All procedures relating to the maintenance and use of animals were approved by the Institutional Animal Care and Use Committee at the Northeast Ohio Medical University. Adult Mongolian gerbils (Meriones unguiculatus) ranging between postnatal (P) day 54–125 were tested in one of two procedures. Cortical responses to signals in noise were measured with intracellular recordings from a group of control (CTR) animals (n = 7). Perceptual detection thresholds were obtained from separate groups using an operant conditioning procedure. Control animals (n = 8) received normal auditory experience during development and were compared to animals with developmental CHL (n = 10). All animals were weaned at P30 and housed with litter mates in a 12 h light/12 h dark cycle. Groups were comprised of animals from multiple litters and included both males and females.

Behavioral thresholds for signal detection in noise were obtained from two groups of adult gerbils: animals reared with CHL prior to hearing onset, and age-matched controls (CTR). Animals were trained to detect a brief 4 kHz signal embedded in a longer noise masker (30% bandwidth centered around the signal frequency). Once animals were reliably performing the task, they received several days of procedural testing to determine threshold (defined as the quietest tone level that could be reliably detected in the presence of the masker, reported here as SNR in dB). Thresholds were compared across groups for each animal’s best day (Figure 2A) and mean across the best 3 days (Figure 2B). CTR animals displayed significantly lower detection thresholds compared to CHLs (Best day: p < 0.001; Best 3 days: p = 0.01, Wilcoxon rank-sum). Differences in thresholds were not due to differences in training or performance on the task. Groups received a comparable number of procedural training trials (Figure 2C: p = 0.97, Wilcoxon rank sum) and learned the task at a similar rate (days of training: CTR 5.6 ± 0.9 vs. CHL 5.8 ± 0.6, p = 0.69, Wilcoxon rank-sum). Furthermore, the amount of procedural training on the task was not correlated to threshold for either group (Figure 2D: CTR: R² = 0.192, p = 0.28, CHL: R² = 0.021, p = 0.69).

One might expect that early hearing loss would affect learning or improvement on perceptual tasks. For this simple simultaneous masking task that was not the case. In addition to equivalent time-courses of training, both groups improved but did so equivalently across testing days (Figure 2E; First day minus last day threshold (dB SNR): CTR 7.8 ± 2.7 vs. CHL 4.7 ± 4.6, p = 0.15, Wilcoxon rank-sum). Furthermore, the rate of learning did not differ between groups, as animals reached best performance over an equivalent number of testing days (number of testing days to reach threshold (dB SNR): CTR 4.6 ± 0.2 vs. CHL 3.7 ± 0.4, p = 0.19, Wilcoxon rank-sum).

Higher thresholds for the CHL animals did not appear to be due to poorer attention to the task. Poor attention can be indicated by high FA rates, measured here as withdrawal from the spout during SAFE trials (which minimizes the chance of receiving a shock during poor attention to the signal). CTR and CHL animals did not differ in their FA rates (Figure 3A: p = 0.26, Wilcoxon rank-sum) and this measure of attention did not predict threshold for either group (Figure 3B: CTR: R² = 0.172, p = 0.31, CHL: R² = 0.159, p = 0.25).

Attention can also be indicated by licking consistency. During testing, animals ideally maintain constant contact with the spout, and withdraw only upon detecting a signal. Another strategy is to drink continuously but make poor contact with the spout, with continuous micro-withdrawals to minimize the magnitude of shock received. This hesitant contact can be an indicator of poor attention or performance anxiety and can be quantified as the number of times an animal breaks contact with the spout between WARNs. CTR animals displayed significantly more breaks in contact between WARNs compared to CHLs (Figure 3C: p < 0.01, Wilcoxon rank-sum) indicating group differences in overall performance strategy. However, this was independent of signal detection thresholds (Figure 3D: CTR: R² = 0.06, p = 0.57, CHL: R² = 0.001, p = 0.93). Notably, this performance strategy would be likely to increase the false alarm rate in CTR animals, thus increasing their thresholds as calculated by the d′ measure. Despite this, CTR thresholds were significantly lower than those of CHLs, suggesting that, if anything, the threshold differences measured here may underestimate perceptual differences across groups.

Task proficiency and performance consistency can also affect behavioral thresholds. As an indicator of task proficiency, we measured performance at the easiest level on the best testing day (Figure 4A). As an indicator of performance consistency we measured the range of d′ scores for the easiest level presented across days; a wider range indicates increased variability in performance (Figure 4B). Neither the d′ score for the easiest level on the best performance day (CTR: R² = 0.35, p = 0.12, CHL: R² = 0.11, p = 0.34) nor the range of d′ scores at the easiest level (CTR: R² = 0.17, p = 0.30, CHL: R² = 0.42, p = 0.57) correlated with behavioral threshold for either group. Thus perceptual deficits in CHL animals are independent from their ability to perform the task.

