Unlike terrestrial mammals that have unambiguous aerial sound transmission pathways via the outer ear and tympanum, sound reception pathways in most odontocetes are not well understood. Recent studies have used auditory brainstem response (ABR) measurements to examine sound reception pathways. This study sought to determine how sound source placements, recording electrode arrangements, and ABR peak analyses affect interpretations of sound reception in the harbor porpoise (Phocoena phocoena). Click stimuli were delivered in air from a contact transducer (“jawphone”). Early ABR peaks (representing auditory nerve responses), and later peaks reflecting higher brainstem activity, were analyzed across jawphone and recording electrode positions. Auditory nerve responses were similar for jawphone placements from the ipsilateral posterior mandible to the tip of the rostrum. Later peaks, however, suggested a possible region of highest sensitivity midway between the posterior mandible and the rostrum tip. These findings are generally similar to previous data for porpoises. In contrast to auditory nerve responses that were largest when recorded near the ipsilateral meatus, later ABR peaks were largest when recorded with a contralateral (opposing) electrode. These results provide information on the processes underlying peaks of the ABR, and inform stimulus delivery and ABR recording parameters in odontocete sound reception studies.

The auditory systems of odontocete cetaceans display morphological adaptations for aquatic functions including underwater hearing and echolocation (Au, 1993; Cozzi et al., 2017). Although the exact mechanism of tissue-based sound transduction from the water to the odontocete ear is not yet completely understood (especially in more exotic species), behavioral evidence, primarily with bottlenose dolphins (Tursiops truncatus), has shown that the mandible is receptive to ultrasonic sound (>20 kHz) used for echolocation (Norris, 1968; Brill et al., 1988; Brill and Harder, 1991). These results have been supported by intracranially recorded brainstem potentials or cochlear microphonics evoked using transducers pressed against the skin surface [bottlenose dolphins (McCormick et al., 1970; McCormick et al., 1980), striped dolphins (Stenella coeruleoalba, Bullock et al., 1968), Pacific white-sided dolphins (Lagenorhynchus obliquidens, McCormick et al., 1980), and harbor porpoises (Phocoena phocoena, Voronov and Stosman, 1982)].

More recent hearing pathway studies with the bottlenose dolphin (Møhl et al., 1999; Brill et al., 2001), beluga (Delphinapterus leucas, Mooney et al., 2008; Popov et al., 2016), Yangtze finless porpoise (Neophocaena asiaeorientalis, Mooney et al., 2014), and Risso's dolphin (Grampus griseus, Mooney et al., 2015) have used non-invasive methods to record the auditory brainstem response (ABR) or the auditory steady-state response (ASSR) that likely comprises repetitive occurrences of ABR peaks [also called the envelope following response (EFR; Popov and Supin, 1998; Supin et al., 2001)]. Unlike earlier intracranially recorded potentials, the ABR and ASSR recorded at the skin surface represent far-field summations of potentials from many structures with specific dipole patterns (Supin et al., 2001; Hall, 2007). The earliest peaks of the ABR represent activity generated by a single ear (as recorded from an electrode close to the cochlea/eighth nerve), and later peaks potentially include input from both the ipsilateral and contralateral ears relative to the sound source (Buchwald and Huang, 1975; Møller et al., 1995). At near threshold stimulus levels it is likely that the ABR is generated solely by the ear ipsilateral to a sound source (Mulsow et al., 2014), providing a clear picture of how a single ear is stimulated by a source. Higher stimulus levels, however, increase the likelihood for acoustic crosstalk to stimulate both ears. This may lead to recordings that reflect binaural activity at a neural level, or peaks that are formed from the far-field voltage summation of responses from both ears. For either case, this can complicate the interpretation of how sound travels to a single ear.

This study examines how sound source position on the mandible and recording electrode location affect the ABR recorded from the skin surface of the harbor porpoise (for a review of information on the harbor porpoise ABR, see Bibikov, 2004). Methods similar to those of previous studies with bottlenose dolphins were used to measure early ABR peaks that are potentially most representative of direct source-to-ear transmission (the auditory nerve response or P1, see Supin et al., 1991; Popov and Supin, 1992; Supin and Popov, 1993; Popov et al., 2006; Mulsow et al., 2014) as well as longer-latency peaks generated at higher levels of the brainstem (P4 and N5, Popov and Supin, 1990; Popov and Supin, 1998). The resulting data provide insight into the processes that underlie these peaks of the ABR, and highlight considerations for hearing pathway studies using non-invasive electrophysiological methods. This study also provides additional data regarding acoustic sensitivity along the mandible to complement previous findings with this species and other odontocetes.

