over optical responses.

At 200 and 400 pps, electrical stimulation responses demonstrated significantly better temporal spread than the responses to combined stimulation using suprathreshold optical stimulation (p < 0.001, Wilcoxon-Mann-Whitney test) except for H134R mice at 200 pps (p = 0.14, Wilcoxon-Mann-Whitney test) (Fig. 4C). The stability of electrical responses was also significantly higher at 200 and 400 pps for H134R mice (p < 0.05, Wilcoxon-Mann-Whitney test), but not for ChIEF mice (p > 0.19, Wilcoxon-Mann-Whitney test) (Fig. 4D).

The fidelity of combined stimulation responses using suprathreshold optical stimulation was similar between H134R and ChIEF mice, except at 200 pps (H134R: 95 ± 72%, ChIEF: 56 ± 90%, p < 0.05, Wilcoxon-Mann-Whitney test) (Fig. 4B). Temporal spread followed a similar trend, with responses at 200 pps demonstrating a significant difference (H134R: 36 ± 56 µs, ChIEF: 110 ± 182 µs, p < 0.05, Wilcoxon-Mann-Whitney test), but no difference for other rates (Fig. 4C). The stability of responses was significantly different at 100 and 200 pps (p < 0.01, Wilcoxon-Mann-Whitney test) but not at 400 pps (p = 0.1, Wilcoxon-Mann-Whitney test) (Fig. 4D).

Opsin Expression Along the Cochlea

Histology showed high transduction in the auditory neurons of the left ears of H134R (n = 7), ChIEF (n = 8), and C128A (n = 9) mice, as evidenced by the fluorescent YFP tag (Fig. 5A). The percentage of YFP-positive neurons was not significantly different between opsin cohorts (p > 0.1, Kruskal-Wallis test; Fig. 5B). A significant apical-basal gradient of YFP-positive cells was evident in most ears (Fig. 5B) (p < 0.05, RM-ANOVA), and appeared to have high variability of opsin quantity between cells, based on the varying YFP brightness, even within the same cochlear turn. A number of cells also demonstrated a clumping phenotype, where bright YFP spots could be identified within cells or along axons, but not on the plasma membrane (Fig. 5A).

This study compared the temporal characteristics of electrical, optogenetic, and combined stimulation of the auditory nerve using clinically relevant stimulation rates and a virus-transduced mouse model that has translational potential. Critically, it identified the subtle differences in responses of auditory neurons expressing opsins with different channel kinetics and light sensitivity.

Optical responses

Robust optogenetic responses to blue light were elicited using both H134R and ChIEF opsins, but not the C128A opsin. This is an unsurprising result, as C128A is part of the step-function opsin family, which exhibits reduced photocurrent amplitudes and evoked voltage changes compared to the rapid opsins used in this study^12,16. Step function opsins are best suited to elevate native activity or to raise excitability for combined stimulation through their long channel closing kinetics rather than eliciting responses directly. For example, one study using the step-function opsin SOUL successfully modulated spiking activity in the prefrontal cortex of macaques for minutes after the end of the light stimulus¹⁷. Another study used the step-opsin ChR2-C128A/D156A to increase activity in inhibitory or excitatory neurons of the medial prefrontal cortex of mice and thereby modulate social and learning behaviours¹⁵. In contrast, H134R and ChIEF both exhibit shorter channel closing kinetics and are capable of eliciting responses directly with their relatively higher photocurrents^7,10–12.

