Research ArticleOPTOGENETICS

Optogenetic stimulation of cochlear neurons activates the auditory pathway and restores auditory-driven behavior in deaf adult gerbils

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Science Translational Medicine  11 Jul 2018:
Vol. 10, Issue 449, eaao0540
DOI: 10.1126/scitranslmed.aao0540
  • Fig. 1 AAV-CatCh–mediated optogenetic manipulation of cochlear SGNs in adult gerbils.

    (A) Scheme of the AAV construct used to transduce SGNs with CatCh-eYFP. hSyn, human synapsin promoter. (B) Depiction of retroauricular injection of the AAV-CatCh. (C) Photo of the retroauricular approach to the middle ear of a gerbil (r, rostral; d, dorsal): The bullostomy provides a view into the middle ear. (D) Montage of images showing the round window (RW) niche and dye-filled glass capillary, which points to the manually drilled hole. (E) Confocal images of immunolabeled midmodiolar cochlear cryosections (representative section of a middle turn) of an AAV-CatCh–injected adult gerbil. eYFP (green) marks transduced SGNs, and calretinin (magenta) generically marks SGNs. Scale bars, 100 μm. It should be noted that the apparent abundance of CatCh in the cytoplasm results from the maximum projection as individual z sections demonstrates a clear membrane expression. Inset (top left): Calretinin-positive but eYFP-negative IHC (arrowhead), with associated eYFP-positive, peripheral SGN neurites. Scale bar, 10 μm. Inset (top center): Close-up single z section of SGNs, highlighting the apparent plasma membrane expression of the construct. Scale bar, 10 μm. (F) Box plot showing the fraction of eYFP-expressing SGNs for the apical, middle, and basal cochlear turn of the injected left ear. No expression was found in the noninjected right ear (n = 9). (G) Box plot showing SGN density for the apical, middle, and basal cochlear turn of the injected left ear and the noninjected right ear (n = 9; *P < 0.05, t test). Data in (F) and (G) are shown as mean ± SEM. n.s. not significant. ITR, inverted terminal repeat; bGH, bovine growth hormone derived polyadenylation site; WPRE, woodchuck hepatitis virus posttranscriptional regulatory element.

  • Fig. 2 Characterization of optical and acoustic ABR in gerbils.

    (A) Experimental workflow: 4 to 12 weeks after AAV-CatCh injection, oABRs were recorded and compared to aABRs of the same gerbils and to those of noninjected animals. Light of a 473-nm laser was coupled in by a 200-μm optical fiber via cochleostomy on the middle cochlear turn or through the round window. (B) oABRs from a representative AAV-CatCh–injected adult gerbil with varying light intensities (radiant flux, 1 ms at 10 Hz). Colors code the stimulus parameters. (C) Amplitude of oABR p1-n1 as a function of light intensity (1-ms light pulses at 10 Hz). (D) Latency of oABR p1 as a function of light intensity for the same 20 gerbils (gray) as in (C). (E) oABRs from a representative AAV-CatCh–injected adult gerbil with varying stimulus rates (32.2 mW for 1 ms). (F) Amplitude of oABR p1-n1 as a function of stimulus rate (1-ms light pulses with high light intensities of 21 to 32 mW). (G) Latency of oABR p1 as a function of stimulus rate for the same 20 gerbils (gray) as in (F). Gray lines in (C), (D), (F), and (G) symbolize different animals (n = 20), and blue line represents mean with vertical error bars indicating SD. Horizontal error bars in (C) and (D) represent the SD of the mean light intensity in the respective bin. (H) aABRs (80-dB SPL, 0.3 ms) recorded at increasing acoustic click stimulation rate. (I) Quantification of aABR (n = 13) p1-n1 amplitude as a function of stimulation rate for acoustic clicks. Gray lines symbolize different animals; black line represents mean with vertical error bars indicating SD. (J) aABR p1 latency plotted against stimulation rate of auditory clicks (n = 13). (K) oABRs (blue; 32.2 mW, 1 ms at 10 Hz) and aABRs (black; click of 0.3 ms, 70 dB, at 10 Hz) recorded in three representative AAV-CatCh–injected adult gerbils. (L) p1-n1 amplitude as a function of stimulus rate normalized against p1-n1 amplitude of 10-Hz aABRs (black) and oABRs (blue); data are shown as mean ± SD. (M) Latency of p1 as function of stimulation rates normalized against p1 latency of 10 Hz aABRs (black) and oABRs (blue) (mean ± SD). Data in (C), (D), (F), (G), (L), and (M) are pooled for oABR measurements in acutely and chronically implanted animals.

  • Fig. 3 Characterization of SGN responses to optogenetic stimulation.