For this study, ABRs were measured using three “active” non-inverting electrodes placed on harbor porpoises' heads following stimulation using a contact transducer (“jawphone”) at one of five positions (Fig. 1). Details of the jawphone and experimental procedure are given in the following sections; however, terms related to the positions of the jawphone and electrodes will be highlighted here for clarity. The locations of the jawphone are identified (and given abbreviations) according to its placement on the mandible: on the posterior margin near the oral commissure (POS), immediately underneath the tip of the rostrum (TIP), and midway between these two points (MID). These points are further specified by the left or right side of the head (i.e., the left posterior placement is POS-L). The recording electrodes are identified as left or right based on the external auditory meatus where they were placed (i.e., MEA-L for the left meatus), and vertex (VER) for the electrode placed near the porpoises' midline immediately behind the blowhole. Relative electrode locations are also used according to the position relative to the jawphone: an ipsilateral electrode is the recording electrode on the same side of the head as the jawphone, and a contralateral electrode is on the opposite side of the head. The VER electrode is not designated as either ipsilateral or contralateral.

FIG. 1.

(Color online.) Schematic of the experimental setup. The jawphone was placed at one of five positions on the porpoise's mandible for each series of data collection (lower right detail).

FIG. 1.

(Color online.) Schematic of the experimental setup. The jawphone was placed at one of five positions on the porpoise's mandible for each series of data collection (lower right detail).

Close modal

Testing was conducted in June 2015 with two harbor porpoises that were rescued, rehabilitated, and housed at the Vancouver Aquarium in Vancouver, British Columbia, Canada: Jack (male, 35 kg, 4 yr/old estimated) and Daisy (female, 50 kg, 7 yr/old estimated). Experiments were conducted in air, with the porpoises resting sternally on a foam mat. Results of prior electrophysiological hearing tests conducted in March 2014 demonstrated that both porpoises were sensitive to frequencies >160 kHz, consistent with the normal hearing range for this species (Andersen, 1970; Kastelein et al., 2017).

The electrical waveforms used as stimuli were approximately 5 μs square wave pulses (clicks) presented at a rate of ∼50 s−1. Clicks were digitally synthesized using a laptop-based computer and EVREST software (Finneran, 2008, 2009). A USB-6259 data acquisition (DAQ) card (National Instruments Corporation, Austin, TX) converted the digital waveforms to analog at a rate of 2 MHz with 16-bit resolution. The analog clicks were attenuated up to 20 dB using custom hardware, then low-pass filtered at 200 kHz using a 3 C filter module (Krohn-Hite Corporation, Brockton, MA). The conditioned signals were delivered through a jawphone placed on the mandibles of the porpoises (Fig. 1). The jawphone was composed of a 4013 hydrophone (Teledyne Reson, Slangerup, Denmark) embedded in a suction cup made of room temperature vulcanizing silicone rubber that was degassed to remove air bubbles. The jawphone was 4.5 cm in length with a suction cup diameter of 4.5 cm. Figure 2 shows an acoustic waveform and spectrum for the click, where 1 V peak excitation produced 126 dB peak-equivalent sound pressure level (peSPL) recorded underwater at a distance of 5 cm [the approximate distance between the jawphone and the porpoise inner ear at the closest positions (Pos. 1 and 5)]. Calibrations using this distance are not comparable to direct field stimulus calibrations, and the true received levels by the porpoise were not precisely known. Thus the 126-dB peSPL value is given as only a reference, and stimuli in the study are reported in terms of outgoing voltage level during data collection, ranging from 10 to −20 dBV in 10 dB steps (with 0 dBV equal to 1 V peak).

FIG. 2.

Acoustic waveform (main panel) and spectrum (inset) of the click stimulus. The figure represents the stimulus properties based on an underwater recording at a distance of 5 cm.

FIG. 2.

