Auditory brainstem responses in Cope's gray treefrog (Hyla chrysoscelis): effects of frequency, level, sex and size

Abstract

Our knowledge of the hearing abilities of frogs and toads is largely defined by work with a few well-studied species. One way to further advance comparative work on anuran hearing would be greater use of minimally invasive electrophysiological measures, such as the auditory brainstem response (ABR). This study used the ABR evoked by tones and clicks to investigate hearing in Cope's gray treefrog (Hyla chrysoscelis). The objectives were to characterize the effects of sound frequency, sound pressure level, and subject sex and body size on ABRs. The ABR in gray treefrogs bore striking resemblance to ABRs measured in other animals. As stimulus level increased, ABR amplitude increased and latency decreased, and for responses to tones, these effects depended on stimulus frequency. Frequency-dependent differences in ABRs were correlated with expected differences in the tuning of two sensory end organs in the anuran inner ear (the amphibian and basilar papillae). The ABR audiogram indicated two frequency regions of increased sensitivity corresponding to the expected tuning of the two papillae. Overall, there was no effect of subject size and only small effects related to subject sex. Together, these results indicate the ABR is an effective method to study audition in anurans.

Fig. 1

a Placement of recording electrodes depicted by “+”s. b Schematics of stimulus trains used in experiments. Depicted are examples of (top) a train of 5 clicks (0.1 ms, 24.9 ms inter-click interval) followed by a 100-ms period of silence broadcast at the beginning and end of a session to verify the presence of a signal (during clicks) and to obtain a recording in the absence of stimulus (during silence), (middle) a train consisting of 9 tones (5 ms in duration, 20-ms inter-tone interval) of increasing intensity, and (bottom) a train of 9 clicks of increasing intensity. Stimulus level is indicated above each sound in the train, in dB SPL (for tones) or dB pSPL (for clicks). c A typical ABR waveform indicating the three measures quantified in this study, including (i) amplitude, measured from maximum of the first positive deflection (P1) to minimum of the subsequent negative deflection (N1) and (ii) latency, measured from the time sound impinged on the tympanic membranes, indicated by the arrow, to the time of P1.

Fig. 2

Representative recordings of ABRs from a single individual in response to tones at a 1.3 kHz, b 1.625 kHz, c 2.6 kHz, and in response to d clicks presented at different sound pressure levels. Downward pointing arrows depict arrival times of sound at the tympanic membranes. The right-pointing arrowheads depict the visually detected thresholds for each frequency or for clicks

Fig. 3

a-d Average traces temporally aligned to P1 (time = 0 ms) in response to tones of 75 dB SPL at frequencies of a 1.3 kHz, b 1.625 kHz, and c 2.6 kHz and to d clicks of 75 dB pSPL. Shaded areas depict ± 1 s.d. e-h Cross correlation coefficients between the average response to tones presented at frequencies of e 1.3 kHz, f 1.625 kHz, and g 2.6 kHz or to h clicks and average responses to all tone frequencies. Legend in e applies to e-h and is in units of dB SPL (for tones) or dB pSPL (for clicks)

Fig. 4

Characterization of the ABR in terms of a-b amplitude (absolute voltage difference between P1 and N1) and c-d latency (time to P1 from sound arrival at tympanic membranes). a & c Mean amplitude and latency (averaged over all individuals) depicted in the form of a contour plot across all frequencies and all levels. Arrows indicate the range of frequencies (1.5 – 1.75 kHz) within which there is a sharp discontinuity in the values of the response measure. b & d Mean (±s.e.m.) amplitude and latency of click-evoked responses and tone-evoked responses across frequencies at five stimulus levels. Data are shown separately for males (open circles) and females (filled circles). The data depicted in b and d represent reduced datasets that included responses to clicks at levels of 65 to 85 dB pSPL and tones of frequencies from 0.75 kHz to 3.0 kHz presented at levels of 65 dB to 85 dB SPL. Plotted data were pooled across multiple imputations of the reduced datasets (see text). Values for some click-evoked responses are slightly displaced along the x-axis to reveal symbols otherwise hidden

Fig. 5

a A representative example of threshold detection based on predicted values of RMS amplitudes of ABRs in response to stimuli presented at different levels. RMS amplitudes of responses were computed over a 10 ms analysis window that began when the stimulus arrived at the tympanic membranes. Depicted here is the best-fit sigmoid curve fit to RMS data for responses from one frog to tones of 2.6 kHz. Thresholds determined from the fits for each stimulus for each frog were averaged to construct the audiogram based on automatically detected thresholds plotted in b. Different threshold criteria based on the mean (solid bold line) and s.d. of the RMS amplitude of the biological signal recorded in the absence of a stimulus are indicated by dashed lines. b Comparison of audiograms and click-evoked response thresholds based on visually detected (VD) thresholds (filled diamonds) and automatically determined thresholds based on different criteria (open triangles and squares). To improve clarity of the plot, error bars are not shown for individual data. Error bars in the legend depict the s.e.m. averaged across frequencies and clicks for each threshold determination method. c Comparisons of mean ± s.e.m. visually detected thresholds for males (open circles) and females (filled circles) for responses to tones of different frequencies and to clicks

Fig. 6

a Visually detected ABR thresholds from this study (unweighted means averaged across all individuals; filled circles) compared with the frequency spectrum of a mixed-species chorus recorded in central Minnesota during the peak of the gray treefrog breeding season (shaded area). Peaks of the chorus spectrum are contributed by i) northern leopard frogs (Rana pipiens), ii) Cope's gray treefrogs (Hyla chrysoscelis), iii) American toads (Bufo americanus), and iv) boreal chorus frogs (Pseudacris maculata). b Visually detected ABR thresholds from this study (filled circles) compared with average multiunit thresholds from invasive recordings from the midbrain of Cope's gray treefrogs (open circles) reported by Hillery (1984b)