    (A and B) Representative juxtacellular SGN recordings during the first 200 ms of a 900 ms pulse train (1 ms, 20 mW) with a repetition rate of 10 Hz of an exemplary single spike–responding (A) and a multiple spike–responding (B) SGN. (C) Quantification of the first spike jitter elicited per light pulse for single spike–responding (green) and multiple spike–responding (red) SGNs. (D to I) Raster plots and peristimulus time histogram of a single spike–responding (D to F) and a multiple spike–responding (G to I) SGN in response to 900-ms train pulses of 10, 100, and 300 Hz. (J) Vector strength as a function of repetition rate (n = 29). Blue lines correspond to the population mean, green lines to single spike–responding SGNs, red lines to multiple spike–responding SGNs, and dashed black line to acoustic stimulation of control SGN (see fig. S4 for details). (K and L) Distribution (K) and quantification (L) of the cutoff frequency (highest frequency with a significant vector strength) for single spike–responding (green) and multiple spike–responding (red) SGNs. (M) Discharge rate as a function of repetition rate (n = 29). (N) Adaptation ratio as a function of the number of spikes per light pulse in response to 10-Hz light pulse train. A color scale is used to represent the repetition rate. Data are expressed as mean ± SEM, and the statistical difference between groups were tested by Mann-Whitney U test (***P ≤ 0.001).

  • Fig. 4 Chronic fiber-based, single-channel oCI.

    (A) Left: Chronic fiber-based single-channel oCI (arrow) housed in a stainless steel capillary. Scale bar, 1 cm. Center left: oCI (I) placed through the round window into the scala tympani just above the stapedial artery (SA). Dashed line represents bullostomy. Center right: oCI 3 weeks after surgery. Right: Gerbil 3 days after implantation. Arrowhead indicates ferrule for optical fiber connection. (B) oABRs of a AAV-CatCh–injected animal (top) at the day of implantation (day 0), and 1, 3, 5, and 7 days after implantation, no oABR was found in PBS-injected animals (bottom). Blue bars indicate optical stimuli. (C) oABR amplitudes (p1-n1; top) and latencies (p1; bottom) during the course of behavioral experiments (n = 7 up to day 40 after implantation; two animals to day 115); each line corresponds to a single animal. (D) X-ray tomography of the cochlea including implant to confirm the fiber’s position after behavioral experiments. Green, basilar membrane; purple, Rosenthal’s canal; black, steel capillary; blue, optical fiber; star, cochlear apex. Scale bar, 500 μm.

  • Fig. 5 Optogenetically cued avoidance behavior.

    (A) Outline of the task: After an initial training period, the gerbil changes the compartments of the shuttlebox upon stimulus presentation (acoustic or optogenetic). (B and C) Picture of the shuttlebox (front wall removed) in a sound-attenuating chamber (B) and of a gerbil in the setup (C). Arrowhead indicates the interface between ferrule and optical fiber. (D and E) Behavioral performance during the course of shuttlebox training for a single habituation session using acoustic stimuli (D) and the subsequent training period using optogenetic stimulation (E). Solid and dashed lines show mean response rates for target and nontarget trails ± SEM for AAV-CatCh–injected (blue; n = 7) and PBS-injected (orange; n = 2) animals. Filled and empty markers show response rates for target and nontarget trails, respectively, for each individual animal. (F) Response rates in response to target (blue) and nontarget (purple) trials during a control experiment with a blocked (left) and reopened (right) beam path. (G) Transfer from optogenetic to acoustic cues for avoidance behavior. (H) Second control performed after transfer to acoustic stimulation. Different marker shapes correspond to different animals and are consistent throughout the figure. (I to L) Average hit rates and behavioral thresholds of individual animals for light power (I and J) and pulse duration (K and L). Solid and dashed lines indicate mean response rates for target and nontarget trails ± SEM across all animals [n = 7 (I) and 5 (K)]. Filled and empty markers show hit rates for target and nontarget trials, respectively. Behavioral thresholds were defined as the weakest stimuli eliciting significant performance in each individual animal (chi-square test, P < 0.01).

  • Fig. 6 Responses of single AI neurons to optogenetic SGN stimulation.

    (A) Schematic shows the experimental approach: After initial mapping of the tonotopic representation to identify field AI of the contralateral auditory cortex, a single tungsten electrode was tangentially inserted and advanced to study the activity of single AI neurons (single unit) within the thalamorecipient layer IV. (B) Neuronal responses of three individual neurons to pure tones along a representative recording track. The BFs of neurons varied systematically along each electrode track where BFs were lower dorsally and increased ventrally. (C) Neuronal responses to individual laser pulses (1 ms at 1 Hz) applied to the contralateral cochlea in a nonmonotonic (left) and monotonic unit (right). (D) Distribution of monotonicity indices of all recorded units. Dashed bar indicates a monotonicity index of 0.5 to discriminate between nonmonotonic and monotonic units. (E) Comparison of acoustic and optical response properties. BFs are plotted against the corresponding laser threshold for each single unit (n = 20). AAF, anterior auditory field; DP, dorsoposterior field; d, dorsal; m, medial.

