Two-channel brain-stem frequency-following responses to pure tone and missing fundamental stimuli

Electroencephalography and clinical Neurophysiology , 92 (1994) 321-330 © 1994 Elsevier Science Ireland Ltd. 0168-5597/94/$07.00

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Two-channel brain-stem frequency-following responses to pure tone and missing fundamental stimuli Gary C. Galbraith * UniL~ersity of California, Los Angeles, School of Medicine, Mental Retardation Research Group, Lanterman Decelopmental Center, P.O. Box IO0-R, Pomona, CA 91769 (USA)

(Accepted for publication: 24 February 1994)

Summary In 2 separate experiments the brain-stem frequency-following response (FFR) was recorded to a pure tone (200 Hz) and complex "missing f u n d a m e n t a l " (MF) stimuli differing in temporal fine structure and envelope modulation depth. F F R s were simultaneously recorded in 2 channels with horizontal and vertical dipole orientations. Horizontal electrodes were identical in both experiments (right-left ear), but the vertical configuration was varied (vertex-left ear; vertex-linked mastoids). The horizontal channel yielded a well defined F F R to tone stimulation at a latency consistent with an origin along the auditory nerve. However, there was no horizontal response to MF stimulation. This latter finding provides electrophysiological support for the conclusion that MFs are not directly coded in the peripheral neural response. Vertical recordings, however, showed equally well defined F F R s to tone and MF stimuli. Thus, a representation of the missing fundamental frequency is registered in the brain-stem. Vertical latencies were consistent with a source at the level of the lateral lemniscus. The F F R is well suited to elucidate certain brain-stem mechanisms of auditory information processing. Important additional information results when responses are compared in horizontal and vertical dipole orientations. Thus, the present results provide the first evoked response demonstration of a peripheral-brain-stem dichotomy of M F coding. Key words: Brain-stem; Frequency-following response; Missing fundamental; Horizontal-vertical dipole orientations

"Missing fundamental" (MF) stimuli, which contain only higher harmonic frequencies of the MF, provide unique opportunities to study sensory coding and higher perceptual processing. For example, in the auditory system it is possible to extract "low" pitch information (also referred to as "periodicity" or "residue" pitch) from complex tones devoid of a fundamental frequency component (Schouten 1940; Bilsen 1977). A compelling example of this phenomenon is the accurate perception of a musical note at the fundamental frequency even though the signal consists of just 2 randomly chosen successive upper harmonics (Houtsma and Goldstein 1972). There is evidence that MF perception is a pervasive phenomenon. Thus, studies of fish, birds and mammals suggest that periodicity pitch perception may be a general process in vertebrate hearing (Cynx and Shapiro 1986). Moreover, in human vision, MF stimuli generated by summing higher harmonic optical gratings have been used to study such phenomena as dichoptic motion sensing (Georgeson and Shackleton 1989; Georgeson and Harris 1990) and visual persistence (May and Ritter 1990).

* Corresponding author. Tel.: 909-595-1221, ext. 2442; Fax: 909-5944709. SSDI 0 0 1 3 - 4 6 9 4 ( 9 4 ) 0 0 0 7 2 - S

An important feature of auditory MF perception is that low pitch cannot be directly attributed to peripheral neural response patterns. This is due to the fact that the fundamental frequency does not exist in the frequency spectrum of the stimulus, nor is it represented in non-linear distortion products along the basifar membrane (Greenberg 1980). Thus, auditory masking at the missing frequency has no effect on low pitch perception (Smith et al. 1978). Furthermore, a central origin of low pitch is confirmed by the fact that correct frequency matches are possible even when stimuli are presented dichotically, i.e., one harmonic to each ear (Houtsma and Goldstein 1972; Bilsen 1973). Given that MF stimuli are not directly coded in the periphery, it is of interest to determine the possible central site(s) of low pitch processing, which conceivably could range from brain-stem to cortex. However, there is evidence that MF coding is mediated at subcortical levels. This was shown by Pantev et al. (1989), who studied the tonotopic organization of the auditory cortex by means of evoked neuromagnetic measurements. Stimuli consisted of pure tones (250 or 1000 Hz) or a complex MF based on harmonics of a 250 Hz fundamental. Their results (100 msec component) showed that the equivalent current dipole for the 250 Hz pure tone and MF occurred at the same cortical depth. However, the response to the 1000 Hz pure

