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Fast-spiking GABA circuit dynamics in the auditory cortex predict recovery of 1 sensory processing following peripheral nerve damage 2 3 Jennifer Resnik1,2 and Daniel B. Polley1,2* 4 5 1 – Eaton-Peabody Laboratories, Massachusetts Eye and Ear Infirmary, Boston MA 02114 USA 6 2 – Department of Otolaryngology, Harvard Medical School, Boston MA 02114 USA 7 * - Contact daniel_polley@meei.harvard.edu 8 9 Abstract: 148 words 10 Figures: 4 11 Supplemental files: 1 12 Supplemental figures: 3 13 14 15 16 17 18 19 20 21 22 23

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Abstract 24 Cortical neurons remap their receptive fields and rescale sensitivity to spared peripheral inputs 25

following sensory nerve damage. To address how these plasticity processes are coordinated over the 26 course of functional recovery, we tracked receptive field reorganization, spontaneous activity, and 27 response gain from individual principal neurons in the adult mouse auditory cortex over a 50-day period 28 surrounding either moderate or massive auditory nerve damage. We related the day-by-day recovery of 29 sound processing to dynamic changes in the strength of intracortical inhibition from parvalbumin-30 expressing (PV) inhibitory neurons. Whereas the status of brainstem-evoked potentials did not predict 31 the recovery of sensory responses to surviving nerve fibers, homeostatic adjustments in PV-mediated 32 inhibition during the first days following injury could predict the eventual recovery of cortical sound 33 processing weeks later. These findings underscore the potential importance of self-regulated inhibitory 34 dynamics for the restoration of sensory processing in excitatory neurons following peripheral nerve 35 injuries. 36

37 Introduction 38

The enduring plasticity of the adult brain supports a remarkable recovery of perceptual and 39 motor capabilities following peripheral nerve injury. In sensory cortex, reorganization following injury 40 involves a rapid unmasking of inputs from adjacent, undamaged regions of the sensory periphery and, in 41 cases of incomplete injury, a slower, progressive increase in responsiveness to surviving nerve fibers 42 within the damaged region1–8. The initial stages of this reorganization may be enabled by homeostatic 43 processes that compensate for dramatic swings in afferent input to maintain neural excitability around a 44 set point9. Recordings from cortical pyramidal neurons in culture or acute brain slices following sudden 45 shifts in excitatory input have revealed a coordinated sequence of changes at glutamatergic and 46

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GABAergic synapses that normalize firing rates and rebalance network activity10–15. Whether and how 47 synaptic and extra-synaptic modifications underlie reorganized sensory processing in deafferented zones 48 of the adult cortex remains to be determined, but accumulating evidence from intact preparations 49 suggests that the stabilization of new synaptic inputs from spared regions of the sensory periphery may 50 emerge through a combination of rapid disinhibition and increased structural motility15–22. 51

In the auditory system, a loss of cochlear hair cells and/or cochlear nerve afferent synapses is 52 associated with reduced GABA signaling and cortical hyperexcitability, as manifest in elevated 53 spontaneous firing rates, enhanced central gain and unmasking of sound-evoked responses from 54 undamaged regions of the cochlea6,7,23–25. However, these biomarkers have not been compared with one 55 another over the full course of functional recovery with fine-grain temporal resolution at the level of 56 individual neurons. As a result, it is unclear whether dynamic changes in intracortical inhibition precede, 57 follow, outlast or recede ahead of functional changes in receptive field mapping and neural 58 hyperexcitability. Nor is it clear whether the decline in GABA markers following peripheral injury arise 59 from a reduced influence of PV-expressing fast-spiking interneurons, which synapse onto pyramidal 60 neuron somata and have a well-recognized role in gating activity-dependent cortical plasticity, or instead 61 from other GABA neuron subtypes, which can synapse onto other GABA neurons and exert a net 62 excitatory effect on network activity26. Here, we address these unanswered questions by measuring 63 changes in PV-mediated intracortical inhibition alongside hyperexcitability and receptive field plasticity 64 from individual regular spiking (RS) putative pyramidal neurons over a several month period 65 surrounding varying degrees of auditory nerve damage. These findings highlight a rapid loss and 66 recovery in PV-mediated inhibition that may compensate for a sudden drop in afferent drive following 67 cochlear afferent loss to support a progressive recovery of sensory processing from spared nerve fibers. 68 69