In order to attribute deficits in masked thresholds to central changes, it is essential to demonstrate that the attenuation provided by CHL is not sufficient to explain these deficits. It is possible that increased signal detection thresholds for CHL animals could be attributed to the stimuli not being presented at sensation levels equivalent to CTRs. To account for this, the two groups were tested with stimuli that differed by 35 dB (for both masker and signal). This level difference was based on behaviorally-measured level thresholds for separate groups of CHL animals in two tasks: AM detection (Rosen et al., 2012) and tone-detection (Buran et al., 2014b), which indicated 35 and 30 dB shifts respectively. We then tested to ensure that the amount of hearing loss at 4 kHz did not predict detection thresholds for our 4 kHz masked tones. Figure 4C shows that hearing levels for a 4 kHz tone, as measured by ABRs for each animal, did not correlate with behavioral thresholds in either CTRs (R² = 0.01, p = 0.84) or CHLs (R² = 0.10, p = 0.55). As there was no differential difficulty in hearing the signal, non-equivalent sensation levels could not account for the increased masked thresholds seen in CHL animals. That is, the characteristics of the perceptual deficit are not what would be predicted based solely on the amount of signal attenuation caused by the CHL.

Since the mechanisms by which central areas contribute to simultaneous masking are not resolved, intracellular responses to noise-masked signals were measured via sharp electrode recordings in auditory cortex. We focused on signal-evoked hyperpolarizations, as these are of cortical origin and reflect central processing (Somogyi et al., 1983; DeFelipe and Jones, 1985; Matsubara, 1988; Albus et al., 1991; Albus and Wahle, 1994; Tomioka et al., 2005; Higo et al., 2007). The spikes that occurred when hyperpolarizations returned to baseline were examined because they were likely to be generated by post-inhibitory rebound. Such spikes can be attributed to local cortical processes.

The subset of cells (n = 16) that exhibited hyperpolarization in response to the signal were examined. For each cell, the response to the signal alone was compared with the response to the masked signal. An example of overlaid traces in Figure 5A (top) shows a FM signal-evoked spiking onset response followed by a hyperpolarization, with rebound spikes upon return to baseline. The bottom trace shows a reduced hyperpolarization with no clear rebound spikes in response to the FM signal during a masker. For the sample of cells tested, the magnitude of the signal-evoked hyperpolarization (measured as the area under the curve relative to the membrane resting potential) was significantly reduced during presentation of the masker (Figure 5B; Wilcoxon signed rank, p = 0.049). Furthermore, those cells that spiked on rebound from the FM signal-evoked hyperpolarization fired significantly fewer rebound spikes during masker presentation (Figure 5C; Wilcoxon signed rank, p = 0.008). A reduced firing response to a masked signal is one measure of a neural correlate of perceptual masking. As this signal-evoked hyperpolarization must arise locally, from intrinsic cortical cellular mechanisms or local inhibitory circuitry, these data are evidence for a cortical contribution to simultaneous masking.

The CHL induced here, via malleus removal, attenuates sound transmission to the inner ear but leaves the cochlea intact: ossicle removal does not alter hair cell counts on the basilar membrane, and bone conduction thresholds are normal (Tucci and Rubel, 1985). Here, visual postmortem analyses excluded the possibility of accidental cochlear damage. In SNHL listeners, reduced cochlear nonlinearities are often used to explain raised thresholds for simultaneous masked signals (Moore and Glasberg, 1986; Oxenham and Bacon, 2003), but this should not be the source of the raised thresholds seen here. The possibility exists that the permanent CHL induced compensatory changes at the periphery, perhaps via efferent alterations of the middle ear reflex (Munro and Blount, 2009). However, signal detection thresholds were not correlated with hearing thresholds at the same frequency (Figure 4C). Another possible peripheral contribution would be the induction of a hearing loss that was not consistent across the frequency range used in the behavioral masking task. Hearing loss with malleus removal is relatively flat across the frequency spectrum (Rosen et al., 2012), mimicking the flat CHL that occurs during otitis media in children (Kokko, 1974; Anteby et al., 1986; Hunter et al., 1994). Here, signals of 4 kHz were masked with a noise spanning 3.4–4.6 kHz, which approximates the gerbil critical band at that center frequency (~1 kHz; Kittel et al., 2002). Finally, it is worth noting that CHL induced via malleus removal is not the best mimic of otitis media, but is well-suited to examine central contributions to hearing loss deficits. Our CHL is permanent, whereas early otitis media typically produces a fluctuating CHL that resolves after childhood. Our model does not reproduce the effusion viscosity that occurs with otitis media, which alters sound transmission delays (Hartley and Moore, 2003). It therefore avoids introducing an element of altered peripheral processing that may affect temporal processing in listeners with otitis media.

Our evidence in combination with previous work indicates that responses to simultaneous masked signals evolve from the periphery to central regions. In the periphery, simultaneous masking functions in auditory nerve fibers are based upon cochlear nonlinearities. For example, with a continuous broadband noise masker, auditory nerve fiber responses to tones shift in a manner consistent with two-tone suppression (the reduction of the response to one tone by simultaneous presentation of another tone) (Costalupes et al., 1984; Ruggero et al., 1992). This suppression is attributable to outer hair cell interactions with the basilar membrane (Robles and Ruggero, 2001). SNHL reduces those nonlinearities, which affects simultaneous masked thresholds (Oxenham and Bacon, 2003). In comparison, CHL, which does not alter cochlear nonlinearities, also affects simultaneous masked thresholds, and this is presumably due to central processes.