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tone occurred at significantly greater cortical depth. This led Pantev et al. (1989) to conclude that " . . . the tonotopic organization of the primary auditory cortex reflects the pitch rather than the frequency of the stimulus (and that) the pitch formation process must take place in subcortical regions" (p. 486). The question addressed in the present paper is whether the implicated subcortical regions might involve brain-stem loci, and whether the brain-stem frequency-following response (FFR) might be useful in elucidating properties of MF coding. The adequate stimulus for the FFR consists of tones with sufficient duration to evoke a sensation of pitch (Moushegian et al. 1973). FFRs to fundamental tones are best defined at approximately 250 Hz (Greenberg 1980), which is also the frequency that produces the strongest sensation of MF pitch (Bilsen and Ritsma 1970; Plomp 1975). The FFR reproduces the frequency of pure tone stimuli (although on- and off-responses can augment this pattern), and is delayed according to the site of origin within the auditory pathway. Importantly, when the stimulus is a complex MF, the resulting FFR reproduces the "non-existent" fundamental frequency (Marsh et al. 1975; Smith et al. 1975, 1978; Greenberg et al. 1987; Galbraith and Brown 1990). Indeed, FFRs to MF stimuli are often more robust than when the missing fundamental frequency is presented as a pure tone. In the present study, 2-channel FFR recording techniques were used to contrast horizontal and vertical dipole configurations (cf., Stillman et al. 1978). When applied to the brain-stem auditory (click) evoked response (BAER), horizontal and vertical dipoles have been shown to reflect earlier and later response components, respectively (Picton et al. 1974; Scherg and Von Cramon 1985). Thus, it seemed reasonable that a similar dipole comparison of the FFR might prove useful in addressing the question of MF coding along the auditory pathway. Two experiments were conducted to increase the generalizability of the observed results. These separate studies involved different subjects, different MF stimuli, and a variation in the reference electrode configuration used to define the vertical dipole. By this means it was possible to demonstrate consistent and meaningful FFR patterns in horizontal and vertical dipole recordings as a consequence of MF and pure tone stimulation.

Experiment I

Method Subjects Ten young adult subjects participated in the study (mean age = 18.8 years). Subjects were recruited from

G.C. GALBRAITH

the local community and each received a total payment of $20.00 for 2 separate recording sessions that lasted approximately 1 h each. All subjects gave their informed consent.

Auditory stimulation Subjects rested in a supine position within a single walled IAC sound room. Auditory stimuli were digitally synthesized and presented via digital-to-analog converter at a sample rate of 12,000/sec. Monaural stimulation to the right ear was delivered through an Etymotic Research ER-3A ear insert tubephone. The left ear was also fitted with a tubephone, resulting in a total of 32 dB external noise exclusion and 70 dB isolation between ears. In the first recording session 3 types of stimuli were used: (1) square wave rarefaction click (70 dB re: SPL, 83/.~sec duration) to elicit the BAER, (2) 200 Hz pure tone to elicit the FFR, and (3) a complex MF stimulus derived by summing the third through fifth harmonics of a 200 Hz fundamental (i.e., 600, 800, and 1000 Hz, all harmonics in sine phase). Tone and MF stimulus duration was 25 msec with a 3 msec linear ramp attenuating onset and offset; intensity was 70 dB re: SPL. Click stimuli were delivered alone, but tone and MF stimuli were randomly intermixed during the FFR condition. Each stimulus type was presented a total of 2000 times at a rate of approximately 6 stimuli/sec. In the second recording session 3 different MF stimuli were presented, and only vertical channel responses were recorded. The stimuli, all based on a 200 Hz fundamental, varied in modulation depth of the wave form envelope as well as temporal fine structure (Greenberg 1980; Galbraith and Brown 1990). This was accomplished by altering the relative phase of only the middle component: (1) all harmonics coherent in sine phase (0°; replicate of MF in first session), (2) middle harmonic in counterphase to the 2 side-band harmonics (180°; same stimulus modulation depth as 0° MF, but complementary temporal fine structure), and (3) middle harmonic in quadrature-phase (90°; reduced modulation depth, temporal fine structure markedly different from 0° and 180° stimuli). The results of this recording session served as a basis for selecting the MF stimulus used in Experiment II.

EEG recording Two channels of electroencephalographic (EEG) activity were recorded as described by Stillman et al. (1978): (1) a horizontal derivation with the active electrode placed on the right earlobe referenced to left earlobe, and (2) a vertical derivation with the active electrode placed at the vertex (Cz) referenced to left

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Fig. 1. Brain-stem auditory evoked responses (BAERs) to click stimuli recorded simultaneously from horizontal and vertical electrode derivations. Displays presented separately for 10 subjects, as well as group average (AVG). Electrical stimulus occurs at origin of each trace, but auditory stimulus arrives 1.0 msec later due to air conduction delay. Time line (vertical dashed line in each column) passes through an absolute latency of 5.8 msec (peak of AVG wave V, vertical derivation). Voltage calibration equals 0.25/,V.

earlobe. 1 The forehead served as ground. Electrodes were applied with the aid of skin prepping paste which resulted in inter-electrode impedance typically below 2.5 kO; impedance was measured before and after the experiment to verify electrode contact. E E G amplifier gain was 200K with an analog frequency bandpass of 30-3000 Hz (6 dB down). Precise temporal correspondence between stimulus and F F R resulted by alternating between the digitalto-analog output of each successive stimulus wave form value and the time-correlated analog-to-digital input from the two E E G channels (skew between channels was 25 ~sec). Each epoch included prestimulus (9.3 msec) and poststimulus (19.0 msec) data. To avoid any possibility of systematic amplifier differences between channels (e.g., frequency response characteristics that might introduce phase distortions), half of the data were recorded through each amplifier system by

t Strictly speaking, this is as an "angled" dipole of approximately 30°, which might affect certain properties of the FFR. As will be shown, however, the overall qualitative pattern of results is similar in Experiments I and II, even though a different reference is used in the vertical channel. Thus, while the term "vertical" in this case is not geometrically precise, its use should not lead to false interpretations of the data.

