Auditory nerve responses to combined optogenetic and electrical stimulation in chronically deaf mice

Since it is possible for hair cells to be activated by electrical- [26], optogenetic- [27] or optoacoustic-stimulation [28], we developed a technique to thoroughly inactivate inner and outer hair cells and so mitigate the effect of hair cell-mediated responses on auditory nerve recordings. In our studies, mice were unilaterally deafened by perfusing neomycin sulphate through the left cochlea over 20–30 min. Auditory brainstem response (ABR) thresholds were recorded before and after deafening. A significant shift in thresholds was observed within one hour of neomycin sulphate exposure (p < 0.05 for all tones; RM-ANOVA, n = 4). Three mice were recovered and their hearing reassessed 2 weeks later; hearing thresholds remained significantly elevated in these animals (figure 1(B); p < 0.05 for all tones; RM-ANOVA, n = 3), and histology showed complete ablation of inner and outer hair cells in the left cochlea of all mice (figure 1(C)). Sporadic hair cell loss was also observed in the outer hair cell region of the right ear, although it appeared to follow no obvious pattern throughout the cochlea.

Hybrid stimulation relies on the interaction of simultaneous electrical and optogenetic stimulation of the auditory nerve. In our stimulation set up, optical stimulation (452 nm, 1 ms pulses) was provided by a 105 µm core optical fibre inserted into the round window of the cochlea and oriented towards the modiolus to maximise the spread of activated neurons, and so increase the probability of evoking hybrid responses. Charge balanced biphasic electrical stimulation was provided by two 100 µm platinum wires inserted into the round window in the base of the cochlea and a fenestration in the apical turn of the cochlea, respectively (figures 2(A) and (B)). Microcomputed tomography (micro-CT) scans confirmed the optical fibre was successfully directed towards the modiolus, projecting light on the auditory nerve where it exited the cochlea (figure 2(C)). To confirm the activation area, we recorded spiking activity in the contralateral inferior colliculus using a 32-channel recording array in response to 1 ms light pulses or the biphasic electrical pulses. The inferior colliculus is a midbrain structure of the auditory pathway that preserves the tonotopic arrangement of the cochlea and, as such, reflects the spread of activation in the cochlea. Indeed, multi-unit responses to optogenetic stimulation indicated broad activation across most frequencies of the adult mouse cochlea (activation range at 3 dB above threshold: 7.8–64.5 kHz for electrical, 26.4–59 kHz for optogenetic), in line with the positioning of the optical fibre, and high overlap between the two stimulation modalities (figure 2(D)).

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After confirming that our deafening and stimulating techniques were effective, we investigated how optogenetic stimulation facilitated activation of the auditory nerve by electrical stimulation at a rate of 4 pps. Compound action potentials in response to optogenetic, electrical, and hybrid stimuli were recorded, and activation thresholds compared across the different stimulation modalities. Activation thresholds were defined as the minimum power level presented that produced a visually identifiable response.

Despite our best efforts to maximally overlap the optogenetic and electrical stimuli, they did not overlap perfectly—i.e. some portions of the auditory nerve were only stimulated by optogenetic or electrical stimuli. As a result, there were three distinct responses to hybrid stimulation: responses of neurons activated by electrical stimulation only, those activated by optogenetic stimulation only, and those activated by interaction of electrical and optogenetic stimulation (i.e. hybrid responses). To isolate the hybrid responses, the responses at the equivalent electrical and optogenetic levels were subtracted from combined recordings. Finally, the difference between activation threshold current for electrical stimulation and hybrid stimulation were measured to evaluate the interaction of optogenetic and electrical stimulation (figure 3(A)).

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Hybrid stimulation was presented at several levels of sub- and supra-threshold optogenetic stimulation each combined with a range of electrical current levels. As the light power was increased, the activation thresholds for electrical stimulation decreased. In some cases, responses were observed at the lowest level of electrical stimulation presented above zero, and so the shift in activation thresholds was likely larger than presented here (figure 3(B)). The magnitude of this shift was significantly greater for acutely deafened animals compared to chronically deafened animals at all light levels (figure 3(B)). For example, at 15–20 dB suprathreshold optical stimulation, the electrical activation threshold was reduced by 328 ± 56 µA in acutely deafened mice compared to 7 ± 7 µA for chronically deafened mice (p < 0.05; t-test; n = 4 acute deaf, n = 4 chronic deaf). Indeed, the overall degree of facilitation was significantly different between the two groups, as measured by comparing the slopes of the change in electrical activation thresholds (p < 0.005; t-test, n = 6 acute deaf, n = 6 chronic deaf).

