Optogenetic Infection and Optical Stimulation: A Study on Auditory Responses in Guinea Pig Cochlear Neurons

Abstract

Objective

This study aims to establish an economically viable and easily accessible adult animal model for optogenetic activation of auditory neurons using adeno-associated viruses (AAVs) carrying ChR2(H134R) to explore the potential of cochlear optogenetics as a hearing restoration technology.

Methods

Healthy adult guinea pigs were used in the experiments. The viral vector AAV2/8-ChR2(H134R)-hSyn-eYFP was administered to the right cochlea via the round window membrane. The confocal microscopy and reverse transcription polymerase chain reaction (RT-PCR) were utilized to analyze the ChR2(H134R) expression localized to spiral ganglion neurons (SGNs). The auditory pathway activation was assessed by recording the optical compound action potential (oCAP) and acoustic compound action potential (aCAP) at various laser intensities.

Results

The ChR2(H134R)-eYFP expression was confirmed in 90% of the tested animals, localized to the SGNs of the injected ear. Higher mRNA levels of ChR2(H134R) and eYFP were observed in the injected ear compared to the non-injected ear, while actin (Actb) mRNA levels were not significantly different. The oCAP was successfully elicited by a 470 nm blue light laser stimulus, with similar amplitudes and latency periods to those of aCAPs when the oCAP was evoked by 5.80 mW blue light and the aCAP was evoked by a 40 dB SPL click. The amplitudes of oCAPs increased with increasing laser intensity.

Conclusion

This study demonstrates the viability of optogenetic activation of the auditory system in adult guinea pigs through the transduction of AAV-ChR2(H134R) in SGNs. Cochlear optogenetics demonstrates potential as a hearing restoration technology, providing a basis for further clinical research and opening new avenues for investigation.

1. Introduction

Hearing impairment is a global health issue, affecting an estimated 466 million people worldwide, representing 6.1% of the global health community and the number is anticipated to rise in the future(Zeng et al., 2008; WHO, 2020). Cochlear implants (CIs) are regarded as the most effective intervention for hearing restoration in individuals with severe to profound hearing loss, achieved through electrical stimulation of SGNs. (Zeng et al., 2008). However, current CIs have several limitations that need to be addressed. For instance, CIs typically provide up to 20 frequency channels, which is significantly fewer than the normal physiological frequency channels in the human auditory system(Moser, 2015). This limitation in frequency resolution results in reduced spectral resolution, ultimately contributing to reduced auditory sensitivity, particularly for speech, in noisy environments. (Friesen et al., 2001). By integrating genetic and optical techniques, optogenetics has demonstrated the potential to specifically target SGNs with high accuracy( Zeng and Galvin III, 1999; Fenno et al., 2011; Hegemann and Nagel, 2013; Packer et al., 2013), thereby reducing channel crosstalk caused by the widespread current around each electrode contact in CIs. Previous studies have demonstrated that genetic manipulation and the expression of transgenes encoding exogenous proteins are well-tolerated, with no significant adverse effects, including immune responses, neuronal toxicity, or cell loss.(Shimano et al., 2013).

The development of several animal models has enabled the study of the activation of the auditory pathway using optogenetics. These include genetically engineered mice(Arenkiel et al., 2007) and rats(Tomita et al., 2009), as well as models utilizing in viral injections into the otocyst of developing embryos were performed in utero (Reisinger et al., 2011). While these models have provided valuable insights, they are often technically complex and expensive.

The goal of our study is to establish a more economically viable and easily accessible adult animal model for optogenetic activation of auditory neurons. AAVs are ideal candidates for this purpose due to their lack of pathogenicity, low immunogenicity, and replication deficiency. AAV-mediated gene transfection has become a standard approach in optogenetic studies (Yizhar et al., 2011; Wrobel et al., 2018).