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Results 70 We implanted optetrode assemblies into the primary auditory cortex (A1) of adult PV-Cre:Ai32 71

mice (Fig. 1a) to isolate regular spiking (RS) putative pyramidal neurons based on their spike shape and 72 estimate the strength of local inhibition on RS units during optogenetic activation of PV–expressing 73 GABA neurons (Fig. 1b-c). The mechanical stability of the optetrode assembly combined with the thin, 74 flexible tetrode wires allowed us to isolate single units (Fig. 1d) and hold them for many weeks, 75 according to a conservative, objective statistical standard (Fig. 1e-f). With this approach, we could 76 measure auditory responsiveness and local inhibitory tone from individual RS neurons in awake, head-77 fixed adult mice over a 7-8 week period surrounding varying degrees of auditory nerve injury (Fig. 1g). 78 Although we could occasionally isolate PV-expressing FS interneurons on our tetrodes (Fig. 1b-c), we 79 could rarely hold these neurons for more than a single recording session (refer to Figure 1 – 80 Supplemental Figure 1). Therefore, all descriptions of long-term changes in single unit response 81 properties were limited to RS, putative pyramidal neurons. 82

Most forms of cochlear injury induce an intractable set of changes to the auditory nerve as well 83 as sensory and non-sensory cells within the cochlea. In cases of widespread cochlear damage, it is 84 impossible to attribute abnormal cortical tuning to a central plasticity versus abnormal cochlear filtering 85 or amplification. We circumvented this problem by selectively eliminating afferent nerve fibers without 86 permanently affecting cochlear mechanics using a well-characterized noise exposure protocol that 87 lesions approximately 50% of high-frequency afferent nerve synapses in the 16-45 kHz region of the 88 cochlea without damaging hair cells (n = 4, Fig. 2a)27,28. This protocol induces only a temporary 89 elevation of auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) 90 thresholds, but a permanent reduction in ABR wave 1 amplitude, which has been shown to reflect the 91

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permanent elimination of high-frequency cochlear afferent synapses (Fig. 2b, the reader is referred to 92 the figure legends and supplementary file 1 for most statistical reporting)29. 93 We implanted optetrode assemblies into the 32 kHz region of the A1 tonotopic map and recorded 94 RS units with high-frequency receptive fields, low-intensity thresholds to broadband noise stimulation 95 and strong feedforward inhibition from PV neurons (Fig. 2c-e, top row). Several hours after noise 96 exposure that affects high-frequency regions of the cochlea, the same unit exhibited elevated response 97 thresholds for a broadband noise burst, slightly reduced PV-mediated inhibition and the appearance of a 98 new, well-tuned low-frequency receptive field that was absent just hours earlier (Fig. 2c-e, 2nd row). 99 We quantified the invasion of spared, low-frequency inputs and the eventual restoration of the original 100 receptive field by measuring daily changes in frequency response area (FRA) overlap with the baseline 101 FRA as well as tone-evoked firing rates from the spared (4-6 kHz) and partially denervated (32-48 kHz) 102 cochlear regions (Fig. 2f, left). We compared these metrics of receptive field reorganization against the 103 typical biomarkers of hyperexcitability in the auditory pathway: decreased response thresholds, 104 increased spontaneous firing rate and increased gain, defined here as the slope of the stimulus input-105 output function (Fig. 2f, middle). Finally, we related these markers of RS unit hyperexcitability and 106 receptive field plasticity to changes in the strength of PV-mediated inhibition (Fig. 2f, right; n = 208). 107

Several hours after noise exposure, firing rates evoked by stimulation of spared, low-frequency 108 cochlear regions had nearly doubled (increased by 96.7%, p = 0.04) with a small, but significant 109 decrease in inhibition (-9.8%, p = 0.02) and no significant change in gain or spontaneous rate (Fig. 2g). 110 Six to ten days after noise exposure, inhibition had decreased by 40% (p<0.0001), receptive fields 111 remained focused on spared regions of the cochlea (p< 0.01) and spontaneous firing rate was 112 significantly elevated (p<0.05). Central gain, spontaneous firing rate and frequency tuning all shifted 113 back towards baseline values as response thresholds in the cochlea, ABR and cortical neurons recovered 114