We did not observe any significant differences between the optogenetic nor electrical activation thresholds of the acutely deafened and chronically deafened mice.

To understand if facilitation occurs at higher stimulation rates and, furthermore, to understand how the temporal characteristics of the auditory nerve response compares across the different stimulation modalities, we repeated our experiments using 100 pps burst stimulation over 300 ms, presented at a rate of 1 Hz (figure 4). As before, hybrid responses were identified as the residual of the combined stimulation recordings, and the electrical- and optical-only recordings. Three measures were taken from the responses: the percentage of responses elicited across the burst, the temporal spread (i.e. the time difference between the earliest and latest response of the burst), and the ratio (i.e. the relative change in peak-to-peak response amplitude from the first response to the last response). Recordings were conducted across a range of supra- and sub-threshold levels per animal and measures were taken from recordings that demonstrated more than two responses across the duration of the burst stimulus.

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For both acutely deafened and chronically deafened animals, electrical-only stimulation evoked a high median percentage of responses (acute median ± interquartile range = 100 ± 2%, chronic: 100 ± 0%). This was also true for optogenetic-only stimulation in acutely deafened animals (86.7 ± 47%), but not for optogenetic-only stimulation in chronically deafened animals, which was significantly lower at 56.7% ± 43% (p = 0.001, Wilcoxon–Mann–Whitney test, n = 17 for electrical, n = 13 for optogenetic). The median percentage of residual hybrid responses elicited using suprathreshold electrical stimulation was consistently high in acutely deaf mice (figure 5(A) purple bars). Medians between 87% and 100% were observed at all levels, except for the highest optogenetic powers used, where the median dropped to 63%. In contrast, the median percentage of residual hybrid responses elicited using suprathreshold electrical stimulation was consistently low in chronically deaf mice, peaking at 86.7% for <15.5 dB suprathreshold optogenetic stimulus.

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The temporal spread of electrical-only responses was low for both acutely and chronically deafened mice (94 ± 14 µs and 35 ± 15 µs, respectively, median ± variance), whereas optogenetic-only responses were significantly higher for both groups (315 ± 27 µs and 285 ± 26 µs for acutely and chronically deafened, respectively; p < 0.001 vs electrical; Wilcoxon–Mann–Whitney test, n = 17 for electrical, n = 13 for optogenetic) (figure 5(B)). Despite the observed differences in percentage of responses elicited for optogenetic-only responses, the temporal spread was not significantly different between acutely and chronically deafened mice. The temporal spread of hybrid responses evoked using suprathreshold electrical stimulation (purple bars) increased with increasing optogenetic stimulus power. However, similar to the trend observed with the percentage of responses elicited, the temporal spread of hybrid responses dropped at optogenetic stimulation powers above 18 dB re threshold (figure 5(B)). This trend was less clear in the chronically deafened mice, but temporal spread was generally higher at suprathreshold levels of optogenetic stimulation compared to sub- or threshold levels of optogenetic stimulation.

The amplitude of the response to electrical stimulation was stable across the burst, with both acutely and chronically deafened groups achieving median ratios (i.e. ratio of the response sizes of the last and first responses of the burst) near 1 (1.04 ± 0.05 and 1.02 ± 0.01, respectively, median ± variance) (figure 5(C)). In contrast, the amplitude of responses to optogenetic stimulation diminished during the burst, resulting in a ratio of 0.36 ± 0.08 in the acutely deaf mice and 0.54 ± 0.07 in the chronically deafened mice. These ratios were significantly lower than the ratio for electrical stimulation within the same group (p < 0.001, Wilcoxon–Mann–Whitney test, n = 17 for electrical, n = 13 for optogenetic) but were not significantly different to each other (p = 0.09, Wilcoxon–Mann–Whitney test, n = 21 for acutely deafened mice, n = 13 for chronically deafened mice). The ratio of hybrid responses followed a similar trend to temporal spread, with increasing optogenetic power eliciting responses with decreasing ratios, except at very high optogenetic stimulation levels.

