Hearing Research, 45 (1990) 123-136 Elsevier
HEARES
123
01347
Estimation of surviving spiral ganglion cells in the deaf rat using the electrically evoked auditory brainstem response Robert D. Hall Auditory Prosthesis Research Laboratory,
(Received
Department of Otolatyngology, Massachusetts, U.S.A. 24 August
1989; accepted
Massachusetts
8 November
Eye and Ear Infirmary,
Boston,
1989)
A procedure was developed to record the electrically evoked auditory brainstem response (EABR) in the rat with sufficiently little stimulus artifact to permit systematic measurements of the first positive wave (Pi), the compound action potential (CAP) of the auditory nerve. Our principal aim was to verify the theoretical prediction that maximum Pi amplitude is directly proportional to the number of excitable auditory nerve fibers. This was carried out in animals with graded lesions of the spiral ganglion induced by perfusion of the cochlea with different concentrations of neomycin. Two series of observations confirmed the theoretical prediction. Several measures of Pi, including maximum amplitude, and slopes of the Pi and Pi-N, growth functions, were highly correlated with the number of surviving spiral ganglion cells. Correlation coefficients (r) ranged from 0.75 to 0.92. Amplitudes of the later waves exhibited much lower correlations with spiral ganglion cell counts. These findings suggest that measurement of the CAP in deaf humans, possibly as wave I of the EABR, should provide quantitative information about the status of the nerve, which could be useful in screening candidates for cochlear implants, prescribing the optimum device for individual patients, and determining how benefits derived from such devices relate to the condition of the auditory nerve. Auditory
nerve; Spiral ganglion;
EABR;
Rat; Evoked potentials
Introduction The benefits derived from cochlear implants vary widely from patient to patient, regardless of the prosthesis that is used. One potentially important factor underlying this variability is the condition of the auditory nerve. Implant users who show marked improvements in speech reading and some ability to understand speech when the only input is through their processors may have many surviving auditory nerve fibers that are optimally distributed in the cochlea with respect to the location of stimulating electrodes. Those who reap very little benefit in speech communication from their implants may have relatively few
Correspondence to: Robert D. Hall, Auditory Prosthesis Research Laboratory, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, U.S.A.
037%5955/90/$03.50
0 1990 Elsevier Science Publishers
or poorly distributed fibers. This hypothesis could be tested in presently implanted patients if it were possible to assess the status of the auditory nerve. A method for estimating the number and distribution of auditory nerve fibers would also have appreciable value in screening candidates for cochlear prostheses and in prescribing the optimum device for individual patients. A number of workers have recognized the possibility of using the electrically evoked auditory brainstem response (EABR) to assess the status of the auditory nerve in deaf people (e.g., Brightwell et al., 1985; Chouard et al., 1985; Simmons et al., 1984; Stypulkowski et al., 1986), and there have been several attempts to relate the EABR to the number of surviving spiral ganglion cells in animals (Black et al., 1983; Shepherd et al., 1983; Walsh and Leake-Jones, 1982; Yamane et al., 1981). However, no quantitative relationship between the EABR and surviving spiral ganglion cells were described until Smith and Simmons
B.V. (Biomedical
Division)
124
(1983) reported that maximum EABR amplitudes and the slopes of EABR growth functions were both directly related to the number of surviving spiral ganglion cells in cats whose cochleas had been perfused with neomycin or damaged by mechanical or thermal insults. Stypulkowski et al. (1986) were unable to replicate that finding, however, and there were other reports suggesting that EABR amplitudes bore no strong relationship to the number of surviving spiral ganglion cells, including one by Simmons (1979) that a deaf cat with a 90% loss of spiral ganglion cells had an essentially normal EABR. Miller et al. (1983) found no relationship between the number of surviving spiral ganglion cells and the thresholds, latencies or amplitudes of the EABR in five guinea pigs. There is a sound theoretical basis for believing that the first positive wave of the EABR, generally recognized as the compound action potential (CAP) of the auditory nerve, should reflect the number of excitable auditory nerve fibers. Goldstein and Kiang (1958) presented a quantitative model of the round window response, including N,, which is also the auditory nerve CAP. In that model N, is seen as the convolution of two functions: (1) the action potential of a single auditory nerve fiber, and (2) the probability density function of spike discharges for the whole population of fibers. Two simplifying assumptions were made and later verified experimentally by Kiang et al. (1976): (1) The unit contributions, i.e., the spike waveforms, are the same for all auditory nerve fibers. (2) All of the units contribute equally to the CAP, regardless of their origin along the cochlear spiral. Thus, the model predicts that with nearly complete synchronization resulting from very brief maximum electric pulses the maximum amplitude of the CAP should be directly related to the number of excitable fibers in the nerve. Strictly speaking, it is the maximum area of the CAP that should be proportional to the number of excitable fibers. However, with the extremely high degree of synchrony achieved by maximum electrical stimulation the area of Pi is directly related to its peak amplitude. There is little if any change in the duration or waveform of Pi over its dynamic range. The duration, approximately 0.25 ms is very close to that of a single unit action potential or the unit
contribution to the CAP (N,) determined by the spike-triggered averaging technique (Kiang et al., 1976). Peak amplitude has been adopted as a substitute for the area measure because the electrical artifact often distorts the leading edge of the wave, especially at high stimulus levels, making the area measure problematic. The main reason that this seemingly simple relationship has been difficult to establish is that artifacts resulting from the electrical stimulation have generally obscured the earliest components of the EABR, especially the first positive wave, making quantitative measurements of the CAP very difficult. With some difficulty it has been possible to make such measurements in the rat. Two sets of observations are presented here, both confirming the theoretical prediction that maximum P, amplitude is directly proportional to the number of excitable spiral ganglion cells. Observations in the first set were made on small groups of rats in three pilot studies; observations in the second set were made on a larger group of animals and constitute the main experiment. Methods Subjects Thirty-five male rats of the Long-Evans strain were used in these experiments. They weighed 265-465 g when they were implanted with stimulating and recording electrodes.. The animals were anesthetized with sodium pentobarbital during the implant operation and all recording sessions (70 mg/kg with additional hourly doses of approximately 13 mg). Rectal temperature was maintained at 36.5-37.5 o C during recording sessions. Implant and lesion methods Unilateral lesions of the spiral ganglion were made by perfusing the left cochlea with different concentrations of neomycin sulphate, 4% to 35%, in boluses of 2-5 ~1 presented at 5-min intervals until a total of 20-30 ~1 had been administered. The perfusions were made through two small holes drilled in the basal and apical turns or through the oval window and an apical hole. To expose the cochlea the ear canal was detached from the bulla and the external meatus was enlarged using a dental bur. The tympanic membrane, tensor
125
tympani, malleus and incus were removed. The stapes was also removed when the cochlea was perfused through the oval window. Stimulating and recording electrodes were in most cases implanted immediately after the co&leas were perfused. In most of the animals a single electrode was implanted in Scala vestibuli through a small hole in the wall of the capsule near the juncture of the first and second turns. The electrode was a Teflon-insulated Pt-Ir wire, 75 pm in diameter, cut off squarely and stripped of insulation for approximately 250 pm at the tip. The electrode was secured in the cochlea with a carboxylate dental cement. The return electrode was a loop of stainless steel wire, 250 pm in diameter, stripped of insulation for 3-4 mm and laid against the occipital bone beneath the dorsal neck muscles. A ‘vertex’ electrode consisted of an O-80 stainless steel screw, l/4 in. long, threaded into the skull midway between bregma and lambda slightly off the midline to avoid damaging the sagital sinus. The EABR was recorded differentially between the vertex screw and the biteboard of the headholder used to secure the animal. The reference electrode was a needle inserted subcutaneously just below the contralateral ear. The stimulating electrode leads were attached to a Plastics One connector that was secured to the skull by a dental acrylic and screws. In the first set of observations reported here EABRs were also recorded from five rats used in behavioral experiments. These animals had three or four cochlear stimulating electrodes inserted into Scala vestibuli through small holes in the lateral wall of the capsule. Stimulating and recording leads were attached to a 9-conductor ITT Cannon Micro-D connector. Also included in the first set of measurements are two rats implanted with ball-tipped electrodes inserted into Scala tympani through the round window. Stimulating and recording conditions To reduce stimulus artifacts very brief, 20-ps, monophasic constant current pulses were used to stimulate the cochlea. They were generated by a Grass S-11 stimulator and PSIU6 isolation units. Averaging responses to equal numbers of positive and negative pulses, 2000 of each in most of the data reported here, also served to cancel much of
the electrical artifact. Each intensity series consisted of the following stimuli: 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 and 1000 PA. Current levels were monitored continuously across a lOOO-ohm series resistor. Stimulus artifact was also minimized by the use of an Ithaca 1201 preamplifier, which has a very rapid recovery from brief periods of saturation. High- and low-pass filters were set at 0.3 Hz and 300 kHz, respectively. A gain of 10,000 was used, and the output was led directly to the A-D converter of an Amplaid MK6 average response computer. Five hundred data points were sampled at 10 ps intervals for each average. Amplitudes of the responses have been measured in two ways: (1) peak amplitude, the difference between the prestimulus baseline and the peak, and (2) peak-to-peak amplitude, the difference between the peak and the preceding trough or the peak and the following trough. Which of these last two measures has been used will be clear in the way the measure is designated. Slopes of growth functions have been calculated as the linear regression of amplitude with intensity, from the last subthreshold intensity (the last intensity at which the amplitude measure was zero) up to 500 I-LA. Spiral ganglion cell counts Immediately following the last EABR recording session the animals were perfused through the heart with a buffered solution of glutaraldehyde and paraformaldehyde. The scalae were perfused with a 2% osmium tetroxide solution, and the cochleas were decalcified and embedded in araldite using the procedures described by Keithley and Feldman (1979). Sections 5 pm thick were cut in a plane perpendicular rather than parallel to the horizontal rnidmodiolar plane used by Keithley and Feldman, that is, in a plane approximating that of surface preparations. This was an attempt to minimize sampling errors resulting from differences in the areas of cross sections that vary from transverse to tangential when the cutting plane is parallel to the modiolus. Every tenth section was saved and stained with toluidine blue. Nuclei of type I and type II spiral ganglion cells were counted separately in each of those sections. To estimate the total number of surviving cells of
126
the two types, the counts were multiplied by 10 and divided by the average number of sections in which type I and type II cells appear in our material. These values, 2.8 and 2.2, respectively, were determined empirically in a series of 17 rats in the following way: Drawings of spiral ganglion cells and their nuclei were made in as many serial sections as were needed, usually about 25, to identify approximately 100 cells. The identification of each cell in successive sections was made primarily on the basis of superposition criteria. The number of sections in which the nucleus of a cell appeared was counted and averaged for all of the identified cells in each rat. Counts for the nuclei of type I and type II cells were made separately. The correction factors used in estimating the total populations of each type are the group averages of the two measures from each animal. In all of the data presented below the P, measures have been related to the counts of only type I cells. It seems unlikely that the thin unmyelinated type II fibers contribute significantly to the CAP. Very little is known about the physiological properties of type II fibers, but Kiang et al. (1983) recorded long-latency unit responses to electrical stimulation of the cat’s cochlea that they conjectured might be from type II units. The latencies were greater than 5 ms, compared to latencies of less than 1 ms for type I units, and the amplitudes of the potentials were quite small. Similarly long latencies in the rat would rule out any contribution of such fibers to the CAP recorded as part of the EABR. As it turns out it mattered little in the present study whether counts of only type I spiral ganglion cells or of type I and type II were used to calculate the correlation coefficients for several measures of Pi. Only a few of the coefficients were altered at all, and none by more than a single point. Nevertheless, it seems appropriate to relate the physiological measures to what is most likely their morphological substrate.
