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Effects of a calcium channel blocker on spontaneous neural noise and gross action potential waveforms in the guinea pig cochlea
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Hearing Research 188 (2004) 117^125 www.elsevier.com/locate/heares
E¡ects of a calcium channel blocker on spontaneous neural noise and gross action potential waveforms in the guinea pig cochlea T. Sueta b
a;b
, S.Y. Zhang c , P.M. Sellick b , R. Patuzzi b , D. Robertson
b;
a Department of Otolaryngology, Fukuoka University, Fukuoka, Japan The Auditory Laboratory, Physiology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia c Department of Otolaryngology, Guangdong Provincial People’s Hospital, Guanzhou, PR China
Received 8 September 2003; accepted 11 November 2003
Abstract The effects of the L-type Ca2þ channel blocker nimodipine on the spectrum of the spontaneous neural noise (SNN) and the waveform of the gross sound-evoked compound action potential (CAP) were investigated by perilymphatic perfusion in the guinea pig cochlea. Both the SNN and the CAP were reversibly suppressed by nimodipine. The percentage reduction in SNN was dosedependent in a manner very similar to the results obtained with the measures of CAP threshold changes. The reduction in the peak SNN caused by 10 WM nimodipine was the same as that caused by 500 WM kainic acid, which totally eliminated any neural responses. For 1 WM nimodipine there was an apparent dissociation between the SNN and CAP changes such that the SNN could be markedly suppressed with only very small changes in CAP thresholds. These results imply that spontaneous release of neurotransmitter from the inner hair cell is more sensitive to block of calcium channels than evoked release. There was no evidence for any marked shift caused by nimodipine, in the position of the main (900 Hz) spectral peak in the SNN. Comparison of the CAP waveform before and after nimodipine perfusion showed that the CAP waveforms were unchanged despite the change in sensitivity. These data do not support the notion of any significant postsynaptic site of action of nimodipine. The data hence provide further support for an exclusively presynaptic role for L-type Ca2þ channels in the regulation of both evoked and spontaneous neurotransmitter release from inner hair cells. 7 2003 Elsevier B.V. All rights reserved. Key words: Cochlea; Transmitter; Calcium channel; Neural noise; Compound action potential; Nimodipine
1. Introduction The excitation of auditory nerve ¢bers in mammals is achieved by release of excitatory neurotransmitter from the inner hair cells of the cochlea. It has been shown that this neurotransmitter release, in conformity with other chemical synapses, is a calcium-dependent process
* Corresponding author. Tel.: +61 (8) 9380 3291; Fax: +61 (8) 9380 1025. E-mail address: [email protected] (D. Robertson). Abbreviations: CAP, compound action potential; SP, summating potential; DC, direct current; SNN, spectrum of the spontaneous neural noise; DMSO, dimethylsulfoxide; N1 , ¢rst negative peak of CAP; N2 , second negative peak of CAP; P1 , ¢rst positive peak of CAP
governed by the operation of voltage-gated calcium channels in the basolateral walls of the hair cells (Robertson and Johnstone, 1979; Siegel and Relkin, 1987; Issa and Hudspeth, 1994). There is now good evidence from a variety of experimental models that these calcium channels belong to the major group of dihydropyridine-sensitive channels known as ‘L-type’ (Bobbin et al., 1990; Zhang et al., 1999; Robertson and Paki, 2002; Platzer et al., 2000; Engel et al., 2002; Kollmar et al., 1997a,b; Zidanic and Fuchs, 1995). The role of these hair cell calcium channels in exerting presynaptic control over both the acoustically driven and the spontaneous ¢ring of auditory nerve ¢bers is of considerable interest because their abnormal function could be a basis for conditions such as tinnitus (see for example Evans et al., 1981; Evans and Borerwe, 1982;
0378-5955 / 03 / $ ^ see front matter 7 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00374-5
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Jastrebo¡ and Sasaki, 1986) and some forms of auditory dysfunction in which hair cell responses appear normal but neural responses are a¡ected (Shivashankar et al., 2003). However, previous studies investigating this question had limitations. For example, isolated hair cell experiments cannot directly test the functional role of calcium channels in determining neural output. On the other hand, in vivo experiments measuring the e¡ects of dihydropyridines on single or gross neural potentials in the cochlea, while they have been used successfully to localize the site of action to the inner hair cell-a¡erent neuron synapse, cannot distinguish between presynaptic and postsynaptic disturbances of neurotransmission. The possibility of a postsynaptic action of nimodipine has to be considered in view of the demonstration that spiral ganglion neuron somata possess voltage-gated calcium channels (Hisashi et al., 1995). In this paper measurements of the electrical noise recorded from the unstimulated cochlea (Cazals and Huang, 1996; Cazals et al., 1998; Dolan et al., 1990; McMahon and Patuzzi, 2002; Patuzzi et al., 2004) were used as an index of spontaneous ¢ring of auditory nerve ¢bers in the guinea pig during intracochlear perfusion with the calcium channel blocker nimodipine. At the same time, detailed measurements were made of the waveform of the compound action potential (CAP) response to tone bursts. The results indicate that nimodipine can suppress all ¢ring of auditory neurons by an action that is most likely to be purely presynaptic in nature.