switching input leads midway through each stimulus session. An on-line artifact rejection routine automatically repeated a stimulus if the raw E E G sample contained 5 or more data values exceeding + 20 /zV (equivalent to + 4 V in the amplified signal, which is less than the + 5 V level which blocked the amplifier). In addition, recording artifacts were reduced since auditory stimuli were delivered through a 28 cm air tube, thus removing the electrical signal from the E E G electrodes. Calibration using a Briiel and Kj;er condenser microphone with flat frequency response to 20 kHz showed an acoustic delay of 1.0 msec (all reported latencies take this delay into account). Single trial B A E R and F F R data were stored during the experiment and the averages computed off-line. A physiological origin of the various responses was evidenced by (1) appropriate temporal delays (air plus neural conduction), and (2) absence of a response when the tubephone was blocked or not inserted.

Digital filtering of FFR wave forms The E E G amplifier bandpass did not allow for more precise filtering of the data without attenuating the 200 Hz response. Therefore, in order to better evaluate the frequency of interest, average F F R wave forms were

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further processed by a finite impulse response (FIR) digital filter (Rabiner and Gold 1975). The symmetrical 127-weight filter had a relative gain of 21.9 dB at 200 Hz. The filtered wave form consisted of 512 data points (42.67 msec) which began 4.85 msec before and ended 12.82 msec after the stimulus. Although not precisely centered, the filtered wave form always encompassed the FFR, and provided a nearly symmetric bracketing of the response, depending, of course, upon the actual delay relative to the stimulus.

Fast Fourier analysis of FFR amplitude Digitally filtered F F R s were quantified by means of Fast Fourier spectral analysis for the 4 conditions (horizontal and vertical dipole x tone and MF stimulus). Power spectral estimates at 200 Hz were computed for a 21.33 msec epoch (256 data points) beginning 5 msec after stimulus onset and extending 1.33 msec beyond stimulus termination. This data window encompassed most, but not all, of the response. (Assuming a 5 msec F F R onset latency, this analysis would include 21.33/25 = 85.3% of the response.) The exclusion of a portion of the data was dictated by the Fast Fourier transform requirement of modulo 2 N data points.

and Brown 1990). This is done by successively computing correlation coefficients as the stimulus template is shifted relative to FFR. Positive peaks in the resulting cross-correlogram thus identify positions of maximum alignment between stimulus and response wave forms, and the number of shifts necessary to achieve this alignment defines the latency, or phase shift, of the F F R relative to the stimulus. In actual practice, particular components of the F F R may be poorly defined, or initial components may include an onset response similar to the B A E R (Galbraith and Brown 1990). Thus, in order to focus on the most clearly defined segments of the response, the present analysis used a 3-cycle template, instead of the full 5 cycles of the stimulus. This often resulted in alignment with later components of the FFR. In such cases, however, absolute latencies are obtained simply by computing the phase shift relative to correspondingly later stimulus cycles. This takes advantage of the cyclical nature of the F F R wave form, but it should be noted that the resulting latencies thereby represent an overall best fit to 3 successive F F R waves.

Results Click-evoked BAER

Cross-correlation analysis of latency (phase) shift between stimulus and FFR Phase delays were quantified by computing lagged cross-correlograms between the 200 Hz stimulus wave form and individual F F R s (Galbraith 1984; Galbraith

Horizontal and vertical B A E R recordings are presented in Fig. 1. Both channels show well defined components, but the clarity of particular components depends on the derivation. Thus, as would be expected from the vertical channel, which is typical of B A E R

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Fig. 2. Superimposed individual FFR wave forms (left panel) and corresponding group averages (right panel). Top: 200 Hz pure tone stimulus wave form and horizontal and vertical dipole FFRs. The group average plots also illustrate successive stimulus cycles ("1-5") as well as corresponding response components ("extra" components in the horizontal response are identified as "0" and "6"). Bottom: 0° MF stimulus and horizontal and vertical FFRs. Horizontal recording is right-left ear; vertical recording is vertex-left ear. Voltage calibration equals 0.5 /xV for individual FFRs and 0.25/zV for group averages.