Acoustic waveform (main panel) and spectrum (inset) of the click stimulus. The figure represents the stimulus properties based on an underwater recording at a distance of 5 cm.

Close modal

ABRs were recorded using 10-mm diameter gold cup electrodes (Viasys Healthcare, Madison, WI) embedded in 40-mm diameter silicone suction cups. The study was designed such that three independent electrophysiological recording channels, each sharing a common reference point and ground were used (Fig. 1). The electrode array thus consisted of the following electrode types: three non-inverting, a single inverting, and a single common ground. The non-inverting electrodes were placed on the porpoises' heads: the VER electrode was placed immediately behind the blowhole, and the MEA-L and MEA-R electrodes were placed immediately under the external auditory meatus on each side. The inverting (“reference”) electrode was placed approximately 10 cm behind the active electrode on the porpoises' midline. The porpoise ABR is still likely projected to this location and it therefore does not represent a true inactive reference point. Nonetheless, differential amplification using this location with the non-inverting electrodes provided a high-amplitude ABR, and the use of differential amplification no doubt enhanced common mode rejection of noise and enhanced the quality of the recording. The common ground electrode was placed near the midline next to the dorsal fin, such that the inverting was approximately midway between the VER non-inverting and the ground.

The incoming electroencephalographic (EEG) activity was amplified using three IP511 biopotential amplifiers (Grass Technologies, Warwick, RI). Each IP511 corresponded to one of the non-inverting channels, with the inverting and ground shared across all of the channels. The EEG signal was amplified 94 dB and band-pass filtered 0.3 to 10 kHz prior to analog/digital conversion at 40 kHz with the USB-6251 DAQ card. Signal processing was conducted with EVREST software, prior to hard disk storage for later analysis.

A single test series consisted of all four click levels (−20 to 10 dBV) being presented while the jawphone was maintained in a single position. Following completion of a test series, the jawphone was moved to another position, and another test series was conducted. This continued until a test series had been completed for each of the five jawphone positions. This entire procedure was conducted four times on two separate days. Therefore, for each porpoise and active electrode position, there were eight ABR measurements for each combination of stimulus level and jawphone position. Each porpoise spent 35−40 min on the foam mats to complete the procedures on each recording day. Animal care and veterinary staff monitored all procedures and regularly recorded heart and respiratory rates. All procedures followed the guidelines of the Canadian Council on Animal Care and were approved by the Vancouver Aquarium's institutional animal care committee (permit #2014-01).

Averaged ABR waveforms were obtained for each click level in a test series using a weighted-averaging procedure (Elberling and Wahlgreen, 1985) based on 512 time-windows representing an approximately 20-ms EEG record following the presentation of each click. The eight ABR waveforms were then coherently averaged to yield a single grand average ABR (based on 4096 time-windows) for each combination of subject, active electrode position, stimulus level, and jawphone position.

The amplitudes and latencies of three peaks within the ABR were chosen for analysis based on their sites of generation in the auditory system and their relation to the ASSR. The earliest peak of the ABR, P1 (wave I in Bibikov, 1992), is a negative peak at the meatus, but a positive peak when recorded at the vertex of the skull using a bipolar configuration, and based on results with dolphins arises from the action potential in the auditory nerve (Ridgway et al., 1981; Popov and Supin, 1991; Supin et al., 1991; Supin et al., 2001). It is therefore indicative of the response of a single ear to the click. The second and third peaks analyzed, P4 and N5 [wave IV and the following negative deflection in Bibikov (1992), respectively] form a vertex positive-negative ABR complex. Based on the similarity to the ABR of bottlenose dolphins and other odontocetes, this complex likely underlies the ASSR that has been measured in hearing pathway studies (i.e., Mooney et al., 2008; Mooney et al., 2014; Mooney et al., 2015; Popov et al., 2016, see Popov and Supin, 1998; Supin et al., 2001).

