Superior temporal resolution of Chronos versus channelrhodopsin

Superior temporal resolution of Chronos versus channelrhodopsin
Hearing Research 322 (2015) 235e241
Contents lists available at ScienceDirect
Hearing Research
journal homepage: www.elsevier.com/locate/heares
Superior temporal resolution of Chronos versus channelrhodopsin-2
in an optogenetic model of the auditory brainstem implant
Ariel Edward Hight a, c, 1, Elliott D. Kozin a, b, 1, Keith Darrow d, Ashton Lehmann a, b,
Edward Boyden e, M. Christian Brown a, b, Daniel J. Lee a, b, *
a
Eaton-Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston, MA, USA
Department of Otology and Laryngology, Harvard Medical School, Boston, MA, USA
Program in Speech and Hearing Bioscience and Technology, Harvard Medical School, Boston, MA, USA
d
Department of Communication Sciences and Disorders, Worcester State University, Worcester, MA, USA
e
Departments of Brain and Cognitive Sciences and Biological Engineering, MIT Media Lab and McGovern Institute, MIT, Cambridge, MA, USA
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 August 2014
Received in revised form
6 November 2014
Accepted 8 January 2015
Available online 15 January 2015
Contemporary auditory brainstem implant (ABI) performance is limited by reliance on electrical neurostimulation with its accompanying channel cross talk and current spread to non-auditory neurons. A
new generation ABI based on optogenetic technology may ameliorate limitations fundamental to electrical stimulation. The most widely studied opsin is channelrhodopsin-2 (ChR2); however, its relatively
slow kinetic properties may prevent the encoding of auditory information at high stimulation rates. In
the present study, we compare the temporal resolution of light-evoked responses of ChR2 to a recently
developed fast opsin, Chronos, to ChR2 in a murine ABI model. Viral mediated gene transfer via a
posterolateral craniotomy was used to express Chronos or ChR2 in the cochlear nucleus (CN). Following a
four to eight week incubation period, blue light (473 nm) was delivered via an optical fiber placed
directly on the surface of the infected CN, and neural activity was recorded in the contralateral inferior
colliculus (IC). Both ChR2 and Chronos evoked sustained responses to all stimuli, even at high pulse rates.
In addition, optical stimulation evoked excitatory responses throughout the tonotopic axis of the IC.
Synchrony of the light-evoked response to stimulus rates of 14e448 pulses/s was higher in Chronos
compared to ChR2 mice (p < 0.05 at 56, 168, and 224 pulses/s). Our results demonstrate that Chronos has
the ability to drive the auditory system at higher stimulation rates than ChR2 and may be a more ideal
opsin for manipulation of auditory pathways in future optogenetic-based neuroprostheses.
This article is part of a Special Issue entitled “Lasker Award”.
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
The cochlear implant (CI) is the most successful of neuroprostheses, and provides meaningful auditory benefits to pediatric
and adult patients with severe to profound hearing loss. In the past
50 years, over 300,000 individuals worldwide have received a CI
(NIDCD, 2014). Over this period, CI technology has evolved from a
crude single channel implant to a multi-channel auditory neurostimulator providing sound and speech perception to the majority
* Corresponding author. Wilson Auditory Brainstem Implant Program, Massachusetts Eye and Ear Infirmary, Department of Otology and Laryngology, Harvard
Medical School, 243 Charles Street, Boston, MA 02114, USA.
E-mail address: [email protected] (D.J. Lee).
1
Contributed equally.
http://dx.doi.org/10.1016/j.heares.2015.01.004
0378-5955/© 2015 Elsevier B.V. All rights reserved.
of deaf users. Cochlear implants have proven highly beneficial for
several etiologies of hearing loss, including genetic causes of
deafness (Vivero et al., 2010). The recent Lasker Award highlights
the development of the CI and illustrates the profound success of
this device and its positive impact on society (Williams, 2013).