T W O - C H A N N E L FFRs

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recordings in general, all 5 B A E R waves are evident. This is seen in the individual data as well as the group average. Horizontal recordings, on the other hand, appear more variable. Yet, there is a clear pattern of well defined early components (I-IV), but diminished wave V. Indeed, several subjects (e.g., nos. 1, 2, 4, and 5) show larger early components in the horizontal than in the vertical derivation. This trend is reflected in the group average plot, which also shows well defined early components and reduced wave V. These results indicate that there are obvious differences in click-evoked brain-stem responses recorded in horizontal and vertical dipole orientations. Moreover, the results support the conclusion that the late component (wave V) is best seen in the vertical channel, while earlier components are well defined in the horizontal channel. These results are treated here only qualitatively, as opposed, for example, to the rigorous spatiotemporal dipole model of Scherg and Von Cramon (1985). Nevertheless, the present B A E R results suggest that other forms of auditory brain-stem responses might also show important dipole differences. FFRs to tone and M F stimuli

F F R data are presented in Fig. 2. Superimposed individual averages (Fig. 2, left panel) demonstrate viable responses and reasonable intersubject wave form synchrony during pure tone stimulation in both the horizontal and vertical channel, and in the vertical channel during 0 ° MF stimulation. By contrast, responses to MF stimulation in the horizontal channel

200 Hz and phase-modified MF stimuli

are poorly defined, or non-existent. What little activity can be seen in the horizontal channel occurs well after MF stimulus termination a n d / o r shows poor intersubject synchrony. Overall trends are best seen in the group average data (Fig. 2, right panel), where synchronous waves in the individual data are reflected as distinct F F R components. Consistent with the individual data, group averaged FFRs to the 200 Hz tone show well defined responses in both dipole configurations. Qualitatively speaking, each of the 5 stimulus cycles is clearly and individually represented in the vertical response. In the horizontal response, however, additional waves are evident. Thus, it may be possible to identify an additional late component ("6"), but the earliest wave ("0") must be considered unreliable since it precedes the stimulus. These extra waves in the horizontal channel may implicate a compound response a n d / o r the presence of noise. It is apparent that the horizontal F F R to tone is phase delayed relative to the stimulus, and that the vertical F F R is further delayed relative to the horizontal response (quantitative latency analyses are presented below). Unlike the B A E R data, where all waves are evident to some degree in both dipole configurations, the non-overlapping F F R latencies suggest successive levels of processing as the signal passes through neural systems reflected first in the horizontal and then the vertical dipole. The vertical F F R to the 0 ° MF also shows a well defined 5-component response. Indeed, the amplitude of this response is as robust as the vertical FFR to tone

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Fig. 3. Left panel: complex missing fundamental (MF) stimulus wave forms (shown in bold) superimposed on a 200 Hz fundamental frequency. Top: all MF harmonics (600, 800, 1000 Hz) phase-coherent (0°). Center: middle harmonic in counterphase (180 °) to the 2 side-band harmonics. Bottom: middle harmonic in quadrature-phase (90 °) to the side-bands. Note the differing phase relationships between MF wave form maxima (arrows) and the peak of the fundamental wave form. Right panel: group average FFRs to the 3 MF stimuli (in bold) superimposed on the same FFR recorded to a 200 Hz pure tone. Note that relative F F R phase shifts reflect the temporal pattern seen for the stimuli (left panel).

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stimulation. However, no organized response is apparent in the horizontal channel as a result of MF stimulation.

Vertical FFRs to MF stimuli differing in middle component phase MF stimuli are constructed by summing only the harmonics of a missing fundamental frequency. However, wave form p a r a m e t e r s such as envelope modulation depth and temporal fine structure can be dramatically altered simply by phase shifting the middle component of a 3-harmonic MF. Thus, the 0 ° MF is constructed by adding all harmonics in sine phase. This configuration results in maximum modulation of the stimulus envelope, with a temporal fine structure in which spectral maxima occur before the peak of the missing fundamental sinusoid (phase lead). A 180 ° MF, on the other hand, is constructed by shifting the middle harmonic 180 ° relative to the two side bands. When this is done, the stimulus envelope has the same modulation depth as the 0 ° MF, but a complementary phase which now lags the fundamental. This is clearly seen by superimposing MF stimulus wave forms on the fundamental frequency (Fig. 3, left panel). Thus, in the case of the 0 ° M F (top) the major stimulus peaks (arrows) are centered on the rising phase of the 200 Hz wave form, while peaks of the 180 ° MF (middle) are centered on the falling phase. However, a 90 ° MF (bottom) appears temporally symmetric to the fundamental wave form, although the envelope modulation depth is greatly reduced, and the temporal fine structure is significantly altered, c o m p a r e d with the other MFs. Since MF temporal fine structure is altered by mid-

Individual FFRs

die component phase, F F R latencies to such stimuli may be problematic. Fig. 3 (right panel) presents F F R s to the 3 phase-shifted M F stimuli, each superimposed on the same F F R wave form evoked by a 200 Hz tone (from the first recording session). It is noteworthy that all MF responses, including the 90 ° FFR, show similar amplitudes and replicate the missing fundamental frequency. However, the results show a pattern of F F R latencies that is consistent with the temporal fine structure of each M F stimulus. Thus, the 0 ° and 180 ° MF responses lead and lag the 200 Hz FFR, respectively, while the 90 ° and 200 Hz responses are nearly synchronous. On this basis, the 90 ° MF was chosen for further study in the following experiment.