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to pre-exposure levels. By the time of the final recording session, approximately 7 weeks after noise 115 exposure, reduced PV-mediated inhibition (-19%, p < 0.001) was the only enduring compensatory 116 plasticity marker for the putative loss of high-frequency auditory nerve synapses. 117 Exposure to ototoxic drugs can cause a sudden and extreme loss of afferent signaling from the 118 cochlea. Would homeostatic adjustments support as complete a recovery of function following a 119 massive loss of auditory nerve fibers? We addressed this question by applying ouabain bilaterally to the 120 cochlear round window membrane (Fig. 3a). In mice, ouabain selectively and evenly eliminates > 95% 121 of Type-I spiral ganglion afferent neurons across the frequency map, without affecting other types of 122 sensory and non-sensory cells in the cochlea or auditory nerve30. Bilateral ouabain treatment virtually 123 eliminated the ABR without any adverse DPOAE effects in all mice (n = 6, Fig. 3b), but, interestingly, 124 was associated with a slow, partial recovery of sound-evoked cortical spiking in half the mice (Fig. 3c) 125 but an unremitting loss of auditory responsiveness in the other half (Fig. 3d). 126

When DPOAE thresholds are normal, ABR wave 1b amplitude is an accurate proxy for the 127 number of cochlear afferent synapses27–29. However, the ABR wave 1b amplitude was not significantly 128 different between mice that recovered auditory processing following ouabain treatment versus those that 129 did not (unpaired t-test, p > 0.4; see also Figure 3 – Supplemental Fig. 1). Whereas our estimate of 130 peripheral denervation bore no relationship to the mode of cortical recovery, early dynamics in 131 inhibitory tone and spontaneous firing rate were linked to the recovery of cortical processing. RS units 132 that eventually recovered auditory thresholds exhibited a transient spike in spontaneous firing rate, a 133 sustained increase in central gain and a steep drop in PV-mediated inhibition that began to recover as 134 high-threshold sound-evoked activity returned (Fig. 3e-h, top row, n = 156). By contrast, in RS units 135 that never recovered auditory sensitivity, changes in spontaneous rate were comparatively sluggish, 136 while inhibitory strength declined gradually and monotonically (Fig. 3e-h, middle row, n =169). This 137

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stark dichotomy in the cortical response to nerve injury was shared among all units recorded from a 138 given mouse; if one unit recovered auditory responsiveness, all units recovered auditory responsiveness 139 (Fig. 4). As a negative control in a separate group of mice (n=3), we applied sterile water to the round 140 window membrane rather than ouabain and confirmed that cortical response properties were stable over 141 the recording period in mice with intact auditory nerves (Fig. 3e-h, bottom row, n = 156). 142 These findings highlight the coordination of short- and long-term changes in adult A1 following 143 peripheral nerve injury. Spontaneous rate, central gain and inhibition all scaled with the degree of nerve 144 damage (Fig. 3f-h versus Fig. 2g, Fig. 4), but varied substantially both over time and between mice. 145 Restored auditory sensitivity was associated with a sharp loss and partial recovery of inhibition from PV 146 interneurons, where the degree of eventual recovery could be predicted from changes in inhibition ~1 147 week after nerve damage (Fig. 4a). Not only was PV-mediated inhibition a predictor of sensory 148 recovery for both mild and severe peripheral damage, but it was a better predictor than were 149 modifications in spontaneous activity (Fig. 4b) or central gain, the biomarkers most commonly 150 employed to assess central auditory plasticity following sensorineural hearing loss (refer to Figure 4 – 151 Supplemental Figure 1 for an expanded analysis of predictors of recovery). 152

153 Discussion 154 In this study, we tracked a plasticity in PV-mediated inhibition and single neuron responsiveness 155 in the primary auditory cortex of adult mice following a sudden loss of input from the auditory nerve. 156 Just hours after noise exposure, we observed the appearance of new, low-threshold receptive fields tuned 157 to undamaged regions of the cochlea, accompanied by a small, significant drop in PV-mediated 158 inhibition. Over the ensuing weeks, as cochlear thresholds and cortical receptive field tuning returned to 159 baseline conditions, we noted only a brief spike in traditional biomarkers of central auditory 160