Our intracellular data reveal a cortical correlate of simultaneous masking. In neurons that exhibited signal-evoked hyperpolarization, the responses to FM tones showed a reduced hyperpolarization and fewer subsequent spikes in the presence of a noise masker (Figures 5B,C). The action potentials that immediately follow the hyperpolarization are likely to be rebound spikes, resulting from intrinsic cellular properties and thus of cortical origin. Cortical inhibition is known to arise locally, via intrinsic horizontal, intralaminar, and some long-range projections (Somogyi et al., 1983; DeFelipe and Jones, 1985; Matsubara, 1988; Albus et al., 1991; Albus and Wahle, 1994; Tomioka et al., 2005; Higo et al., 2007). As the hyperpolarizations seen here arise locally, our data are an example of the cortex modifying inherited subcortical information. The two possible sources of this hyperpolarization are either inhibition from local interneurons, or intrinsic cellular properties that generate afterhyperpolarizations. Intracellular in vivo recordings of ACx pyramidal neurons show responses similar to those seen here: signal-evoked depolarization often followed by hyperpolarization. The hyperpolarization has characteristics of a chloride-mediated IPSP that is likely mediated by GABA_A receptors (De Ribaupierre et al., 1972; Ojima and Murakami, 2002). Here, the hyperpolarization time courses were similar to those previously described. Therefore they are likely to arise from local inhibition rather than intrinsic hyperpolarizing currents activated by depolarization. If the hyperpolarization indeed arises from local cortical interneurons, the reduced hyperpolarization seen during masking may arise from reduced excitatory input onto those interneurons. Reduced neural responses are seen in subcortical regions during simultaneous masking, and may be a source of these excitatory feedforward inputs (Costalupes et al., 1984; Rees and Palmer, 1988; May and Sachs, 1992).

To our knowledge, the only other direct demonstration of inhibitory reduction of spiking during simultaneous masking in ACx is by Volkov and Galazyuk (1992), where a background sound evoked a local inhibitory current that reduced the response to an overlaid signal. However, they did not directly compare unmasked and masked conditions and only tested binaural interactions. Previous neurophysiological analyses of ACx and inferior colliculus have provided some evidence for central contributions to simultaneous masking involving local inhibition. In the inferior colliculus, tone-evoked responses were shifted during simultaneous noise in a manner consistent with local inhibition, providing responses that differed from auditory nerve fibers (Rees and Palmer, 1988). In primary ACx, monotonic cells (those whose discharge rate increases with sound level) responded like auditory nerve fibers, dominated by whichever sound (signal or masker) elicited a stronger response alone. In contrast, nonmonotonic cells (those with more complex level-sensitivity) were suppressed by a noise masker, suggestive of inhibitory influences (Phillips and Cynader, 1985; Phillips and Kelly, 1992). Consistent with this idea, nonmonotonic response profiles do not reflect inputs inherited from lower hierarchical areas. Instead, they arise from local excitatory and inhibitory interactions (Wu et al., 2006). Thus our result is expected: local cortical inhibition shapes responses to signals masked by simultaneous noise. We did not explicitly test for monotonicity, but would predict that the neurons analyzed here were nonmonotonic.

A challenging element is connecting changes in neural discharge with altered auditory perception. The reduced spiking shown in this study indicates a reduced output to a masked signal and would be expected to increase signal detection thresholds. This idea is supported by recordings of cortical auditory evoked potentials (CAEPs) in humans, which show a correlate for the detection of masked signals in the P1/N1/P2 waveform. The amplitude of CAEP peaks is correlated with detectability of signals in noise (Martin et al., 1997). Since the CAEP primarily reflects synchronous activity arising from ACx (Näätänen and Picton, 1987; Eggermont, 2007), the reduction in ACx spiking seen here during the presence of a masker could manifest as reduced CAEP amplitude, and may thus contribute to poor perception in noise.

Cortical changes known to occur with hearing loss suggest a simple mechanism for impaired signal detection. Early CHL directly affects the central auditory system, generally reducing inhibition relative to excitation in order to maintain homeostasis (Sanes, 2013). For example, animals raised with early CHL have reduced IPSC amplitude, decreased firing rates in fast-spiking interneurons, and facilitating rather than the normal depressing short-term plasticity (Takesian et al., 2010, 2012). Inhibition is known to sharpen neural tuning curves at the level of cortex (Ojima, 2011). In normal listeners, inhibition would reduce the magnitude of noise-evoked responses. With reduced cortical inhibition seen from early hearing loss, the frequency components of a noise surrounding a neuron’s BF would increase the response magnitude to noise. At the same time, early CHL reduces the magnitude of ACx firing to tones (Rosen et al., 2012). Concurrently, these effects would reduce the SNR of tones presented in a noise masker. In this manner, changes to the cortical network may subserve the impaired simultaneous masked thresholds seen in this study. This prediction can be directly tested in our animal model. Future experiments that measure neural activity during perceptual detection tasks in CHL animals can connect changes in central auditory regions with perceptual impairments arising from developmental deprivation.