ABR peak amplitudes and latencies for P1, P4, and N5 were measured in EVREST software by placing a cursor on the “shoulder” of the peak: immediately following the peak and preceding the voltage deflection toward zero (Hall, 2007). Peak amplitudes were defined in relation to the mean amplitude of the first 500 μs of each grand average ABR record in order to account for any DC offset in the record (i.e., ABR amplitudes are baseline-to-peak measures). In some cases, P1 displayed peak splitting where two smaller deflections were superimposed on the main peak. The earliest peak was used for latency and amplitude measurements in these cases, as the second peak was not sufficiently defined for all conditions. Peak latencies were defined relative to the electrical generation of a click. Reported values for ABR peak amplitudes and latencies were based on the average of the values obtained for both Jack and Daisy.

The ABRs evoked by the clicks followed patterns typically seen for this species and other odontocetes, with a complex of positive and negative peaks mostly occurring within the first 5 ms following acoustic stimulation (Fig. 3). The peaks in the waveform typically decreased in amplitude and increased in latency with decreasing stimulus level. At the VER electrode, P1 and P4 both appear as vertex-positive deflections, and N5 as a vertex-negative deflection. In contrast, the records from the MEA electrodes feature P1 and P4 peaks that are negative deflections and N5 is positive. This difference roughly indicates the orientation of the dipoles for these three peaks: positive in the dorsal and negative in the ventral directions for P1 and P4 (although P1 is not strongly projected to the vertex) and reversed for N5. Also of note are the similar waveform morphologies on the MEA-L and MEA-R when the jawphone is placed at TIP. This suggests that, as expected, stimulation is similar at the left and right ears with the jawphone projection at that position. The more dissimilar morphologies of the ABRs obtained with the jawphone at POS-R, however, suggest more asymmetry in the relative stimulation at the left and right ears when sound projection occurs from this posterior position.

FIG. 3.

Representative grand-average ABR waveforms for Jack (N = 4096), recorded at VER and MEA electrodes. The top trace in each panel corresponds to a click level of 10 dBV, and records descend in 10 dB steps. The polarities of the ABRs for the MEA electrodes have been inverted (negativity at vertex plotted upwards) for direct comparison to those recorded at the VER electrode (vertex positive upwards). The records correspond to jawphone locations immediately under the rostrum tip (TIP, top) and right mouth commissure (POS-R, bottom). Circles indicate the peaks identified as P1, P4, and N5. Results were similar for the second porpoise, Daisy.

FIG. 3.

Representative grand-average ABR waveforms for Jack (N = 4096), recorded at VER and MEA electrodes. The top trace in each panel corresponds to a click level of 10 dBV, and records descend in 10 dB steps. The polarities of the ABRs for the MEA electrodes have been inverted (negativity at vertex plotted upwards) for direct comparison to those recorded at the VER electrode (vertex positive upwards). The records correspond to jawphone locations immediately under the rostrum tip (TIP, top) and right mouth commissure (POS-R, bottom). Circles indicate the peaks identified as P1, P4, and N5. Results were similar for the second porpoise, Daisy.

Close modal

In Figs. 4 and 5, the amplitudes of P1 and P4 are shown, respectively, as a function of click level for all electrode positions, while the jawphone projected sound from each of the five locations. Because records were coherently averaged to obtain a final grand average, variability in terms of standard deviation or standard error are not reported. Instead, noise estimates were based on residual background noise within each of the eight 512-record measurements. This average level was approximately 160 nV root-mean-square, and resulted in high signal-to-noise ratios for the final coherent averages. Taking into account the coherent averaging of the eight records and assuming the ABR is a deterministic signal and noise random, the error for the peak amplitude values can be approximated as 57 nV (i.e., noise decreases proportional to 1/√8). This noise estimate is markedly smaller than the majority of peak amplitudes and highlights differences between patterns.

FIG. 4.

(Color online) Average amplitudes (n = 2) of P1 as a function of click level. Each panel shows ABR peak amplitudes for the three “active” electrodes at one of five jawphone positions. The squares, downward triangles, and upward triangles correspond to the MEA-L, VER, and MEA-R electrodes, respectively.

FIG. 4.

(Color online) Average amplitudes (n = 2) of P1 as a function of click level. Each panel shows ABR peak amplitudes for the three “active” electrodes at one of five jawphone positions. The squares, downward triangles, and upward triangles correspond to the MEA-L, VER, and MEA-R electrodes, respectively.

Close modal
FIG. 5.