Experiment II Method Subjects Ten young adult subjects (mean age = 19.1 years) received a payment of $10.00 each for participating in a single recording session. Stimulation and recording The complex stimulus consisted of a 90 ° MF. Otherwise, tone and MF stimulation was identical to the first recording session in Experiment I (except that no click stimuli were presented). The only E E G p a r a m e t e r varied was the reference electrode used in the vertical channel, which consisted of the midpoint in a fixed 3 k O shunt between left and right mastoids.

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Fig. 4. Superimposed individual FFR wave forms (left panel) and corresponding group averages (right panel). Top: 200 Hz pure tone stimulus and horizontal and vertical dipole FFRs. Bottom: 90° MF stimulus and horizontal and vertical FFRs. Horizontal recording is right-left ear; vertical recording is vertex-linked mastoids. Voltage calibration equals 0.5 pV for individual FFRs and 0.25/~V for group averages.

TWO-CHANNEL FFRs

Results The results of this experiment (Fig. 4) are entirely consistent with those of Experiment I (Fig. 2). Indeed, qualitative trends are even clearer in the present experiment. Thus, well defined 5-component F F R s to the pure tone are observed in both the horizontal and vertical channel. Moreover, there is again the increasing delay in component latencies as signals are successively reflected in horizontal and vertical dipole configurations. In response to MF stimulation, a well defined F F R is registered in the vertical channel. Once again, however, no consistent MF response is apparent in the horizontal channel.

Quantitative analyses of data from Experiments I and II

FFR spectral intensity Individual F F R spectra were transformed (log e) and submitted to a repeated measures analysis of variance (ANOVA; Dixon 1990). It should be noted that this is a conservative approach since individual wave forms that are out of phase, and thus would cancel in a group averaged FFR, will nevertheless contribute to the spectral variance (examples of this can be seen in the horizontal response to M F stimulation in Fig. 2). Possible differences between Experiments I and II were tested as a between-group factor in the A N O V A . Results showed that the overall group difference between Experiments I and II was not significant. This is not surprising considering the high degree of similarity in the pattern of F F R amplitudes evident in Figs. 2 and 4. T h e r e were, however, several significant firstorder within-group comparisons. Thus, pure tones evoked larger overall amplitudes than MFs ( F (1, 18) = 41.01, P < 0.0001), and amplitudes were larger in the vertical dipole configuration ( F (1, 18) = 6.85, P = 0.0174). However, much of the variance of these first-order effects is explained by a significant Stimulus × Dipole interaction ( F (1, 18) = 5.33, P = 0.0330). This interaction is graphed in Fig. 5, which shows a pattern consistent with the trends so readily apparent in the F F R plots. Thus, F F R spectral power is significantly reduced in the horizontal dipole recording during M F stimulation.

Dipole latencies No latency data could be obtained in the horizontal channel during M F stimulation due to poor response

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Between-group comparisons (Experiment I us. II) In response to pure tone stimulation, there was no difference in the horizontal channel ( F (1, 1 8 ) = 0.01, P = 0.9371). Horizontal latencies were 2.15 and 2.13 msec for Experiment I and II, respectively (grand average = 2.14 msec). Latencies in the vertical channel were delayed well beyond horizontal values, and were more varied. Thus, vertical latencies were 5.23 and 4.80 msec for Experiment I and II, respectively (grand average = 5.02 msec). This difference, however, was not significant ( F (1, 18) = 2.74, P = 0.1151). The failure to demonstrate significant between-group differences in either channel is consistent with the fact that identical pure tone stimuli were used in both experiments. Moreover, since the vertical channel latencies were not significantly different, these results suggest that the two reference electrode configurations did not appreciable alter the F F R to tone. In the case of M F stimulation, however, there was a significant difference in vertical channel latencies ( F (1, 1 8 ) = 5.81, P = 0.0268). Latencies were 4.62 and 5.73 msec for Experiment I and II, respectively. The direction of these latency differences, with the 90 ° F F R delayed 1.11 msec beyond the 0 ° response, is consistent with the relative phase and magnitude of separation between these stimuli (from Fig. 3 this appears to be one-fourth of the 5 msec period, or 1.25 msec).

Within-group comparison (200 Hz us. MF) Due to the relative phase difference of M F stimuli in Experiments I and II, comparisons are more problematic than for tone stimuli. Thus, vertical channel data confound both M F stimulus type and E E G reference electrode. It is more appropriate, therefore, to compare each MF response with the corresponding 200 Hz F F R obtained under identical recording conditions.

328 Results showed that the means for Experiment I (5.23 and 4.62 msec for tone and 0 ° MF, respectively) were not significantly different ( F (1, 9)--2.41, P = 0.1551), although the results were in the expected direction (MF leads tone). However, the difference for Experiment II (4.80 and 5.73 msec for tone and 90 ° MF, respectively) was significant ( F (1, 9) = 27.65, P = 0.0005). This result is at variance with Experiment I (Fig. 3), which showed good alignment between the 90 ° MF and tone responses. The delay in Experiment II may reflect a greater sensitivity of the MF response to the vertical reference electrode. This result suggests the need for further study to systematically clarify such issues.

