Spiral ganglion cell site of excitation I: Comparison of scala tympani and intrameatal electrode responses

Hearing Research

Hearing Research 215 (2006) 10–21

www.elsevier.com/locate/heares

Research paper

Spiral ganglion cell site of excitation I: Comparison of scala tympani and intrameatal electrode responses Lianne A. Cartee a

a,*

, Charles A. Miller b, Chris van den Honert

c

Joint Department of Biomedical Engineering, North Carolina State University and the University of North Carolina at Chapel Hill, Box 7625, Raleigh, NC 27695, United States b Department of Otolaryngology, Head and Neck Surgery University of Iowa, Iowa City, IA, United States c Cochlear Americas Englewood, CO, United States Received 21 February 2006; accepted 28 February 2006 Available online 18 April 2006

Abstract To determine the site of excitation on the spiral ganglion cell in response to electrical stimulation similar to that from a cochlear implant, single-fiber responses to electrical stimuli delivered by an electrode positioned in the scala tympani were compared to responses from stimuli delivered by an electrode placed in the internal auditory meatus. The response to intrameatal stimulation provided a control set of data with a known excitation site, the central axon of the spiral ganglion cell. For both intrameatal and scala tympani stimuli, the responses to single-pulse, summation, and refractory stimulus protocols were recorded. The data demonstrated that summation pulses, as opposed to single pulses, are likely to give the most insightful measures for determination of the site of excitation. Single-fiber summation data for both scala tympani and intrameatally stimulated fibers were analyzed with a clustering algorithm. Combining cluster analysis and additional numerical modeling data, it was hypothesized that the scala tympani responses corresponded to central excitation, peripheral excitation adjacent to the cell body, and peripheral excitation at a site distant from the cell body. Fibers stimulated by an intrameatal electrode demonstrated the greatest range of jitter measurements indicating that greater fiber independence may be achieved with intrameatal stimulation.  2006 Elsevier B.V. All rights reserved. Keywords: Auditory nerve; Cats; Cochlear implant; Electrical stimulation; Single fiber recording

1. Introduction A topic of considerable interest to cochlear-implant research is determining the sites of neural excitation resulting from prosthetic (electrical) stimulation. Given that different excitation sites of auditory nerve fibers have different response properties (van den Honert and Stypulkowki, 1987) and that numerical models have shown that the presence of the cell body alters the expected extracellular potential predicted on the neural surface in response to electrical *

Corresponding author. Address: Joint Department of Biomedical Engineering, North Carolina State University, 2110 Faucette Dr., 118 Weaver Labs, Raleigh, NC 27695, United States. Tel.: +919 515 6726; fax: +919 515 7760. E-mail address: [email protected] (L.A. Cartee). 0378-5955/$ - see front matter  2006 Elsevier B.V. All rights reserved. doi:10.1016/j.heares.2006.02.012

field stimulation (Finley et al., 1990), the control of the excitation site could lead to improvements in the neural coding of electric stimuli. Since site-of-excitation may also be an indicator of the degree of spatial specificity of cochlear stimulation, knowledge of the site-of-excitation could assist in developing clinical stimulus protocols that offer better spatial control of the excitation process. 1.1. Prior studies of site of excitation Physiological data from animal experiments indicate that excitation at different longitudinal sites on auditory nerve fibers results in different response properties. By comparing temporal dispersion of spike times, spike latency, and rate of response growth in single units, four groups of responses have been identified and interpreted

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to represent electrophonic excitation, direct excitation of inner hair cells, peripheral excitation sites, and a central excitation site (Brownell et al., 1985; Hartmann et al., 1984; Javel and Shepherd, 2000; Moxon, 1965, 1971). Because the primary focus of this study is to understand the response of the deaf cochlea to electrical stimulation from a cochlear implant, the experiments were conducted on deafened cats. In deafened preparations, the electrophonic responses and direct excitation of the inner hair cell responses are presumably eliminated limiting possible excitation sites to the spiral ganglion neuron. Evoked compound action potential measurements at the auditory nerve trunk and intracochlear evoked potentials (IEPs) have given evidence that the site of excitation can shift with changes of stimulus level (Miller et al., 2003b; Stypulkowski and van den Honert, 1984). Through growth functions, Miller et al. (2003a) provided evidence that excitation of the spiral ganglion at a peripheral site was more likely to occur with bipolar as opposed to monopolar stimulation. Bipolar stimulation, in six out of seven cases, produced growth functions with small amplitudes at low-level stimuli over a 10– 20 dB dynamic range. At higher stimulation levels, the slope of the growth function rapidly increased. The stimulation level at which the growth function slope experienced rapid growth corresponded to levels where latency shifts occurred in the evoked compound action potential (ECAP) recorded on the nerve trunk at the round window. The results were consistent with a change of site of excitation from a peripheral area with response from a selective population of fibers to a more central excitation site with response from a large population of fibers. 1.2. Cochlear neuron anatomy If excitation were to occur on the peripheral process in a site that requires propagation across the cell body, it may be reasonable to expect a different stimulus response than if excitation were to occur on the central process. The mammalian spiral ganglion consists primarily of Type I cells with bipolar cell bodies. The central processes of the spiral ganglion neurons pass through the internal auditory meatus as part of the auditory nerve and the peripheral processes project to the organ of Corti where they synapse with the hair cells. In addition to the presence of the cell body, the spiral ganglion cell exhibits other anatomical characteristics such as constrictions in the axonal processes adjacent to the cell body, decrease in the distance between nodes at the cell body internode, thinning of the myelin on the cell body internode, and smaller peripheral diameter as compared to the central process diameter adjacent to the cell body (Adamo and Diagneault, 1973; Goycoolea et al., 1990; Kiang et al., 1982; Liberman and Oliver, 1984; Robertson, 1976). Response properties such as threshold, latency and jitter may be significantly affected by propagation across the peripheral process and cell body.