However, there is a small subset of deaf individuals who will not
benefit from the CI due to 1) a small or absent cochlea, 2) a small or
absent auditory nerve, or 3) injury or scarring of the inner ear or
auditory nerve secondary to meningitis, trauma, or tumor, such as
bilateral
vestibular
schwannomas
that
arise
from
Neurofibromatosis-2 (NF-2) (Asthagiri et al., 2009). An auditory
brainstem implant (ABI) is an option to provide hearing sensations
in these patients who are not candidates for the CI due to these
considerations. More than 1000 patients worldwide have been
implanted with an ABI (Lin et al., 2012). The ABI bypasses the
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A.E. Hight et al. / Hearing Research 322 (2015) 235e241
damaged or absent cochlea and auditory nerve to transmit electrical stimuli to the cochlear nucleus (CN) in the brainstem
(Hitselberger et al., 1984; Sennaroglu et al., 2009).
Hearing outcomes of ABI users are highly variable across similar
cohorts of patients (Colletti et al., 2012; Colletti and Shannon, 2005;
Nevison et al., 2002), and overall performance of ABI users lags
behind that seen for CI users. Further, many ABI users experience
side effects, such as facial pain, tingling, and twitching, as well as
dizziness, due to activation of non-auditory neurons (Colletti et al.,
2010). One possible explanation for limited outcomes and side effects may be the spread of electric current (Eisen and Franck, 2005;
Nardo et al., 2008; Venter and Hanekom, 2014). One approach to
improve speech perception is to increase the number of electrode
channels. However, due to current spread, this may result in
€x et al., 2003; Karg et al., 2013; Qazi et al.,
channel cross talk (Boe
2013).
Optical stimulation of the nervous system is now being used as a
novel stimulus paradigm in research laboratories. For the central
auditory system, light-based activation offers a theoretical advantage over traditional electric-based neural stimulation as focused
light may be able to excite a select set of neurons, increasing the
density of independent stimulation channels while reducing the
unintended consequence of current spread (Fu and Nogaki, 2005;
Fu et al., 1998). These properties could address the limitations
seen with the electrically based ABI. Over the past decade, infrared
neural stimulation (INS) of the auditory system has been investigated as an alternative means to stimulate neurons; however, INS
may have limited applications in the central auditory system.
Recent efforts employing INS in the central auditory system have
failed to elicit an evoked response in a deafened animal model,
limiting its potential clinical utility (Verma et al., 2014).
In contrast to INS, optogenetics uses light from the visible
spectrum to stimulate the nervous system, and it has been used to
investigate a host of neural systems (Ayling et al., 2009; Boyden
et al., 2005; Huff et al., 2013; Rolls et al., 2011). Viral-mediated
infection is a common approach to deliver genes encoding for microbial opsins, light-gated transmembrane channels that enable
neurons to respond to optical stimulation. ChR2 is the most widely
used opsin in neuroscience (Bernstein et al., 2008; Boyden et al.,
2005; Chow et al., 2010; Han and Boyden, 2007; Zhang et al.,
2006), however, only a few recent studies have applied optogenetics to the auditory system (Hernandez et al., 2014; Shimano
et al., 2013). Shimano et al. introduced ChR2 into the CN and
demonstrated light-evoked increases in auditory neural activity
locally in the CN. Building on the results of Shimano et al., we
previously showed optogenetic stimulation of the CN results in
activation of the auditory pathway, including the inferior colliculus
and auditory cortex (Darrow et al., 2014). In a recent optogenetics
study of the peripheral auditory system, transgenic mice expressing
ChR2 in spiral ganglion neurons (SGN) of the cochlea showed
neural responses in SGNs themselves and CN and the in neurons of
the central auditory pathway (IC) (Hernandez et al., 2014). Overall,
these studies demonstrate that optogenetics can be used to activate
the auditory system from the periphery throughout the central
pathway.