(Color online) Average amplitudes (n = 2) of P4 as a function of click level. Each panel shows ABR peak amplitudes for the three “active” electrodes at one of five jawphone positions. The squares, downward triangles, and upward triangles correspond to the MEA-L, VER, and MEA-R electrodes, respectively. Amplitude trends for N5 behaved in a similar fashion.

FIG. 5.

(Color online) Average amplitudes (n = 2) of P4 as a function of click level. Each panel shows ABR peak amplitudes for the three “active” electrodes at one of five jawphone positions. The squares, downward triangles, and upward triangles correspond to the MEA-L, VER, and MEA-R electrodes, respectively. Amplitude trends for N5 behaved in a similar fashion.

Close modal

Multiple interactions between ABR peak, electrode montage and jawphone placement are seen in Fig. 4. First, although peak amplitudes increased with increasing stimulus level, there did appear to be some degree of saturation in P1 amplitudes above 0 dBV (and the slopes in some conditions appear somewhat shallower at the higher stimulus levels). Amplitudes for P1 were always lower than P4 and N5 amplitudes for a given stimulus level and position (e.g., Fig. 4 vs Fig. 5), and P1 as recorded from VER was the lowest amplitude of all peaks. These results reflect both the distance from the ABR peak generators, and the magnitude of the equivalent dipole for each peak generator. The amplitudes of P1 recorded on MEA electrodes were largest on the ipsilateral MEA electrode for the POS positions. For the MID and TIP positions, P1 amplitudes were more similar for the two MEA electrodes. In contrast to P1, P4 (Fig. 5) and N5 (data not shown) amplitudes recorded on the MEA electrodes were larger with contralateral (not ipsilateral) stimulation. This pattern held for the POS and MID jawphone placements. At the TIP position, P4 and N5 amplitudes were approximately the same from both the MEA electrodes at a given stimulus level.

Figure 6 shows the superimposition of homologous non-midline responses for both subjects. For further analyses, within-subject averaged ABRs were created from opposing jawphone projection sites (i.e., each pair of overlaid time-domain traces in Fig. 6 were averaged together). This assumes that data for comparable positions on each side of the midline should be mirror images. This was not strictly the case as evidenced by some small asymmetries in ABR waveforms in Fig. 6. These asymmetries probably did not result from hearing deficiencies, however, as both of the porpoises appeared to have normal hearing. Each set of overlaid ABR traces in Fig. 6 are sufficiently similar to justify combining them into a single waveform.

FIG. 6.

ABRs recorded at MEA electrodes for relative jawphone positions at a click level of −10 dBV. Jawphone position and relative MEA electrode position are given in the middle of the figure. For example, the traces at the top represent data recorded on the MEA-L electrode with the jawphone at POS-R, and those recorded on the MEA-R with the jawphone on the POS-L. Data from MEA-R are indicated with dashed lines and data from MEA-L with solid lines in order to demonstrate the similarity of data for comparable conditions.

FIG. 6.

ABRs recorded at MEA electrodes for relative jawphone positions at a click level of −10 dBV. Jawphone position and relative MEA electrode position are given in the middle of the figure. For example, the traces at the top represent data recorded on the MEA-L electrode with the jawphone at POS-R, and those recorded on the MEA-R with the jawphone on the POS-L. Data from MEA-R are indicated with dashed lines and data from MEA-L with solid lines in order to demonstrate the similarity of data for comparable conditions.

Close modal

The data in Fig. 7 represent mean P1 amplitudes and latencies as a function of click level for the five conditions illustrated in Fig. 6: (1) The ipsilateral and (2) contralateral MEA electrodes while the jawphone was at a POS position, (3) the ipsilateral and (4) contralateral MEA electrodes while the jawphone was at a MID position, and (5) the MEA electrodes with the jawphone at the TIP position. The top panel of Fig. 7 shows the amplitudes of P1 as a function of voltage for each of these five conditions. The data for the contralateral ear with the jawphone in a POS position were clearly smaller in amplitude than the other four conditions.

FIG. 7.