Importantly, this study is the first to evaluate the reliability of optogenetic stimulation following day-long, high-rate stimulation. We observed substantial reduction of response size and elevation in optogenetic activation thresholds over the course of repetitive optical stimulation for ChIEF transduced mice. Optogenetic activation thresholds for H134R responses also became elevated over the day, but responses were not entirely lost. These changes did not correlate strongly with changes in electrical responses, and so are likely to be evidence of opsin bleaching. Spectroscopy studies suggest that the bleaching phenomenon reflects a branched pathway that occurs between the ‘open’ state and ‘ground’ state of opsin configurations^18,19. This phenomenon manifests as a reduction in photocurrents and elevation of optogenetic activation thresholds or as a complete loss of response to light stimulation. Although bleaching can last many minutes to hours, extended periods of darkness can recover the opsins back to the ‘ground’ state. Bleaching has been reported for slow opsins such as C128A²⁰, and Matarazzo et al.²¹ noted high intensities (15 mW) or long pulse durations (5 ms) consistently resulted in loss of the optically evoked compound response in the sciatic nerve of H134R expressing transgenic mice. However, bleaching has not been studied in detail for rapid opsins (i.e., ChIEF and H134R), possibly due to the relatively short duration of other studies and/or minimisation of the stimuli delivered. Indeed, bleaching of ChIEF and H134R appeared to correlate with the number of light stimuli presented in our study. These results emphasise the need for extended and/or chronic stimulation studies for opsins currently undergoing clinical translation. Techniques and encoding strategies that minimise the energy would thus be more valuable for the clinical translation of optogenetics, such as combined stimulation.

Combined facilitation

Unlike previous studies using transgenic H134R mice, we did not observe reliable facilitation of electrical activation thresholds at any light intensity for combined stimulation for any opsin. In the case of H134R, and possibly the other opsins in our study, this is likely a consequence of the difference in the method of mediating opsin expression in the auditory neurons. Compared to transgenic mice, using an AAV-based transduction method resulted in fewer auditory neurons expressing the opsin. In addition, the opsin was not optimally trafficked to the cell membrane compared to transgenic mice used in previous studies. Indeed, similar results have been observed in previous studies of the inferior colliculus response. Thompson et al.⁶ demonstrated facilitation of the electrical response using optogenetic stimulation in a transgenic model. However, Richardson et al.⁸ showed that, in a virus transduced model, facilitation could do not be consistently achieved. The variability of opsin expression in the auditory nerve resulting from viral transduction decreases the likelihood of overlapping the electrical and optogenetic stimuli and diminishes the success of combined stimulation at low rates.

Lack of facilitation in ChIEF and C128A mice at 100 pps stimulation rates may also reflect a difference in the ion preference of those opsins compared to H134R. A study by Hart et al.²² suggested that it is not merely the optogenetic depolarising currents that facilitate electrical activation thresholds, but rather the increase in intracellular calcium, which reduces the activation threshold of voltage-gated ion channels. The ion preferences of ChIEF and C128A are assumed to be similar to H134R, but there is little direct evidence to support this.

C128A appeared to have no effect on facilitation of responses to electrical stimulation, indicating the opsins were ineffective – either conducting too weakly or conducting the wrong ions to permit combined stimulation. As mentioned above, C128A is prone to a phenomenon known as bleaching, where opsins enter a non-functional ‘lost’ state, rather than returning to a ready, closed state following light stimulation. Schoenenberger et al.²⁰ reported that C128A transfected brain slices had to be dissected under orange light to prevent complete loss of the opsin in the tissue under white light. Although best efforts to mitigate bleaching of the opsins were taken by conducting surgery under orange light, gradually increasing the intensity of light across trials, and using an amber light to accelerate opsin closing, these efforts were insufficient to mediate any facilitation of electrical stimulation. Unfortunately, the techniques used in this study are not suitable to uncover the root cause, and further investigation using in vitro patch clamp techniques may be useful to understand why C128A was ineffective in this application.

Regardless, it is possible that, similar to other applications, C128A may be more suited to improving hearing where there are some residual functional hair cells.

High-rate stimulation

Although facilitation was not observed at low stimulation rates (1–4 pps), three characteristics of facilitated combined stimulation responses (fidelity, temporal precision and latency) were evident at the higher stimulation rates (100–400 pps). The presentation of many stimuli close together improves the probability of eliciting a combined response. For example, at 400 pps, the tissue is exposed to light for 40% of the burst stimulus.