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hyperactivity, such as increased spontaneous firing rate and increased central gain. By contrast, PV-161 mediated inhibition remained reduced through our final recording session and was the sole physiological 162 indicator for the permanent, presumed loss of high-frequency cochlear synapses. Importantly, this is not 163 to say that auditory response normalization was mediated by disinhibition; our observations are 164 correlative and do not speak to the necessity nor sufficiency of changes in PV networks for functional 165 recovery. Compensation for peripheral damage could have arisen from post-synaptic changes in GABA 166 receptors in the RS principal neurons recorded here31 or from presynaptic changes in the GABA neurons 167 themselves16,19,32, as well as many other homeostatic plasticity mechanisms in the RS neurons including 168 upward scaling of glutamatergic synapses11,12, changes in extrasynaptic GABA or glutamate receptors 169 that mediate tonic currents33–35, or changes in intrinsic membrane response properties36–38. 170

In classic and contemporary studies of adult cortical plasticity following deafferentation, fine-171 scale reorganization varied widely between local subnetworks of RS neurons and the completeness of 172 global remapping was negatively correlated with the spatial extent of peripheral lesions5,39–41. Here we 173 report that the mode of cortical sensory recovery (complete, partial or none) was roughly shared between 174 all RS units recorded in a given mouse and did not strictly depend on the estimated auditory nerve 175 damage. Unlike focal ablations of the retina, basilar membrane or skin surface, cochlear ouabain 176 treatment eliminates 95% of primary afferent synapses without affecting sensory transduction 177 mechanisms7,30. This provided us with an uncommon opportunity to track the restoration of sensory 178 responses transmitted through the small fraction of surviving nerve fibers, rather than the typical 179 approach of describing competitive reorganization from ectopic, spared inputs neighboring the lesion. 180 As compared to the rapid unmasking of excitatory inputs from neighboring undamaged regions of the 181 sensory organ, which occurs within hours (Fig. 2, see also3,8), the recovery of function for spared 182 afferent inputs interspersed within a damaged portion of the nerve, if it occurred at all, unfolded over 183

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several weeks. The variable modes of recovery between mice with carefully matched nerve damage may 184 relate to earlier observations that cortical sensory reorganization is not only determined by the pattern 185 and density of peripheral innervation, but may also be directed by activity-dependent differences that are 186 orchestrated through behavioral use42–44. 187

Following either type of nerve damage, the cortical networks that recovered function were those 188 that matched the sudden drop in bottom-up excitatory drive with a rapid dip in PV-mediated inhibition. 189 Precisely balanced excitation and inhibition enables a wider dynamic range of sensory information 190 coding26,45–47 and supports adaptive plasticity without forsaking network stability48–51. These findings 191 suggest that self-regulating PV circuits may not only play an important role in rebalancing network 192 activity in response to reduced afferent drive during developmental critical periods13,52, but in the adult 193 cortex following sensory nerve damage as well. 194 195 196 Materials and methods 197 Animals and cochlear denervation: 198 All procedures were approved by the Animal Care and Use Committee at the Massachusetts Eye and Ear 199 Infirmary and followed guidelines established by the National Institutes of Health for the care and use of 200 laboratory animals. Subjects included 13 PV-Cre:Ai32 mice of either sex (a cross between B6;129P2-201 Pvalbtm1(cre)Arbr/J and Ai32 (RCL-ChR2(H134R)/EYFP), Jackson Laboratory), aged 16 weeks at the 202 time of optetrode implantation. 203 204 Bilateral auditory nerve damage 205

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Acoustic exposure: The acoustic over-exposure stimulus was an octave band of noise (8–16 kHz) 206 presented at 98 dB sound pressure level (SPL) for 2 h. During exposures, animals were awake and 207 unrestrained within a 12x10 cm, acoustically transparent cage. The cage was suspended directly below 208 the horn of the sound-delivery loudspeaker in a small, reverberant chamber. Noise calibration to target 209 SPL was performed immediately before each exposure session. 210

Ouabain: Selective elimination of Type-I spiral ganglion neurons was achieved by applying a 1 211 mM solution of ouabain octahydrate (Sigma) and sterile water to the left and right round window niche, 212 as described previously30. Animals were anesthetized with ketamine (120 mg/kg) and xylazine (12 213 mg/kg), with half the initial ketamine dose given as a booster when required. The connective tissue and 214 underlying muscle were blunt dissected and held away from the bulla with retractors. A small opening 215 was made in the bulla with the tip of a 28.5-gauge needle. The exposed round window niche was filled 216 with 1-2 µL of the ouabain solution using a blunted needle. Ouabain was reapplied five more times at 217 15-minute intervals, wicking the remaining solution away with absorbent paper points before each 218 application. For control experiments, sterile water was placed on the cochlear round window using an 219 identical procedure. Measurements of the auditory brainstem response (ABR) and distortion product 220 otoacoustic emission (DPOAE, see subsequent Experimental Procedures) were made during the ouabain 221 application to confirm functionality and ABR threshold shift without changes in DPOAE thresholds or 222 amplitudes. Additional ouabain was applied, as necessary, until the ABR threshold at 16 kHz was 223 greater than 55 dB sound pressure level (SPL). The incision was sutured and the mouse was given an 224 analgesic (Buprenex, 0.05 mg/kg) before being transferred to a heated recovery chamber. 225 226 Cochlear function tests: 227