Hybrid responses in acutely deafened animals appeared to demonstrate both good temporal fidelity and precision when optogenetic powers were close to activation threshold, whereas chronically deafened animals showed the best outcomes at higher powers (<15.5 dB). This trend was consistent across subthreshold and suprathreshold electrical stimulation.

Although combined stimulation could elicit hybrid responses at subthreshold levels of electrical stimulation, the degree of facilitation at 100 pps was much lower than that observed at 4 pps (data not shown). Subthreshold hybrid responses were observed in all animals but could not be elicited at levels lower than 55 µA below threshold.

Hybrid responses at subthreshold electrical stimulation levels typically evoked fewer responses across the burst than their supra-threshold counterparts. In this context, these responses rarely exceeded the temporal fidelity of their supra-threshold counterparts. However, at optogenetic stimulation levels close to threshold, these subthreshold electrical hybrid responses demonstrate good temporal precision, similar to that of their suprathreshold counterparts (figure 5).

Following final electrophysiology experiments, chronically deafened cochleae were processed to confirm exposure to neomycin sulphate under isoflurane anaesthesia induced hair cell loss as effectively as under the injectable anaesthesia used for the data in figure 1. Histology confirmed complete hair cell ablation in five of the six mice, with only one cochlea showing 42 remaining IHCs in the basal hook-turn region (data not shown).

To gain deeper insight into the temporal properties of the three stimulation modalities, we conducted single unit recordings of the auditory nerve in acutely deafened mice. Glass micropipettes were inserted through the brain towards the auditory nerve where it enters the dorsal cochlear nucleus (DCN) and ANs identified by their response to optogenetic or electrical stimulation. Optogenetic-only (1 ms), electrical-only (58 µs or 230 µs), or hybrid stimuli were presented as a burst over 250 ms at rates of 4–400 pps at 1–2 Hz. Power levels presented were relative to threshold, defined as the power level that resulted in 80% spike probability at 4 pps.

Generally, spike probabilities in response to suprathreshold optogenetic stimulation decreased with increasing stimulation rate. Temporal precision also diminished, as evidenced by increased jitter and decreased vector strength, at high stimulation rates. Optogenetic responses demonstrated a sharp drop off in spike probability and increased temporal precision around 100 pps even at very suprathreshold power levels. Increasing the power of optogenetic stimulation by 2–3 dB improved the average spike probability at 100 Hz (p < 0.05; RM-ANOVA, n = 6) (figure 6(E)), but did not significantly improve temporal precision.

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Electrical-only stimulation at threshold resulted in significantly greater temporal precision than optogenetic-only at stimulation rates between 20–200 pps (jitter: p < 0.005, vector strength: p < 0.05; two-tailed t-test, n = 5 for electrical, n = 6 for optogenetic). Even at the low electrical stimulation levels used here, the mean spike probability was similar to optogenetic stimulation at high stimulation rates. For example, the mean spike probability for threshold electrical stimulation at 200 pps was 14.9 ± 3.4%, compared to 13.7 ± 5.8% for suprathreshold optogenetic stimulation.

A subset of neurons were stimulated with both optogenetic-only and hybrid (using suprathreshold optogenetic and subthreshold electrical) stimulation, allowing direct comparison of measures. We observed an improvement in the spike probabilities in a subset of these neurons in response to hybrid stimulation compared to optogenetic-only stimulation (figure 6(E)), but not an improvement in temporal precision. The degree of improvement was dependent on the optogenetic power used, with the most improvement seen at lower optogenetic stimulation powers.

Following electrophysiology experiments, we confirmed deafening in the chronic deaf mice via whole mount preparations (cochlea pairs n = 2) or whole cochlea imaging (n = 3). Whole mount preparations and whole cochlea imaging showed no surviving inner or outer hair cells except for one animal, which had 42 surviving IHCs in the basal hook turn region (figure 7(A)).

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AN densities in left (deafened) and right (control) ears were evaluated in Rosenthal's canal of the apical, middle, and basal turns (averaged over 2–4 mid-modiolar sections per turn per animal; n = 3). There was significant degeneration of ANs in the middle turn Rosenthal's canal of deafened ears compared to the control ears (p < 0.05; paired t-test). Although degeneration was also evident in the apical turn of deafened ears, the reduction in AN density was not significant due to the small sample count, as the apical turn of one animal could not be assessed due to severe fibrosis. The remaining animals showed only minor fibrosis near the site of fenestration for the deafening procedure. No difference in AN density between deafened and control ears was observed in the basal turn (figure 7(C)).