(Color online) Average amplitudes (top) and latencies (bottom) for P1 at the MEA electrodes for relative jawphone conditions (e.g., the POS data represent the average of values with the jawphone at POS-L and POS-R). Data are pooled for Jack and Daisy. Solid lines indicate values from the MEA electrode ipsilateral to the jawphone, and dashed lines represent values from the MEA electrode contralateral to the jawphone. Note that TIP placement lacks contralateral conditions due to its central location. For the latency data, lines represent the best-fit functions for the data with a shared slope of −3.84 μs/dB. The values next to each dataset represent the y-intercept of the best-fit line (i.e., the P1 latency of each function at a click level of 0 dBV). The asterisk indicates the same intercept value for the TIP and MID contralateral conditions.

FIG. 7.

(Color online) Average amplitudes (top) and latencies (bottom) for P1 at the MEA electrodes for relative jawphone conditions (e.g., the POS data represent the average of values with the jawphone at POS-L and POS-R). Data are pooled for Jack and Daisy. Solid lines indicate values from the MEA electrode ipsilateral to the jawphone, and dashed lines represent values from the MEA electrode contralateral to the jawphone. Note that TIP placement lacks contralateral conditions due to its central location. For the latency data, lines represent the best-fit functions for the data with a shared slope of −3.84 μs/dB. The values next to each dataset represent the y-intercept of the best-fit line (i.e., the P1 latency of each function at a click level of 0 dBV). The asterisk indicates the same intercept value for the TIP and MID contralateral conditions.

Close modal

Latencies of P1 for all five conditions decreased with increasing click level (Fig. 7, bottom panel). There was more separation of the data among conditions for the latencies than for the amplitudes. The ipsilateral conditions for the POS and MID locations displayed the shortest latencies (with the POS ipsilateral latency showing the shortest latency). To estimate the time differences between each of the P1 latency-intensity functions, linear polynomials with a shared slope and independent y-intercepts were fit to the data using nonlinear regression in OriginPro 2017 software (OriginLab Corporation, Northampton, MA). The resulting fits are shown along with the data in the bottom panel of Fig. 7. The shared slope converged on a value of −3.84 μs/dB, and y-intercepts ranged between 0.97 to 1.13 ms. The R2 values ranged from 0.85 to 0.98 for each of the datasets, indicating that the parameters from the model fits should be sufficient for a first-order approximation of the time differences (i.e., y-intercepts) between each data set. The POS and MID data were separated by approximately 0.04 to 0.05 ms for both the ipsilateral and contralateral conditions. Interestingly, the TIP data and the corresponding regression fit were nearly identical to those for the MID/contralateral condition.

Amplitude trends for P4 and N5 peaks (Figs. 8 and 9, respectively) were similar to each other in most regards, although quite different than those observed for P1. Where P1 amplitudes for a given ear were fairly similar with the exception of the contralateral electrode with the POS jawphone position, there was a fair degree of separation in P4 and N5 amplitudes across jawphone positions. The amplitudes were generally highest at the contralateral electrode when the jawphone was at the MID positions, and smallest at the ipsilateral electrode with the POS positions. Amplitude patterns for P4 and N5 measured at the VER electrode were similar for the MID and TIP positions, with lower amplitudes at the POS positions. The N5 amplitudes as recorded at the VER electrode were substantially larger than all other peak values.

FIG. 8.

(Color online) Average amplitudes (top) and latencies (bottom) for P4 for relative jawphone conditions (e.g., the POS data represent the average of values with the jawphone at POS-L and POS-R). Data are pooled for Jack and Daisy. For the MEA electrodes, solid lines indicate values from the electrode ipsilateral to the jawphone, and dashed lines represent values from the electrode contralateral to the jawphone. Note that the TIP placement lacks contralateral conditions due to its central location.

FIG. 8.

(Color online) Average amplitudes (top) and latencies (bottom) for P4 for relative jawphone conditions (e.g., the POS data represent the average of values with the jawphone at POS-L and POS-R). Data are pooled for Jack and Daisy. For the MEA electrodes, solid lines indicate values from the electrode ipsilateral to the jawphone, and dashed lines represent values from the electrode contralateral to the jawphone. Note that the TIP placement lacks contralateral conditions due to its central location.

Close modal
FIG. 9.