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Mice were anesthetized with ketamine and xylazine (as above), and placed on a homeothermic 228 heating blanket during testing. ABR stimuli were 5-ms tone pips (8, 16 and 32 kHz pure tones) with a 229 0.5 ms rise-fall time delivered at 30 Hz. Intensity was incremented in 5-dB steps, from 20-80 dB SPL. 230 ABR threshold was defined as the lowest stimulus level at which a repeatable waveform could be 231 identified. DPOAEs were measured in the ear canal using primary tones with a frequency ratio of 1.2, 232 with the level of the f2 primary set to be 10 dB less than f1 level, incremented together in 5-dB steps. 233 The 2f1-f2 DPOAE amplitude and surrounding noise floor were extracted. DPOAE threshold was 234 defined as the lowest of at least two continuous f2 levels, for which the DPOAE amplitude was at least 2 235 standard deviations greater than the noise floor. All treated animals underwent rounds of DPOAE and 236 ABR testing before, 2 days and approximately 30 days after the procedure. 237 238 Immunohistochemistry 239

Mice were perfused with 0.01M phosphate buffered saline (PBS) (pH = 7.4) followed by 4% 240 paraformaldehyde in 0.01 M PBS. Brains were removed and stored in 4% paraformaldehyde for 12 hrs 241 before transferring to cryoprotectant (30% sucrose) for 48 hr. Sections (40 μm) were cut using a cryostat 242 (Leica CM3050S). Sections were washed in PBS containing 0.1% Triton X-100 (3 washes, 5 min each), 243 incubated at room temperature in blocking solution (Super Block) for 6 min at room temperature and 244 then incubated in primary antibody (PV 27 rabbit anti-parvalbumin, Swant,1:1,000 dilution) at 4 degrees 245 C overnight. The next day, slices were washed and incubated in secondary antibody (Alexa 647 goat 246 anti-rabbit immunoglobulin G, Invitrogen, 1:200 dilution) for 1.5 hrs at room temperature. Sections 247 were mounted on gelatin-subbed glass slides (BBC Biochemical) and coverslipped. Fluorescence images 248 were obtained with a confocal microscope (Leica). 249 250

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Chronic optetrode implantation: 251 Mice were brought to a surgical plane of anesthesia with ketamine/xylazine, as described above. 252

Body temperature was maintained at 36.5°C with a homeothermic blanket system. Using a scalpel, a 253 small craniotomy was centered over the right auditory cortex leaving the dura mater intact. The brain 254 surface was covered with sterile ointment. Chronic implants consisted of a modified microdrive array 255 (VersaDrive 4, Neuralynx Inc) containing 4 independently moveable, closely spaced tetrodes and an 256 optic fiber (0.2 mm, Thorlabs, Inc.). The optic fiber was mounted into the microdrive assembly so that it 257 rested several millimeters above brain the surface. Tetrodes were four twisted nichrome wires (12.5 μm 258 in diameter, Stablohm 650 wire, California Fine Wire Company), electroplated with gold to reach an 259 impedance of 0.5–1 MΩ (nanoZ and ADPT-NZ-VERSA adapter, Neuralynx Inc). Microdrive arrays 260 were positioned over the right primary auditory cortex based on an initial mapping of tonotopic gradient 261 orientation using independently moveable tungsten microelectrodes53. Tetrodes were lowered into the 262 middle cortical layers to match the approximate depth of tungsten microelectrodes recordings. Silver 263 wire was fixed in place atop the left frontal cortex to serve as a ground. A titanium head plate was 264 affixed to the skull once the microdrive assembly was firmly in place with dental cement (C&B 265 Metabond). 266 267 Neurophysiology data collection: 268