2. Materials and methods Experiments were performed on 46 young pigmented guinea pigs (207^430 g) of either sex. They were anesthetized with a combination of Nembutal and Hypnorm (fentanyl/£uanisone). Subsequently, the depth of anesthesia was maintained by hourly administration of Hypnorm and half doses of Nembutal every 2 h (Lowe and Robertson, 1995; Zhang et al., 1999). All procedures conformed to the Code of Practice of the National Health and Medical Research Council of Australia and were approved by the Animal Ethics Committee of The University of Western Australia. For initial evaluation of the cochlear condition, the tympanic bulla was opened via a dorsolateral approach and a silver wire hook electrode was placed on the bony shelf near the round window. A chloride silver wire reference electrode was inserted into the animal’s neck muscles. The threshold of the auditory nerve CAP was measured at tone burst frequencies ranging from 2 to 20 kHz. If the CAP thresholds determined by this initial screening were within normal limits as determined by previous work in the laboratory, preparations were
made for intracochlear perfusion and recording from within scala tympani, as detailed below. The whole cochlear perfusion method was employed. A small hole was made in the basal turn scala tympani and the outlet hole was drilled in the apex. Care must be taken to avoid blockage of the perfusion holes by blood clots. A perfusion pipette (V80 Wm tip diameter) was sealed into the basal turn hole with a small bead of silicone ¢xed to the shank of the pipette. Perfusion was performed at a rate of 3 Wl/min for 10 min. Control arti¢cial perilymph consisted of (in mM) 137 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 1 NaH2 PO4 , 12 NaHCO3 , and 11 glucose (Zhang et al., 1999). The solution pH was adjusted to 7.4 at 37‡C. Nimodipine was mixed with arti¢cial perilymph after being dissolved in dimethylsulfoxide (DMSO). The solutions containing nimodipine at concentrations ranging from 0.5 to 10 WM contained DMSO concentrations ranging from 0.005 to 0.1%. We have previously shown that 0.1% DMSO in arti¢cial perilymph does not a¡ect cochlear neural threshold (Zhang et al., 1999). Kainic acid was used at 500 WM only. All salts and chemicals were obtained either from Sigma or BDH chemicals. Measurements of the CAP, the direct current (DC) receptor potential, or summating potential (SP) and the spectrum of the spontaneous neural noise (SNN), before, during and after perfusion, were made using a ¢ne etched tungsten wire inserted into the shank of the perfusion pipette. Acoustic stimuli consisted of computergenerated gated tone bursts (10 ms duration with 1 ms rise^fall times, repetition period 201 ms) delivered in a calibrated closed sound delivery system. Potentials were ampli¢ed 100 times and ¢ltered (low- and high-frequency cuto¡s 1 Hz and 3 kHz respectively) before display and storage in computer memory. All acoustic stimuli were generated using custom software with a PC and stereo sound card (DigitalAudio, CardDeluxe, 24 bits, 96 kHz). One channel of the sound card was used for recording electrical activity. The CAP and the DC receptor potential (SP) were recorded by averaging the responses to 20 tone burst presentations and the visual detection thresholds of both the CAP and the SP were estimated from these recordings at 1 min intervals during the experiment. The rationale for recording both CAP and SP thresholds is that the SP threshold is a reasonable measure of the sensitivity of the outer hair cells, whereas the CAP threshold is a measure of the sensitivity of the excitatory synaptic drive from the inner hair cells to the afferent neurons. Hence it has been shown that these two measures covary in response to treatments that a¡ect the outer hair cell cochlear ampli¢er, whereas treatments that a¡ect the inner hair cell synapse cause larger changes in CAP than SP thresholds (Zhang et al., 1999).