In our study, the channelrhodopsin-2 (ChR2) gene expression in the adult guinea pig cochlea was successfully identified at the levels of both molecule and cell, confirming the successful transduction of the viral vector. The transduction efficiency of ChR2 in cochlear SGNs was quantified(Yizhar et al., 2011; Wrobel et al., 2018). Furthermore, we used electrophysiological methods to analyze the relationship between laser intensity and auditory electrophysiology by measuring the amplitude of the compound action potential (CAP). This approach provides a biologically safe and efficient protocol for manipulating SGNs in adult animals, which is essential for further experimental studies in auditory restoration.

2. Materials and Methods

2.1. Animal Preparation

For the experiments, healthy guinea pigs of adult age (25–30 weeks of age, either sex) were sourced from Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). Prior to surgery, the animals were anesthetized via intraperitoneal injection with pentobarbital sodium (40 mg/kg; Merck Company, Kenilworth, NJ, USA). The animals’ body temperature was maintained at approximately 37°C using a thermostatically controlled heating plate. Ethical approval for all experimental procedures was obtained from the Institutional Animal Care and Use Committee (IACUC) of the Chinese PLA General Hospital.

2.2. Vector Preparations

The viral vector AAV2/8-ChR2(H134R)-hSyn-eYFP consists of AAV2/8 carrying ChR2(H134R) fused to eYFP under the control of the human synapsin (hSyn) promoter. (Fig.1A). The vector was obtained from Obio Technology Corp., Ltd. (Shanghai, China). A standard concentration of 1.10E+13 v.g./ml was used for the viral inoculum, and a volume of 5 µl was applied as the standard dose.

Figure 1.

Optogenetic activation of SGNs in adult guinea pigs using AAV2/8-ChR2(H134R).(A) Schematic illustration of the AAV vector used for transducing SGNs with ChR2(H134R).(B) Depiction of the retroarticular surgical route to the round window (RW) in guinea pigs.(C) Image of the retroarticular incision, highlighting the access to the RW.(D) Procedure for virus injection: The catheter is connected to a micro-syringe, and a micro-injection pump is used to deliver the virus into the round window.

2.3. Viral Vector Injection

Injections were performed using micropipettes (Thermo Fisher Scientific) that were pulled from glass capillaries after heating to achieve a sufficiently thin inner diameter. The micropipettes were linked to a microinjector (Eppendorf, Germany) and operated by a microinjection pump (Eppendorf, Germany). A 5 µl volume of the viral vector suspension was administered into the right cochlea of adult guinea pigs (n = 10) via the round window membrane, ensuring precise targeting. The left cochlea, which was not injected, served as the control group (n = 10). The animals were allowed a 6-week recovery period before subsequent experiments.

2.4. Surgical Procedure for Inner Ear Injection

After anesthesia, the retroauricular area was shaved to prepare for the incision. The retroauricular approach was selected to access the bulla (the air-containing middle ear cavity). The muscles covering the bulla were dissected and removed using a mucous membrane knife. A hole was created above the round window on the bulla using a micro-electric drill. Subsequently, the cochlea’s round window and promontory were exposed. A small-diameter tubing was inserted into the round window, and the viral vector was precisely introduced into the inner ear using a micro-injection pump over a period of 1 minute. The surface of round window was then covered with a gelatin sponge. The muscles and skin were sutured. (Fig. 1B–D)

2.5. Optical Stimulation Protocol

A 470 nm continuous wave emitter was employed for laser stimulation, with a light-conducting fiber coupled to the laser device to ensure precise control. Intracochlear stimulation was achieved by inserting the fiber into the inner ear’s scala tympani. Laser pulses with a duration of 5 ms and a power of 3.70 mW (as measured at the fiber tip) were utilized to evoke the oCAP. The prominent oCAP amplitude made it possible to readily observe optically elicited potentials on the oscilloscope, which in turn allowed for the fine-tuning of the fiber’s position and orientation. The amplitude of the oCAP served as the read-out. The fiber was adjusted in position until the oCAP waveform reached stability, after which it was secured in place.