Pilot studies The first set of data was obtained from three small groups of rats treated in different ways described below. Combined they were the first evidence of a strong positive relationship between the maximum amplitude of Pi and the number of surviving spiral ganglion cells. They are presented
here to show that the relationship is robust and not peculiar to a specific set of experimental procedures. Two animals had electrodes implanted in Scala tympani through the round window. Their cochleas were not perfused with neomycin, and their survival time, i.e., the interval between implantation and the last EABR recording session, was only 43 days. Five animals were implanted with three or four Scala vestibuli electrodes. Their cochleas were perfused with either 9% or 17% solutions of neomycin, and survival times ranged from 110 to 273 days. Three had been run daily for several months in behavioral experiments and received stimulation through their implants for about an hour each day. Six animals were part of a pilot study to determine whether graded lesions of the spiral ganglion could be obtained by perfusing the cochlea with different concentrations of neomycin. The left cochlea of each rat was perfused with a 4%, 9% or 17% solution of neomycin, and the animals were allowed to survive for either 30 or 60 days before a single stimulating electrode was implanted in the cochlea. I~ediately afterward the EABR was recorded for a single stimulus intensity series, and the animals were sacrificed.
Main experiment The left cochleas of 22 rats were perfused with one of four concentrations of neomycin (48, 9%, 178, 35%) or the saline vehicle during the same operation in which a single electrode was implanted in the cochlea. The EABR was recorded for a stimulus intensity series immediately afterward and then four more times at approximately 30-day intervals. In order to ensure that there would be little spiral ganglion cell survival in a few subjects, approximately 1000 or fewer cells, the cochleas of three animals that were perfused with the 35% neomycin solution were also damaged mechanically by probing with a pin in the apical hole used for the perfusion. For 12 of the 22 rats the final recording session contained a second intensity series in which the animals were paralysed by a single dose of tubocurarine (1.5 mg) to eliminate muscle contaminants of the EABR resulting from stimulation of
127
the facial and vestibular nerves at relatively high stimulus levels. Tracheal canulas were installed before the recording session so that the animals could be respired during paralysis.
TABLE I NUMBER OF SURVIVING SPIRAL GANGLION IN RATS WITH NEOMYCIN LESIONS Rat
Results Pilot studies Illustrative EABR waveforms for part of an intensity series are shown in Fig. 1. Successive positive and negative peaks have been numbered serially in a purely descriptive way. P1 is clearly the CAP of the auditory nerve. With high current levels its peak latency can be as short as 80 ps; with near threshold stimuli the peak latency does not exceed 290 ps. Section of the eighth nerve in the internal meatus abolishes all of the later waves immediately and P1 after some minutes. The P1 peak is clearly resolved in most records, although the stimulus artifact encroaches on the rising limb of the potential at high stimulus levels. The six animals whose cochleas were perfused with different concentrations of neomycin had spiral gangion cell losses that were directly related
NM-8 NM-9 NM-6 NM-5 NM-10 NM-7
Neomycin Concentration
Survival Period
4% 4% 9% 9% 17% 17%
30 60 31 59 33 60
days days days days days days
CELLS
Spiral Ganglion Cells Type1
Type11
Total
15,432 9,879 14,217 10,224 4,485 6,928
1,228 740 700 1,026 245 316
16,600 10,619 14,917 11,250 4,730 7,244
to the concentration of the drug. The spiral ganglion cell counts for these animals are presented in Table I, where there is some suggestion that the longer survival time might also have led to greater spiral ganglion cell loss, except at the highest neomycin concentration. A measure of maximum P1 amplitude was not available for one of these animals (NM-5) because the intensity series was terminated at 600 PA
50
-
p1
P2
msec
msec
Fig. 1. Electrically evoked auditory brainstem responses (EABR) comprising a partial intensity series from one subject. Traces are averages of 2000 responses. Stimulus onset is at time 0, and the initial deflection before Pi is stimulus artifact. The distance between successive tick marks on the ordinates equals 2 PV for all responses except the last at 400 PA where it equals 4 pV.
lL7
.
i
L
c
2
Number
4
6
of
Spiral
8
10
Ganql~on
12
Cells
14
16
between the correlation coefficients for the slope and maximum amplitude measures is real, but data from the main experiment suggest it probably is not. The observations described below for the main experiment were made to confirm the strong relationship indicated by these initial observations between the amplitude of P, and the number of excitable spiral ganglion cells. Such a confirmation based on more extensive systematic data seemed desirable as a basis for the development of a similar measurement technique for humans.
‘8
ilOOO’s)
Fig. 2. Slopes of P, growth functions plotted against the number of surviving spiral ganglion cells for the six rats whose spiral ganglion cell counts are presented in Table I.
because the facial muscles were strongly excited by the electrical stimulus. Another measure of P,, the slope of its growth function, was therefore examined initially so that all six subjects could be included in the analysis. That measure is plotted against the number of surviving spiral ganglion cells in Fig. 2 where a rather strong positive correlation, r = 0.79, is apparent. The correlation was high enough to suggest that the slopes of PI growth functions might be a useful index of surviving spiral ganglion cells if the correlation could be confirmed in a larger sample of the deaf rats. A similarly strong relationship was found in the regression of maximum PI amplitude on the number of surviving spiral ganglion cells shown in Fig. 3. Data from five of the animals included in Fig. 2 (excluding the rat for which this measure was not available) are presented together with data from other animals for which the EABR measures and spiral ganglion cell counts were available prior to completion of the main experiment. The correlation is high, r = 0.92, and is confirmation of the expected relationship between maximum P, amplitude and the number of excitable auditory nerve fibers. The relationship appears to hold for a rather wide range of experimental conditions, that is, with differences in the type and location of stimulating electrodes, with differences in survival times following implantation and in the amount of stimulation the animals received. The sample sizes are too small to know whether the difference
Main experiment Satisfactory data were not obtained from 6 of the 22 animals for the following reasons: One rat died, apparently of a respiratory infection. The lead to the cochlear electrode broke in two rats shortly before the final session and could not be repaired. P, could not be measured with confidence in three rats because of unusually long stimulus artifacts. The unusual artifacts did not appear to be related to spiral ganglion cell survival, which ranged from 7% to 48% in the three animals. EABRs recorded as part of an intensity series from one animal are shown in Fig. 4 to illustrate one kind of muscle contaminant that made it
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4
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14
16
18
(1000’s)
Fig. 3. Maximum P, amplitude plotted as a function of the number of surviving spiral ganglion cells for animals treated in different ways. See text for details. The neomycin pilot rats are five of the six animals represented in Fig. 2. The regression line is a least squares best fit as it is in all plots of this kind presented here.
129
Stim.
450 400 350 300 250 200 150 100
1
0
msec
4.5
Fig. 4. EL4BR.s from part of an intensity series for rat NM-11. What appears to be a large increase in waves N, and P4 in the top trace (for the 500 PA stimulus) is a myogenic response originating in the digastric muscle as a result of facial nerve activation.
necessary to paralyse some animals in order to measure the late waves of the EABR. The N, and P4 waves were supplanted at the highest stimulus intensity shown (500 PA) by a diphasic response from the posterior belly of the digastric muscle, which is innervated by motoneurons of the facial nerve (Hall, 1989). The presence of such responses is readily documented if one continues to measure the amplitudes of the N, and P4 peaks, with which the muscle potentials coincide, as shown for the same animal in Fig. 5. The N, and P4 peaks are typically asymptotic before the digastric potentials appear. The large and rapid increases in what appear initially to be the N3 and P4 waves reach amplitudes much in excess of any auditory brainstem responses. The latencies of the muscle potentials are very close to those of the N, and P4 peaks, as measured in animals following eighth nerve section or in normal animals at high intensities when the responses are extremely large and the N3-P4 contribution is negligible. In each of the four recording sessions represented in the figure the relatively small N3-P4 auditory responses gave way to the much larger muscle potentials at high current levels. The growth of the digastric response in Fig. 5 appears to be associated with a second period of
growth at high current levels in the functions for the Ni and P2 waves. These increases in Ni and PZ, however, do not reflect synaptically mediated muscle responses because they are not abolished by curare, as shown in Fig. 6. The origins of these increases in Ni and P2 are uncertain. They do not appear to be of facial nerve origin as stimulation of the nerve at its exit from the stylomastoid foramen does not result in a prominent negativepositive complex of short latency in the vertex response, even in the presence of large digastric potentials. Our working hypothesis is that the Ni and Pz increases at high current levels are related to vestibular activation. The digastric potentials, however, were eliminated by curare, as evidenced by the absence of any large increase in the N,--P, measure following administration of the drug. Curarization had no systematic effect on any other components of the EABR. The EABR was quite stable over a period of four months or more when no intentional damage was done to the cochlea. This is illustrated in the growth functions of Fig. 5 for subject NM-11, a control animal whose cochlea was not perfused with neomycin or saline. In contrast, neomycin poisoning led to systematic decreases with time in most animals, as illustrated in the growth functions of Fig. 7 from subject NM-26 whose cochlea was perfused with a 35% neomycin solution. Several other features of the curves in Fig. 7 are of interest. First the increases in N, and P2 at high intensities, like those shown in Figs. 5 and 6, are not accompanied by similar increases in Pi. In nearly every record maximum Pi amplitude could be determined because the P1 functions had reached asymptote. That was equally true of the later waves, P3 and P4, as long as there were no muscle contaminants. The initial asymptote of P2 was always clear, even when the growth function had a second rising limb at high current levels. On the other hand, although the initial asymptote of N1 was sometimes clear, the growth function frequently revealed no clear maximum, as in Figs. 5 and 6, and this was usually reflected in the Pi-N, peak-to-peak amplitude measure, as illustrated for NM-26 in Fig. 7. The measure of Pi-N, amplitude appeared to have several advantages over peak amplitude measurements of Pr, but the absence of a clear asymptote in many Pi-N, growth func-
130 50
50 PI
940
P2
40
3 ; 30 .% E
30
$ 20 Y : fk 10
20
Subject
NM-l
1
10
0 400 N3-P4 300 0 Day Implanted 0 64 Days Later l
200
96 Days Later
a 131 Days Later 100
0 200
400 Stimulus
600 Current
1000
(~~
0
200
400 Stimulus
Fig. 5. Growth functions for the same subject whose EABRs appear in recording sessions. Data from the second session for this subject were appearance and rapid growth of the digastric potentials is indicated different scale on the ordinate
tions made it necessary to examine other metrics to characterize PI-N, amplitude and its relationship to surviving spiral ganglion cells. Maximum PI amplitude is the measure of the auditory nerve CAP that is related most directly in theory to the number of excitable spiral ganglion
TABLE
1
600
800
Current
1000
(~)
Fig. 4. The functions in each graph are from four different marred by technical difficulties and are not presented. The in the N,-P, peak-to-peak amplitude measure. Note the for the measure.