Single unit recordings were made from awake, head-fixed mice. Mice were continuously video 269 monitored during recording. Raw neural signals were digitized at 32-bit, 24.4 kHz (RZ5 BioAmp 270 Processor; Tucker-Davis Technologies) and stored in binary format for offline analysis. The signal was 271 bandpass filtered at 300–3000 Hz with a second-order Butterworth filter and movement artifacts were 272 minimized through common mode rejection. Spike waveforms were extracted based on a threshold and 273

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sorted offline into single units using Wave_clus, a semiautomatic clustering algorithm54. Sorts were 274 performed on concatenated data files from all recording sessions. Neurons were considered continuously 275 recorded if spike sorting from data across the entire experiment yielded a single unit by the following 276 criteria: 1) Waveforms constituted a statistically isolated cluster when all waveforms from all recording 277 sessions were considered; 2) Biophysical properties consistent with single units, such as an absolute 278 refractory period, were met; 3) High uniformity in spike shape was maintained, as assessed by a 279 comparison of the sum of squared errors (SSE) across days (fractional difference in amplitude squared); 280 and 4) Consistently high signal to noise ratios. 281

The first recordings after auditory nerve injury were made approximately 2 hours after the end of 282 noise exposure and approximately 14 hours after ouabain application (to allow for extra recovery from 283 injectable anesthetics). Recordings were made every 1-3 days thereafter. We only included units that 284 were active in at least 75% of recording sessions and whose firing rate was stable for the two baseline 285 recording sessions prior to peripheral damage. After offline sorting the mean waveform of neurons was 286 calculated, the tetrode wire on which the amplitude of the spikes was largest was used. The trough-to-287 peak interval and the peak trough ratio were calculated and units were classified as fast-spiking PV+ 288 units or RS-putative excitatory units. The classification was confirmed by laser response in the pre-289 condition. All subsequent analyses were performed in MATLAB 2015a (MathWorks). 290

291 Acoustic stimuli: 292

Stimuli were generated with a 24-bit digital-to-analog converter (National Instruments model 293 PXI-4461). For DPOAE and ABR tests, as well as during electrode implant surgery, stimuli were 294 presented via in-ear acoustic assemblies consisting of two miniature dynamic earphones (CUI 295 CDMG15008–03A) and an electret condenser microphone (Knowles FG-23339-PO7) coupled to a 296

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probe tube. Stimuli were calibrated in the ear canal in each mouse before recording. In awake 297 recordings, stimuli were presented via a free-field electrostatic speakers (Tucker-Davis Technologies) 298 facing the left (contralateral) ear. Free-field stimuli were calibrated before recording with a wide-band 299 ultrasonic acoustic sensor (Knowles Acoustics, model SPM0204UD5). 300 301 Neurophysiology data analysis: 302

Rate-level functions. Broadband noise tokens (4-64 kHz, 0.1 s duration, 4 ms raised cosine 303 onset/offset ramps) were presented at 0-80 dB SPL in 5 dB increments. Threshold was defined as the 304 lowest of at least 3 continuous stimulus levels for which the response to sound was significantly higher 305 than the spontaneous activity. Gain was defined as the relationship between sound level (input) and 306 firing rate (output). The gain was measured as the slope of the linear fit of the initial rising phase from 307 the rate-level function. 308

Frequency response areas. FRAs were delineated using pseudorandomly presented tone pips (50 309 ms duration, 4 ms raised cosine onset/offset ramps, 0.5–1 s intertrial interval) of variable frequency (4–310 48 kHz in 0.1 octave increments) and level (0–75 dB SPL in 5 dB increments). Each tone pip was 311 repeated 3 times and responses to each iteration were averaged. The start of the spike collection window 312 was set to be the point when the firing rate began to consistently exceed the spontaneous rate by at least 313 3 SD. The offset of the spike collection window was the first bin after the response decreased to less 314 than 4 SD above the spontaneous rate. Additional details on spike windowing and FRA boundary 315 determination are described elsewhere55. Receptive field overlap was defined as the number of 316 frequency–intensity combinations in the intersection of the reference and comparator FRA divided by 317 the number of points contained within their union. The reference FRA was the average of the pre-318 damage FRAs for a given unit and the comparator was selected from each individual measurement time. 319

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320 Optogenetic activation: 321 Collimated blue light (473 nm, 500 ms duration, 12mW) was generated by a laser (DPSS, 322 LaserGlow Co.) and delivered to the brain surface via an optic fiber coupled to the tetrode assembly. 323 Precise timing of the light pulse was controlled via a custom shutter system. The intensity of the blue 324 laser was calibrated for each implant prior to the start of recording with a photodetector (Thorlabs, Inc.). 325 Percentage of inhibition in excitatory cells was calculated as the percentage of 20 ms bins, during the 326 500ms laser duration, that were significantly suppressed when compared to spontaneous firing rate 327 (paired t-test corrected for multiple comparisons). 328

