in guinea pigs [16] and rats [18] show a lack of high-sync VCN neurons in their recordings (i.e., rmax#0.9). It is shown here that the strength of phase-locking in the chinchilla’s VCN resembles the cat’s, at least for CFs,600 Hz, thus enhancing the synchro- nization capabilities of ANFs. In other words, there exist highly synchronized neurons in chinchilla and some of those neurons also exhibit significant entrainment to the stimulus. The improvement in synchronization, however, comes with greater distortion in the representation of the stimulating waveform. Methods Surgical procedures Chinchillas (average weight = 500 grams) and cats (average weight = 3 Kg) were initially anesthetized with intraperitoneal injections of sodium pentobarbital (75 mg/Kg, i.p., and 50 mg/ Kg, i.p., for chinchillas and cats, respectively). A level of surgical anesthesia was maintained by additional i.p. injections of pentobarbital in chinchillas and via a catheter inserted in the femoral vein in cats. The rest of the surgical procedure was very similar for both species. A thermostatically controlled heating blanket maintained body temperature at 37uC. After insertion of a tracheal cannula, the left ear was removed and the bulla was vented with 20 cm of a 1-mm plastic tube. Following removal of the overlying cerebellum, the cochlear nucleus was covered with agar. To further reduce brain pulsations, a chamber was mounted over the skull opening and filled with mineral oil. Two scales were positioned along the cat CN, one scale in chinchilla, to record the position of the electrode. Auditory nerve recordings were performed in some experiments (mostly cats). For this purpose, the auditory nerve was approached following retraction of the cochlear nucleus by inserting cotton balls between the CN and the temporal bone. Several recordings in chinchillas were obtained from axons in the ventral acoustic stria (VAS), which is one of the three output tracts of the CN. The VAS was accessed dorsally by inserting a microelectrode at least 4 mm through the floor of the fourth ventricle [22]. Some of the data were collected from cats that were part of labeling experiments published elsewhere [23,24]. Based on histological reconstructions in some of the animals, it was judged that the recordings originated from the nerve root area and the caudal end of the AVCN. Experimental protocol A RadioShack SuperTweeter speaker was used to present the acoustic stimuli in a closed ear coupler. A calibrated probe tube was inserted very close to the eardrum. The other end of the probe tube was connected to a K0 Bruel and Kjær condenser microphone and was amplified 60 dB. Acoustic stimuli were calibrated from 50–24000 Hz in 50-Hz steps in the chinchilla and from 50–40000 Hz in 50-Hz steps in the cat. KCl-filled micropipettes, with impedances of 10–40 MV, were used to record single-unit activity from either CN units or ANFs. Spike times relative to stimulus onset were determined using a level detector (i.e., the output of an oscilloscope trigger) and stored digitally at a 1-ms resolution. A high-intensity FM sweep was used as search stimulus for neurons. Responses to this search stimulus yielded an initial estimate of the CF of the neuron. The neuron’s receptive field (i.e., a family of iso-intensity curves) was determined using five or ten 50-ms tone pips with intensities varying between 0 and 90 dB SPL, in 10 dB SPL increments. Frequency separation was usually 100 Hz for CFs,2000 Hz or 200 Hz for CFs.2000 Hz. Threshold and final CF estimates were deter- Information Transmission in the Auditory Brainstem mined by analysis of the receptive field. Post-stimulus time histograms (PSTHs) at CF were obtained using 250 repetitions of a 50 ms tone pip, usually at 60 dB SPL. For most neurons in this study, this level corresponds to 30 to 50 dB relative to threshold. If necessary, a PSTH at CF was also obtained using an 80-dB SPL tone. Rate-vs.-level curves at CF were also obtained from 0 to 90 dB SPL in 5-dB SPL steps. Analysis of the responses of chinchilla VCN neurons (CF,1000 Hz) to synthetic vowel stimuli are presented here. These data have not been published before but were collected as part of a previous study [22]. Vowels were generated using the cascade branch of a Klatt synthesizer (1980). The fundamental frequency, f0, was 100 Hz for most neurons except in one case (f0 = 181.6 Hz). Only responses to vowels /i/ (as in heed) and /e/ (as in bet) will be discussed here. First (F1) and second (F2) formant frequencies for /i/ are 300 and 2300 Hz, respectively. F1 and F2 frequencies for /e/ are 500 and 1800 Hz, respectively. Each vowel stimulus (dura- tion = 150 ms) was presented 200 times, with a repetition period of 350 or 500 ms at three intensity levels: 20, 40 and 60 effective dB SPL. (The measure of sound intensity, effective dB SPL, is computed by summing energy contributions from each frequency band in the speech waveform. Each energy contribution is a proportion of the maximum dB SPL taken from the acoustic tone calibration.) Whispered vowels (/i/, /u/ and /a/) were recorded in a soundproof room from two native speakers of American English (male and female) and presented to anesthetized cats. The microphone used for the recordings was a K0 Bruel and Kjær condenser microphone and the sampling rate was 20 kHz. Each vowel (duration = 300 ms) was presented 100 times at a rate of 1/ sec at 40 and 60 dB effective SPL at a playback rate of 50 kHz (following resampling). Only responses to the female /i/ sound will be presented in this report. Temporal analysis and unit classification Because glass micropipettes were used to record neural activity, the segregation of primary and secondary neurons posed a problem. (This was not a concern for VAS recordings.) Shapes of PSTHs as well as certain response statistics of CN neurons are well known [11,13], but that classification is based on responses to moderate- to high-frequency stimulation (.1 kHz). A neuron was classified as originating from the VCN if it satisfied at least one of the three criteria described here: 1) 2) 3) Responses to CF tones yielded r.0.9. The 0.9 value was used as a criterion based on published rmax for ANF responses in cats [5,6] and chinchillas [9,10].The statistical significance (p,0.001) of r was evaluated using the Rayleigh test [25]. The shape of the action potential was bipolar [26]. There was a rate reduction below the spontaneous rate activity in response to single off-CF tones with increasing intensity [24]. Responses of VCN neurons with CFs in the 1–2 kHz range are also reported here. In response to CF tones, these neurons have PSTHs with one of the two following shapes: primarylike-with- notch (PLN) or an onset with an L-shaped PSTH (OnL). Based on labeling experiments [6,11,24], those PSTHs are primarily associated with globular bushy cells. Units deemed VCN neurons using the criteria expressed above were classified into three mutually exclusive groups: – Group 1: Neurons with r.0.9 in their responses to CF tones. – (Group 1 corresponds to highly synchronized neurons.) PLOS ONE | www.plosone.org 2 September 2012 | Volume 7 | Issue 9 | e44286