cells; maximum PI amplitude should be directly proportional to the number of excitable auditory nerve fibers. This kind of relationship is shown in Fig. 8 were it is clear that the theoretical prediction is confirmed again. The correlation coefficient (r = 0.77) is slightly lower than the one found
II
CORRELATIONS
(R) BETWEEN
EABR WAVES
AND SURVIVING
TYPE 1 SPIRAL Peak-to-peak
Peak amplitudes
GANGLION
CELLS
amplitudes
9
p2
P3
P4
PI-N,
W
amplitude at 500 pA
0.77 0.75
0.47 0.45
0.40 0.55
0.27 0.43
0.81
0.50 0.69
0.59 0.61
0.41 0.72
Slope of growth function to 500 pA
0.84
0.42
0.18
0.41
0.90
0.66
0.62
0.65
Maximum Amplitude
-P2
N2
-9
W-P‘,
Pl
Subject
30-
-
400’
’
*
*
’
’
’
*
*
)
N3-P4
Nl l
NM-22
300
I
Before Curarization
0 After Curarizotion
0
200
400 Stimulus
1000
0
CuGPeent ($)O
200
400
600
Stimulus Current
600
1000
(pA)
Fig. 6. Growth functions for rat NM-22 obtained in the final session before and after the animal was curarized to eliminate the digastric muscle response, as indicated by the plots for the N3-P4 amplitude measure. Amplitudes of the auditory components of the EABR were not influenced by the curarkation. Increases in the NI and P2 waves at high stimulus intensities are not of myogenic Oligill.
in the initial observations described above, due apparently to a few deviant measures. It seemed worthw~~, therefore, to examine other measures of Pi that might be less sensitive to errors of measurement. The correlations between four other measures of Pr and the number of surviving spiral ganglion cells are shown in Fig. 9. EABRs at 500 PA, where nearly maximum values of Pi are obtained in most animals, appear to be freer of residual stimulus artifacts than responses at higher intensities, and the regression of Pr peak amplitude at 500 I_LAon the number of spiral ganglion cells, as shown in the lower left of Fig. 9, is at least as strong as that for maximum P, amplitude. A subm~um measure of P, amplitude, moreover, is the kind that may be most useful in human subjects who are not
likely to tolerate electrical stimuli strong enough to evoke the maximum CAP. The slopes of growth functions have the same advantage, and in addition may be less ‘noisey’ in as much as they are derived from a series of measurements rather than a single measure of amplitude. The advantage of the P,-N, peak-to-peak amplitude measure is that it seems less sensitive to residual tails of artifacts that may shift the Pt peak upward or downward from the baseline if the cancellation of the electrical artifact is not complete, as is frequently the case at high stimulus intensities. The high correlation between the slope of Pr-N, growth functions and the number of surviving spiral ganglion cells (r = 0.90) may reflect the advantages of both peak-to-peak amplitude measures and the use of slope as the final metric. In any case, it is clear
r
60
PI
5o
Pl-Nl Subject
NM-26
Nl
Nl -P2
0 Day Implanted /
0 34 Days Later n
66 Days Later
a 99 Days Later A 132 Days Later
200
400
Stimulus
600
Current
600
1000
(pA)
Fig. 7. Growth functions for the early components of the EABR recorded at approximately monthly intervals from rat NM-26 whose cochlea had been perfused with a 35% neomycin solution. The systematic decline of the response presumably reflects a progressive loss of spiral ganglion cells, as indicated by the diminishing P, amplitude over time.
r =
.
.77
.
/
.
. . 0
.
2
4
Number
6
8
10
12
of Spiral Ganglion
14
16
IS
20
22
Cells (1000’s)
Fig. 8. Maximum P, amplitude plotted as a function of the number of surviving type I spiral ganglion cells for 16 rats whose cochleas had been perfused with different concentrations of neomycin.
from all of the plots in Fig. 9 that the amplitude of Pi and the slopes of its growth functions, measured in various ways, are highly correlated with the number of surviving type I spiral ganglion cells in deaf rats. Because the earliest waves of the EABR can be difficult to record one would like to know how the later components of the response are correlated with the number of surviving spiral ganglion cells. Correlation coefficients for several amplitude measures of the later waves are presented in Table II. The correlations for the Pi and Pi-N1 measures are also presented to facilitate comparisons between the coefficients for the auditory nerve responses and the later brainstem waves. The correlations between ganglion cell counts and peak amplitudes of P2, P3 and P4, measured in three different ways, are low; only one, P3 amplitude at
133
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l
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Fig. 9. Four different measures of Pt plotted as a function of the number of surviving spiral ganglion cells. The different measures are indicated on the ordinates of the four graphs. Slopes of the P, and PI-N, growth functions were based on response amplitudes up to a stimulus intensity of 500 PA_ All four measures show a strong relationship between Pr amplitude and the number of spiral ganglion cells.
Number
of
Spiral
Ganglion
Cells
(1000’s)
Fig. 10. Ns-P4 peak-to-peak amplitude plotted as a function of the number of surviving spiral ganglion cells reveals the best correlation between any of the later EABR waves and spiral ganglion cell counts in the present data.
a stimulus intensity of 500 ,uA, is statistically reliable. The picture is somewhat better for the peak-to-peak ~plitude measures, although it is clear that the correlations are for the most part much weaker than those for Pi. It is possible, however, that even the weaker relationships might be useful under some circumstances. The strongest of these relationships in the present data is shown in Fig. 10 as the regression of NJ-P4 amplitude at 500 yA on the number of surviving type I ganglion cells. It remains to be seen whether this relationship is sufficiently stable to have practical utility. Discussion Reduction of the electrical stimulus artifact has made it possible to make systematic measurements
134
of the auditory nerve CAP when it is recorded as the first positive wave of the EABR. The measurements in both sets of data presented here clearly confirm the theoretical prediction that the maximum amplitude of P, is proportional to the number of excitable auditory nerve fibers. The assumption underlying the present measurements is that counts of spiral ganglion cell bodies provide good estimates of the number of excitable fibers. There is some indication in the literature that counts of spiral ganglion cells in the rat, like those reported by Keithley and Feldman (1979), yield estimates of the co&ear nerve population that are smaller than those obtained by counting fibers in the trunk of the nerve (Hoeffding and Feldman, 1988). Hoeffding and Feldman thought it likely that the differences reflected differences in the precision attainable with the two counting methods. Losses occurring with age, however, were percentagewise quite similar in the two studies, which suggests that counts of cell bodies in Rosenthal’s canal are a relatively constant proportion of fiber counts. That kind of constant error would have no effect on the correlations reported here between the various measures of P, and surviving spiral ganglion cells. It would lead to a systematic underestimation of surviving auditory nerve fibers from the regression equations based on the present data. The discrepancy between cell body and fiber counts in the rat should be resolved by making both types of measurements in the same animals. The measures of P1 amplitude used to determine the correlations with surviving spiral ganglion cells were measures of absolute amplitude, not measures that were normalized to reduce the variability resulting from differences in recording conditions or in other factors that influence the measurement of gross neural potentials. Stimulating and recording conditions in the rat were sufficiently uniform to permit this. Nevertheless, there was enough variability in some of the measures to make estimations of spiral ganglion cell populations possible only within rather large error bounds. In the main experiment the slopes of P,-N, growth functions yielded the highest correlation with surviving spiral ganglion cells and, consequently, the smallest standard error of estimate. The data from the upper righthand graph in
”
“2
!J4
Siope of Pl
1)6
Nl
Growth
08
13
Function
Fig. If. The regression of spiral ganglion cell counts on the slope of P,-N, growth functions (thick line) plus and minus the standard error of estimate (thin lines). The data from the upper righthand graph in Fig. 9 have been replotted here to emphasize the potential for predicting spiral ganglion cell populations from the physiological index.