This study is the first to examine optogenetic responses in a model of chronic deafness with near-complete inactivation of both inner and outer hair cells, ensuring no indirect activation of the auditory nerve via the hair cells. Previously published techniques, such as acoustic deafening and systemic aminoglycoside treatments (e.g. intraperitoneal kanamycin injections) achieve incomplete deafening, with many IHCs remaining at safe doses [29–32]. By directly perfusing the aminoglycoside neomycin sulphate directly through the left cochlea over 20–30 min, an extended time-period compared to our previous study [20], both inner and outer hair cells were effectively inactivated. Despite the unilateral ablation of hair cells in the left cochlea, ABR recordings indicated responses at high acoustic power (figure 1(B)). This is likely due to contamination from responses from the non-deafened ear, despite occlusion of the ear canal. Consequently, hearing thresholds presented here likely underestimate the severity of deafening. Deafening under isoflurane anaesthesia was less effective than deafening under combined ketamine/xylazine anaesthetic, consistent with a previous study that demonstrated the protective effects of isoflurane [33]. We only observed surviving hair cells in the basal region of one animal, and these hair cells showed gross morphological changes, suggesting their contribution to the recorded responses was negligible. Even allowing for the known protective effects of isoflurane anaesthesia, direct perfusion of neomycin was effective at mitigating hair cell mediated responses to stimuli, providing an effective model to test auditory nerve responses with little impact of hair cell mediated responses.

Additionally, this deafening technique was sufficient for the development of nerve degeneration in the 2–3 weeks following treatment. We observed substantial loss of auditory neuron cell bodies in Rosenthal's canal in the apical and middle turns, but not the basal turn. This aligns with the observation of surviving hair cells in the basal region of the cochlea, indicating that the sensory hair cells and neurons in this region are slower to degenerate following aminoglycoside exposures.

A major limiting factor of cochlear implant user outcomes is poor spatial precision of electrical stimulation. Previous studies have shown that in combining electrical and optogenetic stimulation, both the spread of activation and energy needs can be minimised [20, 34]. These studies investigated combined stimulation in the cochlea using in vitro techniques or in vivo recordings from the auditory midbrain. The results presented here are the first to investigate hybrid stimulation at the level of the auditory nerve in vivo. Our results confirm that optogenetic and electrical stimulation can combine at the level of the auditory nerve and evoke responses from individual neurons. Similar to results from the auditory midbrain and in vitro auditory nerve culture, we found the addition of optogenetic stimulation reduces the amount of electrical charge needed to activate the auditory nerve. Although we did not measure the spatial precision in this study (rather, we intentionally maximised the region of activation of our separate stimuli), a previous study using similar stimulation equipment established that combined stimulation with a more focused optical stimulus can improve the spatial precision of activation over electrical and optogenetic stimulation alone by reducing the electrical charge used to activate the auditory nerve [20].

We observed a clear difference between the degree of facilitation in acutely deafened and chronically deafened mice, with acutely deafened mice demonstrating a significantly greater increase in facilitation. This is most likely a consequence of the degenerating auditory nerve resulting from chronic deafness. Indeed, a study of the auditory nerve following a 10 d regime of intraperitoneal aminoglycoside deafening treatment in guinea pigs showed a significant decrease in auditory neuron density within 7 d of completing treatment [35].