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Previous studies have shown that CAP threshold measurements for di¡erent tone burst frequencies are relatively unprejudiced by electrode position (Johnstone et al., 1979), but it is important that SP recordings are obtained at a frequency appropriate for the position of the recording electrode (Cheatham and Dallos, 1984). In this experiment, our recording electrode was placed near the high-frequency end of the cochlea in the basal turn, and a 16 kHz tone burst was therefore employed to measure both neural and hair cell function at the characteristic frequency of the recording electrode position. On several occasions, we also measured the CAP thresholds at 4 and 10 kHz in order to check for e¡ects ‘downstream’ from the site of the cochlear perfusion pipette. These CAP thresholds to lower frequencies behaved similarly to the 16 kHz thresholds for all perfusions in which they were investigated, and hence they were not recorded routinely. In addition to CAP and SP measurement, the SNN was also recorded before and after perfusion with control arti¢cial perilymph and with perilymph containing kainic acid and nimodipine. The SNN was averaged with a Blackman^Harris window using a Labview program. In most experiments, the spectrum was obtained from the average of 30 separate 1 s samples, using a sampling rate of 22 050 Hz. In a small number of early experiments a di¡erent sound card (Crystal semiconductor corporation, 16 bits, 44 kHz) and an earlier version of the Labview program were used for SNN data acquisition. Although this resulted in slightly altered appearance of the SNN recordings, these di¡erences did not a¡ect the interpretation of the results obtained.
3. Results 3.1. Control perfusions and SNN We have previously shown that perfusion of the scala tympani with arti¢cial perilymph caused no signi¢cant
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changes in the CAP and SP thresholds (Zhang et al., 1999). Fig. 1A shows an example of the spectrum of the SNN before and at the end of a 10 min perfusion with arti¢cial perilymph. It is clear from this example that arti¢cial perilymph caused insigni¢cant changes in the amplitude and shape of the SNN. In contrast to the results obtained with arti¢cial perilymph, perfusion with arti¢cial perilymph containing 500 WM kainic acid caused a large change in the SNN (Fig. 1B). This concentration of kainic acid rapidly eliminated all neural responses to tone burst stimuli, as assessed by the CAP response. Changes were seen in di¡erent regions of the SNN, but for the remainder of this paper, attention is focused on those changes occurring between about 300 and 2000 Hz, because this region of the spectrum has been shown to be most closely associated with primary auditory a¡erent discharge (McMahon and Patuzzi, 2002). Changes at very low frequencies are hard to interpret, especially in view of the fact that it has been shown that at least some of this part of the SNN emanates from extracochlear, and probably non-neural sources such as EMG and electrical contamination. 3.2. Nimodipine perfusion and SNN Perfusion of the scala tympani with arti¢cial perilymph containing nimodipine at concentrations ranging from 0.5 to 10 WM caused varying amounts of alteration of the SNN (Fig. 2). For a concentration of nimodipine of 0.5 WM, the e¡ects of perfusion did not di¡er signi¢cantly from control perfusions (Fig. 2A). At a concentration of 1 WM, the peak of the SNN centered at around 700^1000 Hz was substantially reduced in amplitude after perfusion, but the peak of the spectrum was still visible and remained at the same frequency (Fig. 2B). 5 and 10 WM perfusions (Fig. 2C, D) further reduced the SNN amplitude, with e¡ects mostly evident in the 700^1000 Hz region of the SNN spectrum. 10 WM nimodipine in fact, produced a reduction similar to that seen after 500 WM kainic acid perfusion. We
Fig. 1. Examples from one animal showing the e¡ects of perfusion on the SNN recorded from basal turn scala tympani. Solid lines, before perfusion, gray lines, after 10 min perfusion with A: arti¢cial perilymph, B: 500 WM kainic acid.