Discussion

The present results demonstrate different response patterns evoked early in the auditory pathway as a function of stimulus type and dipole orientation. Thus, B A E R responses to click stimulation (Fig. 1) showed that wave V was best defined in the vertical channel, while earlier waves were often better developed in the horizontal channel. B A E R results are treated only qualitatively in the present paper, and were obtained mainly to demonstrate the existence of dipole differences for the more commonly studied BAER, thus anticipating the possibility that tones and MF stimuli might yield dipole differences as well. It should be noted, however, that the present B A E R results are consistent with those of Picton et al. (1974). Support for a neural origin of the F F R along the auditory pathway is evidenced by latencies that lag beyond the stimulus by an amount appropriate to the putative anatomical site. In almost all conditions where a response was measurable, it consisted of a delayed 5-component F F R that replicated the number of stimulus cycles. An exception occurred in the horizontal channel of the pure tone response in Experiment I (Fig. 2), where extra components are apparent. However, it is difficult to attach much significance to these extra components since comparable data in Experiment II showed exact stimulus-response agreement. It is possible, of course, for far-field F F R recordings to reflect multiple generator sources in the form of a compound response (Huis in 't Veld et al. 1977; Stillman et al. 1978). Stillman et al. (1978) recorded horizontal and vertical FFRS (apparently the only other study to do so, but only with pure tones), and found evidence that the F F R is composed of 2 distinct wave forms. Thusl there were double peaks separated by 1.4-1.8 msec, with differing amplitudes in the two channels. This pattern was not observed in the present study. However, it must be appreciated that the pres-

G.C. GALBRA1TH ent data were subjected to digital filtering which attenuated higher frequency components and emphasized only 200 Hz activity. Thus, results are limited to a consideration of the fundamental frequency only. The 200 Hz pure tone evoked well defined FFRs in both the horizontal and vertical channel, while MF stimuli evoked a response only in the vertical channel. The most conspicuous feature of these results, therefore, is the absence of an F F R in the horizontal channel during MF stimulation. Although Experiments I and II differed in the vertical channel reference electrode (earlobe vs. linked mastoids), and MF stimulus (0 ° vs. 90°), the overall pattern of results was consistent. The results thus appear to generalize across several stimulus and recording conditions (additional studies are currently in progress to determine if these patterns also generalize to other fundamental frequencies). The absence of a response to MF stimulation in the horizontal channel, in association with a robust response in the vertical channel, provides strong evidence that these two dipole configurations are measuring different events. This was further evidenced by non-overlapping latencies in response to pure tone stimulation in which horizontal and vertical latencies (average of both experiments)were 2.14 and 5.02 msec, respectively. By comparison with BALER reference norms (Chabot and John 1986), these latencies would be analogous to a delayed wave I (1.22-2.16 msec) and wave IV (4.32-5.50 msec). Using a detailed spatio-temporal dipole model, Scherg and Von Cramon (1985) demonstrated a predominately horizontal orientation underlying B A E R waves I-III. These horizontal dipoles appeared to reflect transverse propagation along the auditory nerve (dipoles I and I - ) to the ipsilateral cochlear nucleus (dipole III) and thence via second order neurons crossing the midline in the trapezoid body (dipole III-). At this point the second order neurons assume a vertical orientation upon entering the contralateral lateral lemniscus (dipoles IV and V). (Although the dipole model was unable to provide finer distinctions for the IV-V complex, anatomical considerations suggest that some vertically oriented third order neurons may also emanate from ipsi- and contralateral superior olive, and contralateral medial nucleus of the trapezoid body.) Further, by assuming that a triphasic compound action potential would be represented in the probability function of neural firing along the first few relays in the brain-stem, Scherg and Von Cramon (1985) demonstrated that wave II resulted from the second peak of dipole I. This is consistent with the findings of Moiler et al. (1988), based on direct intracranial recordings in man, that the first two B A E R peaks, occurring at approximately 1.9 and 2.9 msec, are generated by distal and proximal auditory nerve, respec-