One unique property of the auditory system is its capability of
providing a highly synchronous response with the rapidly varying
features of an acoustic waveform, a property necessary to encode
the also rapidly varying characteristics of speech. Original studies of
the kinetics of ChR2 and its variants (Boyden et al., 2005; Zhang
et al., 2006) suggest that ChR2 may be too slow for optimal function in the auditory system (Darrow et al., 2014). Over the last
several years, a host of new opsins have become available with
variable activation thresholds, wavelengths of stimulation, and
most importantly, kinetic properties (Yizhar et al., 2011). One of the
most recently developed opsins, Chronos (Klapoetke et al., 2014),
appears to have faster kinetic properties that may be better suited
for conveying temporal cues to the auditory system. Herein, we
compare the temporal characteristics of ChR2 and Chronos in a
translational murine ABI model.
2. Methods
2.1. Animal protocol
All experimental procedures were performed in accordance
with the National Institutes of Health guidelines for the care and
use of laboratory animals as well as the approved animal care and
use protocols at the Massachusetts Eye & Ear Infirmary, Boston, MA.
2.2. Surgical exposure of the dorsal cochlear nucleus
Methods to expose the dorsal cochlear nucleus (DCN) have been
described (Kozin et al., 2015). Direct exposure of the DCN, rather
than stereotaxic injection, was used to minimize the chances of
missing the desired injection site in addition to replicating the
potential surgical approach used during human ABI operations.
Normal hearing CBA/CaJ mice aged 4e6 weeks were anesthetized
with xylazine (10 mg/kg) and ketamine (100 mg/kg) via an intraperitoneal (IP) administration. Following anesthesia, the overlying
scalp was exposed to provide unobstructed access to the surgical
site. The mouse was placed in a Kopf small animal stereotaxic
holder (Tujunga, CA), and held in place by a snout clamp. The left
parietal, interparietal, and occipital bones of the skull are exposed
and rongeurs are used to make a craniotomy over the interparietal
bone, left of midline, ~2 mm caudal to the lambda suture line.
Following craniotomy, using a 5 French suction, aspiration of
lateral-most portion of the left cerebellum reveals the underlying
DCN (Fig. 1).
2.3. Pressure microinjection for gene transfer
After the DCN was clearly visible, pressure microinjections are
made into the DCN using a 5 ml Hamilton syringe. Between 1.5 and
2.0 ml of adeno-associated virus with ChR2 (AAV2.8-ChR2 fused
with GFP or mCherry and CAG promoter, courtesy of Dr. Edward
Boyden's lab) or Chronos (AAV 2/8) were infused over a 2e4 min
period. (Plasmid for Chronos was provided by Dr. Edward Boyden,
Massachusetts Institute of Technology, Cambridge, MA. Amplification took place at the Boston Children's Viral Core, Boston, MA.
Measured titers of Chronos were 1.21 1014 GC/ml.) Immediately
following injection, the incision was closed and the scalp was sutured. Four additional mice were used as either ‘sham’ or control
cases. These included sham-injected mice (n ¼ 2) that underwent
the exact surgical protocol as AAV injected mice, including injection
of saline into the brainstem over 2e4 min, and control mice (n ¼ 2),
which had no history of manipulation.
2.4. Re-exposure of dorsal cochlear nucleus and exposure of
contralateral inferior colliculus
After a four to eight week survival time to allow expression of
either ChR2 or Chronos, the mice were prepared for acute surgery
to characterize responses to optical stimulation. Mice were reanesthetized and underwent the above-described surgical procedure for performing the craniotomy and the cerebellar aspiration
for direct visualization of the DCN. After the injected region was reexposed for optical stimulation, a craniotomy was made over the
right IC and the exposed brain surface was covered with highviscosity silicon oil. During the course of physiological recordings,
A.E. Hight et al. / Hearing Research 322 (2015) 235e241
237
Fig. 1. Surgical approach to the DCN for opsin injection and IC for neural recording with a multichannel electrode probe. A: The skin and muscle are retracted laterally to expose the
lambda and the coronal suture lines. B: Placement of NeuroNexus recording probe into the IC in a vertical direction. C: Left-sided posterior craniotomy and partial cerebellar
aspiration have been performed and a 400 mm diameter optical fiber mounted on a micromanipulator is introduced through the craniotomy and onto the CN surface. D: Schematic
representation of the recording probe positioned along the tonotopic axis of the central nucleus of the inferior colliculus (ICc) so that each of the 16 recording electrodes (spaced
50 mm apart) records a different characteristic frequency.
the core body temperature of the animal was maintained at 36.8 C
with a homeothermic blanket system.