Fig. 9 have been replotted in Fig. 11 to show the standard error in estimating the number of spiral ganglion cells from the slope of P,-N, growth functions. The plot says that about two thirds of the time our predictions should not err by more than 2300 cells. Additional experience in recording the rat’s EABR will probably reduce that error, but even this kind of predictability may be difficult to achieve in humans. The variability in EABR amplitudes is likely to be greater than it is in the rat or other laboratory animals as a consequence of greater individual differences in subjects as well as greater variability in stimulating and recording conditions. Slopes of growth functions, which should be independent of differences in absolute amplitude, may provide a way to normalize the human data, all the while retaining other advantages of that metric mentioned above. The high correlation between the slopes of Pi-N, growth functions and spiral ganglion cell counts found in the present study suggest that such measures may be useful for estimating spiral ganglion cell populations in deaf people. The generally low correlations between the amplitudes of the later EABR waves and the counts of spiral ganglion cells may explain, in part at least, why earlier attempts to demonstrate a relationship between EABR amplitudes and the status
135
of the auditory nerve met with mixed success (e.g., Miller et al., 1983; Smith and Simmons, 1983; Stypulkowski et al., 1986). There is clearly a positive relationship between the amplitudes of the later components and the amplitude of Pi, as is obvious in the growth of the entire response with increasing stimulus intensity. But the intrasubject correlations seen in growth functions for the various waves recorded from an individual are not the correlations of interest. Across-subject correlations are. In the main experiment presented here across-subjects correlations between Pi amplitude and the amplitudes of the later waves, measured both as peak and peak-to-peak excursions, ranged from 0.12 to 0.59. The amplitudes were measured at a stimulus level of 500 PA for this analysis. If Pi is a good predictor of surviving spiral ganglion cells, other measures of the EABR that show only modest correlations with Pi are not likely to provide much information about the status of the auditory nerve. The electrically evoked middle latency response (EMLR) has been the subject of a series of studies concerned with its possible utility in assessing the status of the auditory pathways, including the auditory nerve, in humans (e.g., Kileny and Kemink, 1987) and animals (Burton et al., 1989). Jyung et al. (1989) have recently reported that following aminoglycoside poisoning the slopes of EMLR growth functions from individual guinea pigs showed orderly decreases with time that reflected reductions in spiral ganglion cell densities. Different animals were sacrificed at progressively longer intervals following the administration of kanamycin and ethacrynic acid, and the longer survival times were associated with progressively greater reductions in spiral ganglion cell densities. It is not yet clear how robust the correlation is between the slopes of EMLR growth functions and spiral ganglion cell densities in the animal model. The EMLR may be subject to the same kinds of limitations as the later waves of the EABR in assessing the status of the auditory nerve. The MLR, moreover, is more sensitive than the ABR to state variables, including anesthetics, (e.g. Smith and Kraus, 1987) and it remains to be seen how that sensitivity might compromise its use in humans where it is generally more variable than the ABR (e.g., Ozdamar and Kraus, 1983). Predic-
tions of spiral ganglion cell populations from EMLR measures are bound to err when central lesions interupt ascending auditory pathways in the absence of peripheral lesions. Stimulating and recording conditions were quite favorable for obtaining good quantitative measures of the auditory nerve CAP in the present experiments. Such conditions may be harder to realize in human subjects, especially when the CAP is used for screening purposes under the assumption that only extracochlear stimulation is permissible. Intracochlear stimulation in the rat permitted the use of relatively small currents of very brief duration, tending thereby to minimize stimulus artifacts. Similarly, intracranial recording electrodes yielded much higher signal levels and better signal/noise ratios than do scalp electrodes used with human subjects, and those ratios as well as the general stability of the recording conditions were enhanced by the use of anesthesia. The advantages of intracochlear electrodes have already been realized in several EABR studies of humans with cochlear implants (Abbas and Brown, 1988; Thornton and Hermann, personal communication; van den Honert and Stypulkowski, 1986). Unfortunately, wave I was not a consistent feature of the responses found in any of those studies. More recently, however, Brown et al. (1989) have been able to extract the CAP from potentials recorded intracochlearly from subjects implanted with Symbion electrodes. They modified a forward masking paradigm first described by Charlet de Sauvage et al. (1983) to eliminate much of the stimulus artifact. Using an electrical masking stimulus and a subtraction procedure they were able to extract from the potentials recorded intracochlearly at one electrode the CAP evoked by stimulation at two other intracochlear electrodes. Modest correlations were found between the slopes of the growth curves and scores on two tests of word recognition. Somewhat stronger correlations were found between the word recognition scores and the slopes of recovery functions measured with increasing intervals between masking and probe stimuli. The findings, regarded as tentative in view of the small sample, emphasize nevertheless the possibility that measures of auditory nerve responses in the deaf may have appreciable utility in predicting success with cochlear implants. Be
136
that as it may, there is still the problem of obtaining good measures of the CAP in candidates for cochlear implants. Whether or not any of the procedures used in the present study will prove useful for that purpose, we hope that the demonstration of the relationship between P, and the number of surviving spiral ganglion cells will add impetus to the search for a suitable method. Acknowledgements The author is most grateful to Elizabeth P. Sonnabend and Jennifer L. Massengill for their technical assistance, to Elizabeth M. Keithley for teaching us how to prepare histological sections of the cochlea, to Joseph B. Nadol, Jr. for his continuous encouragement and support, and to Donald K. Eddington, Nelson Y.-S. Kiang and Aaron R. Thornton for many helpful discussions throughout this research. References Abbas, P.J. and Brown, C.J. (1988) Electrically evoked brainstem potentials in cochlear implant patients with multielectrode stimulation. Hear. Res. 36, 153-162. Black, R., Clark, G., Shepherd, R., O’Leary, S. and Walters, C. (1983) Intracochlear electrical stimulation. Brainstem response audiometric and histopathological studies. Acta Otolaryngol., Suppl. 399, 5-17. Brightwell, A., Rothera, M., Conway, M. and Graham, J. (1985) Evaluation of the status of the auditory nerve: psychophysical tests and ABR. In: R.A. Schindler and M.M. Merzenich (Eds.), Cochlear Implants, Raven Press, New York, pp. 343-349. Brown, C.J., Abbas, P.J. and Gantz, B. (1989) Electrically evoked whole-nerve action potentials recorded in cochlear implant users. Assoc. Res. Otolaryngol. Abst., p. 274. Burton, M.J., Miller, J.M. and Kileny, P.R. (1989) Middlelatency responses. I. Electrical and acoustic excitation. Arch. Otolaryngol. Head Neck Surg. 115, 59-62. Charlet de Sauvage, R., Cazals, Y., Erre, J.P. and Aran, J.M. (1983) Acoustically derived auditory nerve action potential evoked by electrical stimulation. An estimation of the waveform of a single unit contribution. J. Acoust. Sot. Am. 73, 616-627. Chouard, C.H., Meyer, B. and Gegu, D. (1985) Pre- and per-operative electrical testing procedure. In: R.A. Schindler and M.M. Merzenich (Ed%), Cochlear Implants, Raven Press, New York, pp. 365-374. Goldstein, M.H. and Kiang, N.Y.-S. (1958) Synchrony of neural activity in electric response evoked by transient acoustic stimuli. J. Acoust. Sot. Am. 30, 107-114. Hall, R.D. (1989) Electrically evoked auditory brainstem re-
sponse in the rat: Its use in estimating spiral ganglion cell populations and the effects of facial nerve activation. Assoc. Res. Otolaryngol. Abst. p. 24. Hoeffding, V. and Feldman, M.L. (1988) Changes with age in the morphology of the cochlear nerve in rats: Light microscopy. J. Comp. Neurol. 276, 537-546. Jyung, R.W., Crowther, J.A., Marsal, S. and Miller, J.M. (1989) Prediction of eighth nerve survival using the electricallyevoked middle latency response. Assoc. Res. Otolaryngol. Abst., p. 29. Keithley, E.M. and Feldman, M.L. (1979) Spiral ganglion cell counts in an age-graded series of rat cochleas. J. Comp. Neurol. 188, 4299442. Kiang, N.Y.S., Keithley, E.M. and Liberman, M.C. (1983) The impact of auditory nerve experiments on cochlear implant design. AM. N.Y. Acad. Sci. 405, 114-121. Kiang, N.Y.S., Moxon, E.C. and Kahn, A.R. (1976) The relationship of gross potentials recorded from the cochlea to single unit activity in the auditory nerve. In: J.B. Ruben, C. Elberling and G. Salomon (Eds.), Electrocochleography, University Park Press, Baltimore, pp. 95-115. Kileny, P.R. and Kemink, J.L. (1987) Electrically evoked middle-latency auditory potentials in cochlear implant patients. Arch. Otolaryngol. Head Neck Surg. 113, 1072-1077. Miller, J.M., Duckert, L.G., Malone, M.A. and Pfingst, B.E. (1983) Cochlear prostheses: Stimulation induced damage. Ann. Otol., Rhinol. Laryngol. 92, 599-609. Ozdamar, 0. and Kraus, N. (1983) Auditory middle-latency responses in humans. Audiology 22, 34-49. Shepherd, R.K., Clark, G.M. and Black, R.C. (1983) Chronic electrical stimulation of the auditory nerve in cats. Acta Otolaryngol., Supp. 399, 19-31. Simmons, F.B. (1979) Electrical stimulation of the auditory nerve in cats. Long-term electrophysiological and histological results. Ann. Otol. 88, 533-539. Simmons, F.B., Lusted, H.S., Meyers, T. and Shelton, C. (1984) Electrically induced auditory brainstem response as a clinical tool in estimating nerve survival. Ann. Otol. Rhinol. Laryngol. 112,97-100. Smith, D.I. and Kraus, N. (1987) Effects of chloral hydrate, pentobarbital, ketamine, and curare on the auditory middle latency response. Am. J. Otolaryngol. 8, 241-248. Smith, L. and Simmons, F.B. (1983) Estimating eighth nerve survival by electrical stimulation. Ann. Otol. Rhinol. Laryngol. 92, 19-23. Stypulkowski, P.H., Van den Honert, C. and Kvistad, S.D. (1986) Electrophysiologic evaluation of the cochlear implant patient. Otolaryngologic Clin. N. Am. 19, 249-257. Van den Honert, C. and Stypulkowski, P.H. (1986) Characterization of the electrically evoked auditory brainstem response (ABR) in cats and humans. Hear. Res. 21, 109-126. Walsh, S.M. and Leake-Jones, P.A. (1982) Chronic electrical stimulation of auditory nerve in cat: physiological and histological results. Hear. Res. 7, 281-304. Yamane, H., Marsh, R.R. and Potsic, W.P. (1981) Brain stem response evoked by electrical stimulation of the round window of the guinea pig. Otolaryngol. Head Neck Surg. 89, 117-124.