Compared to transgenic animals, fidelity at 100 pps for H134R was significantly lower, possibly as a result of the poorer quality and/or quantity of opsin expression in transduced animal models⁵. Studies have shown that improving trafficking to the plasma membrane improves activation thresholds and the reliability of responses at high stimulation rates. Keppeler et al.⁴ demonstrated that the inclusion of a trafficking peptide motif to the end of the Chronos opsin increased the maximum stimulation rate at which optical auditory brainstem responses could be elicited. Similarly, Richardson et al.⁸ demonstrated that a minimum number of transfected auditory neurons were necessary to elicit an optically evoked response at the inferior colliculus. Developments to gene therapy techniques to improve the consistency of transduction along the cochlea and successful opsin trafficking are likely to benefit all applications of optogenetic and combined stimulation.

ChIEF optogenetic-only responses demonstrated significantly higher reliability at 100 pps over H134R, likely a consequence of faster channel closing kinetics. Despite this, ChIEF responses were not significantly more stable nor temporally precise. Furthermore, reliability of ChIEF was not significantly improved over H134R responses at 200 and 400 pps, demonstrating that the improvements granted by faster channel kinetics are limited. Similar results have been observed with Chronos and vf-Chrimson, which exhibit channel closing kinetics of 3.6 ms²³ and 2.7 ms²⁴, respectively, much shorter than the ~ 9 ms of ChIEF. Studies of auditory neuron responses mediated by these opsins indicate that spike probability drops below 50% by 200 pps even with the addition of trafficking peptide motifs to the opsins^4,25. These opsins require significant higher stimulating power for functional responses, casting further doubt on feasible clinical translation of optogenetics for cochlear implants.

Combined stimulation using suprathreshold optogenetic stimulation, however, showed very high reliability and stability even up to 400 pps for both opsins. Although the temporal precision was poorer at these levels compared to electrical stimulation, combined stimulation still outperformed optogenetic-only stimulation whilst maintaining electrical-like reliability and stability. Interestingly, the temporal properties of the combined stimulation responses for the two opsins were similar, indicating that the closing kinetics do not strongly influence combined stimulation outcomes. Rather than merely faster closing kinetics, improvements to the temporal precision and fidelity of optogenetic and combined stimulation will likely result from multifaceted opsin characteristic changes.

Opsin expression

Using AAV gene therapy, we were able to successfully transduce a high proportion of auditory neurons with the opsin genes. Similar to previous studies, we observed large variability of opsin expression between cells within the same turn, but also along the apical-basal gradient of the cochlea^7,8. We also report here a sub-cellular clumping of fluorescence indicative of sub-optimal opsin expression or trafficking in some cells. This may be caused by a number of cellular processes, including protein degradation, or endosomal/lysosomal uptake. The cause and consequence of this phenotype requires further investigation.

Limitations & Clinical Translation

For the best outcomes of combined stimulation, high spatial overlap of the two stimuli is critical. This is not easily achieved with viral methods of auditory neuron transduction in mice. Improving the quantity of transduced auditory neurons and the quality of opsin expression will improve both optogenetic-only and combined stimulation, but combined stimulation will also require the clever design of devices that can maximise overlap between the two stimuli. In our study, stimulation was confined to the basal turn where expression was worst, although typically ~ 60% of neurons in that turn were transduced.

It is important to recognise that these studies were conducted in acutely deafened ears. Consequently, the auditory nerve has not undergone degeneration as many chronically deaf ears do. Previous studies have shown that degeneration resulting from chronic deafness can negatively impact the performance of both optogenetic and combined stimulation, demanding higher powers of optogenetic stimulation to achieve electrical-like temporal fidelity⁵. These results emphasise the importance of developing therapeutics to maintain or restore auditory nerve health, such as neurotrophins.

Novel stimulation methods are necessary for advances in cochlear implant recipient outcomes, and it is critically important to validate these methods in clinically translatable models. This study is the first to show that optogenetic responses can be severely diminished or lost during day-long, high-rate stimulation of auditory neurons expressing the ChIEF or H134R opsins. This has important implications for the preclinical assessment and translation of optogenetics to clinical applications.