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1.3. Modeling predictions If the site of excitation on the cell is peripheral to the cell body, one might expect the capacitive effects of the cell body to influence the summation stimulus response. Because summation is a predominantly linear behavior, we assume that it is governed by the passive, linear properties of the neural membranes such as membrane resistance and capacitance. The RC (resistive/capacitive) circuit nature of the membrane determines the rate of charge and discharge of the membrane. The effects of a large cell body capacitance would be expected to hold charge longer resulting in a longer time constant for summation. Similarly, the cell body capacitance might be expected to influence the refractory behavior in response to the 2nd pulse of a two-pulse stimulus if the cell was stimulated peripheral to the cell body. If activation and repolarization were linear processes, the greater capacitance of the cell body would be expected to hold charge over a longer period of time, similar to the behavior for summation. If the greater charge carrying ability of the cell body ultimately delays repolarization, the absolute and relative refractory periods will be prolonged resulting in a larger refraction time constant. In contrast to summation, however, the behavior of the excited membrane is no longer linear. Once excitation occurs, the non-linear behavior of the action potential plays an important role in the cellular response. Variations in nodal area, channel density, and channel response rates add a non-linear, stochastic component to the membrane response. In addition, the possibility of failure of pulses initiated during the relative refractory period to propagate across the cell body may affect the response to the two-pulse stimuli for evaluating refractory behavior (Kohllo¨ffel, 1974; Robertson, 1976; Stypulkowski and van den Honert, 1984). Using a computational model, Cartee (2006) demonstrated, that the increased cell diameter of the cell body increases the summation time constant of the cell at the peripheral node adjacent to the cell body. When excitation occurred at peripheral node sites (other than the adjacent node), the summation time constant was not significantly influenced by the presence of the cell body and behaved similarly to the response when the excitation site was located at nodes central to the cell body. The model indicated that the influence of the cell body on the summation time constant prolonged the summation time constant only when the site of excitation was the single node adjacent and peripheral to the cell body. For pulse pairs producing a refractory response, the influence on the cell body on the refraction time constant was also highly localized. The computational model showed a decrease in the refraction time constant at the node peripheral to the cell body. At more peripheral sites, an increase in the refraction time constant was observed.

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1.4. Study design

2.1. Surgical preparation

In a previous paper, Cartee et al. (2000) described the response of auditory neurons to stimulation through an electrode within the internal auditory meatus, hereafter referred to as intrameatal (IM) stimulation. An intrameatal stimulus delivered in the central nerve trunk was assumed to excite the neuron on the central portion of the axon. In this study, the response of scala tympani (ST) stimulation to that of intrameatal stimulation was compared using matched protocols. This allowed comparison of response properties designed to infer the site of excitation resulting from scala tympani stimulation to properties recorded from a controlled site of excitation on the central portion of the spiral ganglion axon. Previous studies have associated patterns of spike latency and jitter with sites of excitation based on expected behavior and known anatomical features of the spiral ganglion cell. Some studies were complicated by the use of biphasic stimuli (Javel et al., 1987). With biphasic stimuli, excitation may occur in response to either phase of the current stimulus. While excitation typically occurs at a lower threshold during the cathodic phase, it is possible for stimulation to occur in response to a ‘‘virtual cathode’’ during the anodic stimulus phase (van den Honert and Stypulkowki, 1987; Rattay, 1990). The following studies attempt to eliminate as many complicating factors as possible by stimulating with a monopolar, charge-balanced pseudo-monophasic stimulus and by comparing data to control data from a presumably known site of excitation. We evaluated the response to single pulses and pulse pairs. Pulse pairs with small (0.5 ms or less) interpulse intervals (IPIs) were used to evaluate the ability of the membrane to integrate stimuli, and pulse pairs with larger IPIs (0.75 ms or greater) were used to record the refractory behavior of cells. The response to each type of stimulus was evaluated to determine if the single fibers behaved differently or similarly in response to IM and ST stimulation, indicating that the site of excitation for ST stimulation was either peripheral or central.

Cats free of middle-ear infection were anesthetized and studied in acute experimental sessions. The tympanic bulla was opened posterolaterally and the round window membrane removed. Acute deafening was performed by infusion of a 10% w/v solution of neomycin sulfate through the opened round window. A partial occipital craniotomy was performed, and the cerebellum overlying cochlear nucleus was aspirated or retracted. Glass micropipettes were placed into the auditory nerve under visual control and were advanced through the nerve with a microdrive. Experiments continued until the EABR threshold was elevated compared to the threshold recorded at the beginning of the experiment. At the conclusion of the experiment, animals were euthanized by an overdose of sodium pentobarbital.

2. Materials and methods Experimental preparations C031–C037 (group 1) were studied at the Duke University Hearing Research Laboratories. The methods used in this laboratory are described in more detail in Cartee et al., 2000. For this group, two preparations were implanted exclusively with an IM electrode, and two preparations were implanted with both IM and ST electrodes. Additional data were collected for the ST stimulation using a second group of animals (group 2). This group, C82–C96, was studied at the University of Iowa Department of Otolaryngology – Head and Neck Surgery. Methods used in this laboratory are described in more detail in Miller et al. (2001). Protocols for both groups were as closely matched as possible and were identical in terms of electrical pulse shape, delivery, and stimulus protocol.

2.2. Electrode placement For group 1 animals, a stimulating electrode was placed in the internal auditory meatus. The intrameatal electrode was fabricated from a traditional tungsten microelectrode (Frederick Haer, catalog UEWSFESE) by removing 1 mm of insulation at the tip to produce a large stimulating surface area of approximately 0.6 mm2. A bead of cynoacrylate was formed on the needle 1 mm from the tip. The electrode was inserted by hand into the internal auditory meatus through a small hole drilled by hand in the modiolar wall underlying the round window. The electrode was advanced until the cynoacrylate bead rested against the modiolar wall insuring an insertion no greater than 1 mm from the modiolar wall. Once placed, the intrameatal electrode was secured with cynoacrylate. Placement of the intrameatal electrode within the internal auditory meatus was verified post-mortem by gross dissection. The scala tympani electrode was a Pt/Ir ball electrode. The electrode was placed by hand insertion into the first turn of the cochlea beyond the hook and cemented into place with cynoacrylate. A subcutaneous needle in the neck served as the return electrode for both scala tympani and intrameatal stimulation. A subcutaneous needle in the forepaw served as return electrode. 2.3. Stimulus generation Stimuli were generated through a custom software controlled D/A system (200,000 samples/s, group 1; 100,000 samples/s, group 2) connected to a capacitively coupled, optically isolated current source. All stimuli were ‘pseudomonophasic’. Pseudo-monophasic waveforms are rectangular current pulse follow by a small, prolonged current of the opposite polarity. The pseudo-monophasic current pulse was produced by passing monophasic stimulus currents through a series 5 lF capacitor. An equation defining the magnitude of the 2nd phase of the pseudo-monophasic