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Fig. 2. Examples from individual animals of the e¡ects of nimodipine perfusion on the SNN. Solid lines, spectrum before perfusion, gray lines, after 10 min perfusion with A: 0.5 WM nimodipine, B: 1 WM nimodipine, C: 5 WM nimodipine, D: 10 WM nimodipine.
were able to quantitatively estimate the peak frequency of the SNN for eight separate perfusions with 5 WM nimodipine. The mean peak frequency was 907 Hz (S.E.M. 6.5 Hz) before perfusion and 910 Hz (S.E.M. 7.9 Hz) after perfusion with the peak after perfusion being sampled when there was a substantial reduction in the peak amplitude compared to before perfusion. The di¡erence in mean peak frequency was not signi¢cant. Such estimates were not possible for 10 WM perfusions because of the loss of a discrete peak in the relevant frequency region. The e¡ects of nimodipine perfusion were only slowly reversible if the natural replacement of perilymph was used as the means of wash-
Fig. 3. Example of reversibility of nimodipine e¡ects. Solid black line, before perfusion; gray line, after perfusion with 5 WM nimodipine; broken line, after a 10 min wash with control arti¢cial perilymph.
out of the drug. However, reversibility was rapid if the nimodipine perfusion was followed by a second perfusion with normal arti¢cial perilymph (Fig. 3). To quantify the reduction in SNN, we obtained a single measure of the SNN amplitude, by averaging the amplitude readings at all spectral frequencies between 400 and 1000 Hz. Fig. 4 shows the percent reduction in this measure seen at the end of the various 10 min perfusions. The average reduction in the SNN measure after arti¢cial perilymph was only 2.2%. After 0.5 WM nimodipine, the reduction was only 0.6% and
Fig. 4. Mean SNN reduction at the end of 10 min perfusions with various solutions. Arti¢cial perilymph (n = 11); 0.5 WM nimodipine (n = 9); 1 WM nimodipine (n = 7); 5 WM nimodipine (n = 9); and 10 WM nimodipine (n = 8); KA, 500 WM kainic acid (n = 10). Note absence of signi¢cant di¡erence between arti¢cial perilymph and 0.5 WM nimodipine and between 10 WM nimodipine and 500 WM kainic acid.
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Fig. 5. Example of time course of CAP and SP threshold changes from one animal with a 10 WM nimodipine perfusion. Thick line at top shows perfusion length (10 min). Solid circle: CAP thresholds, open circles: SP thresholds. Note substantially larger e¡ect on CAP threshold 60 dB change maximum possible limit of sound delivery system.
this was not signi¢cantly di¡erent from the result with arti¢cial perilymph (unpaired t-test, P s 0.9). The reductions in the measure of the SNN peak were 20.5, 30.5, 40.7% in 1, 5, 10 WM nimodipine respectively and these were all signi¢cantly di¡erent from perfusion with arti¢cial perilymph or with 0.5 WM nimodipine. After perfusion with 500 WM kainic acid the mean SNN reduction was 43.3%. The reduction in SNN caused by 10 WM nimodipine was not signi¢cantly di¡erent from that caused by 500 WM kainic acid (unpaired t-test, P s 0.9). 3.3. Correlations between SNN, CAP and SP thresholds For all the above perfusions in which the SNN was
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Fig. 6. Mean maximum CAP threshold changes with arti¢cial perilymph (n = 11), 0.5 WM nimodipine (n = 9), 1 WM nimodipine (n = 7), 5 WM nimodipine (n = 9) and 10 WM nimodipine (n = 8).
measured, we also estimated the CAP and SP thresholds before, during and after perfusion. The mean maximum CAP threshold change after arti¢cial perilymph was 2.7 dB. Typically, arti¢cial perilymph caused very small depression of CAP thresholds in the ¢rst few minutes of perfusion, but this rapidly recovered over the course of the perfusion. In agreement with previous studies, nimodipine perfusions caused marked changes. Fig. 5 shows an example of the e¡ects of 10 WM nimodipine on CAP and SP thresholds, illustrating the classical result of a much greater e¡ect on CAP compared to SP thresholds (see for example Zhang et al., 1999; Bobbin et al., 1990). Fig. 6 shows the average e¡ects on CAP thresholds for the di¡erent perfusions. After ni-
Fig. 7. Example of changes produced by perfusion with 1 WM nimodipine and washout on CAP and SP thresholds (E) (solid circle: CAP, open circle: SP) and SNN (A^D). Arrows indicate time points at which each SNN measurement was obtained. In E, dark horizontal line shows length of nimodipine perfusion, dotted line shows change of perfusion pipette, gray line shows washout. Note signi¢cant reversible change in SNN peak in absence of substantial change in CAP and SP thresholds.