How degeneration of the auditory nerve leads to poorer hybrid facilitation is not clear. One hypothesis is that there is reduced opsin expression and/or trafficking to the plasma membrane. Assuming that the mechanism of facilitation depends on the optogenetically-induced depolarising current, reduced plasma membrane expression would decrease the resulting optogenetic depolarisation, and thus the probability of facilitation. We expect this would also lead to an increase in the optogenetic activation threshold, which was not observed. Alternatively, degeneration of peripheral dendrites following chronic deafness has been shown to move the site of electrical activation centrally [21–24], where channel expression and myelination may be sufficiently different to impact the overlap of optogenetically and electrically induced depolarising currents. Studies investigating voltage gated ion channel distribution across auditory neurons show that ion channels are distributed evenly along the unmyelinated peripheral dendrites, but are localised at the nodes of Ranvier along the myelinated central axon [36, 37]. Although opsins show similar spread of expression across dendrites, they do not appear to show any localisation to the nodes of Ranvier in auditory neurons. This disparity in expression of opsins and voltage gated ion channels along the central axon may reduce the overlap between optogenetically induced and voltage gated ion channels, and thus reduce facilitation. A third hypothesis is that degeneration may impact the ability of the neuron to restore its resting potential following optogenetic stimulation. Natively evoked depolarising currents along the axon of a neuron rely on the controlled flow of sodium and potassium ions across the cell membrane. In contrast, the opsin used in this study, ChR2-H134R, is a non-selective cation channel, permitting the flow of calcium, sodium, potassium, and hydrogen ions across the cell membrane. This abnormal ion flow may take longer to restore in a degenerated neuron than in a healthy neuron.

Our results provide preliminary insight into the effects of degeneration on optogenetic responses of the auditory nerve. Although previous studies have demonstrated the changes in the auditory nerve response to electrical stimulation following chronic deafening [21–24], this study presents the first examples of optogenetic responses in such a model. Further research into the mechanism linking degeneration and optogenetic stimulation outcomes will be useful towards the clinical application of optogenetics and hybrid stimulation as novel stimulation strategies for next generation cochlear implants.

Traditionally, electrical stimulation has been the primary means of stimulation for neuroprostheses due to its safety and reliability in evoking responses from neural tissue, especially at high stimulation rates [38]. In contrast, optogenetic stimulation is unable to achieve consistent responses at high rates due to the limited kinetics of opsins [19, 39]. Efforts to engineer opsins and improve trafficking of opsins to the plasma membrane have demonstrated improvements to nerve responses at high stimulation rates [19]. These improvements, however, have not been sufficient to achieve good spike probability at the rates of contemporary cochlear implants (e.g. 400 pps or greater).

A previous study investigating hybrid stimulation found adding subthreshold electrical stimulation to suprathreshold optogenetic stimulation improved entrainment rates at the level of the auditory midbrain (inferior colliculus) compared to optogenetic stimulation alone [20]. However, the responses evoked purely from the interaction of the combined stimulation (i.e. hybrid responses) could not be extracted from responses evoked by electrical-only nor optogenetic-only stimulation alone. Using compound action potential recordings, we were able to investigate the stability and fidelity of hybrid responses from the auditory nerve. In contrast to the Thompson et al [20] study, responses evoked using suprathreshold optogenetic stimulation and subthreshold electrical stimulation demonstrated poor fidelity, but good stability compared to optogenetic-only stimulation. On the other hand, suprathreshold electrical stimulation with subthreshold optogenetic stimulation could achieve both better stability and following ability than optogenetic-only stimulation. Single unit recordings confirmed this finding in a subset of neurons, however, the improvement to temporal precision and spike probability was limited. Further research to uncover the precise mechanism of hybrid facilitation is critical to unlock further improvements in this stimulation paradigm.

The probability of evoking sustained responses in chronic deaf mice was generally lower with compound action potentials showing poor percentage of elicited responses. This appears to be a consequence of prolonged deafness, affecting the ability of the nerve to sustain responses to optogenetic stimulation. As discussed above, the precise mechanism of this remains unclear.

Additionally, optogenetic responses in four of the six chronically deafened mice showed an increased latency and decreased response size when retested following 2–4 h of combined stimulation. Similar behaviour was seen in the acutely deafened animals, although only a subset was retested. The cause of this change is unclear but may be due to a number of factors including the surgical procedure or opsin deactivation following over exposure to light as seen with other opsins [40, 41].

Not only high stimulation rates, but also high temporal precision is necessary to encode the fine temporal information of pitch and sound quality. There has been little success to date in achieving the temporal precision of electrical stimulation using optogenetic stimulation techniques. Indeed, in our study we observed a similar outcome, with optogenetic stimulation performing poorly at all measures of temporal precision compared to electrical stimulation, reflecting the slower kinetics of the opsin mediated responses. The opsin used in this study, ChR2-H134R, has relatively slow kinetics compared to other excitatory opsins, with many other opsins exhibiting much faster kinetics and better temporal precision [42]. However, even these 'rapid' opsins demonstrate poor temporal precision compared to electrical stimulation. In our study, we found hybrid responses demonstrated electrical-like temporal precision when the concurrent optogenetic stimulus was near threshold but worsened with increasing optogenetic stimulus power. Indeed, there appears to be a trade-off between facilitation and temporal precision, suggesting the best outcomes can be achieved at an intermediate level that both facilitates electrical activation without sacrificing temporal precision. The decreased temporal precision of hybrid responses with increasing light power suggests the optogenetic stimulation and opsin kinetics dominates the activation of the nerve at these higher powers.