2.5. Optical stimulation
Blue light (473 nm) stimuli were produced by a laser (BL473T100FC, Shanghai Laser & Optics Century Co.) targeting the peak
wavelength sensitivities of ChR2 (~475 nm) and Chronos (~500 nm)
(Klapoetke et al., 2014). Light stimuli were delivered via an optical
fiber (400 mm diameter) that was held in place by a micromanipulator and placed directly on the exposed surface of the DCN
(Fig. 1). Pulses of 1 ms duration were presented at pseudorandomized rates from 14 to 448 pulses/s for train durations of 300
or 500 ms followed by 300 or 500 ms of no stimulation, respectively. Either 50 or 80 trials were presented at light intensity
ranging from 0 to 13 mW. The laser was calibrated by positioning
the 400 mm diameter optical fiber ~2 mm from a standard Si
photodiode power sensor (9.5 mm diameter, Thorlabs S121C)
connected to a USB power and energy meter interface (Thorlabs
PM100USB). The voltage command parameters were systematically
varied based on the range of pulse amplitudes, widths, and rates
used experimentally. Resulting measured laser intensity (radiant
exposure, mW) was calibrated to the voltage input.
2.6. Contralateral inferior colliculus recordings
Multiunit recordings were made from the central nucleus of the
IC using a penetrating 16-channel linear silicone probe (NeuroNexus Technologies, Fig. 1). The position of the recording probe was
first inserted perpendicular to the exposed surface above the IC,
approximately ~1 mm lateral to the midline and immediately
caudal to the transverse sinus. The probe was repositioned until a
complete tonotopic map across the recording channels was obtained (Guo et al., 2012; Malmierca et al., 1993) using acoustic
frequencies from 8 to 45.25 kHz in 0.5 octave steps and from 0 to
80 dB in 10 dB steps, using 20 ms duration tone bursts with a
repetition rate of 10 bursts/s. Injected mice had acoustic thresholds
(<40 dB SPL) comparable to non-injected mice. Raw voltage signals
were band-pass filtered (0.3e3 kHz, 5 pole) and sampled at 25 kHz.
Common mode rejection was performed across all 16 channels and
then the signal was digitally filtered (zero-phase butterworth
band-pass filter, 0.5e3 kHz, 5-pole).
To compute driven firing rate, average spike count was
computed over the pulse train duration and spontaneous firing
rate, computed from spikes collected during the no stimulation
period, was subtracted from it. Paired t-tests were performed
between the firing rates and spontaneous rates from every trial
for each electrode and stimulus intensity. For each electrodeintensity combination in which the driven rate was not significant (p > 0.01), the driven rate was assigned to be 0 spikes/
second. Driven rateeintensity curves were generated from the
average of driven rate across all electrodes collected during each
stimulus intensity presented at 28 pulses/s and normalized to the
highest driven rate evoked by any stimulus intensity. To investigate temporal properties of laser-evoked spiking we computed
the synchronization index (SI, magnitude of the vector of averaged spikes collected during the period between stimulus pulses;
the SI varies between 0 (no synchronization) and 1 (all spikes
occurring exactly at the same phase of the stimulus period);
Dynes and Delgutte, 1992). For all stimulus electrode-intensities
in which driven rate was calculated to be zero spikes/s SI was
also set to zero.