2.6. Immunofluorescent staining and Confocal microscopy

Slices were treated with 0.25% Triton X-100 solution for 30-minute incubation at 25°C and then rinsed with PBS in three 5-minute intervals to remove excess solution. The samples were involved a 2-hours incubation after adding a blocking reagent with diluted primary antibodies (mouse anti-NF 1:400 and rabbit anti-mCherry 1:400, Abcam). After removing the primary antibodies, the samples were subjected to three 5-minute PBS rinses Secondary antibodies diluted in the blocking reagent (sheep anti-mouse Alexa-488 1:400 and sheep anti-rabbit Alexa-568 1:400, Abcam) were added, and the samples were involved a 1-hour incubation in the dark, followed by three 5-min rinses with PBS, DAPI staining solution (Abcam) was added to the specimens. The antibody expression was observed with a Leica SP8 laser confocal fluorescence microscope at 20× and 40× magnifications to capture detailed images.

2.7. Real-time Quantitative PCR

Trizol reagent (Invitrogen) was used to lyse the cells, and total RNA was isolated as described by the manufacturer. Subsequently, first-strand cDNA was synthesized using the Superscript III RT Mix (dNTP Mix, DEPC-treated water) for qPCR (Thermo Fisher). Quantitative real-time PCR was performed using Mix II (10× PCR Buffer (-), Mg²⁺, dNTPs, forward primer, TaqMan probe, reverse primer, Platinum® Taq DNA Polymerase, ddH₂O) (Thermo Fisher) on a 7500-qPCR apparatus (Thermo Fisher). The PCR primers for amplifying the target genes are provided in Supplementary Table 1. Actin (Actb) functioned as the internal reference gene. Each sample was tested in triplicate to minimize stochastic error.

2.8. Pathophysiology of Auditory Pathway

Initially, we assessed the impact of surgical and viral interventions on auditory function by analyzing the reduction in acoustic CAP or auditory brainstem responses (ABRs). Subsequently, we determined the auditory threshold and latency of the suprathreshold aABR in virus-injected animals, comparing these metrics to those of normal saline controls. To characterize the optical stimulation of the auditory system, we recorded oCAPs using optical stimulation via fiber-coupled laser, following established blue light stimulus protocols. We compared the waveform, number of waves, and amplitude of oCAPs with those of aCAPs elicited by 40 dB clicks. Additionally, we quantitatively compared the aCAPs (stimulus frequency: 21 Hz; pulse duration: 10 ms; sound intensity: 40 dB SPL) recorded from noninjected guinea pigs to the oCAPs (stimulus frequency: 3.1 Hz; pulse duration: 10 ms; power: 5.80 mW) obtained from virus-injected animals.

The impact of middle ear surgery and viral injection on auditory function was assessed by examining the reduction in acoustic auditory brainstem responses (ABR) or compound action potentials (CAP). Then, the auditory threshold and latency of the suprathreshold ABR were evaluated in the virus-injected group, with comparisons made to the normal saline control group. To characterize the laser-induced activation of the auditory pathway, oCAPs were recorded using fiber-coupled laser stimulation, following established blue light stimulus protocols. The waveform, number of waves, and amplitude of oCAPs were compared with those of aCAPs elicited by 40 dB clicks. Additionally, the aCAPs (stimulus rate: 21 times/sec; duration: 10 ms; intensity: 40 dB SPL) recorded from non-injected guinea pigs(n=10) were quantitatively compared with the oCAPs (stimulus rate: 3.1 times/sec; duration: 10 ms; intensity: 5.80 mW) obtained from virus-injected ones(n=10).

2.9. oCAP recording

The auditory pathway was tested using both acoustic and optogenetic stimulation by recording the neuronal population responses. For acoustic stimulation, the aCAP was recorded with an average of 1,024 trials and the acoustic stimulation frequency was 20 Hz. For optogenetic stimulation, the oCAP was captured with an average of 100 trials. A silver-based probe was positioned on the round window surface, with the reference electrode positioned in the surrounding ear muscles. The oCAP was recorded using a Tucker-Davis ABR workstation (Tucker-Davis Technologies, Alachua, FL, USA) in a double-walled sound-isolated room. The sampling rate was set at 3.1 Hz with a bandpass filter of 300–3,000 Hz. The potentials were monitored on an oscilloscope adjacent to the recording setup to fine-tune the placement of the optical stimulator. Signal averaging (100 times) was performed, and the data were stored for further analysis. The average amplitudes of oCAPs were recorded under different laser intensities (ranging from level 1 to level 10), and the correlation between amplitudes and laser intensities was analyzed.