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current as a function of time for this size capacitor is given by Cartee (2000). 2.4. Recording techniques Potentials from the micropipette were amplified and bandpass filtered (30 or 100 Hz low frequency corner and 3 or 10 kHz high frequency corner) prior to sampling (group 1: 200,000 samples/s; group 2: 100,000 samples/s). Artifact subtraction was implemented using a template subtraction technique. Templates were acquired from stimuli that did not generate a response. Spike latency was detected by a level descriminator and stored (group 1), or determined off-line from digitally stored waveforms (group 2). Additional details of the recording techniques are reported in Cartee et al. (2000) for group 1 and in Miller et al. (1999) for group 2. 2.5. Data analysis For all animals, either post-stimulus time histograms (PSTs) or digitized spike waveforms were analyzed to verify that the activity was electrically driven. Events that occurred more than 1 ms following stimulus onset were rare and were ignored as latencies greater than 1 ms are typically attributed to electrophonic or synaptic excitation (van den Honert and Stypulkowski, 1984). Single pulse threshold (SPT) was defined as the stimulus magnitude of a single pulse resulting in a 50% response probability. Stimulus magnitude was varied over a range of values resulting in low, moderate, and high response probabilities. From the resulting I/O function, threshold was calculated by linear interpolation. Threshold was calculated from both single pulses and from the response to the first pulse of each pulse pair with an IPI of 1 ms or longer. (IPI was defined as the time from onset of the first pulse until onset of the second pulse of the pulse pair.) If the threshold was not stable throughout the recording period, shifting by more than 1 dB, the fiber was not included in the analysis. For all protocols using paired stimuli, the magnitude of the two stimulus pulses in the pair were identical and covaried over a range of amplitudes producing low, moderate, and high response probabilities. As for threshold determination, I/O curves were recorded, and the summation and refraction thresholds were computed by linear interpolation. Summation threshold was defined as the stimulus magnitude resulting in a 50% response probability to the summation pulse pair. Refraction threshold was defined as the stimulus magnitude resulting in a 50% response probability for the 2nd pulse of a pulse pair given that the first pulse of the pair generated an action potential. For each fiber with summation data at more than one IPI, a time constant relating the change in summation threshold to the IPI was determined. The data were fit to the following exponential equation:

IPI

ST ¼ 0:5essum þ 1:0

13

ð1Þ

where ST is the summation threshold normalized to SPT; IPI is the interpulse interval; and ssum is the summation time constant. The form of the equation assumes that the summation threshold will be half the SPT for overlapping pulses (IPI = 0) since the two equal-amplitude pulses would sum to a value equivalent to the magnitude of the SPT. At large IPIs, it is assumed that the summation threshold will be equal to the SPT. Similarly, for fibers with refractoriness data at more than one IPI, a refraction time constant relating refraction threshold to IPI was determined from the following equation: RT ¼

1 1e

sIPI

ð2Þ

ref

where RT is the refraction threshold normalized to SPT; IPI is the interpulse interval; and sref is the refraction time constant. The form of the equation assumes that the refraction threshold will equal the SPT at large IPIs, and will be infinite (absolutely refractory) for very small IPIs. For animals C82–C96, latency was defined as the time from stimulus onset until the occurrence of the maximum first derivative of the rising phase of the action potential. This point has previously been related to the time of excitation (Spach and Kootsey, 1985) and approximated the level of the voltage discriminator used to detect spike times in animals C031–C037. For animals C031–C037, latency was defined as the time from stimulus onset until spike discrimination. Reported latencies are the mean predicted latencies at threshold (50% firing efficiency) determined from linear interpolation of the measured latencies at low, medium, and high stimulus levels with 100 trials at each level. Jitter was defined as the standard deviation of the latencies predicted at threshold determined from the linear interpolation of the jitter calculated for each stimulus level tested. 2.6. Stimulus protocol For group 1, SPT was determined using a series of single 50 ls monopolar, cathodic, pseudo-monophasic pulses. For group 2, SPT was found from the threshold of the first pulse in an equal-amplitude pulse pair with IPI of 2 ms or greater. Each pulse of the pair was a 50 ls monopolar, cathodic, pseudo-monophasic pulse. Stimuli were presented in sets of 100 single pulses or pulse pairs at a repetition rate of 13 Hz. SPT was always determined before the response to other pulse pairs was evaluated. Data are reported only for stable fibers where it was possible to maintain contact with the single fiber and continue recording activity after the initial threshold measurement. All pulse pairs were equal-magnitude, monopolar, cathodic and pseudo-monophasic. For the summation protocol, interpulse intervals (IPIs) of 100, 200 and 300 ls were used. For the refractoriness protocol, the responses to IPIs of