Combined stimulation, previously shown to increase spectral resolution compared to electrical stimulation due to facilitation of the response, was shown here to improve reliability and temporal precision of responses at 400 pps in mice transduced with the ChIEF and H134R opsins. However, facilitation of the electrical response was not evident in these mice nor the C128A mice. We reason that very high viral transduction efficiency is necessary for the priming effect of combined stimulation. This study provides important insight into the use of optogenetics for clinical applications.

- Virus preparations & Pup injections

Adeno-associated virus (AAV)-Anc80 and AAV-PhP.eB were chosen as the viral vectors due to their high specificity for spiral ganglion neurons following intra-cochlear injection in neonates. The pAAV-hSyn1-ChR2(H134R)-EYFP (Addgene plasmid #26973) was packed as an Anc80 AAV by the Vector and Genome Engineering Facility, Children’s Medical Research Institute (New South Wales, Australia) and prepared in phosphate buffered saline (PBS; pH7.4) supplemented with 50 mM NaCl and 0.001% pluronic F-68. AAV-PhP.eB with a hSyn1-driven ChIEF-EYFP fusion or ChR2(C128A)-EYFP fusion gene and Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) plasmids were prepared by VectorBuilder and prepared in PBS supplemented with 200 mM NaCl and 0.001% pluronic F-68. Viral titre was estimated at 1.76e¹² vg/mL for AAV-Anc80-hSyn1-ChR2(H134R-EYFP), 1.10e¹³ vg/mL for AAV-PhP.eB-hSyn1-ChIEF-EYFP, and 6.38e¹³ vg/mL for AAV-PhP.eB-hSyn1-ChR2(C128A). Under hypothermic anesthesia, the left cochlea of C57BL/6 neonates aged P2-4 days was exposed via a post-auricular incision and blunt dissection of the soft tissues to access the bulla, which was opened using a sharp needle and microscissors. One microlitre of the vector was then delivered through the round window into the cochlear perilymph via rapid injection using a Hamilton syringe positioned using a micropositioner. Tissues were replaced to their original position and the skin layer was sutured. Pups were warmed until breathing returned, rubbed with nesting material, and returned to the mother. Experimenters could not be blinded to which virus the mice received due to litter naming conventions perpetuated throughout each stage of the experiments.

- Deafening and CAP recordings

Electrophysiology recordings of auditory nerve compound action potentials (CAPs) were made in mice (n = 5; 2 female, 3 male; 81.6 ± 12.6 days old). Prior to the recordings, mice were unilaterally deafened with neomycin using a similar technique to that described in ⁵. In summary, mice were anesthetised (0.25-3% gaseous isoflurane), and analgesia (lignocaine hydrochloride; 0.1 mL 1% V/V, or bupivicaine; 0.1 mL 0.5% V/V) was applied subcutaneously at the site of incision. An incision was made ~1 mm caudal to the pinna, and soft tissues dissected to expose the bulla. A bullostomy was performed to expose the round window and the mid-apical turn of the cochlea. A fenestration in the mid-apical turn was hand-drilled using a sharp probe and the round window membrane was pierced with a ⌀ 0.22 mm steel wire. 10-15 µL of 10% neomycin sulfate (w/V) in 9% saline solution (w/V) was perfused through the round window while gentle aspiration was applied to the mid-apical fenestration over 20-30 minutes.

Approximately 30 minutes after the deafening procedure, and still under anesthesia, the mice underwent compound action recordings of the auditory nerve. The mice were placed in a stereotaxic frame and rotated for better lateral access to the exposed cochlea. Stimulating equipment was then inserted into the cochlea. Electrical stimulation was delivered via two 50 µm platinum wires (PTFE insulated; tips exposed), one positioned ~1 mm into the mid-apical fenestration used in the deafening procedure and one placed ~1 mm into the round window. Optical stimulation was delivered using an optical fibre (105 µm core, NA 0.22) attached to the round window platinum wire. Amber light was delivered using an optical fibre (200 µm core, NA 0.22) placed against the cochlea wall.