TWO-CHANNEL FFRs

tively. Thus, it would appear that the present horizontal FFR to tone, with a latency of 2.14 msec, reflects a process of transverse propagation localized to distal auditory nerve. For the vertical FFR to tone, with a latency (5.02 msec) equivalent to BAER wave IV, the dipole model suggests mainly second order neurons of the contralateral lateral lemniscus. Even for vertical latencies to MF stimulation which varied between experiments (4.62 and 5.73 msec), due in part to phase characteristics of the 0° and 90° stimuli, the different latencies are still within the range of BAER waves IV-V. However, further studies are required to determine if some of the MF latency variation between experiments is due to differences in the reference electrode in the vertical channel. This is suggested by the fact that in Experiment I (left ear reference) the FFR responses to pure tone and 90 ° MF were closely aligned (Fig. 3), but in Experiment II (linked mastoids reference) the 90° response was significantly delayed relative to a pure tone response. This may reflect a greater sensitivity of the MF response to the difference between a slightly angled and a vertical dipole orientation. If so, then the results of Experiment II would suggest that MF coding may in fact be somewhat more rostral (i.e., longer latency) to the site reflecting the FFR to a pure tone. Of course, it may not be possible to fully generalize BAER component latencies and polarities to the FFR, since different neuron subtypes and processes are involved. Thus, the FFR depends on phase-locking neurons (Marsh et al. 1972; Smith et al. 1975; Stillman et al. 1978; Gardi et al. 1979), which has implications for important differences in anatomy and physiology. For example, at least 22 anatomically distinct neuron types have been identified in the (cat) cochlear nucleus, nearly all of which are capable of encoding amplitude modulation (AM) information in terms of temporal response properties (Rhode and Greenberg 1992). For example, so-called "pauser-buildup units" of the dorsal cochlear nucleus phase lock exceedingly well to lowfrequency AM signals, but show poor synchronization to sinusoidal stimuli of comparable frequency. And in describing particular neurons in the posteroventral cochlear nucleus, Rhode and Greenberg (1992) state: "Onset choppers can also exhibit an exceptionally high degree of phase locking to low-frequency signals, as well as excellent temporal coding of amplitude modulation ... Thus, these units may play an important role in encoding pitch and spectral maxima of complex signals, including speech" (p. 122). It is evident from such findings that neuronal mechanisms exist to encode the unique properties of complex MF stimuli as early as the first synapse within the brain-stem auditory pathway. Assuming similar processing in humans, the vertically oriented FFR would

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thereby reflect signals encoded principally in cochlear nucleus and passing via second-order neurons through the lateral lemniscus. With regard to MF stimuli, it should be appreciated that changes in temporal fine structure alter the timbre, or quality, of the stimulus. However, it has been shown that such changes do not alter pitch perception, which still matches the missing fundamental frequency (Wightman 1973). In a parallel fashion, the present FFR results showed essentially equal amplitude responses to the different MF stimuli. Robust FFRs to 90° MF stimuli, even though the modulation depth of the stimulus envelope is greatly reduced, have been previously reported (Greenberg 1980; Greenberg et al. 1987; Galbraith and Brown 1990). Taken together, these perceptual and FFR results are an important demonstration that the neural code depends on temporal properties of the MF, and not some simple attribute of the overall stimulus wave form envelope. Such an interpretation is consistent with the temporal encoding of complex signals by phase-locking neurons, as discussed above. It thus appears that the frequency-following response is well suited to elucidate brain-stem mechanisms of auditory information processing. The FFR offers distinct advantages because it is evoked not only by tones, but also by temporally complex stimuli such as missing fundamentals and even synthetic vowels (Greenberg 1980). Thus, unique and important dimensions of auditory information processing can be studied. For example, stimuli that elicit the FFR are ideally suited for use in dichotic listening tasks, thereby facilitating the study of such phenomena as selective attention effects at the level of the brain-stem (Galbraith and Arroyo 1993). In the present study it was also shown that important additional information is obtained by simultaneously recording data with horizontal and vertical dipole orientations. By this means, it has been possible to provide the first electrophysiological demonstration of a peripheral-brain-stem dichotomy in the coding of complex auditory missing fundamental stimuli. In conclusion, the present results provide a more focused interpretation of the conclusion reached by Pantev et al. (1989), based on evoked neuromagnetic measurements, that the process of low pitch formation takes place subcortically. The absence of a horizontal response to MF stimulation demonstrates that such stimuli bypass peripheral coding mechanisms, as would be expected since the fundamental frequency is not present in the stimulus, nor is it represented in non-linear distortion products along the basilar membrane (Greenberg 1980). Yet, the presence of a robust FFR in the vertical channel, occurring at latencies consistent with BAER waves IV-V, indicates that a representation of the missing fundamental frequency is fully

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developed relatively early in the brain-stem auditory pathway. I acknowledge the assistance of Soham P. Jhaveri and Joo H. Kim, as well as important corrections to the manuscript by three anonymous reviewers. The data were collected in the UCLA Clinical Neurophysiology Laboratory located at the Lanterman Developmental Center. The ideas expressed are those of the author and are not to be construed as necessarily reflecting the policy of the California Department of Developmental Services. The present study was partially supported by NIH Grant HD 04612.