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0.75, 1, 2, 3 and 5 ms were evaluated. The order of the two subsets within the protocol (refractoriness and summation) was randomly varied with the exception of one animal. For C96, the summation protocol was always completed before beginning the refractoriness protocol. Within the summation and refractoriness subsets, the order of the IPIs delivered was randomly varied except that for group 2, the IPI used for threshold detection was typically not repeated in the refractoriness protocol. 2.7. Animal care and handling The care and use of the animals reported in this study were approved by Duke University IACUC 8574-9411R5, the University of Iowa protocol #0105095 and Research Triangle Institute IACUC A525-96-11R7. Use of animals was approved and supported by NIH/NIDCD (grant R29 DC02822). 3. Results Single pulse threshold, latency, and jitter values for fibers stimulated by IM and ST electrodes are shown in Fig. 1 in response to a single, 50 ls stimulus. Stable threshold data was measured from 33 IM stimulated and 64 ST stimulated fibers. The histograms are reported as the percentage of fibers responding for each stimulus location (IM or ST). IM stimulated threshold values are significantly lower than ST stimulated threshold values, as expected given the placement of the stimulating electrode within the neural tissue. However, some fibers responded to ST stimulation with thresholds within the range of those seen with IM stimulation. Presumably, the overlap in threshold ranges occurs for fibers relatively distant from the IM stimulating electrode and fibers relatively near the ST stimulating electrode. Latency values at single-pulse threshold were not significantly different (P = 0.37, T = 0.9, DF = 95) for IM and ST stimulation. The similar values for latencies of IM stimulated and ST stimulated fibers can be seen in the center panel of Fig. 1. The bottom panel of Fig. 1 shows jitter values for single-pulse threshold. The IM stimulated fibers demonstrate some of the higher jitter values at threshold level. While the jitter values show a great deal of overlap, a t-test reflects a significant difference in the distribution of jitter for the two populations (P = 0.005, T = 2.86, DF = 95). Based on the single-pulse threshold, latency, and jitter values, there is no compelling evidence for different sites of excitation for fibers stimulated by ST electrodes in comparison with those stimulated at a central excitation site with an IM electrode. We assessed single fiber membrane integration and refractoriness for each electrode location by calculating the summation and refraction time constants of individual fibers. Fig. 2 shows the data recorded for a representative fiber excited by ST stimulation. The top panels show the I/O functions recorded for IPIs falling within the range

Fig. 1. Threshold, latency and jitter values in response to single-pulse stimulation. Bars show the percent of either IM or ST stimulated fibers with values falling within the designated range.

of either the summation protocol (top left) or the refractory protocol (top right). The summation and refraction thresholds are shown on the figure as the point where the interpolating line connecting data points at a given IPI intersects the 50% response rate line. For refraction thresholds, the spike probability is the probability of activation occurring in response to the 2nd pulse of the pulse pair given that the 1st pulse of the pair resulted in activation of the fiber. The bottom panels of Fig. 2 show the summation and refraction thresholds normalized to SPT calculated from the I/O functions shown in the top panel. For the summation thresholds, the value of ssum resulting in the best-fit curve using Eq. (1) was found. Likewise, for the refraction thresholds, the value of sref resulting in the best fit of Eq. (2) to the data was found. The best-fit curves to the data in Fig. 2 are shown. Fig. 3 shows the summation and refraction time constants vs. latency for the summation and refraction responses calculated for all IM and ST stimulated fibers. Latency was chosen for the ordinate of the plot as latency

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Fig. 2. Data recorded from fiber C037-11. Top right: I/O functions recorded at the IPIs shown for several levels of stimulus magnitude are shown. The summation threshold was defined as the point where the line interpolating adjacent data points intersected the 50% response rate (dotted line) for the pulse pair. SPT is shown by the triangle. Top left: I/O functions recorded at the IPIs shown for several levels of stimulus magnitude are shown. Data points show the rate of response to the 2nd pulse of the pulse pair given that the 1st pulse of the pair resulted in activation. The refraction threshold was defined as the point where the line interpolating adjacent data points intersected the 50% conditional response rate (dotted line) for the pulse pair. SPT is shown by the triangle. Bottom left: summation thresholds normalized to SPT derived from the data in the top left are shown together with the exponential curve fit to the data using Eq. (1). SPT is shown by the dotted line. Bottom right: refraction thresholds normalized to SPT derived from the data in the top right are shown together with the exponential curve fit to the data using Eq. (2). SPT is shown by the dotted line.

may infer site of excitation with more peripheral excitation sites resulting in longer latencies and more central excitation sites resulting in shorter latencies. The latency values shown are the average latencies at summation or refraction threshold for each IPI for which summation or refraction thresholds were recorded. A t-test comparison of the refraction time constants and refractory latencies of the IM and ST electrode data indicated that the two groups were not significantly different (P = 0.35, T = 0.93, DF = 56 for sref and P = 0.77, T = 0.288, DF = 80 for latency). However, for the summation data, the time constant and latency data were both significantly different for the two groups (P = 0.001, T = 4.08, DF = 64 for ssum and P = 0.2 · 106, T = 5.01, DF = 89 for latency). Summation time constants for IM stimulated fibers were consistently in the

lower range of the summation time constants measured. Likewise, the latencies of IM stimulated data were in the lower range of measured latencies. There was overlap between IM and ST electrode latencies for mid-range latencies. Many ST stimulated fibers exhibited summation time constants that were higher than any measured for IM stimulated fibers. Of the single-pulse (threshold), summation and refraction data sets, only the summation data set clearly demonstrated differing fiber response properties across the two modes (IM and ST) of stimulation. Modeling results (Cartee, 2006) indicated that the additional charge holding ability of the large capacitance present in the cell body may elevate the summation time constant. This effect was localized to the peripheral excitation site located at the node adjacent to the cell body. Based on the modeling data, we hypothesized that the

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Fig. 3. Top: mean summation latency averaged across each IPI measured versus the summation time constant for IM and ST stimulated fibers. Bottom: mean refraction latency averaged across each IPI measured versus the refraction time constant for IM and ST stimulated fibers.

fibers with high summation time constants were stimulated at the peripheral node adjacent to the cell body. Additional analysis, using a clustering algorithm (Theodoridis and Koutroumbas, 2003), was conducted to objectively group the fibers according to their physiological responses to electrical stimulation. The clustering algorithm calculated the Euclidian distance between points and divided natural groupings of points into clusters. The clustering algorithm in the Matlab (v.6) statistics toolbox was used to calculate clusters. As the summation time constant and summation latency data showed the clearest distinction between the ST and IM stimulation conditions, these two data sets were used for the cluster analysis. To prevent dominance of one variable over another due to the different magnitudes of the values, the summation time constant and summation latency data were individually normalized using the following equation: Z¼