(Color online) Average amplitudes (top) and latencies (bottom) for N5 for relative jawphone conditions (e.g., the POS data represent the average of values with the jawphone at POS-L and POS-R). Data are pooled for Jack and Daisy (from Figs. 4 and 5). For the MEA electrodes, solid lines indicate values from the electrode ipsilateral to the jawphone, and dashed lines represent values from the electrode contralateral to the jawphone. Note that TIP lacks contralateral conditions due to its central location.

FIG. 9.

(Color online) Average amplitudes (top) and latencies (bottom) for N5 for relative jawphone conditions (e.g., the POS data represent the average of values with the jawphone at POS-L and POS-R). Data are pooled for Jack and Daisy (from Figs. 4 and 5). For the MEA electrodes, solid lines indicate values from the electrode ipsilateral to the jawphone, and dashed lines represent values from the electrode contralateral to the jawphone. Note that TIP lacks contralateral conditions due to its central location.

Close modal

The latency-intensity functions for VER-recorded P4 and N5 had longest latencies when the jawphone was at TIP, and the shortest when at POS positions. The latency patterns of P4 and N5 at the MEA electrodes differed, however. For P4, latencies were shortest at the ipsilateral electrodes, and contralateral patterns at the POS and MID positions were similar to each other and to those for the TIP placement. The N5 latencies were longest for the ipsilateral electrodes (and for TIP placements) at the highest click levels. At the lowest click level for POS placements; however, the latencies at the ipsilateral MEA electrode became shorter than all other conditions.

The ABR peak amplitudes in the present study were approximately an order of magnitude larger than those previously recorded at the skin surface by Bibikov (1992); however, as noted by Bibikov (2004), peak amplitudes of ABRs recorded at the skin surface by Klishin and Popov (2000) were also approximately an order of magnitude larger than those measured by Bibikov (1992) and more comparable to measures made in this study.

As the jawphone placement progressed to more anterior positions, P1 amplitudes generated by the auditory nerves of the ipsilateral and contralateral ears became more similar (for lower click levels where saturation effects were not observed). This suggests increased interaural attenuation when the jawphone is located laterally (and closer to the POS region). This is likely due to stimulus spreading and/or the influence of intervening structures with differing acoustic impedances [i.e., the skull and air spaces, a comparable result to a study with bottlenose dolphins Mulsow et al. (2014)]. Assuming similar received levels at the ipsilateral ear under POS and MID conditions (i.e., Fig. 7, top panel) the differences in y-intercept values for the P1 data regression lines should give an idea of the acoustic travel time between the two points. The calculated difference of 36 μs between the y-intercepts and an approximate difference in location of 5.5 cm yields a sound speed of approximately 1500 m/s. This is a reasonable value based on the previous estimates of sound speed in acoustic fats (Norris and Harvey, 1974; Aroyan et al., 2000). A similar calculation for the latency difference between the ipsilateral MID and TIP conditions; however, yields a lower sound speed of approximately 750 m/s. This is much lower than the expected sound speed value, and may arise from intervening low sound-speed airspaces (i.e., mouth and digestive tract) between the rostrum tip and inner ear.

The P4 amplitudes and latencies recorded at the MEA electrodes could be hypothesized to arise from (1) primarily contralateral input when recorded with the jawphone at contralateral and TIP placements and (2) increasingly ipsilateral input as the jawphone is moved to the same side of the head as the electrode. Interestingly, the latencies of the lower-amplitude N5 responses during ipsilateral stimulation (i.e., at −20 dBV click levels) are markedly shorter than those during contralateral stimulation. This could result from a large-amplitude N5 response for contralateral stimulation primarily arising from activity following crossing of signals across the midline of the brainstem (neural decussation), producing longer latencies. Lower-amplitude ipsilateral N5 responses could become more dominant at lower stimulus levels (at shorter latencies due to shorter, uncrossed neural paths) during ipsilateral stimulation, as decreased N5 amplitudes from the contralateral ear—stimulated through acoustic crosstalk—could allow the emergence of ipsilateral responses.

Inhibition of activity from the contralateral ear by earlier stimulation at the ipsilateral ear may also play a part in emergence of a low-amplitude ipsilateral N5 component. As excitatory−inhibitory interactions from binaural stimulation are known to occur in auditory brainstem nuclei including the superior olivary complex, lateral lemniscus, and inferior colliculus (e.g., see Irvine, 1992), such a thesis is at least possible. However, it is also likely that if multiple stimulation pathways are in play in generating the porpoise ABR, that complex interactions for specific sound entry points, stimulus level and ABR recording channels led to this peculiarity in ABR N5 amplitude.