Recording electrodes comprised a platinum wire placed on the cochlea wall between the stapedial artery and the round window and a steel needle inserted subcutaneously at the back of the neck.

- Brain recordings

Craniotomy for IC recordings

Adult mice (n = 21; 10 female, 11 male; 63.3 ± 2.8 days old) were used for multiunit recordings of the inferior colliculus. The mice were placed under gaseous isoflurane anesthesia and unilaterally deafened on the left side as described above for CAP recordings. Mice were then placed in a stereotaxic frame and analgesia applied (lignocaine hydrochloride; 0.1 mL 1% V/V, or bupivacaine hydrochloride; 0.1 mL 0.5% V/V; subcutaneous) to the surgical site before an incision was made along the midline of the scalp. A diamond dental drill was used to perform a craniotomy over the right inferior colliculus. The dura over the inferior colliculus was carefully removed using a 30g needle. A saline soaked cotton ball was placed over the exposed brain until the recording electrode was inserted.

Craniotomy for auditory nerve recordings

Mice that underwent inferior colliculus recordings also underwent simultaneous auditory nerve recordings. Prior to the craniotomy over the inferior colliculus, the ear canals were exposed and cut close to the skull using sharp microscissors, and ear bars inserted. The muscles overlying the parietal and interparietal bones on the left side of the skull were dissected using sharp forceps. The inferior colliculus of the right side was exposed, as described above. Similarly, a small drill was used to expose the brain under the left interparietal bone, and the dura removed using a 30g needle. A saline soaked cotton ball was placed over the exposed brain until the recording electrode was inserted.

Simulation and recording

Following the craniotomies, the stimulating equipment was inserted into the exposed cochlea as described above for CAP recordings.

A tungsten electrode (TM33B20, World Precision Instruments, USA; impedance ~2 MΩ) was inserted through the left hemisphere towards the auditory nerve using a microdriver (David Kopf Instruments, USA) while electrical stimulation was applied to the cochlea. Similarly, a single shank 32-channel array with 50 µm spacing (NeuroNexus Technologies, USA) was inserted into the inferior colliculus. To provide recording stability, a 1% (w/V) agar solution was applied over the exposed brain and inserted shank electrode.

Recording ground electrodes in the form of steel needles were inserted into the right axillary region and between the eyes on the scalp for shank and tungsten electrode recordings, respectively.

- Stimulating waveforms

Light stimuli were delivered using a custom 452 nm laser (OptoTech, Australia) coupled to an optical fibre (105 µm core, NA 0.22) by an FC connector and/or a 595 nm LED (M595F2, Thorlabs, USA) coupled to an optical fibre (205 µm core, NA 0.22) via an SMA connector. Light was presented at 0.25-60 ms pulses at 0-14.5 mW intensities for blue light, and 1-300 ms pulses at 3.7 mW for amber light. The lasers were calibrated using a photodiode (PDA36A2, Thorlabs, USA) coupled via an optical fibre. Electrical stimuli were delivered by a custom-built stimulator connected to the platinum wires. Biphasic current pulses with a phase of 25 µs (interphase gap of 8 µs) were delivered over a range of current levels (CL), where CL in µA is calculated as: . For combined stimulation trials, the electrical stimuli were delayed such that they finished with the end of the laser pulse.