V  meanðV Þ stdðV Þ

Fig. 4 shows the resulting clusters following cluster analysis of the summation data. While the ST data were distributed across three clusters, all responses to IM stimulation fell within a single cluster, Cluster 1. For clarity, the IM stimulated data are plotted using a different symbol. However, they were all grouped together into a single cluster along with the ST stimulated Cluster 1 data. Fibers grouped together in a second cluster are shown by filled circles. Finally, a single fiber, marked by a triangle, was grouped into its own cluster. The algorithm recognized this fiber as distinct from the other fibers. When the algorithm was forced to form only two clusters, it separated this fiber into a cluster of one fiber and grouped all the remaining fibers into a second cluster. To test the reliability of the clustering algorithm with all animals grouped, the clustering algorithm was repeated on the summation data from each individual animal. In all cases other than C82, the clustering of fibers in individual animals matched that of the grouped data. The fact that the clustering algorithm was not consistent for C82 may be a result of the small sample size (n = 3). In preparations C035 and C037, the responses to both IM and ST stimulation were recorded. Inspection of the summation thresholds demonstrated overlap of Cluster 1 summation thresholds with the IM stimulated summation thresholds. The single Cluster 3 fiber in C96, overlaps with the Cluster 1 fibers that show a short summation time constant. Both the IM and ST stimulated Cluster 1 data and the Cluster 3 data show a short summation time period. For these fibers, the summation threshold for the 0.3 ms IPI condition is typically close to the single-pulse threshold, indicating that little if any charge remains on the membrane 0.3 ms following the initial stimulus. Cluster 2 fibers, on the other hand, show the effects of summation at an IPI of 0.3 ms indicating that the membrane at the site of excitation remains depolarized from the initial stimulus, lowering the amplitude needed for excitation with a two-pulse stimulus. This slower decay of membrane charge is consistent with a

ð3Þ

where Z is the normalized summation or latency data, V is the data set being normalized, mean(V) is the average of the data set and std(V) is the standard deviation of the data set.

Fig. 4. The results of the clustering algorithm for the summation mean latency and summation time constant data are shown. Each cluster is indicated on the graph. IM stimulated and ST stimulated fibers within Cluster 1 are shown by different symbols.

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larger membrane capacitance as would be expected for excitation sites close to the cell body. If the hypothesis that cluster 2 fibers are stimulated on the periphery and cluster 1 fibers are stimulated meatally were to hold, then longer latencies for cluster 2 fibers would be expected in comparison to cluster 1 fibers although some overlap in latency ranges may exist due to varying conduction velocity for different diameter fibers. For animals C035, C037, C82 and C83, Cluster 2 fibers show longer latencies than Cluster 1 fibers. In addition, Cluster 1 fiber latencies overlap with IM stimulated fiber latencies. This data is not as consistent for animal C96, however. In this animal, three Cluster 2 fibers overlap in latency range with Cluster 1 fibers. The single fiber for Cluster 3 had the longest latency recorded for any fiber in the study (>0.7 ms). Given our modeling results (Cartee, 2006), one working hypothesis is that for the ST stimulated fibers in Cluster 1, excitation was initialized on the central part of the axon and therefore, these fibers behave like IM stimulated fibers and cluster with IM stimulated fibers. The fibers in Cluster 2 with the higher summation time constant values, may represent fibers that were excited peripherally at the node adjacent to the cell body where the summation time constant is elevated due to the capacitive load of the cell body. The single, long latency, short summation time constant fiber in Cluster 3 may represent peripheral excitation at a more peripheral site than the node adjacent to the cell body as modeling results predict a shorter time constant for stimulation at this site, and a longer latency would be expected. We hypothesized that neurons stimulated at a peripheral site would have greater jitter than neurons stimulated in the meatus. The unique anatomical attributes of the cell body and nodes adjacent to the cell body were expected to increase the variability in the latency, and, therefore, increase the overall jitter. Comparison within animals of jitter in each cluster as compared to the jitter measured with IM stimulation showed considerable overlap, contrary to the expected result. Fig. 5 shows the summation and refraction jitter values combined for all animals. For both summation and refraction responses as well as the single pulse response (Fig. 1) some of the highest jitter values recorded were for IM stimulation. Low jitter values were also recorded, resulting in a large range of jitter values in response to IM stimulation. The jitter within cluster was evaluated for IM stimulated data and for ST stimulated data, eliminating the IM stimulated data from Cluster 1. The values are shown in Table 1. The average jitter for the summation and refractory conditions was obtained by computing the average jitter values across all IPIs where a jitter value was obtained for each fiber. The jitter values for IM stimulation more closely match the values for Cluster 2 fibers. One possible cause of larger than expected jitter values in IM stimulated fibers may be the use of two different protocols to measure spike latency. For group 1, latency was measured as the time from stimulus onset until the spike was discriminated with a voltage detector. In group 2, the

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Fig. 5. Average jitter responses across IPI in response to both summation (top) and refractory (bottom) pulse pairs are shown. Each bar represents the percentage of either IM or ST stimulated fibers with jitter values within the ranges shown.

Table 1 Mean jitter and standard deviation of jitter (in parentheses) in response to IM and ST stimulation for the single pulse, summation, and refractory stimulus protocols

Single pulse Summation Refractory

Cluster 1 Intrameatal

Cluster 1 Scala Tympani

Cluster 2

Cluster 3

0.07 (0.03) 0.09 (0.03) 0.06 (0.02)

0.04 (0.02) 0.06 (0.02) 0.04 (0.02)

0.08 (0.02) 0.09 (0.02) 0.06 (0.01)

0.04 0.1 *

Responses are shown as a function of Cluster with the Cluster 1 responses separated for IM and ST stimulated responses. Refractory data was not available for Cluster 3 as indicated by the asterisk. Since Cluster 3 contained one unit, it was not possible to calculate the standard deviation.

entire spike waveform was recorded and analyzed offline. Spike latency was computed offline as the time from stimulus onset until the maximum derivative of the upstroke of the action potential. To compare IM and ST stimulated single-pulse jitter, we looked at data from C035 and C037, the two animals in the study where IM and ST stimulated data were both measured in the same animal using identical equipment and protocols. IM and ST stimulated jitter values from these two animals are shown in Fig. 6. In this data set, latency was measured consistently using a voltage discriminator. The data agree with the combined animal data in that some of the highest jitter values are recorded from IM stimulated fibers and IM stimulated jitter values cover a broad range of values. In addition, we simulated a voltage discriminator in software. Latency