References Bilsen, F.A. On the influence of the number and phase of harmonics on the perceptibility of the pitch of complex signals. Acustica, 1973, 28: 60-65. Bilsen, F.A. Pitch of noise signals: evidence for a central spectrum. J. Acoust. Soc. Am., 1977, 61: 150-161. Bilsen, F.A. and Ritsma, R.J. Some parameters influencing the perceptibility of pitch. J. Acoust. Soc. Am., 1970, 47: 469-475. Chabot, R.J. and John, E.R. Normative evoked potential data. In: F.H. Lopes da Silva, W. Storm van Leeuwen and A. R~,mond (Eds.), Handbook of Electroencephalography and Clinical Neurophysiology, Vol. 2 (Revised Set.). Elsevier Science Publishers, Amsterdam, 1986: 263-309. Cynx, J. and Shapiro, M. Perception of missing fundamental by a species of songbird (Sturnus vulgaris). J. Comp. Psychol., 1986, 100: 356-360. Dixon, W.J. BMDP Statistical Software Manual (2V, ANOVA with Repeated Measures). Univ. Calif. Press, Berkeley, CA, 1990: 489-527. Galbraith, G.C. Latency compensation analysis of the auditory brainstem evoked response. E|ectroenceph. clin. Neurophysiol., 1984, 58: 333-342. Galbraith, G.C. and Arroyo, C. Selective attention and brainstem frequency-following responses. Biol. Psychol., 1993, 37: 3-22. Galbraith, G.C. and Brown, W.S. Cross-correlation and latency compensation analysis of click-evoked and frequency-following responses in man. Electroenceph. clin. Neurophysiol., 1990, 77: 295-308. Gardi, J., Merzenich, M. and McKean, C. Origins of the scalp-recorded frequency-following response in the cat. Audiology, 1979, 18: 353-381. Georgeson, M.A. and Harris, M.G. The temporal range of motion sensing and motion perception. Vision Res., 1990, 30: 615-619. Georgeson, M.A. and Shackleton, T.M. Monocular motion sensing, binocular motion perception. Vision Res., 1989, 29: 1511-1523. Greenberg, S. Temporal neural coding of pitch and vowel quality. UCLA Working Papers in Phonetics, 1980, 52: 1-183.

G.C. GALBRAITH Greenberg, S., Marsh, J.T., Brown, W.S. and Smith, J.C. Neural temporal coding of low pitch. I. Human frequency-following responses to complex tones. Hear. Res., 1987, 25: 91-114. Houtsma, A.J.M. and Goldstein, J.L. The central origin of the pitch of complex tones: evidence from musical interval recognition. J. Acoust. Soc. Am., 1972, 51: 520-529. Huis in 't Veld, F., Osterhammel, P. and Terkildsen, K. The frequency selectivity of the 500 Hz frequency following response. Scand. Audiol., 1977, 6: 35-42. Marsh, J.T., Smith, J.C. and Worden, F.G. Receptor and neural responses in auditory masking of low frequency tones. Electroenceph. clin. Neurophysiot., 1972, 32: 63-74. Marsh, J.T., Brown, W.S. and Smith, J.C. Far-field recorded frequency-following responses: correlates of low pitch auditory perception in humans. Electroenceph. clin. Neurophysiol., 1975, 38: 113-119. May, J.G. and Ritter, A.B. What determines the visual persistence of complex stimuli. Bull. Psychon. Soc., 1990, 25: 27-29. M011er, A.R., Jannetta, P.J. and Sekhar, L.N. Contributions from the auditory nerve to the brainstem auditory evoked potentials (BAEPs): results of intracranial recordings in man. Electroenceph. clin. Neurophysiol., 1988, 71: 198-211. Moushegian, G., Rupert, A.L. and Stillman, R.D. Scalp-recorded early responses in man to frequencies in the speech range. Electroenceph. clin. Neurophysiol., 1973, 35: 665-667. Pantev, C., Hoke, M., Liitkenh6ner, B. and Lehnertz, K. Tonotopic organization of the auditory cortex: pitch versus frequency representation. Science, 1989, 246: 486-488. Picton, T.W., HiUyard, S.A., Krausz, H.I. and Galambos, R. Human auditory evoked potentials. I. Evaluation of components. Electroenceph, clin. Neurophysiol., 1974, 36: 179-190. Plomp, R. Auditory psychophysics. Annu. Rev. Psychol., 1975, 26: 207-232. Rabiner, L.R. and Gold, B. Theory and Application of Digital Signal Processing. Prentice-HaU, Hillsdale, NJ, 1975: 177-180. Rhode, W.S. and Greenberg, S. Physiology of the cochlear nuclei. In: A.N. Popper and R.R. Fay (Eds.), The Mammalian Auditory Pathway: Neurophysiology. Springer, New York, 1992: 94-152. Scherg, M. and Von Cramon, D. A new interpretation of the generators of BAEP waves l-V: results of a spatio-temporal dipole model. Electroenceph. clin. Neurophysiol., 1985, 62: 290299. Schouten, J.F. The residue, a new component in subjective sound analysis. Proc. Kon. Ned. Akad. Wetensch., 1940, 43: 356-365. Smith, J.C., Marsh, J.T. and Brown, W.S. Far-field recorded frequency-following responses: evidence for the locus of brainstem sources. Electroenceph. clin. Neurophysiol., 1975, 39: 465-472. Smith, J.C., Marsh, J.T., Greenberg, S. and Brown, W.S. Human auditory frequency-following responses to a missing fundamental. Science, 1978, 201: 639-641. Stillman, R.D., Crow, G. and Moushegian, G. Components of the frequency-following potential in man. Electroenceph. clin. Neurophysiol., 1978, 44: 438-446. Wightman, F.L. Pitch and stimulus fine structure. J. Acoust. Soc. Am., 1973, 54: 397-416.