- Histology

Following acute electrophysiology experiments, mice were euthanised (cervical dislocation under gaseous isoflurane anesthesia) the left cochlea was dissected and processed for cryo-sectioning. Cochleae were immediately fixed following dissection in 10% (V/V) neutral buffered formalin for 2-16 hours and then rinsed in PBS before 2-3 days of decalcification in 0.12M ethylenediaminetetraacetic acid (EDTA). Processed cochleae were embedded in optimum cutting temperature compound (OCT, Tissue-Tek, Saruka, Japan) and snap-frozen for cryo-sectioning serial sections at 12 µm. Mid-modiolar sections were immunostained using mouse anti-HuD (1:1000, Santa Cruz Biotechnology, SC-48421) or anti-Tuj1 (1:500, Biolegend, 801201) for spiral ganglion neurons, and mounted with 4′,6-diamidino-2-phenylindole (DAPI) to visualise nuclei. The Rosenthal’s canal in three turns of each cochlea were imaged using a fluorescent microscope and processed using ImageJ (National Institute of Health, USA). Opsin-positive spiral ganglion neurons were identified as EYFP positive cells and counted as a percentage of all spiral ganglion cells (HuD positive cells).

- Analysis/Statistical methods

Auditory nerve thresholds were identified as the minimum level at which a response could be confidently identified. Inferior colliculus threshold was identified as the power level that produced activity at 33% of the maximum evoked activity. Spatial tuning curves were fitted for the 33% level at each recording electrode. Activation widths at any level were calculated using the crossings of the spatial tuning curve.

The first response in the burst was always discarded due to a large and variable onset response. Burst recordings with at least two additional responses (i.e., two responses after the first discarded response) were required for inclusion in the analysis of fidelity. Recordings with fidelity of at least 50% were required for the analysis of stability and temporal spread. Individual responses were identified as having a P1-N2 size greater than the quiet period following the burst, and a P1 occurring within 1-2.5 ms following stimulation.

Numerical results stated are average ± standard error mean, except for burst stimulation fidelity, temporal spread, and stability, which state median ± interquartile range.

Optogenetic activation: Statistical tests for optogenetic activation thresholds and dynamic range used Student’s t-test. Normality was confirmed using the Shapiro-Wilk test.

Burst measures: Statistical tests for fidelity, temporal spread, and stability used Wilcoxon-Mann-Whitney test, as Shapiro-Wilk test showed data was not normally distributed.

Histology: Statistical tests for expression differences between turns used repeated measures analysis of variance (RMANOVA), and differences between cohorts used the Kruskal-Wallis test, as variances were not consistently equal according to the Levene’s test.

Data availability

The data generated during the current study are available from the corresponding author on reasonable request.

Acknowledgements

We gratefully acknowledge Ella P. Trang and Philippa Kammerer for their histological expertise and research support. The authors acknowledge our funding sources: the National Health and Medical Research Council (NHMRC; #2002523), the Australian Government Research Training Program, and the Bionics Institute Incubator Fund. The Bionics Institute acknowledges the support it receives from the Victorian Government through the operational infrastructure program.

Author contributions

R.T.R., J.F.B., and D.B.G. contributed to the conception and design of the study; E.A.A. and A.A.A. performed the experiments; E.A.A. analysed the data; A.W. and A.C.T. contributed to interpretation of results; E.A.A. and R.T.R. wrote the manuscript. All authors reviewed the manuscript.

Competing interests

The authors declare no competing interests.

Ethics statement

The use of animals in this study was reviewed and approved by St Vincent’s Hospital Animal Ethics Committee (#20-004, #23-001). All animal use and care followed the Guidelines to Promote the Wellbeing of Animals used for Scientific Purposes (2013), the Australian Code for Care and Use of Animals for Scientific Purposes (8^th edition, 2013), and the Prevention of Cruelty to Animals Amendment Act (2015). This study is reported in accordance with ARRIVE guidelines (https://arriveguidelines.org).

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No competing interests reported.

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Impact of opsin kinetics on high-rate stimulation of the auditory nerve in mice

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Journal Publication

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Abstract

Figures

Introduction

Results

Optical responses

Facilitation of auditory nerve responses

Facilitation of inferior colliculus activity

Temporal fidelity, precision and stability of high-rate stimulation

Opsin Expression Along the Cochlea

Discussion

Optical responses

Combined facilitation

High-rate stimulation

Opsin expression

Limitations & Clinical Translation

Conclusion

Methods

Declarations

References

Additional Declarations

Status:

Journal Publication

Version 1