Taken together, the observed patterns for P4 and N5 are consistent with studies of the lateral lemniscus and inferior colliculus in humans and cats. These structures probably include neural pathways both from the ipsilateral and contralateral ears following neural decussation, which occurs in the pons (at least in terrestrial mammals) at the level of the superior olivary complex (Buchwald and Huang, 1975; Møller et al., 1995). Recordings from the harbor porpoise lateral lemniscus by Voronov and Stosman (1982) also demonstrated that the lowest thresholds for this brainstem structure occurred with acoustic stimulation on the contralateral side of the head. It is worth noting that the differences in latency patterns at the MEA electrodes, and overall amplitudes at the VER electrode suggest that P4 and N5 may have separate generators. This is potentially explained by evidence that the lateral lemniscus and inferior colliculus may both be involved in the generation of these later ABR waves (Bullock et al., 1968; Voronov and Stosman, 1982; Supin et al., 2001; Bibikov, 2004).

The differences in reception based on P1 amplitudes at the five jawphone locations in this study appear modest in comparison to similar results for delphinids, both in early studies that measured either intracranial or far-field monaural responses (Bullock et al., 1968; McCormick et al., 1980), and to more recent studies that measured later peaks of the ABR, including the rhythmic ASSR (Møhl et al., 1999; Mooney et al., 2008; Mooney et al., 2015; Popov et al., 2016). The P4 and N5 functions for the MEA electrodes display more separation than those for P1, suggesting that the most efficient point of stimulation may be at the MID location. This is particularly interesting, as P1 was initially hypothesized to provide the most unambiguous information on signal-to-ear transmission. Best sensitivity at the MID position aligns well with results from the only comparable study with the harbor porpoise (Voronov and Stosman, 1982), where intracranially recorded potentials had lowest (best) thresholds for stimuli delivered at a 5−6 cm region near the current study's MID jawphone placement. This location, which is roughly the size of the current jawphone cup, is likely the best position for eliciting the ABR and ASSR in audiometric studies with this species. Like this study, the contact transducer measurements of Voronov and Stosman (1982) were conducted in air. Direct comparisons of underwater and aerial measurements would be quite useful in determining the extent to which aerial measurements can be used to predict underwater sensitivity patterns.

That P4 and N5 at the MEA electrodes provided the most separation of functions and confirmed previous findings suggests that these peaks are of use in evaluating hearing pathways. This is despite the hypothesized shortfall (i.e., binaural contributions) that these peaks might have had relative to P1. There are a few caveats with these peaks, however, that should be noted. Larger responses do not necessarily indicate more efficient reception pathways to the ear, as the summation of voltages corresponding to each ear may contribute to the overall far-field ABR or ASSR amplitudes. This voltage addition may have been why the VER electrode amplitude patterns did not reflect better sensitivity at the MID jawphone placement relative to TIP. Although the VER electrode may provide high P4-N5 amplitudes (especially if the MEA location was used as an inverting electrode due to the inverted peak polarities), it is hypothesized that the MEA electrode referenced to a relatively inactive location provides more accurate ear-specific information on hearing pathways.

It is likely that many of these concerns are reduced at near-threshold stimulus levels where only responses from the most receptive ear (likely ipsilateral) are present (although if extraneous electrical noise limited the determination of threshold, the addition of responses from both ears could complicate the interpretation of sensitivity patterns). A detailed description of near-threshold effects is unfortunately not possible, as there was limited experimental time for data collection with each porpoise, and obtaining supra-threshold responses was prioritized. Based on the current findings, future examination of ABR peaks at lower stimulus levels with multiple electrode and jawphone positions seems warranted. In the absence of such data, a consideration of these issues in future cetacean ABR and ASSR receptivity pattern studies would be of interest.

The authors wish to thank Dr. Martin Haulena, Brian Sheehan, Troy Neale, and the animal care and training staff at Vancouver Aquarium for support and assistance during data collection. Funding was provided by U.S. Navy Fleet Forces Command. This is contribution 174 of the National Marine Mammal Foundation.

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