2.10. Statistical analysis

Data were visualized with SigmaPlot and analyzed statistically with SPSS 25.0 (IBM SPSS Statistics, Chicago, IL, USA). Results are shown as mean ± standard error of the mean (s.e.m.). Statistical analyses were conducted using two-tailed Student’s t-tests or one-way ANOVA. Post hoc comparisons for ANOVA were made using Bonferroni tests to identify significant differences among groups, with a significance level of α = 0.05.

3. Results

3.1. Expression of ChR2 in the Spiral Ganglions via Optogenetics

Four to six weeks post-injection, ChR2 expression in SGNs was analyzed using confocal microscopy and RT-PCR. The expression of ChR2(H134R) in 90% (18 of 20) of the tested animals was reliably and effectively confirmed by both methods, localized to the SGNs of the injected cochlear compared to the non-injected side. Cochlear cryosections were subjected to immunohistochemistry, revealing precise staining in SGNs. (Fig. 2). The yellow regions in the images indicate the colocalization of NF200 and eYFP, confirming the successful expression of ChR2 in the somata and neurites using the Imari software. (Fig. 3).

Figure 2.

Confocal images of SGNs in guinea pigs. (A–D) Confocal microscopy images showing transduced SGNs. eYFP (red) indicates neurons transduced with ChR2(H134R), DAPI (blue) staining cell nuclei, and neurofilament-200 (green) labels SGNs. Scale bars, 50 μm. These images demonstrate robust ChR2(H134R) expression in SGNs, confirming successful transduction. (E–H) Control samples under identical immunostaining conditions, showing no eYFP expression, confirming specificity of the transduction process.

Figure 3.

Concurrent localization of anti-NF200 (red) and eYFP (green)in SGNs.

(A-F) Confocal images showing concurrent localization of anti-NF200 (red) and eYFP (green)in SGNs from different angles. Yellow regions indicate overlap, confirming ChR2 expression in SGNs. Scale bars, 50 μm.

3.2. Injection in SGNs results in the up-regulation of RNA for ChR2 (H134R)-eYFP

Prior to evaluating ChR2 expression in SGNs, a strong fluorescent output was detected in the neuronal somata and fiber tracts of the SGNs (Fig. 2,3). To further validate the ChR2 expression, RT-PCR was conducted to confirm the ChR2 and eYFP expression in the auditory neurons. Gene-specific primers encoding ChR2 (H134R), eYFP, and actin (Actb) were constructed by means of Clone Manager software (Sci Ed Software). Quantitative analysis of mRNA levels in SGNs was performed using qPCR (Fig. 4A). The analysis demonstrated that mRNA levels of ChR2 (H134R) and eYFP were elevated in the the virus-administered ear relative to the non-administered one (Fig. 4B), while the levels of actin (Actb) mRNA did not reveal substantial difference between the two groups (Fig. 4B).

Figure 4.

Expression analysis of ChR2(H134R), eYFP, and actin (Actb) in SGNs.

(A) qPCR assay showing amplification curves for ChR2(H134R), eYFP, and actin (Actb) in SGNs. The cycle number is plotted against normalized fluorescence intensity (Rn) to illustrate PCR amplification. (B) Bar graph summarizing RNA expression levels of ChR2(H134R), eYFP, and actin (Actb) in SGNs of injected ears compared to non-injected controls. Data are presented as mean±SEM, with animal numbers indicated. Actin (Actb) expression was detected in both injected and non-injected ears, while ChR2(H134R) and eYFP RNA were specifically detectable in the SGNs of injected ears.