  • Fig. 7 Restoration of hearing in a gerbil model of ototoxic deafness.

    (A) Experimental workflow: After AAV-CatCh injection, ABRs were recorded, and gerbils were trained in the shuttlebox for 6 days using acoustic stimulation and finally deafened by bilateral intracochlear injections of Kanamycin solution. After deafness was confirmed using aABRs and a single shuttlebox session, animals were implanted with an optical fiber. Subsequently, oABRs were recorded, and shuttlebox experiments were performed using optogenetic stimulation. (B) aABR thresholds (individual data and mean ± SEM, n = 4) for logarithmically spaced pure tones ranging from 1 to 16 kHz and click stimuli, before (black triangles and solid line) and after deafening (red triangles and dashed line). (C) Representative aABR in response to a 60-dB click stimulus before deafening (black; top.). Absence of aABRs in response to 100-dB click stimuli and 1, 4, and 16 kHz pure tones after deafening (gray; middle). oABRs in AAV-CatCh–injected oCI-implanted deafened animal (blue; bottom). Vertical dashed lines indicate stimulus onsets. All traces were recorded from the same animal. (D) Histological verification of IHC loss upon deafening. Overview of the apical turn of the organ of Corti in a control animal (top left) and a kanamycin deafened animal (bottom left). Phalloidin was used to stain actin prominently expressed in hair cells and supporting cells (red), and calretinin was used to stain SGNs and IHCs (cyan): Arrowheads point to the location of IHCs. Note the lack of IHCs in the deafened animals. Scale bars, 100 μm. Right: Magnification of the regions outlined on the left side, respectively. Note that spiral ganglion afferents are still present in the deafened animal (bottom right). Scale bar, 10 μm. Learning curves of shuttlebox behavior using 70-dB acoustic stimulation before deafening (E), 70 dB after deafening (F), optogenetic cues (G), and acoustic cues up to 100 dB after deafening (H). Solid and dashed lines indicate the mean hit rates ± SEM for target and nontarget trials, respectively. Filled and empty markers indicate the individual rates for each animal (n = 4). Different marker shapes correspond to different animals and are consistent throughout the figure.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/449/eaao0540/DC1

    Methods

    Fig. S1. Effects of postnatal transfection of SGNs in adult Mongolian gerbils using the opsins Chronos and CatCh.

    Fig. S2. SGN density of AAV-injected but CatCh-negative animals.

    Fig. S3. oABRs: Dependence on stimulus intensity, rate, and duration—relationship to fraction of CatCh-expressing SGNs.

    Fig. S4. Amplitude and latency comparison between aABRs and oABRs.

    Fig. S5. Single auditory nerve fiber responses to acoustic click trains.

    Fig. S6. oABR amplitudes and thresholds in chronically implanted gerbils.

    Fig. S7. Determination of fiber position with three-dimensional x-ray tomography.

    Fig. S8. Optical thresholds for AI single units with high versus low BFs.

    Fig. S9. Estimating the spread of excitation using Monte Carlo ray tracing.

    Fig. S10. Shuttlebox location detection algorithm.

    Table S1. Number of animals used for each experiment.

    Table S2. Raw data for graphs containing an animal n lower than 20.

    References (3048)

  • The PDF file includes:

    • Methods
    • Fig. S1. Effects of postnatal transfection of SGNs in adult Mongolian gerbils using the opsins Chronos and CatCh.
    • Fig. S2. SGN density of AAV-injected but CatCh-negative animals.
    • Fig. S3. oABRs: Dependence on stimulus intensity, rate, and duration—relationship to fraction of CatCh-expressing SGNs.
    • Fig. S4. Amplitude and latency comparison between aABRs and oABRs.
    • Fig. S5. Single auditory nerve fiber responses to acoustic click trains.
    • Fig. S6. oABR amplitudes and thresholds in chronically implanted gerbils.
    • Fig. S7. Determination of fiber position with three-dimensional x-ray tomography.
    • Fig. S8. Optical thresholds for AI single units with high versus low BFs.
    • Fig. S9. Estimating the spread of excitation using Monte Carlo ray tracing.
    • Fig. S10. Shuttlebox location detection algorithm.
    • Table S1. Number of animals used for each experiment.
    • Legend for table S2
    • References (3048)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Raw data for graphs containing an animal n lower than 20.