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Fig. 6. Average jitter values across IPI in response to single pulses in preparations C035 and C037. Each bar represents the number of IM or ST stimulated fibers with a jitter value within the range shown.

and jitter were computed in a sample data set using both the level detector and the maximum first derivative marker. The latency and jitter computed using both analysis techniques were not significantly different as determined by a paired t-test (P = 0 for latency and P = 0.009 for jitter). Jitter values in response to ST stimulated single-pulse stimulation demonstrated a significant amount of animal to animal variation. To eliminate this effect, the jitter in each animal was analyzed separately. Only one animal, C83, showed a significant correlation between threshold and jitter. For this animal, the correlation was highly significant (P < 0.0001) and negative (0.825) showing an increase in jitter with decreasing threshold. Further analysis revealed that cluster 2 data in this animal had a higher average jitter value than cluster 1 data, and there was no correlation between threshold and jitter when the two clusters of data were analyzed separately. If the two clusters of data correctly predict site of excitation, this implies that the negative correlation between threshold and jitter in this particular animal is a result of decreased jitter with central excitation. The remaining animals showed no correlation between jitter and threshold. Single-pulse threshold values were significantly lower for IM stimulation than for ST stimulation (Fig. 1) due, presumably, to the proximity of the electrode. We would predict Cluster 2 fibers to have lower thresholds if they represent peripheral excitation of fibers close the stimulation electrode and Cluster 1 fibers to have higher thresholds if they represent centrally excited fibers, perhaps fibers descending from higher turns of the cochlea, that are distant from the stimulating electrode. For animals C035, C037, C82, and C83, Cluster 2 fibers have lower thresholds than Cluster 1 fibers. However, this is not the case for C96. Latencies for single-pulses at threshold were clearly shorter for Cluster 1 fibers than Cluster 2 fibers for C035 and C037, supporting the hypothesis that Cluster 1 fibers were centrally stimulated. However, the remaining animals did not show such a clear distinction in latency values when the clusters were compared. Refractory data recorded from the ST stimulation site were evaluated only for fibers that were stable long enough

to record both summation and refractory data are shown, as summation data were needed for the cluster analysis. The theory predicts that Cluster 1 fibers will behave similarly to IM fibers. In general, Cluster 2 fibers require a longer IPI before the refraction threshold returns to the SPT while Cluster 1 fibers and IM fibers recover more quickly. Refraction time constants fit to the data for IM and Cluster 1 fibers average 0.55 ms with a standard deviation of 0.24 ms while for Cluster 2 fibers the average refraction time constant is 0.838 ms with a standard deviation of 0.306 ms. Comparison of refraction latencies within animals showed longer latencies for Cluster 2 fibers for only two of the preparations studied, C035 and C037. There was no evident pattern of refraction latency as a function of cluster for the other preparations. 4. Discussion Of the data collected in these experiments, the summation data most clearly suggests multiple sites of excitation in response to ST stimulation. If central and peripheral excitation sites exist, then the fact that the difference in responses from the two areas is most evident in the summation data is not surprising, as summation relies on the charge holding capacity of the membrane at the site of excitation and, therefore, is highly dependent on the capacitance of the surrounding neural structures. In addition, the summation response is a predominantly linear response depending on the membrane resistance and capacitance without involving non-linear membrane currents underlying action potentials. For the development of a technique to detect stimulus localization and perhaps evaluate electrode configurations designed to control site of excitation, a protocol including summation pulses appears to be the most promising. While refractory behavior also has some dependence on the charge holding capacity of the membrane, it is also dependent on the recovery of the ionic current carrying channels that produce the action potential. The non-linearity of the current channels adds an additional level of complexity to the membrane behavior. The non-linear behavior at each node between the site of excitation and the recording electrode could potentially influence the refractory behavior. It is also possible, with the two-pulse refractory protocol, that the site of excitation may be different for the first and second pulses. Given that the magnitudes of our two pulses were always kept equal, the same electric field and current spread would be expected with each pulse. If the excitation site were to shift, it would be expected to move closer to the stimulating electrode in the direction of the stronger field strength rather than in the area of weaker field strength. Additionally, the refractory properties of the membrane would prevent a shift in excitation to a more central site. As the action potential propagates centrally toward the brainstem, more centrally located sites will be more recently excited and, hence, more refractory. Therefore, excitation would not be expected to shift to a

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more central excitation site because the more central membrane would be more refractory than the initial excitation site. Numerical model simulations (Cartee, 2006) predict an increase in the refraction time constant with a peripheral shift of excitation site. Finally, it is possible that the action potential generated by the second pulse of the pair, being a less stable action potential occurring during the relative refractory period may have failed to conduct across the cell body. Prior indication of conduction failure across the cell body has previously been reported in the literature (Kohllo¨ffel, 1974; Robertson, 1976; Stypulkowski and van den Honert, 1984). Given the central location of our recording electrode, it was not possible for us to detect conduction failure. If failure had occurred for refractory pulses, however, the overall effect would have been to lengthen the overall time needed for membrane recovery that allowed safe propagation, lengthening the refraction time constant. Given these considerations, our data indicate that the summation data is a good choice to examine possible differences in site of excitation with a scala tympani electrode. Cluster analysis resulted in 3 possible populations within the scala tympani stimulated data. We hypothesized that these populations represented peripheral excitation at a node adjacent to the cell body, peripheral excitation at a node distant from the cell body, and central excitation. In general, the prediction of clusters correlated with expected SPT values for most animals. There was also correlation with expected refraction time constants. However, the correlations were not perfect, and no correlation between expected measurements and actual measurements existed for animal C96. Several possibilities exist for the lack of correlation of site of excitation predicted using summation data and the expected threshold and refraction data. The first possibility is that the input variables to the clustering technique may not have been optimal choices. Better methods for predicting site of excitation from the single fiber data may exist. The second possibility is that the site of excitation for summation, refraction, and single-pulse data is not always the same site. Given the different nature of the stimuli, it is possible that the site of excitation differs for each stimulus. Finally, the overlap in measured thresholds and latencies may be large enough that it will not be possible to detect site of excitation using single-fiber recording techniques. One notable finding of this study is that the clusters predicted by the summation data were poorly correlated to single pulse latency. If the clusters predicted by the summation data accurately predict different sites of excitation, then the selection of single-pulse latency only as an indicator of site of excitation would be a poor choice. As previously discussed, Miller et al. (2003) presented evidence suggesting that long-latency, localized activation was more likely to occur with bipolar stimulation than with monopolar stimulation. In this study, only one fiber had a short time constant and long latency that would imply