HEARES
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01347
Estimation of surviving spiral ganglion cells in the deaf rat using the electrically evoked auditory brainstem response Robert D. Hall Auditory Prosthesis Research Laboratory,
(Received
Department of Otolatyngology, Massachusetts, U.S.A. 24 August
1989; accepted
Massachusetts
8 November
Eye and Ear Infirmary,
Boston,
1989)
A procedure was developed to record the electrically evoked auditory brainstem response (EABR) in the rat with sufficiently little stimulus artifact to permit systematic measurements of the first positive wave (Pi), the compound action potential (CAP) of the auditory nerve. Our principal aim was to verify the theoretical prediction that maximum Pi amplitude is directly proportional to the number of excitable auditory nerve fibers. This was carried out in animals with graded lesions of the spiral ganglion induced by perfusion of the cochlea with different concentrations of neomycin. Two series of observations confirmed the theoretical prediction. Several measures of Pi, including maximum amplitude, and slopes of the Pi and Pi-N, growth functions, were highly correlated with the number of surviving spiral ganglion cells. Correlation coefficients (r) ranged from 0.75 to 0.92. Amplitudes of the later waves exhibited much lower correlations with spiral ganglion cell counts. These findings suggest that measurement of the CAP in deaf humans, possibly as wave I of the EABR, should provide quantitative information about the status of the nerve, which could be useful in screening candidates for cochlear implants, prescribing the optimum device for individual patients, and determining how benefits derived from such devices relate to the condition of the auditory nerve. Auditory
nerve; Spiral ganglion;
EABR;
Rat; Evoked potentials
Introduction The benefits derived from cochlear implants vary widely from patient to patient, regardless of the prosthesis that is used. One potentially important factor underlying this variability is the condition of the auditory nerve. Implant users who show marked improvements in speech reading and some ability to understand speech when the only input is through their processors may have many surviving auditory nerve fibers that are optimally distributed in the cochlea with respect to the location of stimulating electrodes. Those who reap very little benefit in speech communication from their implants may have relatively few
Correspondence to: Robert D. Hall, Auditory Prosthesis Research Laboratory, Department of Otolaryngology, Massachusetts Eye and Ear Infirmary, 243 Charles Street, Boston, MA 02114, U.S.A.
037%5955/90/$03.50
0 1990 Elsevier Science Publishers
or poorly distributed fibers. This hypothesis could be tested in presently implanted patients if it were possible to assess the status of the auditory nerve. A method for estimating the number and distribution of auditory nerve fibers would also have appreciable value in screening candidates for cochlear prostheses and in prescribing the optimum device for individual patients. A number of workers have recognized the possibility of using the electrically evoked auditory brainstem response (EABR) to assess the status of the auditory nerve in deaf people (e.g., Brightwell et al., 1985; Chouard et al., 1985; Simmons et al., 1984; Stypulkowski et al., 1986), and there have been several attempts to relate the EABR to the number of surviving spiral ganglion cells in animals (Black et al., 1983; Shepherd et al., 1983; Walsh and Leake-Jones, 1982; Yamane et al., 1981). However, no quantitative relationship between the EABR and surviving spiral ganglion cells were described until Smith and Simmons
B.V. (Biomedical
Division)
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(1983) reported that maximum EABR amplitudes and the slopes of EABR growth functions were both directly related to the number of surviving spiral ganglion cells in cats whose cochleas had been perfused with neomycin or damaged by mechanical or thermal insults. Stypulkowski et al. (1986) were unable to replicate that finding, however, and there were other reports suggesting that EABR amplitudes bore no strong relationship to the number of surviving spiral ganglion cells, including one by Simmons (1979) that a deaf cat with a 90% loss of spiral ganglion cells had an essentially normal EABR. Miller et al. (1983) found no relationship between the number of surviving spiral ganglion cells and the thresholds, latencies or amplitudes of the EABR in five guinea pigs. There is a sound theoretical basis for believing that the first positive wave of the EABR, generally recognized as the compound action potential (CAP) of the auditory nerve, should reflect the number of excitable auditory nerve fibers. Goldstein and Kiang (1958) presented a quantitative model of the round window response, including N,, which is also the auditory nerve CAP. In that model N, is seen as the convolution of two functions: (1) the action potential of a single auditory nerve fiber, and (2) the probability density function of spike discharges for the whole population of fibers. Two simplifying assumptions were made and later verified experimentally by Kiang et al. (1976): (1) The unit contributions, i.e., the spike waveforms, are the same for all auditory nerve fibers. (2) All of the units contribute equally to the CAP, regardless of their origin along the cochlear spiral. Thus, the model predicts that with nearly complete synchronization resulting from very brief maximum electric pulses the maximum amplitude of the CAP should be directly related to the number of excitable fibers in the nerve. Strictly speaking, it is the maximum area of the CAP that should be proportional to the number of excitable fibers. However, with the extremely high degree of synchrony achieved by maximum electrical stimulation the area of Pi is directly related to its peak amplitude. There is little if any change in the duration or waveform of Pi over its dynamic range. The duration, approximately 0.25 ms is very close to that of a single unit action potential or the unit
contribution to the CAP (N,) determined by the spike-triggered averaging technique (Kiang et al., 1976). Peak amplitude has been adopted as a substitute for the area measure because the electrical artifact often distorts the leading edge of the wave, especially at high stimulus levels, making the area measure problematic. The main reason that this seemingly simple relationship has been difficult to establish is that artifacts resulting from the electrical stimulation have generally obscured the earliest components of the EABR, especially the first positive wave, making quantitative measurements of the CAP very difficult. With some difficulty it has been possible to make such measurements in the rat. Two sets of observations are presented here, both confirming the theoretical prediction that maximum P, amplitude is directly proportional to the number of excitable spiral ganglion cells. Observations in the first set were made on small groups of rats in three pilot studies; observations in the second set were made on a larger group of animals and constitute the main experiment. Methods Subjects Thirty-five male rats of the Long-Evans strain were used in these experiments. They weighed 265-465 g when they were implanted with stimulating and recording electrodes.. The animals were anesthetized with sodium pentobarbital during the implant operation and all recording sessions (70 mg/kg with additional hourly doses of approximately 13 mg). Rectal temperature was maintained at 36.5-37.5 o C during recording sessions. Implant and lesion methods Unilateral lesions of the spiral ganglion were made by perfusing the left cochlea with different concentrations of neomycin sulphate, 4% to 35%, in boluses of 2-5 ~1 presented at 5-min intervals until a total of 20-30 ~1 had been administered. The perfusions were made through two small holes drilled in the basal and apical turns or through the oval window and an apical hole. To expose the cochlea the ear canal was detached from the bulla and the external meatus was enlarged using a dental bur. The tympanic membrane, tensor
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tympani, malleus and incus were removed. The stapes was also removed when the cochlea was perfused through the oval window. Stimulating and recording electrodes were in most cases implanted immediately after the co&leas were perfused. In most of the animals a single electrode was implanted in Scala vestibuli through a small hole in the wall of the capsule near the juncture of the first and second turns. The electrode was a Teflon-insulated Pt-Ir wire, 75 pm in diameter, cut off squarely and stripped of insulation for approximately 250 pm at the tip. The electrode was secured in the cochlea with a carboxylate dental cement. The return electrode was a loop of stainless steel wire, 250 pm in diameter, stripped of insulation for 3-4 mm and laid against the occipital bone beneath the dorsal neck muscles. A ‘vertex’ electrode consisted of an O-80 stainless steel screw, l/4 in. long, threaded into the skull midway between bregma and lambda slightly off the midline to avoid damaging the sagital sinus. The EABR was recorded differentially between the vertex screw and the biteboard of the headholder used to secure the animal. The reference electrode was a needle inserted subcutaneously just below the contralateral ear. The stimulating electrode leads were attached to a Plastics One connector that was secured to the skull by a dental acrylic and screws. In the first set of observations reported here EABRs were also recorded from five rats used in behavioral experiments. These animals had three or four cochlear stimulating electrodes inserted into Scala vestibuli through small holes in the lateral wall of the capsule. Stimulating and recording leads were attached to a 9-conductor ITT Cannon Micro-D connector. Also included in the first set of measurements are two rats implanted with ball-tipped electrodes inserted into Scala tympani through the round window. Stimulating and recording conditions To reduce stimulus artifacts very brief, 20-ps, monophasic constant current pulses were used to stimulate the cochlea. They were generated by a Grass S-11 stimulator and PSIU6 isolation units. Averaging responses to equal numbers of positive and negative pulses, 2000 of each in most of the data reported here, also served to cancel much of
the electrical artifact. Each intensity series consisted of the following stimuli: 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800 and 1000 PA. Current levels were monitored continuously across a lOOO-ohm series resistor. Stimulus artifact was also minimized by the use of an Ithaca 1201 preamplifier, which has a very rapid recovery from brief periods of saturation. High- and low-pass filters were set at 0.3 Hz and 300 kHz, respectively. A gain of 10,000 was used, and the output was led directly to the A-D converter of an Amplaid MK6 average response computer. Five hundred data points were sampled at 10 ps intervals for each average. Amplitudes of the responses have been measured in two ways: (1) peak amplitude, the difference between the prestimulus baseline and the peak, and (2) peak-to-peak amplitude, the difference between the peak and the preceding trough or the peak and the following trough. Which of these last two measures has been used will be clear in the way the measure is designated. Slopes of growth functions have been calculated as the linear regression of amplitude with intensity, from the last subthreshold intensity (the last intensity at which the amplitude measure was zero) up to 500 I-LA. Spiral ganglion cell counts Immediately following the last EABR recording session the animals were perfused through the heart with a buffered solution of glutaraldehyde and paraformaldehyde. The scalae were perfused with a 2% osmium tetroxide solution, and the cochleas were decalcified and embedded in araldite using the procedures described by Keithley and Feldman (1979). Sections 5 pm thick were cut in a plane perpendicular rather than parallel to the horizontal rnidmodiolar plane used by Keithley and Feldman, that is, in a plane approximating that of surface preparations. This was an attempt to minimize sampling errors resulting from differences in the areas of cross sections that vary from transverse to tangential when the cutting plane is parallel to the modiolus. Every tenth section was saved and stained with toluidine blue. Nuclei of type I and type II spiral ganglion cells were counted separately in each of those sections. To estimate the total number of surviving cells of
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the two types, the counts were multiplied by 10 and divided by the average number of sections in which type I and type II cells appear in our material. These values, 2.8 and 2.2, respectively, were determined empirically in a series of 17 rats in the following way: Drawings of spiral ganglion cells and their nuclei were made in as many serial sections as were needed, usually about 25, to identify approximately 100 cells. The identification of each cell in successive sections was made primarily on the basis of superposition criteria. The number of sections in which the nucleus of a cell appeared was counted and averaged for all of the identified cells in each rat. Counts for the nuclei of type I and type II cells were made separately. The correction factors used in estimating the total populations of each type are the group averages of the two measures from each animal. In all of the data presented below the P, measures have been related to the counts of only type I cells. It seems unlikely that the thin unmyelinated type II fibers contribute significantly to the CAP. Very little is known about the physiological properties of type II fibers, but Kiang et al. (1983) recorded long-latency unit responses to electrical stimulation of the cat’s cochlea that they conjectured might be from type II units. The latencies were greater than 5 ms, compared to latencies of less than 1 ms for type I units, and the amplitudes of the potentials were quite small. Similarly long latencies in the rat would rule out any contribution of such fibers to the CAP recorded as part of the EABR. As it turns out it mattered little in the present study whether counts of only type I spiral ganglion cells or of type I and type II were used to calculate the correlation coefficients for several measures of Pi. Only a few of the coefficients were altered at all, and none by more than a single point. Nevertheless, it seems appropriate to relate the physiological measures to what is most likely their morphological substrate.