2.7. Histology and immunohistochemistry
Following conclusion of experiments, mice were euthanized
with an overdose of ketamine and perfused with normal saline
followed by 4% paraformaldehyde. Brainstems were extracted from
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A.E. Hight et al. / Hearing Research 322 (2015) 235e241
the skull and post-fixed for 2 h. Brainstems were cryoprotected in
30% sucrose for 48 h, and then sectioned using a cryostat into
30e60 mm sections. Before the staining procedure, sections were
allowed to thaw at room temperature and then rehydrated in PBS
for 10 min. After washing with PBS, tissue was permeabilized and
blocked with blocking solution (0.3% Triton X-100, 15% heat inactivated goat or donkey serum in PBS) for 1 h. Visualization of cell
nuclei was enabled with 4,6-diamidino-2-phenylindole (DAPI;
Vector Laboratories). Staining was analyzed with epifluorescence
microscopy (Axioskop2 Mot Axiocam, Zeiss) and confocal microscopy (Leica).
3. Results
3.1. Expression of Chronos and ChR2 in the cochlear nucleus
Opsin-linked immunofluorescence demonstrated Chronos or
ChR2 gene transfer throughout the DCN and in the ventral cochlear
nucleus (VCN). Chronos-GFP immunofluorescence appeared in an
array of cell types, including morphologies consistent with DCN
fusiform cells (red arrow, Fig. 2B), giant cells, and cartwheel cells.
We did not directly investigate anterograde labeling of axons in
Chronos cases, but such labeling has been observed previously in
ChR2 cases, which employed an identical AAV serotype and promoter (Darrow et al., 2014).
3.2. Synchronization and driven rate of IC neural activity in
response to optical stimulation of CN
Neural activation in the IC was evoked by light stimulation of the
CN. Dot raster and peristimulus time (PST) histogram plots from
one recording site in a Chronos mouse are shown in Fig. 3. High,
sustained rates of firing were observed during the light pulse trains
and low rates of spontaneous firing were observed when there are
no stimuli (second half of traces). For the pulse train of 448 pulses/s
(Fig. 3B), the PST histogram shows that the high initial firing
adapted to steady-state firing over the course of the 500-ms pulse
train. Even during the adapted portion of the evoked response, the
driven rates were sustained and above spontaneous firing. This
adaptation was not present for the low pulse rate (Fig. 3A), even
though the overall firing rate was slightly lower.
The temporal pattern of evoked neural activity in the IC depends
on the stimulus pulse rate. For the train of pulses at 28 pulses/s
(Fig. 3A), evoked activity was synchronized to the pulses, whereas
for the train of pulses at 448 pulses/s (Fig. 3B), evoked activity,
though high, appeared to be less synchronized. The average SI
values are plotted against stimulus rate for Chronos and ChR2 cases
in Fig. 4A. For both opsins, there was a decline in SI with increasing
pulse rate. For all rates, recordings from Chronos mice had higher SI
than those from ChR2 mice. These differences were significant at
rates of 56, 168 and 224 pulses/s (Fig. 4A, asterisks).
The decline in synchrony with increasing pulse rate was not
due to a decline in firing rate and driven rate was significant at all
tested stimulus rates (14e448 pulses/s, Fig. 4B). Even at high pulse
rates (e.g. 448 pulses/s), evoked activity was significantly above
spontaneous (paired t-test), high (average 76 spikes/s), and sustained over the entire stimulus pulse train (Fig. 3B) despite the
compromised kinetics of both Chronos and ChR2. Further, the
firing rates of mice injected with Chronos or ChR-2 were
increasing, monotonic functions of laser intensity, with evidence
of incomplete saturation at the highest tested intensities (Fig. 5A).
At these intensities, average firing rates were significantly lower
(p < 0.001) for ChR2 versus Chronos, average rate 55 spikes/s for
ChR2 versus 127 spikes/s for Chronos (~12 mW, stimulus
rate ¼ 28 pulses/s).