3.3. Optical stimulation activates the auditory pathway

Auditory function was assessed by measuring the aCAP and the threshold and latency period of the acoustic auditory brainstem responses (aABR). Because of the Operative procedure and Virus administration, both aCAP and the threshold and latency period of aABR were mildly compromised (Fig. 5A,B,C) (P < 0.01). When in comparison with the normal saline control group, the virus solution itself did not have an additional effect on the threshold of aABR (Fig. 6A,B) (P > 0.01). Therefore, the hearing loss observed in the injected guinea pigs was not attributed to the virus solution.

Figure 5.

Effects of AAV-ChR2(H134R) injection on auditory responses in guinea pigs (n=20).

(A) Representative aABR and aCAP waveforms prior to and following virus-injection. (B) Average aABR amplitudes before and after injection. (C) Latency of aABR waves before and after injection. (D) Average aCAP amplitudes before and after injection.

Figure 6.

Effects of AAV-ChR2(H134R) injection on ABR thresholds in guinea pigs.

(A) ABR thresholds in guinea pigs following AAV-ChR2(H134R) injection. (B) ABR thresholds in guinea pigs following saline injection. Data are presented as mean ± SEM, with statistical significance indicated (P<0.01)

Since ChR2 expression was confined to SGNs, the optogenetically induced auditory activity is specifically attributable to these neurons. To assess the evocation of the auditory signal pathway, we employed both acoustic and optogenetic stimulus, recording aCAP and oCAP, respectively. The oCAP was successfully elicited by a 470 nm blue light laser stimulus, whereas no standard waveform was observed when the blue light was off (Fig. 7A). The oCAP exhibited significant differences in waveform, number of waves, and amplitude compared to the aCAP，which was elicited by 40 dB clicks (Fig. 7B), indicating distinct characteristics between the two types of CAPs.

Figure 7.

Optogenetic and acoustic stimulation-induced oCAPs in guinea pigs.

(A) Representative oCAP waveforms before and after optogenetic stimulation (470 nm blue light laser; 5.80 mW/mm² at the indicated duration and 5 Hz; 50 trials).(B) Comparison of representative aCAP waveform in response to acoustic stimulation at 20 Hz (average of 1024 trials) and oCAP waveform in response to 470 nm blue light laser stimulation at 3.70 mW and 11.1 Hz (average of 100 trials).

Quantitative comparison was conducted between the aCAP (stimulus frequency: 21 Hz; pulse duration: 10 ms; intensity: 40 dB SPL) of non-injected animals and the oCAP(stimulus rate: 3.1 Hz; pulse duration: 10 ms; intensity: 5.80 mW) (Fig. 7B). The results exhibited comparable amplitudes and nearly parallel latency periods: 0.78±0.023 μV (latency 2.71 ms) for the oCAP activated by 5.80 mW blue light stimulation and 0.70±0.034 μV (latency 2.59 ms) for the aCAP evoked by a 40 dB SPL click. This similarity implies that SGN activation induced by optogenetic and acoustic stimulation is highly synchronous.

We examined the oCAP responses across a range of trans-cochlear irradiance intensities. Our analysis of the correlation between oCAP amplitudes and laser intensities showed that amplitudes rose proportionally with laser intensity (Fig. 8). In contrast, optogenetic stimulation in the control group’s cochlea failed to evoke any action potentials when the laser was applied. Despite minor variations in amplitude, waveform, and latency observed among different experimental guinea pigs, the data remained consistent within individual samples. Collectively, these findings indicate that optogenetic coding can produce action potentials in SGNs comparable to those induced by acoustic stimulation.

Figure 8.

Laser intensity-dependent oCAP amplitudes in ChR2(H134R)-expressing guinea pigs. Average oCAP amplitudes recorded from ChR2(H134R)-expressing adult guinea pigs in response to increasing laser intensities (levels 1 to 10). Data are presented as mean±SEM, with statistical significance indicated where applicable.