Pilot studies The first set of data was obtained from three small groups of rats treated in different ways described below. Combined they were the first evidence of a strong positive relationship between the maximum amplitude of Pi and the number of surviving spiral ganglion cells. They are presented
here to show that the relationship is robust and not peculiar to a specific set of experimental procedures. Two animals had electrodes implanted in Scala tympani through the round window. Their cochleas were not perfused with neomycin, and their survival time, i.e., the interval between implantation and the last EABR recording session, was only 43 days. Five animals were implanted with three or four Scala vestibuli electrodes. Their cochleas were perfused with either 9% or 17% solutions of neomycin, and survival times ranged from 110 to 273 days. Three had been run daily for several months in behavioral experiments and received stimulation through their implants for about an hour each day. Six animals were part of a pilot study to determine whether graded lesions of the spiral ganglion could be obtained by perfusing the cochlea with different concentrations of neomycin. The left cochlea of each rat was perfused with a 4%, 9% or 17% solution of neomycin, and the animals were allowed to survive for either 30 or 60 days before a single stimulating electrode was implanted in the cochlea. I~ediately afterward the EABR was recorded for a single stimulus intensity series, and the animals were sacrificed.
Main experiment The left cochleas of 22 rats were perfused with one of four concentrations of neomycin (48, 9%, 178, 35%) or the saline vehicle during the same operation in which a single electrode was implanted in the cochlea. The EABR was recorded for a stimulus intensity series immediately afterward and then four more times at approximately 30-day intervals. In order to ensure that there would be little spiral ganglion cell survival in a few subjects, approximately 1000 or fewer cells, the cochleas of three animals that were perfused with the 35% neomycin solution were also damaged mechanically by probing with a pin in the apical hole used for the perfusion. For 12 of the 22 rats the final recording session contained a second intensity series in which the animals were paralysed by a single dose of tubocurarine (1.5 mg) to eliminate muscle contaminants of the EABR resulting from stimulation of
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the facial and vestibular nerves at relatively high stimulus levels. Tracheal canulas were installed before the recording session so that the animals could be respired during paralysis.
TABLE I NUMBER OF SURVIVING SPIRAL GANGLION IN RATS WITH NEOMYCIN LESIONS Rat
Results Pilot studies Illustrative EABR waveforms for part of an intensity series are shown in Fig. 1. Successive positive and negative peaks have been numbered serially in a purely descriptive way. P1 is clearly the CAP of the auditory nerve. With high current levels its peak latency can be as short as 80 ps; with near threshold stimuli the peak latency does not exceed 290 ps. Section of the eighth nerve in the internal meatus abolishes all of the later waves immediately and P1 after some minutes. The P1 peak is clearly resolved in most records, although the stimulus artifact encroaches on the rising limb of the potential at high stimulus levels. The six animals whose cochleas were perfused with different concentrations of neomycin had spiral gangion cell losses that were directly related
NM-8 NM-9 NM-6 NM-5 NM-10 NM-7
Neomycin Concentration
Survival Period
4% 4% 9% 9% 17% 17%
30 60 31 59 33 60
days days days days days days
CELLS
Spiral Ganglion Cells Type1
Type11
Total
15,432 9,879 14,217 10,224 4,485 6,928
1,228 740 700 1,026 245 316
16,600 10,619 14,917 11,250 4,730 7,244
to the concentration of the drug. The spiral ganglion cell counts for these animals are presented in Table I, where there is some suggestion that the longer survival time might also have led to greater spiral ganglion cell loss, except at the highest neomycin concentration. A measure of maximum P1 amplitude was not available for one of these animals (NM-5) because the intensity series was terminated at 600 PA
50
-
p1
P2
msec
msec
Fig. 1. Electrically evoked auditory brainstem responses (EABR) comprising a partial intensity series from one subject. Traces are averages of 2000 responses. Stimulus onset is at time 0, and the initial deflection before Pi is stimulus artifact. The distance between successive tick marks on the ordinates equals 2 PV for all responses except the last at 400 PA where it equals 4 pV.
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.
i
L
c
2
Number
4
6
of
Spiral
8
10
Ganql~on
12
Cells
14
16
between the correlation coefficients for the slope and maximum amplitude measures is real, but data from the main experiment suggest it probably is not. The observations described below for the main experiment were made to confirm the strong relationship indicated by these initial observations between the amplitude of P, and the number of excitable spiral ganglion cells. Such a confirmation based on more extensive systematic data seemed desirable as a basis for the development of a similar measurement technique for humans.
‘8
ilOOO’s)
Fig. 2. Slopes of P, growth functions plotted against the number of surviving spiral ganglion cells for the six rats whose spiral ganglion cell counts are presented in Table I.
because the facial muscles were strongly excited by the electrical stimulus. Another measure of P,, the slope of its growth function, was therefore examined initially so that all six subjects could be included in the analysis. That measure is plotted against the number of surviving spiral ganglion cells in Fig. 2 where a rather strong positive correlation, r = 0.79, is apparent. The correlation was high enough to suggest that the slopes of PI growth functions might be a useful index of surviving spiral ganglion cells if the correlation could be confirmed in a larger sample of the deaf rats. A similarly strong relationship was found in the regression of maximum PI amplitude on the number of surviving spiral ganglion cells shown in Fig. 3. Data from five of the animals included in Fig. 2 (excluding the rat for which this measure was not available) are presented together with data from other animals for which the EABR measures and spiral ganglion cell counts were available prior to completion of the main experiment. The correlation is high, r = 0.92, and is confirmation of the expected relationship between maximum P, amplitude and the number of excitable auditory nerve fibers. The relationship appears to hold for a rather wide range of experimental conditions, that is, with differences in the type and location of stimulating electrodes, with differences in survival times following implantation and in the amount of stimulation the animals received. The sample sizes are too small to know whether the difference
Main experiment Satisfactory data were not obtained from 6 of the 22 animals for the following reasons: One rat died, apparently of a respiratory infection. The lead to the cochlear electrode broke in two rats shortly before the final session and could not be repaired. P, could not be measured with confidence in three rats because of unusually long stimulus artifacts. The unusual artifacts did not appear to be related to spiral ganglion cell survival, which ranged from 7% to 48% in the three animals. EABRs recorded as part of an intensity series from one animal are shown in Fig. 4 to illustrate one kind of muscle contaminant that made it
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ipiral
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311s
14
16
18
(1000’s)
Fig. 3. Maximum P, amplitude plotted as a function of the number of surviving spiral ganglion cells for animals treated in different ways. See text for details. The neomycin pilot rats are five of the six animals represented in Fig. 2. The regression line is a least squares best fit as it is in all plots of this kind presented here.
129
Stim.
450 400 350 300 250 200 150 100
1
0
msec
4.5
Fig. 4. EL4BR.s from part of an intensity series for rat NM-11. What appears to be a large increase in waves N, and P4 in the top trace (for the 500 PA stimulus) is a myogenic response originating in the digastric muscle as a result of facial nerve activation.
necessary to paralyse some animals in order to measure the late waves of the EABR. The N, and P4 waves were supplanted at the highest stimulus intensity shown (500 PA) by a diphasic response from the posterior belly of the digastric muscle, which is innervated by motoneurons of the facial nerve (Hall, 1989). The presence of such responses is readily documented if one continues to measure the amplitudes of the N, and P4 peaks, with which the muscle potentials coincide, as shown for the same animal in Fig. 5. The N, and P4 peaks are typically asymptotic before the digastric potentials appear. The large and rapid increases in what appear initially to be the N3 and P4 waves reach amplitudes much in excess of any auditory brainstem responses. The latencies of the muscle potentials are very close to those of the N, and P4 peaks, as measured in animals following eighth nerve section or in normal animals at high intensities when the responses are extremely large and the N3-P4 contribution is negligible. In each of the four recording sessions represented in the figure the relatively small N3-P4 auditory responses gave way to the much larger muscle potentials at high current levels. The growth of the digastric response in Fig. 5 appears to be associated with a second period of
growth at high current levels in the functions for the Ni and P2 waves. These increases in Ni and PZ, however, do not reflect synaptically mediated muscle responses because they are not abolished by curare, as shown in Fig. 6. The origins of these increases in Ni and P2 are uncertain. They do not appear to be of facial nerve origin as stimulation of the nerve at its exit from the stylomastoid foramen does not result in a prominent negativepositive complex of short latency in the vertex response, even in the presence of large digastric potentials. Our working hypothesis is that the Ni and Pz increases at high current levels are related to vestibular activation. The digastric potentials, however, were eliminated by curare, as evidenced by the absence of any large increase in the N,--P, measure following administration of the drug. Curarization had no systematic effect on any other components of the EABR. The EABR was quite stable over a period of four months or more when no intentional damage was done to the cochlea. This is illustrated in the growth functions of Fig. 5 for subject NM-11, a control animal whose cochlea was not perfused with neomycin or saline. In contrast, neomycin poisoning led to systematic decreases with time in most animals, as illustrated in the growth functions of Fig. 7 from subject NM-26 whose cochlea was perfused with a 35% neomycin solution. Several other features of the curves in Fig. 7 are of interest. First the increases in N, and P2 at high intensities, like those shown in Figs. 5 and 6, are not accompanied by similar increases in Pi. In nearly every record maximum Pi amplitude could be determined because the P1 functions had reached asymptote. That was equally true of the later waves, P3 and P4, as long as there were no muscle contaminants. The initial asymptote of P2 was always clear, even when the growth function had a second rising limb at high current levels. On the other hand, although the initial asymptote of N1 was sometimes clear, the growth function frequently revealed no clear maximum, as in Figs. 5 and 6, and this was usually reflected in the Pi-N, peak-to-peak amplitude measure, as illustrated for NM-26 in Fig. 7. The measure of Pi-N, amplitude appeared to have several advantages over peak amplitude measurements of Pr, but the absence of a clear asymptote in many Pi-N, growth func-
130 50
50 PI
940
P2
40
3 ; 30 .% E
30
$ 20 Y : fk 10
20
Subject
NM-l
1
10
0 400 N3-P4 300 0 Day Implanted 0 64 Days Later l
200
96 Days Later
a 131 Days Later 100
0 200
400 Stimulus
600 Current
1000
(~~
0
200
400 Stimulus
Fig. 5. Growth functions for the same subject whose EABRs appear in recording sessions. Data from the second session for this subject were appearance and rapid growth of the digastric potentials is indicated different scale on the ordinate
tions made it necessary to examine other metrics to characterize PI-N, amplitude and its relationship to surviving spiral ganglion cells. Maximum PI amplitude is the measure of the auditory nerve CAP that is related most directly in theory to the number of excitable spiral ganglion
TABLE
1
600
800
Current
1000
(~)
Fig. 4. The functions in each graph are from four different marred by technical difficulties and are not presented. The in the N,-P, peak-to-peak amplitude measure. Note the for the measure.