3.3. Spatial pattern of response for optogenetic-based stimulation
of the cochlear nucleus
There was variability in the spatial pattern of responses from
case to case. Fig. 5B shows a case where nearly all electrodes
recorded laser-evoked activity for stimulus levels at and above
threshold (driven rate > 0 spikes/s, indicated by non-dark blue
coloring, in the web version). Highest spike rates were observed on
electrodes 2e8. Other cases (data not shown) had variable patterns
of neural activation: of the 16 electrodes, the average number
activated across all laser intensities at and above threshold (stimulus rate ¼ 28 pulses/s) was 12.4 with a range of 9e14.9 electrodes
for Chronos (n ¼ 4 mice) and 9.2 with a range of 4.6e14 electrodes
for ChR2 (n ¼ 4 mice). Our data set is insufficient, though, to
correlate the opsin staining pattern with the spatial pattern of responses. Sham and control mice showed no response to optical
stimulation (one example shown in Fig. 5C).
4. Discussion
4.1. Chronos versus ChR2 for light-evoked activation of the auditory
system
Our study is the first to characterize the temporal properties of
opsins expressed in the central auditory system. Of all presently
studied opsins, Chronos has the fastest on/off kinetics based on
Fig. 2. Chronos expression in the cochlear nucleus. A: Mosaic confocal image showing Chronos-GFP expression within the dorsal cochlear nucleus (DCN) and ventral cochlear
nucleus (VCN). B: Confocal image of the DCN demonstrates Chronos-GFP expression within a fusiform cell (red arrow) and in other neuronal populations. DAPI demonstrates cell
nuclei. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
A.E. Hight et al. / Hearing Research 322 (2015) 235e241
239
Fig. 3. IC neural activity evoked by light pulses (Chronos injected mouse). A: Responses at a low stimulus rate (28 pulses/s) elicit synchronized spikes (dots on raster plot at top).
Bottom plot shows PST histogram for the same data. B: Responses at a high stimulus rate (448 pulses/s) show less synchrony (synchronization index indicated on the plots). In
addition, the PST histogram shows the prominent rate adaptation during the pulse train. Driven rates (an average over the 500 ms duration pulse train) are indicated on the plots.
firing rates in in vitro studies (Klapoetke et al., 2014). For in vivo
stimulation of the CN, we found that the use of Chronos resulted in
better neural synchrony to light stimuli compared to ChR2. For both
opsins, there was a decline in SI with increasing pulse rates;
however, the decline was more pronounced for ChR2. Significant
differences in the SI of evoked activity between the two opsins
were found at 56, 168 and 224 pulses/s. Synchronous activity at
these pulse rates are clinically significant because contemporary
clinical ABI processors employing the SPEAK sound processing
strategy use pulse rates of 250 pulses/s; this pulse rate is a carrier
that works well for neural encoding of amplitude modulation
(McCreery et al., 2013).
Although the temporal properties of optogenetic responses have
not been characterized previously, the responses of IC neurons to
electrical stimulation of the cochlea have previously been reported.
These responses also decline with increasing pulse rates, and their
synchrony measures are comparable to the optogenetic responses
reported here (Middlebrooks and Snyder, 2010; Snyder et al.,1995). In
addition, synchrony is even higher in IC recordings from awake animals under comparable stimulus conditions (Chung et al., 2014). In
those studies, differences among types of units were observed, with
some units able to fire synchronously to high rates (>100 pulses/s)
whereas others unable to synchronize to these rates. In the present
study, single recording sites probably sampled from a combination of
these unit types, future studies could employ spike sorting to examine
the synchronization limit of single units to optical stimulation.
During optogenetic stimulation, even for pulse rates for which
responses were non-synchronized, driven rates were substantial
(Figs. 3B, 4B). Such driven rates will signal the presence of a
stimulus even though there is little synchrony to the fine time
structure of the stimulus. Further, since rateeintensity curves
(Fig. 5B) suggest that the entire dynamic range of the response
was not captured due to the limitations of our laser, a higherintensity stimulus would likely produce even higher driven
rates. However, the level at which potential light-induced damage is manifested is not clear.
Fig. 4. Synchronization index and driven rate as a function of pulse rate. A: Average SI (at ~12 mW) is significantly greater for Chronos than ChR2 at stimulus rates of 56, 168 and 224
pulses/s (* ¼ p < 0.05, two-sample t-test). B: Average maximal driven rates over all 16 electrodes.