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peripheral excitation at a site away from the cell body. This same fiber had a shorter latency in response to a singlepulse threshold stimulus implying that the site of excitation for the threshold stimulus was different from that of the two-pulse summation stimulus. This supports the evidence of Miller, suggesting that long-latency, peripheral excitation is unlikely to occur with monopolar stimulation. However, the evidence suggests that peripheral excitation at the node adjacent to the cell body may be common with monopolar stimulation. Stimulation of the auditory nerve using an electrical stimulus results in a highly-synchronized stimulus response in comparison to the more temporally diffuse response of the auditory nerve following acoustic stimulation (Kiang and Moxon, 1972). If the path to a more natural sounding percept with cochlear implants can be achieved by more closely mimicking the response of the auditory neuron to an acoustic stimulus, then it would follow that methods of increasing temporal dispersion of the neural response to an electric stimulus would also increase cochlear implant performance. Methods of increasing jitter have been proposed and tested (Litvak et al., 2003; Rubinstein et al., 1999). If it were possible to control the site of stimulation, then one would hypothesize that stimulation at a peripheral site would be preferable to central stimulation since better spatial control of neural population responding to electrical stimulation should be possible with peripheral excitation sites. In addition, the peripheral axon has smaller nodes with more stochastic independence (Verveen, 1962), and the possibility of conduction failure or summation at the cell body would also increase temporal dispersion of the post-stimulus response. If desynchronization of fibers can be achieved by stimulating the fibers in areas with higher jitter, then the results indicate that peripheral stimulation (as indicated by membership in Cluster 2) has a higher average jitter than central stimulation, as predicted. Mino et al. (2004), using a computational model, predicted an increase in jitter with increasing distance between the neuron and the stimulating electrode. Assuming that threshold increases with increasing distance to the stimulating electrode, a positive correlation between threshold and jitter would be expected. For the IM stimulated data, no significant correlation existed between threshold and jitter within or across animals. For the ST stimulated data, only one preparation showed a correlation between threshold and jitter with jitter decreasing with increasing threshold. This trend was opposite to the predicted correlation. For this preparation, there was no correlation of threshold and jitter when each cluster was analyzed separately. The lack of correlation between jitter and threshold may simply be because threshold is not a good indicator of electrode-to-fiber distance. van den Honert and Stypulkowski (1984) by using characteristic frequency (CF) as an indicator of place of stimulation found that single fiber threshold may vary by a factor of four for fibers at a similar

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electrode-to-fiber distance as indicated by CF. Another possible explanation is that the effect of distance from the stimulating electrode, which is expected to show a positive correlation between threshold and jitter, may offset any influence of site of excitation on jitter, which would be expected to show a negative correlation between threshold and jitter with low threshold, peripherally stimulated fibers having higher jitter than high threshold, centrally stimulated fibers. The unexpected result was that IM stimulation resulted in average jitter values as high or higher than that of ST Cluster 2 fibers. Given the presumed central excitation site of the IM stimulated fibers and the close proximity of the electrode to the fiber, low jitter values would be expected. Increased jitter for IM stimulated fibers was not a function of simply increasing the average jitter for each individual fiber, but resulted from an increase in the overall range of jitter values for fibers with some jitter values higher than the maximum ST stimulated jitter values and some jitter values on the order of the minimum ST stimulated values. The high jitter values recorded in some IM stimulated fibers may be the result of a highly focal stimulus response region with excitation being dominated by a very small number of nodes (possibly even a single node). A decrease in the nodal area producing activation would lead to a decrease in the number of Na+ channels contributing to excitation resulting in highly stochastic responses. Weak action potentials generated by a few Na+ channels would have slower activation phases and therefore longer latencies. A larger number of contributing Na+ channels would lead to a faster activation time and shorter latencies. Since the IM electrode was inserted into the nerve bundle, a highly focal stimulus area may result when the stimulated fiber is adjacent to the stimulating electrode. The large spread in jitter values measured in response to IM stimulation indicates that the greatest desynchronization of fibers as indicated by jitter may be produced by an IM electrode. Some early cochlear implants investigated the placement of electrode in the modiolus (Simmons, 1983). Even though the IM placement had the advantage of lower thresholds, and more focal stimulation, the benefits did not outweigh the increased risk of damage to the auditory nerve during surgical placement. However, current electrode fabrication techniques producing ever smaller electrodes have led to a reinvestigation of the feasibility of modiolar electrodes as an alternative to scala tympani electrode placement (Zappia et al., 1990; Badi et al., 2002). Acknowledgements The authors wish to acknowledge Roger Miller for his assistance in the preparation and data collection for animals C035 and C037 and Charles Finley for his assistance in the data collection for animals C031–C037. The authors would also like to acknowledge the contributions of an