4. Discussion

In this study, the guinea pig was established as an adult guinea pig model for developing optical stimulation. AAVs were utilized as vectors for neural transduction, optimized to express ChR2 (H134R) safely and efficiently. This optimization is crucial as AAVs enable high-level and long-term expression of foreign proteins(Deverman et al., 2016). Utilizing the AAV-ChR2 (H134R) vector with the neuron-specific hSyn promoter, we attained selective transgene expression in neurons in the SGNs of adult guinea pigs, thereby enhancing the transduction rate for ChR2 expression. This approach allowed us to efficiently target the desired neurons and optimize the expression levels of ChR2, which is crucial for our optogenetic studies. Administering the virus into inner ear led to ChR2 expression in about 90% of the virus-administered cochleas, a rate significantly higher than the roughly 50% achieved with intra-modiolar injection. This enhanced transduction efficiency underscores the effectiveness of the round window approach in achieving robust ChR2 (H134R) expression in the cochlear neurons(Deverman et al., 2016; Wrobel et al., 2018). Recent studies have not only enhanced transduction rates throughout the cochlea but also minimized significant SGNs reduction in most experimental animals(Keppeler et al., 2018; Mager et al., 2018). We critically examined whether the injection of the virus through the round window and the subsequent expression of ChR2 (H134R) in SGNs would impair auditory capabilities in mature guinea pigs. Our findings revealed that neither the virus injection nor the expression of ChR2 (H134R) had any significant detrimental impact on the auditory pathway, as shown by the mean aABRs and aCAPs amplitudes recorded prior to and following the injection. This indicates that our approach is safe and effective for studying auditory responses in optogenetically infected cochlear neurons. Additionally, several methods of AAV vector injection have been proven to cause no obvious neuronal loss or decrease in acoustic ABR in the cochlea(Shimano et al., 2013; Hernandez et al., 2014). Thus, our method of virus injection into the cochlea can be considered a viable option for establishing an adult animal model. One of the key benefits of optogenetic technology is its capacity to selectively induce the ChR2 (H134R) expression in SGNs. To verify the expression above, we conducted assessments at both the molecular and cellular levels. Immunohistochemical staining and RT-PCR analyses demonstrated that ChR2 (H134R) is mainly localized in the neurites and somata of SGNs. This selective expression ensures precise control over the neurons, which is crucial for our study of auditory responses.

The results of colocalization and immunohistochemistry supported the high transduction rates of optogenetic stimulation in auditory SGNs. The non-injected ear showed no signs of ChR2 (H134R) expression, confirming the specificity of the viral vector delivery. The development of optogenetic models is crucial for advancing hearing research in both experimental and clinical settings, facilitating a deeper understanding of auditory mechanisms. This method successfully demonstrates adult cochlear transfection. Under direct optogenetic SGN stimulation, the oCAPs exhibited similar amplitudes and expected latency periods to those of aCAPs. Mild variability in terms of amplitudes and latency periods of oCAPs within the experimental cohort of guinea pigs could be attributed to variations in transduction efficiency, ChR2 (H134R) expression levels, or the stability of optical fiber positioning.

Furthermore, the relationship between oCAPs and various optical stimulation intensities revealed that higher light intensities corresponded to greater oCAP amplitudes. The maximum light intensity(5.8 mW) lies well within the non-harmful range for in vivo optogenetic applications, which can reach up to 300 mW/mm²(Han, 2012). Consequently, the oCAPs achieved were confirmed to be biosafe. Optical stimulation holds the potential to significantly enhance the accuracy of auditory prosthetics by providing sufficient individual stimulation channels, thereby improving frequency coding. Our study aims to optimize optogenetic adult models for auditory studies and subsequent clinical applications. To achieve this, we have focused on increasing transduction and expression rates through the optimization of viral vectors and gene sequences. Stable oCAPs can be achieved by better positioning the emitter within the cochlea.

In summary, our study demonstrates the efficacy of optogenetic activation of SGNs in adult guinea pigs. These findings not only provide compelling evidence for the potential of cochlear optogenetics as a novel technique for hearing recovery but also pave the way for future clinical investigations.

Funding Statement

This work was supported by the Beijing Natural Science Foundation of China under Grant 7222185.