thresholds in adulthood, letting us assess nervous system function independent of cochlear function. Methods Animals All procedures relating to the maintenance and use of animals were in accordance with the ‘‘Institutional Animal Care & Use Committee Handbook’’ and were approved by the University Animal Welfare Committee at NYU. Male and female gerbil pups (Meriones unguiculatus) were weaned from commercial breeding pairs (Charles River) at postnatal day (P) 30 and maintained in a 12-h light/12-h dark cycle. Behavioral data were obtained from 24 adult control (CTR) animals (P65–125, 9 males and 15 females) and 23 animals with developmental CHL (P65–125, 15 males and 8 females). The groups were comprised of animals from multiple litters. All animals that entered the behavioral protocol were included in the analyses. No selection criteria were imposed, as is common in behavioral studies. Therefore, poor performers were not eliminated during any phase of the procedure, allowing us to compare both mean performance and between-animal variability within and across treatment groups. Physiological data were obtained from separate groups of adult CTR and developmental CHL gerbils: 15 adult CTR (P60–81, 9 males and 6 females) and 8 CHL (P61–90, 5 males and 3 females). Behavioral Training and Testing Experimental environment. Behavioral data were collected as previously described [22]. Gerbils were placed in a small wire cage in a room lined with echo-attenuating material, and observed in a separate room via a closed circuit monitor. The test cage contained a stainless steel drinking spout and metal floor plate. When the animal contacted both the plate and spout, a circuit was completed that initiated water delivery via a syringe pump (Yale Apparatus). A personal computer, connected to a digital I/O interface (Tucker-Davis Technologies) measured animal contact and controlled the timing of acoustic stimuli, water delivery (0.25 ml/min), and a small current delivered at the end of warning trials. Auditory stimuli were generated by the Tucker-Davis Technologies system and delivered via a calibrated tweeter (KEF Electronics) centered 1 m in front of the test cage, with the speaker ,20 degrees above the animal’s head. Sound level at the test cage was measured with a spectrum analyzer (Bru¨ el & Kjaer 3550) via a 1/4 inch free-field condenser microphone positioned at the head location when in contact with the spout. Noise levels are in units of dB SPL, converted from RMS measurements. Auditory stimuli. CTR and CHL groups were divided into subgroups that were trained on sAM modulation frequencies (MF) of either 5 Hz or 100 Hz. Separate subgroups were used for the two MFs to ensure that all subgroups had equivalent experience with their tasks. The stimulus was broadband noise with a low frequency falloff of 25 dB at 3.5 kHz and a high frequency falloff of 25 dB at 20 kHz. The sound pressure level remained constant during both the pre-trial and warning intervals to exclude the use of an energy cue. Each trial was 2500 ms total length, comprised of a pretrial interval containing 1200 ms of unmodulated noise, 1000 ms of either a modulated noise (warn trial) or a continuation of the unmodulated noise (safe trial), and 300 ms of unmodulated noise. The spout was monitored for contact during the final 500 ms of the pretrial interval, and the trial proceeded only if the animal remained in contact with the spout for .50% of this interval. The warning stimulus was 1000 ms of sinusoidally amplitude modulated noise at a modulation frequency of either 5 or 100 Hz (depending on experimental group) and at varying Hearing Loss Disrupts AM Processing depths. Warning stimuli were followed immediately by an aversive unconditioned stimulus (300 ms electrical current delivered via the lick spout). To determine whether the animal detected the warning stimulus, contact with the spout was monitored during the final 100 ms of the warning stimulus. During this window, a contact time of ,50 ms was scored as a hit. For the same window during safe trials (where the entire 2500 ms duration consisted of unmodulated noise), a contact time of ,50 ms was scored as a false alarm (FA). Warning trials always occurred at the end of a block of 2–4 safe trials, randomized to avoid temporal condition- ing. Sound levels. In order to choose appropriate amplitude levels for the two experimental groups, task-specific sound level thresholds for a 100% MD sAM were assessed in pilot groups of CTR (n = 6) and CHL (n = 6) animals. Trained animals were tested with 100% MD sAM noise at varying amplitudes using a staircase procedure; levels were moved up and down in 3 dB increments based on performance, and threshold was determined as the level where detection dropped below 50%. Experimental levels were then set to be ,20 dB above task-specific threshold: CTR animals were presented signals at 35 dB SPL, and CHL animals were presented signals at 70 dB SPL. For the experimen- tal animals, it was not possible to obtain task-specific level thresholds from each animal prior to sAM detection testing for two reasons: 1) animals needed to be very practiced to yield accurate level thresholds and 2) presenting difficult stimuli (e.g., low sound level) early in training can extinguish the learned behavior. Thus individual experimental animals had their task-specific thresholds measured after completion of all stages of training and testing. Procedural training. All training used a conditioned avoid- ance procedure [29,30]. Animals were first placed on controlled water access, and upon introduction to the experimental cage, learned to obtain water from a lick spout in the presence of an unmodulated noise stimulus, while contact with the waterspout was monitored. Animals were then trained to withdraw from the spout when an acoustic cue (modulation of amplitude) was present. To train the withdrawal response, a low AC current (0.5–1.0 mA, 300 ms; Lafayette Instruments) was delivered through the waterspout immediately after the warning signal. Since animals display large between-subject variability in pain sensitivity [31,32,33], the shock level was adjusted continuously for each animal to reliably produce withdrawal from the spout without dissuading an animal from approaching the spout on subsequent trials. To establish criterion performance on the procedure, warning trials (100% modulation depth) were presented until performance reached a criterion of ,70% correct over 10 consecutive trials. Assessment of sAM detection thresholds. Once animals in each experimental group reached criterion on the conditioned avoidance procedure, we obtained an initial assessment of sAM detection threshold by testing animals on a broad range of sAM modulation depths (MD; 10–100% MD in 10% steps) presented ten times each in a randomized order, with the same order being delivered to each animal. In order to obtain minimum thresholds, animals underwent 7–10 days of additional testing. Each day, a small range of MDs (five in total, divided evenly into steps of 10% depth) was repeatedly presented in descending order, bracketing each animal’s initial detection threshold. On each subsequent day, an animal’s performance on the previous day determined the range of MDs on which it was tested (i.e., always bracketing the previous detection threshold). As animals were challenged with harder values based on their performance, thresholds across training days are an indicator of improvement. Thresholds PLoS ONE | www.plosone.org 2 July 2012 | Volume 7 | Issue 7 | e41514