anonymous reviewer. This work was supported by the NIH/NIDCD Grant R29 DC02822. References Adamo, J.A., Diagneault, E.A., 1973. Ultrastructural features of neurons and nerve fibers of the spiral ganglion of cats. J. Neurocyt. 2, 91–103. Badi, A., Hillman, T., Shelton, C., Normann, R., 2002. A technique for implantation of a 3-dimensional penetrating electrode array in the modiolar nerve of cats and humans. Arch. Otolaryngol. Head Neck Surg. 128, 1019–1025. Brownell, W.E., Bader, C.R., Bertrand, D., de Ribaupierre, Y., 1985. Evoked mechanical responses of isolated cochlear outer hair cells. Science 277, 194–196. Cartee, L.A., 2000. Evaluation of a model of the cochlear neural membrane. II: Comparison of model and physiological measure of membrane properties measured in response to intrameatal electrical stimulation. Hear. Res. 146, 153–166. Cartee, L.A., van den Honert, C., Finley, C.C., Miller, R.L., 2000. Evaluation of a model of the cochlear neural membrane. I: Physiological measurement of membrane characteristics in response to intrameatal electrical stimulation. Hear. Res. 146, 143–152. Cartee, L.A., 2006. Spiral ganglion cell site of excitation. II: Numerical model analysis. Hear. Res. in press, doi:10.1016/j.heares.2006.02.011. Finley, C.C., Wilson, B.S., White, M.W., 1990. Models of neural responsiveness to electrical stimulation. In: Miller, J.M., Spelman, F.A. (Eds.), Cochlear Implants: Models of the Electrically Stimulated Ear. Springer-Verlag, New York, pp. 55–96. Goycoolea, M.V., Stypulkowski, P., Muchow, D.C., 1990. Ultrastructural studies of the peripheral extensions (dendrites) of type I ganglion cells in the cat. Laryngology 100, 19–24. Hartmann, R., Topp, G., Klinke, R., 1984. Discharge patterns of cat primary auditory fibers with electrical stimulation of the cochlea. Hear. Res. 13, 47–62. van den Honert, C., Stypulkowski, P.H., 1984. Physiological properties of the electrically stimulated auditory nerve. II: Single fiber recordings. Hear. Res. 14, 225–243. van den Honert, C., Stypulkowki, P.H., 1987. Temporal response patterns of single auditory nerve fibers elicited by periodic electrical stimuli. Hear. Res. 29, 207–222. Javel, Shepherd, 2000. Electrical stimulation of the auditory nerve. III: Response initiation sites and temporal fine structure. Hear. Res. 140, 45–76. Javel, E., Tong, Y.C., Shepherd, R.K., Clark, G.M., 1987. Responses of cat auditory nerve fibers to biphasic electrical current pulses. Ann. Otol. Rhinol. Laryngol. 96 (Suppl. 128), 26–30. Kiang, N.Y.S., Moxon, E.C., 1972. Physiological considerations in artificial stimulation of the inner ear. Ann. Otol. Rhinol. Laryngol. 81, 714–730. Kiang, N.Y.S., Rho, J.M., Northrop, C.C., Liberman, M.C., Ryugo, D.K., 1982. Hair-cell innervation by spiral ganglion cells in adult cats. Science 217 (9), 175–177. Kohllo¨ffel, L.U.E., 1974. A study of neurone activity in the spiral ganglion of the cat’s basal turn. Arch. Oto-Rhino-Laryngol. 109, 179–202. Liberman, M.C., Oliver, M.E., 1984. Morphometry of intracellularly labeled neurons of the auditory nerve: Correlations with functional properties. J. Comp. Neurol. 223, 163–176. Litvak, L., Delgutte, B., Eddington, D., 2003. Improved neural representation of vowels in electric stimulation using desynchronizing pulse trains. J. Acoust. Soc. Am. 114, 2099–2111. Miller, C.A., Abbas, P.J., Robinson, B.K., Rubinstein, J.R., Matsuoka, A.J., 1999. Electrically evoked single-fiber action potentials from cat: responses to monopolar, monophasic stimulation. Hear. Res. 130, 197–218. Miller, C.A., Robinson, B.K., Rubinstein, J.T., Abbas, P.J., Runge Samuelson, C., 2001. Auditory nerve responses to monophasic and biphasic electric stimuli. Hear. Res. 151, 79–94.

L.A. Cartee et al. / Hearing Research 215 (2006) 10–21 Miller, C.A., Abbas, P.J., Nourski, K.V., Hu, N., Robinson, B.K., 2003a. Electrode configuration influences action potential initiation site and ensemble stochastic response properties. Hear. Res. 175, 200–214. Miller, C.A., Nagase, S., Abbas, P.J., Hu, N., Robinson, B.K., 2003b. The effect of recording electrode position on the electrically evoked compound action potential. Abstract #765 presented at the 2003 Midwinter Meeting of the Association for Research in Otolaryngology. Mino, H., Rubinstein, J.T., Miller, C.A., Abbas, P.J., 2004. Effects of electrode-to-fiber distance on temporal neural response with electrical stimulations. IEEE Trans. BME 51, 13–20. Moxon, E.C., 1965. Electrical stimulation of the cat’s cochlea: A study of discharge rates in single auditory nerve fibers, Master’s Thesis, MIT, Cambridge, MA. Moxon, E.C., 1971. Neural and mechanical responses to electric stimulation of the cat’s inner ear, Doctoral Dissertation, MIT, Cambridge, MA. Rattay, F., 1990. Electrical Nerve Stimulation: Theory, Experiments and Applications. Springer-Verlag, New York. Robertson, D., 1976. Possible relation between structure and spike shapes of neurones in guinea pig cochlea ganglion. Brain Res. 109, 487–496.

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Rubinstein, J.T., Wilson, B.S., Finley, C.C., Abbas, P.J., 1999. Pseudospontaneous activity: stochastic independence of auditory nerve fibers with electrical stimulation. Hear. Res. 172, 108–118. Simmons, B.F., 1983. Percepts from modiolar (eighth nerve) stimulation. Ann. N Y Acad. Sci. 405, 259–263. Spach, M.S., Kootsey, J.M., 1985. Relating the sodium current and conductance to the shape of transmembrane and extracellular potential by stimulation: effects of propagation boundaries. IEEE Trans. BME BME-32 10, 743–755. Stypulkowski, P.H., van den Honert, C., 1984. Physiological properties of the electrically stimulated auditory nerve. I: Compound action potential recordings. Hear. Res. 14, 205–223. Theodoridis, S., Koutroumbas, K., 2003. Pattern Recognition, second ed . Academic Press, Elsevier Science, San Diego, CA. Verveen, A.A., 1962. Axon diameter and fluctuation in excitability. Acta. Morphol. Neerlando-Scand. 5, 79–85. Zappia, J.J., Hetke, J.F., Altschuler, R.A., Niparko, J.K., 1990. Evaluation of a silicon-substrate modiolar eighth nerve implant in a guinea pig. Otolaryngol. Head Neck Surg. 103 (4), 575–582.