Surprisingly, the responses of chronically deafened animals appeared to have better temporal precision compared to acutely deafened animals. This is possibly a reflection of the poorer ability of the nerve to sustain responses to the stimulus (i.e. reduced percentage of elicited responses) thus reducing the sample size, and/or reduced hybrid facilitation. Nevertheless, both good temporal precision and temporal fidelity could be achieved in chronically deafened animals, but required the use of higher optogenetic stimulation powers compared to the acutely deafened animals. This is likely a reflection of the poor facilitation seen in these animals.

In our single-unit recordings, we found optogenetic and hybrid stimulation paradigms resulted in increased jitter relative to electrical stimulation. Although this is described as poor temporal precision, jitter is normal in acoustic auditory nerve responses and may be an important cue for sound localisation. Further to this point, studies have implicated jitter as important for detection of interaural time differences and sound localisation in cochlear implant users [43–45].

In this study, we directed our optical fibre towards the modiolus, illuminating as much of the auditory nerve as possible to improve the likelihood of overlap with the region of electrical stimulation, and hence the probability of successfully eliciting responses to hybrid stimulation. This simplified investigation of the temporal properties of the auditory nerve response to the three modes of stimulation. However, in a clinical translation of this technique, one would try to achieve a narrow region of activation. A previous study investigated the spread of activation of optogenetic, electrical, and hybrid stimulation in the cochlea using measures from the inferior colliculus. Hybrid stimulation not only achieved a reduced spread of activation compared to electrical and optogenetic stimulation, but also an improvement in the entrainment rates compared to optogenetic stimulation. Hence, as long as the region of overlap between the two stimuli—optogenetic and electrical—is sufficient, then hybrid stimulation can achieve both high spatial and temporal precision when the optogenetic stimulus is near activation threshold.

We only recorded compound action potential responses at a single rate of 100 pps, even though contemporary cochlear implants typically use rates of 400 pps or more. We were limited by the ability to consistently extract the auditory nerve response from the electrical artefact at rates higher than 100 pps. It would be ideal to test the temporal properties of the nerve to the three stimulation modalities at clinically relevant rates, but this was not possible with our setup, nor the opsin we used. Indeed, even the fastest opsins to date are unable to maintain good spike probability at rates beyond 100 pps, and this remains the limiting factor of optogenetic and hybrid stimulation.'Rapid' opsins with faster kinetics may combine with electrical stimulation to achieve even higher temporal precision. Future studies should consider the use of a variety of opsins to understand the effect of kinetics on the spatial and temporal precision of hybrid stimulation.

The transgenic mouse model used in this study expressed opsin in all auditory neurons, allowing for optogenetic and hybrid activation. However, clinical translation of optogenetic or hybrid stimulation would require the use of gene therapy techniques to deliver the opsin gene to the auditory nerve. Unfortunately, such techniques do not achieve complete transfection of the nerve, leaving some neurons that do not express the opsin and are incapable of optogenetic and hybrid responses. Indeed, a previous study demonstrated that hybrid stimulation did not have any consistent effect on the spatial precision, although subthreshold optogenetic stimulation did reduce the electrical activation threshold [46]. Techniques to improve transfection rates of gene therapy are likely to benefit not only optogenetic stimulation but also hybrid stimulation. Nevertheless, a hybrid stimulation technique would still allow for electrical activation of non-transfected neurons, and so does not necessarily limit the use of these neurons.

Finally, auditory nerve responses were recorded under anaesthesia, which has been previously shown to impact the behaviour of nerves and the auditory system [47–49]. As such, it is not clear how the results seen here will translate in the awake animal. Development of reliable recording techniques for awake animals will greatly improve our understanding of auditory nerve behaviour and be imperative in the investigation of hearing loss and alternative stimulation strategies.