329 Statistical analysis 330

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neurophysiological signal types. J. Neurosci. 32, 9159–9172 (2012). 460 461 Acknowledgements 462 We thank Prof. Nao Uchida for assistance with optetrode fabrication and Dr. Ken Hancock for support 463 with data collection software. This work was supported by an EMBO postdoctoral fellowship (JR), R01 464 DC009836 (DBP), a research grant for Autifony Therapeutics (DBP) and the Lauer Tinnitus Research 465 Center (DBP). 466 467

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Figure Legends 468 Figure 1. Approach for chronic single unit recordings and optogenetic activation in mouse A1. (A) 469 Immunolabeling of PV neurons in A1 with co-localization of EYFP reporter in Pv-Cre:Ai32 transgenic 470 mice. DAPI labels cell nuclei. Scale bar = 15 μm. (B) Spike raster plots illustrating that optogenetic 471 activation of fast spiking (FS) PV+ units (black, top) inhibits regular spiking (RS) units (gray, bottom). 472 Right, spike waveforms for the RS and FS units. Arrowheads denote spike peak and trough. Scale bars, 473 0.5 ms and 50 µV. (C) Scatter plot showing the bimodal distribution of peak-tough amplitude and 474 timing differences across all RS (gray) and FS (black) units. (D) A random sub-sampling of spike 475 waveforms recorded over 53 days from a single wire of a tetrode projected down into the first three 476 principal components (PC). (E) Spike waveforms from the two units identified in (D) across all 477 recording sessions, color-coded and superimposed chronologically. Waveforms for random units were 478 selected at random from all simultaneously recorded units. (F) The variability in the actual unit 479 waveforms, estimated as the sum of squared errors (SSE), is significantly less than randomly shuffled 480 units (p < 0.001, f(1)=814.73, mean ± SEM). (G) Experimental design. 481 482 Figure 2. Moderate loss of high-frequency auditory nerve fibers induces striking – but partially 483 reversible - receptive field reorganization and inhibition. (A) Adult mice (16 weeks) were exposed 484 to octave-wide noise at 98 dB SPL for 2 hours, which has been shown to eliminate approximately 50% 485 of auditory nerve synapses onto inner hair cells in the 16-45 kHz region of the cochlea27,28. (B) Mean ± 486 SEM ABR (left) and DPOAE thresholds (middle) were temporarily elevated 2 days after noise exposure 487 but recover fully by 30 days. ABR wave 1b amplitude reflects the permanent loss of Type-I spiral 488 ganglion nerve fibers (right). (C) A frequency response area (FRA) from a single unit recorded in the 489 high-frequency zone of the A1 tonotopic map rapidly assumed a low-frequency FRA hours after noise 490

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exposure but shifted back towards baseline tuning over the ensuing month. (D-E) Rasters depict spiking 491 from the same RS unit evoked by broadband noise at varying sound levels (D) or inhibited by 492 optogenetic activation of neighboring PV+ neurons (E). Note temporary threshold shift and change in 493 inhibition strength. (F) Quantification approach illustrated from a different single unit recorded before 494 (black) and after (red) noise exposure. (G) Mean ± SEM (n = 208 units) values for each response 495 property illustrated in (F). Data for each unit are normalized to the mean value measured during the 496 three day period prior to noise exposure. Red symbols are used to visually highlight the rapid plasticity 497 occurring during the first five days following nerve damage. Asterisks = post-hoc pairwise comparison 498 between identified groups (B) or to baseline value (G) p < 0.05 after correcting for multiple 499 comparisons. 500 501 Figure 3. Gradual and variable recovery of auditory processing following a massive, bilateral loss 502 of cochlear afferent nerve fibers can be predicted from early changes in inhibitory strength. 503 Plotting conventions follow Fig. 2. (A) Ouabain was applied bilaterally to the round window membrane. 504 (B) Mean ± SEM ABR thresholds were substantially elevated 30 days after ouabain application (left) but 505 DPOAE thresholds were unaffected, indicating normal outer hair cell function (middle). ABR wave1b 506 was virtually eliminated after ouabain (right). Darker and lighter shading represent measurements from 507 mice that recovered cortical sound thresholds versus mice that did not, respectively. (C-D) Rasters 508 document changes in noise-evoked spiking and laser-induced inhibition, respectively, from single A1 509 RS units recorded over a 53 day period from a mouse that eventually recovered function (C) and a 510 mouse that did not recover function (D). (E-H) Mean ± SEM noise-evoked thresholds (E), rate-level 511 function gain (F), spontaneous firing rate (G) and inhibition strength (H) for all units recorded from 512 three mice that recovered (top row, n = 156), three mice that did not recover (middle row, n = 169) or 513