cells; maximum PI amplitude should be directly proportional to the number of excitable auditory nerve fibers. This kind of relationship is shown in Fig. 8 were it is clear that the theoretical prediction is confirmed again. The correlation coefficient (r = 0.77) is slightly lower than the one found
II
CORRELATIONS
(R) BETWEEN
EABR WAVES
AND SURVIVING
TYPE 1 SPIRAL Peak-to-peak
Peak amplitudes
GANGLION
CELLS
amplitudes
9
p2
P3
P4
PI-N,
W
amplitude at 500 pA
0.77 0.75
0.47 0.45
0.40 0.55
0.27 0.43
0.81
0.50 0.69
0.59 0.61
0.41 0.72
Slope of growth function to 500 pA
0.84
0.42
0.18
0.41
0.90
0.66
0.62
0.65
Maximum Amplitude
-P2
N2
-9
W-P‘,
Pl
Subject
30-
-
400’
’
*
*
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’
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N3-P4
Nl l
NM-22
300
I
Before Curarization
0 After Curarizotion
0
200
400 Stimulus
1000
0
CuGPeent ($)O
200
400
600
Stimulus Current
600
1000
(pA)
Fig. 6. Growth functions for rat NM-22 obtained in the final session before and after the animal was curarized to eliminate the digastric muscle response, as indicated by the plots for the N3-P4 amplitude measure. Amplitudes of the auditory components of the EABR were not influenced by the curarkation. Increases in the NI and P2 waves at high stimulus intensities are not of myogenic Oligill.
in the initial observations described above, due apparently to a few deviant measures. It seemed worthw~~, therefore, to examine other measures of Pi that might be less sensitive to errors of measurement. The correlations between four other measures of Pr and the number of surviving spiral ganglion cells are shown in Fig. 9. EABRs at 500 PA, where nearly maximum values of Pi are obtained in most animals, appear to be freer of residual stimulus artifacts than responses at higher intensities, and the regression of Pr peak amplitude at 500 I_LAon the number of spiral ganglion cells, as shown in the lower left of Fig. 9, is at least as strong as that for maximum P, amplitude. A subm~um measure of P, amplitude, moreover, is the kind that may be most useful in human subjects who are not
likely to tolerate electrical stimuli strong enough to evoke the maximum CAP. The slopes of growth functions have the same advantage, and in addition may be less ‘noisey’ in as much as they are derived from a series of measurements rather than a single measure of amplitude. The advantage of the P,-N, peak-to-peak amplitude measure is that it seems less sensitive to residual tails of artifacts that may shift the Pt peak upward or downward from the baseline if the cancellation of the electrical artifact is not complete, as is frequently the case at high stimulus intensities. The high correlation between the slope of Pr-N, growth functions and the number of surviving spiral ganglion cells (r = 0.90) may reflect the advantages of both peak-to-peak amplitude measures and the use of slope as the final metric. In any case, it is clear
r
60
PI
5o
Pl-Nl Subject
NM-26
Nl
Nl -P2
0 Day Implanted /
0 34 Days Later n
66 Days Later
a 99 Days Later A 132 Days Later
200
400
Stimulus
600
Current
600
1000
(pA)
Fig. 7. Growth functions for the early components of the EABR recorded at approximately monthly intervals from rat NM-26 whose cochlea had been perfused with a 35% neomycin solution. The systematic decline of the response presumably reflects a progressive loss of spiral ganglion cells, as indicated by the diminishing P, amplitude over time.
r =
.
.77
.
/
.
. . 0
.
2
4
Number
6
8
10
12
of Spiral Ganglion
14
16
IS
20
22
Cells (1000’s)
Fig. 8. Maximum P, amplitude plotted as a function of the number of surviving type I spiral ganglion cells for 16 rats whose cochleas had been perfused with different concentrations of neomycin.
from all of the plots in Fig. 9 that the amplitude of Pi and the slopes of its growth functions, measured in various ways, are highly correlated with the number of surviving type I spiral ganglion cells in deaf rats. Because the earliest waves of the EABR can be difficult to record one would like to know how the later components of the response are correlated with the number of surviving spiral ganglion cells. Correlation coefficients for several amplitude measures of the later waves are presented in Table II. The correlations for the Pi and Pi-N1 measures are also presented to facilitate comparisons between the coefficients for the auditory nerve responses and the later brainstem waves. The correlations between ganglion cell counts and peak amplitudes of P2, P3 and P4, measured in three different ways, are low; only one, P3 amplitude at
133
:
r = .90
z.08
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9
.
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-
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Number
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Gonglion
+
l
Nukb:r
of6
Spiral
Cells (1000’s)
Gonglion
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’
(1000’s)’
Fig. 9. Four different measures of Pt plotted as a function of the number of surviving spiral ganglion cells. The different measures are indicated on the ordinates of the four graphs. Slopes of the P, and PI-N, growth functions were based on response amplitudes up to a stimulus intensity of 500 PA_ All four measures show a strong relationship between Pr amplitude and the number of spiral ganglion cells.
Number
of
Spiral
Ganglion
Cells
(1000’s)
Fig. 10. Ns-P4 peak-to-peak amplitude plotted as a function of the number of surviving spiral ganglion cells reveals the best correlation between any of the later EABR waves and spiral ganglion cell counts in the present data.
a stimulus intensity of 500 ,uA, is statistically reliable. The picture is somewhat better for the peak-to-peak ~plitude measures, although it is clear that the correlations are for the most part much weaker than those for Pi. It is possible, however, that even the weaker relationships might be useful under some circumstances. The strongest of these relationships in the present data is shown in Fig. 10 as the regression of NJ-P4 amplitude at 500 yA on the number of surviving type I ganglion cells. It remains to be seen whether this relationship is sufficiently stable to have practical utility. Discussion Reduction of the electrical stimulus artifact has made it possible to make systematic measurements
134
of the auditory nerve CAP when it is recorded as the first positive wave of the EABR. The measurements in both sets of data presented here clearly confirm the theoretical prediction that the maximum amplitude of P, is proportional to the number of excitable auditory nerve fibers. The assumption underlying the present measurements is that counts of spiral ganglion cell bodies provide good estimates of the number of excitable fibers. There is some indication in the literature that counts of spiral ganglion cells in the rat, like those reported by Keithley and Feldman (1979), yield estimates of the co&ear nerve population that are smaller than those obtained by counting fibers in the trunk of the nerve (Hoeffding and Feldman, 1988). Hoeffding and Feldman thought it likely that the differences reflected differences in the precision attainable with the two counting methods. Losses occurring with age, however, were percentagewise quite similar in the two studies, which suggests that counts of cell bodies in Rosenthal’s canal are a relatively constant proportion of fiber counts. That kind of constant error would have no effect on the correlations reported here between the various measures of P, and surviving spiral ganglion cells. It would lead to a systematic underestimation of surviving auditory nerve fibers from the regression equations based on the present data. The discrepancy between cell body and fiber counts in the rat should be resolved by making both types of measurements in the same animals. The measures of P1 amplitude used to determine the correlations with surviving spiral ganglion cells were measures of absolute amplitude, not measures that were normalized to reduce the variability resulting from differences in recording conditions or in other factors that influence the measurement of gross neural potentials. Stimulating and recording conditions in the rat were sufficiently uniform to permit this. Nevertheless, there was enough variability in some of the measures to make estimations of spiral ganglion cell populations possible only within rather large error bounds. In the main experiment the slopes of P,-N, growth functions yielded the highest correlation with surviving spiral ganglion cells and, consequently, the smallest standard error of estimate. The data from the upper righthand graph in
”
“2
!J4
Siope of Pl
1)6
Nl
Growth
08
13
Function
Fig. If. The regression of spiral ganglion cell counts on the slope of P,-N, growth functions (thick line) plus and minus the standard error of estimate (thin lines). The data from the upper righthand graph in Fig. 9 have been replotted here to emphasize the potential for predicting spiral ganglion cell populations from the physiological index.
