– – Group 2: Neurons with r#0.9 in response to CF tones but able to synchronize strongly (i.e., r.0.9) to certain off-CF tones. Group 3: Neurons with r#0.9 in response to single tones, regardless of the stimulus frequency. Regularity analysis [13] was performed on interspike intervals (ISIs) computed from ANFs’ and VCN neurons’ responses to CF tones. The coefficient of variation (CV) of the ISI was used as an indicator of the regularity of the response [13]. CV is defined as the ratio of the standard deviation of the ISI to its mean value. In cats, average CVs obtained from responses of ANFs and high-sync neurons are 0.68 and 0.45, respectively. Statistical analyses show significant differences between the CVs of ANFs and high-sync neurons (Kolmogorov-Smirnoff test, P.0.001). In chinchillas, the combined sample of Groups 1 and 2 neurons also exhibit CVs (m = 0.45) that are smaller than CVs (m = 0.65) computed from ANF responses (Such differences are statistically significant as determined by the Kolmogorov-Smirnoff test, P.0.001). CVs from the population of chinchilla Group 3 neurons are also smaller (m = 0.38) than CV values in ANFs (Kolmogorov-Smirnoff test, P.0.001). CFs of all the units used in the above analyses were below 2 kHz. The fact that units classified as VCN neurons have lower CVs than ANFs validates the aforementioned criteria to segregate primary from second-order neurons. Entrainment indices were computed from ISI histograms. The index is defined as the ratio of the number of intervals that fall in a 1 3 window located at T , T ,where T = 1/CF, to the total 2 2 number of intervals [6]. For responses perfectly entrained to the stimulus, this index equals 1. The quality of the representation of the stimulus waveform in the discharges of ANFs and VCN neurons was evaluated using a measure of distortion. Distortion in the neural response to a CF tone was measured by computing the power spectrum density (PSD) of the spike train. Total harmonic distortion (THD) was defined as the sum of the powers of the first eight harmonics to the power of the spectral component at CF: 9 P A2 n THD(%)~100| n~2 A2 1 1 ð Þ where A21 and A2n represent the power of the spectral component at CF and its harmonics, respectively. For responses to single tones, A1 corresponds to the amplitude at the tone frequency. Similarly for responses to vowel stimuli, A1 corresponds to the amplitude at the F1 frequency. Temporal analysis of neural responses to vowel sounds was performed using period histograms and shuffled autocorrelograms (SACs). The duration of the cycle used to construct period histograms was set to 1/f0. SACs were constructed using MATLAB routines provided in [27]. The analysis windows for the construction of the SACs were 50–150 ms and 50–300 ms for synthetic and whispered spoken vowels, respectively. The duration of the binwidth was 50 ms for all the SACs. Correlation indices (CIs), which correspond to the peak value of the normalized SACs [7], were computed to quantify the temporal structure in the neural responses to vowel sounds. Ethics statement Experiments were performed at the Department of Physiology, University of Wisconsin – Madison, where the author was a research scientist. All procedures were approved by the Animal Information Transmission in the Auditory Brainstem Care and Use Committee of the University of Wisconsin-Madison (Protocol ID: M00205). Results Responses were obtained from VCN neurons and ANFs in 29 chinchillas and 9 cats. Because of the small number of chinchilla ANFs collected for this research, r values from a published study (Fig. 7 in [9]) were used for comparison purposes. Responses to single tones of highly synchronized neurons in chinchilla and cat Figures 1A and 1B show dot raster plots of the responses of an ANF and a VCN neuron, respectively, to a CF tone. Whereas the chinchilla VCN neuron is well locked to one stimulus phase (continuous single trace in Fig. 1B), responses of the ANF in the same species are less synchronized to the stimulus waveform (continuous trace in Fig. 1A). Vector strength values computed from the ANF’s and VCN neuron’s responses to the 200-Hz tone were 0.69 and 0.98, respectively. Because of its vector strength value, the VCN neuron was classified as ‘‘highly synchronized.’’ Discharge patterns of ANFs also show a larger amount of randomness in the interspike intervals than high-sync neurons. A consequence of this jitter can be seen in the broad shape of the period histogram of the responses (Fig. 1C) and in the CV value ( = 0.64). By contrast, the period histogram of the high-sync neuron is narrow (Fig. 1D) and has a small CV value ( = 0.07). Similar differences occur between period histograms of responses of an ANF and a VCN neuron in cat (Figs. 1E and 1F, respectively). The ability of a high-sync neuron to fire approximately one action potential per stimulus phase, and with little variability in the spike’s time of occurrence, is also evident in Fig. 1B. In fact, near perfect entrainment ( = 0.99) to the stimulus waveform was measured from the response of the high-sync neuron. The entrainment index for the ANF’s response was lower ( = 0.57). Insets in Figs. 1C–1F show the results of the PSD analysis of the neural responses. The results consist, approximately, of a series of local peaks located at the stimulus frequency (f0 = CF = 200 Hz in Figs. 1C and 1D) and at their harmonics (e.g., 2*f0, 3*f0, etc.), as indicated by the dots in the insets. (For the insets in Figs. 1E and 1F, f0 = CF = 400 Hz.) The spectral representation at f0 is more predominant for ANFs than for VCN neurons. For example, the amplitudes at f0 and 2*f0 are closer in value for VCN neurons (insets in Figs. 1D and 1F) than for ANFs (insets in Figs. 1C and 1E). In Fig. 2, THDs were higher for the two VCN neurons (291% and 228% in chinchilla and cat, respectively) than for the two ANFs (62% and 52% in chinchilla and cat, respectively). Also notable are the differences in receptive field plots computed from the responses of ANFs and VCN neurons. Although all the receptive field plots in Fig. 2 are asymmetric around CF, such asymmetries are magnified in VCN neuron responses in part because of the large firing rates evoked by some above-CF tones, which can be much larger than rates produced by CF tones. Such differences in firing rates (e.g., DRmax = 85 and 40% in Figs. 2B and 2F, respectively) are responsible for shifts in the ‘‘center of gravity’’ of the receptive field (Dfmax = 1 and 0.8 octaves in Figs. 2B and 2F). Asymmetric receptive fields are also a property of ANFs (Figs. 2A, 2B and 2C) but less noticeable. The large firing rates measured from responses of VCN neurons in Fig. 2 probably arise from the tendency of those neurons to entrain to the stimulus waveform, even for frequencies different from CF [24]. Dot raster plots at several intensity levels displayed in the insets in Figs. 2A and 2B reveal the greater ability of the VCN PLOS ONE | www.plosone.org 3 September 2012 | Volume 7 | Issue 9 | e44286