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three mice that underwent a sham surgery (bottom row, n = 156). Asterisks = post-hoc pairwise 514 comparison between identified groups (B) or to baseline value (E-H) p < 0.05 after correcting for 515 multiple comparisons. 516 517 Figure 4. Early changes in PV-mediated inhibitory strength predict eventual recovery of cortical 518 sound processing after nerve damage. (A-B) PV-mediated inhibition strength (A) and change in 519 spontaneous firing rate (B) timelines for sound exposed, ouabain recovered and ouabain non-recovered 520 mice (red, blue and teal accordingly, plotted for visualization purposes as a 4th order polynomial fit to 521 the data presented Figs. 2 and 3). (Middle and Right) Correlation between broadband noise threshold at 522 day 50 and PV-mediated inhibition (A) or spontaneous firing rate change (B) at days 6-10 for ouabain-523 treated (middle) and sound-exposed mice (right, Spearman’s correlation coefficient, Rs). Each symbol 524 represents a single RS unit. The shape and color correspond to a particular group, as described above. 525 The shading corresponds to a particular mouse within that group. 526 527 Figure 1, figure supplement 1. Long-term tetrode recordings from isolated single units is feasible 528 with RS neurons, but not FS neurons. (A) As per Fig. 1E, single unit spike waveforms across all 529 recording sessions, are shown for a representative RS unit as well as two representative FS units. The 530 RS unit waveform is consistent across the 7-8 week recording period, while the FS unit waveforms are 531 lost after a few recording sessions. (B) Cumulative probability functions depict the number of 532 consecutive recording sessions before each RS and FS unit was lost in noise-exposed and ouabain-533 treated mice. (C) For the analysis provided in the main text, we only analyzed units that were present in 534 at least 75% of all recording sessions for a given mouse. None of the FS units we recorded met this 535

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criterion. Thus, we were unable to contrast changes in the FS neurons themselves with changes reported 536 in the main text for RS neurons or PV-mediated inhibition. 537 538 Figure 3, figure supplement 1. ABR wave 1 amplitude scales with the degree of auditory nerve 539 damage, but does not vary systematically between mice that recover cortical sound thresholds 540 versus those that do not. The amplitude of ABR wave 1b arises directly from the auditory nerve 541 compound action potential and, provided DPOAE thresholds are normal (as is the case here), serves as a 542 useful proxy for the surviving number of afferent nerve fibers onto inner hair cells. The ABR wave 1 543 amplitude is presented alongside the mean auditory response threshold measured in A1 single units for 544 each mouse subjected to auditory nerve damage. ABR wave 1 amplitude does not predict the eventual 545 recovery of sound processing in mice subjected to massive auditory nerve damage with ouabain. 546 547 Figure 4, figure supplement 1. Auditory cortex units show robust PV-mediated inhibition and 548 low-threshold sensory responses prior to nerve damage, both of which are unrelated to the 549 eventual response thresholds after nerve damage. (A) Distribution of best frequency values for 550 primary auditory cortex units recorded before nerve damage. RS units recorded in noise-exposed mice 551 were targeted to a high-frequency region of the tonotopic map, in the zone of putative cochlear 552 denervation. (B-C) Histogram of PV-mediated inhibition strength (B) and response threshold (C) 553 distributions prior to auditory nerve damage. (D) The strength of PV-mediated inhibition and auditory 554 response thresholds are not correlated with each other before nerve damage (Pearson’s correlation 555 coefficient). (E-F) PV-mediated inhibition strength and auditory response thresholds after nerve damage 556 cannot be predicted from the strength of PV-mediated inhibition before nerve damage (Pearson’s 557 correlation coefficient). 558

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559 Supplementary file 1. Statistical reporting. 560 Detailed description of statistical analysis for all figures and figure supplements. 561

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