Fig. 9 have been replotted in Fig. 11 to show the standard error in estimating the number of spiral ganglion cells from the slope of P,-N, growth functions. The plot says that about two thirds of the time our predictions should not err by more than 2300 cells. Additional experience in recording the rat’s EABR will probably reduce that error, but even this kind of predictability may be difficult to achieve in humans. The variability in EABR amplitudes is likely to be greater than it is in the rat or other laboratory animals as a consequence of greater individual differences in subjects as well as greater variability in stimulating and recording conditions. Slopes of growth functions, which should be independent of differences in absolute amplitude, may provide a way to normalize the human data, all the while retaining other advantages of that metric mentioned above. The high correlation between the slopes of Pi-N, growth functions and spiral ganglion cell counts found in the present study suggest that such measures may be useful for estimating spiral ganglion cell populations in deaf people. The generally low correlations between the amplitudes of the later EABR waves and the counts of spiral ganglion cells may explain, in part at least, why earlier attempts to demonstrate a relationship between EABR amplitudes and the status
135
of the auditory nerve met with mixed success (e.g., Miller et al., 1983; Smith and Simmons, 1983; Stypulkowski et al., 1986). There is clearly a positive relationship between the amplitudes of the later components and the amplitude of Pi, as is obvious in the growth of the entire response with increasing stimulus intensity. But the intrasubject correlations seen in growth functions for the various waves recorded from an individual are not the correlations of interest. Across-subject correlations are. In the main experiment presented here across-subjects correlations between Pi amplitude and the amplitudes of the later waves, measured both as peak and peak-to-peak excursions, ranged from 0.12 to 0.59. The amplitudes were measured at a stimulus level of 500 PA for this analysis. If Pi is a good predictor of surviving spiral ganglion cells, other measures of the EABR that show only modest correlations with Pi are not likely to provide much information about the status of the auditory nerve. The electrically evoked middle latency response (EMLR) has been the subject of a series of studies concerned with its possible utility in assessing the status of the auditory pathways, including the auditory nerve, in humans (e.g., Kileny and Kemink, 1987) and animals (Burton et al., 1989). Jyung et al. (1989) have recently reported that following aminoglycoside poisoning the slopes of EMLR growth functions from individual guinea pigs showed orderly decreases with time that reflected reductions in spiral ganglion cell densities. Different animals were sacrificed at progressively longer intervals following the administration of kanamycin and ethacrynic acid, and the longer survival times were associated with progressively greater reductions in spiral ganglion cell densities. It is not yet clear how robust the correlation is between the slopes of EMLR growth functions and spiral ganglion cell densities in the animal model. The EMLR may be subject to the same kinds of limitations as the later waves of the EABR in assessing the status of the auditory nerve. The MLR, moreover, is more sensitive than the ABR to state variables, including anesthetics, (e.g. Smith and Kraus, 1987) and it remains to be seen how that sensitivity might compromise its use in humans where it is generally more variable than the ABR (e.g., Ozdamar and Kraus, 1983). Predic-
tions of spiral ganglion cell populations from EMLR measures are bound to err when central lesions interupt ascending auditory pathways in the absence of peripheral lesions. Stimulating and recording conditions were quite favorable for obtaining good quantitative measures of the auditory nerve CAP in the present experiments. Such conditions may be harder to realize in human subjects, especially when the CAP is used for screening purposes under the assumption that only extracochlear stimulation is permissible. Intracochlear stimulation in the rat permitted the use of relatively small currents of very brief duration, tending thereby to minimize stimulus artifacts. Similarly, intracranial recording electrodes yielded much higher signal levels and better signal/noise ratios than do scalp electrodes used with human subjects, and those ratios as well as the general stability of the recording conditions were enhanced by the use of anesthesia. The advantages of intracochlear electrodes have already been realized in several EABR studies of humans with cochlear implants (Abbas and Brown, 1988; Thornton and Hermann, personal communication; van den Honert and Stypulkowski, 1986). Unfortunately, wave I was not a consistent feature of the responses found in any of those studies. More recently, however, Brown et al. (1989) have been able to extract the CAP from potentials recorded intracochlearly from subjects implanted with Symbion electrodes. They modified a forward masking paradigm first described by Charlet de Sauvage et al. (1983) to eliminate much of the stimulus artifact. Using an electrical masking stimulus and a subtraction procedure they were able to extract from the potentials recorded intracochlearly at one electrode the CAP evoked by stimulation at two other intracochlear electrodes. Modest correlations were found between the slopes of the growth curves and scores on two tests of word recognition. Somewhat stronger correlations were found between the word recognition scores and the slopes of recovery functions measured with increasing intervals between masking and probe stimuli. The findings, regarded as tentative in view of the small sample, emphasize nevertheless the possibility that measures of auditory nerve responses in the deaf may have appreciable utility in predicting success with cochlear implants. Be
136
that as it may, there is still the problem of obtaining good measures of the CAP in candidates for cochlear implants. Whether or not any of the procedures used in the present study will prove useful for that purpose, we hope that the demonstration of the relationship between P, and the number of surviving spiral ganglion cells will add impetus to the search for a suitable method. Acknowledgements The author is most grateful to Elizabeth P. Sonnabend and Jennifer L. Massengill for their technical assistance, to Elizabeth M. Keithley for teaching us how to prepare histological sections of the cochlea, to Joseph B. Nadol, Jr. for his continuous encouragement and support, and to Donald K. Eddington, Nelson Y.-S. Kiang and Aaron R. Thornton for many helpful discussions throughout this research. References Abbas, P.J. and Brown, C.J. (1988) Electrically evoked brainstem potentials in cochlear implant patients with multielectrode stimulation. Hear. Res. 36, 153-162. Black, R., Clark, G., Shepherd, R., O’Leary, S. and Walters, C. (1983) Intracochlear electrical stimulation. Brainstem response audiometric and histopathological studies. Acta Otolaryngol., Suppl. 399, 5-17. Brightwell, A., Rothera, M., Conway, M. and Graham, J. (1985) Evaluation of the status of the auditory nerve: psychophysical tests and ABR. In: R.A. Schindler and M.M. Merzenich (Eds.), Cochlear Implants, Raven Press, New York, pp. 343-349. Brown, C.J., Abbas, P.J. and Gantz, B. (1989) Electrically evoked whole-nerve action potentials recorded in cochlear implant users. Assoc. Res. Otolaryngol. Abst., p. 274. Burton, M.J., Miller, J.M. and Kileny, P.R. (1989) Middlelatency responses. I. Electrical and acoustic excitation. Arch. Otolaryngol. Head Neck Surg. 115, 59-62. Charlet de Sauvage, R., Cazals, Y., Erre, J.P. and Aran, J.M. (1983) Acoustically derived auditory nerve action potential evoked by electrical stimulation. An estimation of the waveform of a single unit contribution. J. Acoust. Sot. Am. 73, 616-627. Chouard, C.H., Meyer, B. and Gegu, D. (1985) Pre- and per-operative electrical testing procedure. In: R.A. Schindler and M.M. Merzenich (Ed%), Cochlear Implants, Raven Press, New York, pp. 365-374. Goldstein, M.H. and Kiang, N.Y.-S. (1958) Synchrony of neural activity in electric response evoked by transient acoustic stimuli. J. Acoust. Sot. Am. 30, 107-114. Hall, R.D. (1989) Electrically evoked auditory brainstem re-
sponse in the rat: Its use in estimating spiral ganglion cell populations and the effects of facial nerve activation. Assoc. Res. Otolaryngol. Abst. p. 24. Hoeffding, V. and Feldman, M.L. (1988) Changes with age in the morphology of the cochlear nerve in rats: Light microscopy. J. Comp. Neurol. 276, 537-546. Jyung, R.W., Crowther, J.A., Marsal, S. and Miller, J.M. (1989) Prediction of eighth nerve survival using the electricallyevoked middle latency response. Assoc. Res. Otolaryngol. Abst., p. 29. Keithley, E.M. and Feldman, M.L. (1979) Spiral ganglion cell counts in an age-graded series of rat cochleas. J. Comp. Neurol. 188, 4299442. Kiang, N.Y.S., Keithley, E.M. and Liberman, M.C. (1983) The impact of auditory nerve experiments on cochlear implant design. AM. N.Y. Acad. Sci. 405, 114-121. Kiang, N.Y.S., Moxon, E.C. and Kahn, A.R. (1976) The relationship of gross potentials recorded from the cochlea to single unit activity in the auditory nerve. In: J.B. Ruben, C. Elberling and G. Salomon (Eds.), Electrocochleography, University Park Press, Baltimore, pp. 95-115. Kileny, P.R. and Kemink, J.L. (1987) Electrically evoked middle-latency auditory potentials in cochlear implant patients. Arch. Otolaryngol. Head Neck Surg. 113, 1072-1077. Miller, J.M., Duckert, L.G., Malone, M.A. and Pfingst, B.E. (1983) Cochlear prostheses: Stimulation induced damage. Ann. Otol., Rhinol. Laryngol. 92, 599-609. Ozdamar, 0. and Kraus, N. (1983) Auditory middle-latency responses in humans. Audiology 22, 34-49. Shepherd, R.K., Clark, G.M. and Black, R.C. (1983) Chronic electrical stimulation of the auditory nerve in cats. Acta Otolaryngol., Supp. 399, 19-31. Simmons, F.B. (1979) Electrical stimulation of the auditory nerve in cats. Long-term electrophysiological and histological results. Ann. Otol. 88, 533-539. Simmons, F.B., Lusted, H.S., Meyers, T. and Shelton, C. (1984) Electrically induced auditory brainstem response as a clinical tool in estimating nerve survival. Ann. Otol. Rhinol. Laryngol. 112,97-100. Smith, D.I. and Kraus, N. (1987) Effects of chloral hydrate, pentobarbital, ketamine, and curare on the auditory middle latency response. Am. J. Otolaryngol. 8, 241-248. Smith, L. and Simmons, F.B. (1983) Estimating eighth nerve survival by electrical stimulation. Ann. Otol. Rhinol. Laryngol. 92, 19-23. Stypulkowski, P.H., Van den Honert, C. and Kvistad, S.D. (1986) Electrophysiologic evaluation of the cochlear implant patient. Otolaryngologic Clin. N. Am. 19, 249-257. Van den Honert, C. and Stypulkowski, P.H. (1986) Characterization of the electrically evoked auditory brainstem response (ABR) in cats and humans. Hear. Res. 21, 109-126. Walsh, S.M. and Leake-Jones, P.A. (1982) Chronic electrical stimulation of auditory nerve in cat: physiological and histological results. Hear. Res. 7, 281-304. Yamane, H., Marsh, R.R. and Potsic, W.P. (1981) Brain stem response evoked by electrical stimulation of the round window of the guinea pig. Otolaryngol. Head Neck Surg. 89, 117-124.