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A.E. Hight et al. / Hearing Research 322 (2015) 235e241
Fig. 5. Neural activity as a function of stimulus intensity and recording position. A: Plot of normalized driven rate across all electrodes as a function of laser intensity for the two
opsins. (Stimulus rate ¼ 28 pulses/s). B: Response map showing driven rate as a function of electrode number (position in IC, see Fig. 1D) and laser intensity. (Chronos injected
mouse, stimulus rate ¼ 56 pulses/s.) C: Control mouse response map showing no response evoked by light. (Stimulus rate ¼ 28 pulses/s.)
4.2. Limitations of viral-mediated gene transfer
There are inherent limitations to viral mediated gene transfer in
the central nervous system that may have influenced our results.
We previously demonstrated that expression of opsins as a result of
gene transfer is variable from case to case (Darrow et al., 2014).
Consequently, the locations and numbers of activated cells differed
somewhat between Chronos and ChR2 cases. This may affect synchrony because, at least for responses to acoustic stimuli, different
types of neurons have different temporal characteristics (Young,
1984). We believe that at least some of the excitatory responses
observed here may have been mediated by fusiform cells of the
DCN because in almost all cases they express Chronos or ChR2 and
because they project directly to the IC (Darrow et al., 2014; Oliver,
1984; Oliver and Morest, 1984). Perhaps the slow kinetics of ChR2
in these cells accounts for both the low SI and lower driven rates
observed in our study. Regardless of the exact etiology, the performance of Chronos is superior in both driven rate and SI.
4.3. Translational models and future optogenetics research in the
central auditory system
In addition to examining the temporal properties of Chronos
and ChR2, our study demonstrates a feasible translational approach
for gene transfer of opsins to the CN. Specifically, our surgical
approach in the murine model allows for visualization of the DCN
and inoculation with a viral gene transcript (Kozin et al., 2015). In
many respects, our model is analogous to surgical approach of the
human ABI placement. While viral mediated gene transfer has
inherent weaknesses and risks, numerous FDA-approved gene
therapy studies are ongoing and may be a potential tool for gene
transfer to the CN of humans.
Further, our study represents the beginning of a path toward
the particular opsin chosen for an eventual prosthesis. We define
the ideal characteristics of an “auditory opsin” for use in a neuroprosthesis: 1) fast temporal kinetics to encode speech information, 2) low activation threshold to decrease energy requirements
from illumination powered by an external source, and 3) a promoter that enables tissue-specificity using virally-mediated gene
transduction to the CN (to minimize non-specific expression of
opsins). Looking forward, our study raises several questions: What
is the ideal gene transfer approach for delivery of opsins to the
auditory system? What is the long-term safety profile of opsins,
and will there be any deleterious effects from their presence?
Finally, can optogenetic-based stimulation function as replacement
to electrical stimulation, and can it be used as an adjunct? The
answers to these questions, both in the central and peripheral
auditory systems, remain to be seen and should be the focus of
future studies.
5. Conclusion
Previous studies have demonstrated the feasibility of optogenetic stimulation for light-based activation of the central auditory system. Currently, the most widely used opsin in
neuroscience is ChR2; however, it may not possess the temporal
properties necessary to encode auditory information. We find, in
an ABI animal model, that Chronos has significantly improved
kinetic properties compared to ChR2. These studies highlight the
need to further examine and identify the ideal opsins that can
support the high stimulation rates needed for the transfer of
temporal cues along the auditory pathways. Future studies may
seek to design opsins optimized for a new generation ABI based on
light.
Acknowledgments
Preliminary results of this study were presented at the Association for Research in Otolaryngology Midwinter Meeting, February
2013 and 2014. This work was supported by a Fondation Bertarelli
grant (DJL and MCB), a MED-EL grant (DJL), and National Institutes
of Health Grants DC01089 (MCB), T32 DC000038 (AEH), T32
DC000020 (EDK).
A.E. Hight et al. / Hearing Research 322 (2015) 235e241
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