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modipine perfusion, average CAP thresholds were elevated by 4.9, 9.2, 24.6, and 51.3 dB (0.5, 1, 5 and 10 WM nimodipine respectively). All except the change after 0.5 WM were signi¢cantly di¡erent from the e¡ect of arti¢cial perilymph perfusion. The CAP threshold changes were dose-dependent in a manner roughly similar to the results obtained with the measures of SNN. However, for concentrations of nimodipine of 1 WM, we sometimes saw a dissociation between the SNN and CAP results, such that there was only a very small change in CAP thresholds, although SNN was markedly suppressed. This is illustrated in Fig. 7. 3.4. CAP waveforms and nimodipine We also investigated whether the shape of the CAP waveform was altered after nimodipine perfusions that caused CAP threshold elevations and SNN reduction. We did this by matching as closely as possible the ¢rst negative peak of CAP^¢rst positive peak of CAP (N1 ^ P1 ) amplitude at di¡erent sound intensities before and after perfusion. Fig. 8 shows examples of this matching procedure from three di¡erent animals after perfusion
with 5 WM nimodipine. The CAP waveforms after perfusion are seen superimposed on a substantial DC component which is the relatively una¡ected SP generated at the higher sound pressures used. The records after perfusion have been shifted vertically to produce the best visual match of the CAP component of the responses. Fig. 8A^C show examples from three di¡erent animals. The CAP threshold changes were 6, 18 and 18 dB respectively and the level of the tone was 6 dB above threshold in each case. Fig. 8D^G illustrate the results of the matching procedure in a single animal at di¡erent points on the CAP input^output function. It is apparent from these results that the overall CAP waveforms are very similar before and after perfusion. One quantitative measure of features of the CAP waveform is the N1 ^N2 latency di¡erence, and in Fig. 9 this is plotted against N1 ^P1 amplitude for all perfusions. For arti¢cial perilymph perfusion, the data before and after perfusions overlap completely because neither the N1 ^P1 amplitude nor the N1 ^N2 latency di¡erences were signi¢cantly altered by this control perfusion. The data from perfusions with 5 and 10 WM nimodipine show a clear leftward shift in the distribution of data
Fig. 8. Examples of comparison of CAP waveforms before and after 5 WM nimodipine perfusion. A^C were obtained from three di¡erent animals. The CAP threshold changes were 6, 18 and 18 dB respectively and the waveforms shown were all obtained at 6 dB above visual detection threshold. D^G were obtained from one animal at a time after perfusion when the CAP threshold change was 6 dB. Solid lines in all panels, CAP waveforms before perfusion; gray lines, after 10 min perfusion with 5 WM nimodipine.
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Fig. 9. Relation between N1 ^N2 latency di¡erence and N1 ^P1 amplitude before (solid circle) and after (open circle) perfusion. A: Arti¢cial perilymph, n = 5. B: 5 WM nimodipine, n = 4. C: 10 WM nimodipine, n = 4.
points after perfusion, re£ecting the reduced CAP amplitude. There is, however, no systematic vertical displacement of the data points, consistent with the lack of obvious change in the shape of the matched CAP waveforms illustrated in Fig. 8.
4. Discussion In the present study we have shown that intracochlear perfusion of nimodipine, a blocker of L-type voltagegated Ca2þ channels, reduces the amplitude of the SNN in a dose-dependent manner, in addition to its welldocumented e¡ect on the CAP threshold evoked by tone burst stimuli (Bobbin et al., 1990; Zhang et al., 1999). In agreement with previous reports, the threshold of the DC receptor potential (SP) was less a¡ected than the CAP and the SNN, consistent with a rather selective action of nimodipine on the ¢nal stage of auditory transduction, the release of neurotransmitter from the inner hair cells and/or its interaction with receptors on the postsynaptic dendrite membrane. The fact that there is a change in SP threshold, albeit less than on the CAP, is explained by an additional action of nimodipine on the outer hair cells which are responsible for determining the normal sensitive mechanical drive to
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the inner hair cells (Bobbin et al., 1990; Zhang et al., 1999). The mechanism of this outer hair cell component to the e¡ect is unknown. The CAP threshold is a measure of sound-evoked neural responses while the SNN is considered to re£ect the spontaneous ensemble ¢ring of auditory a¡erents (Cazals and Huang, 1996; Cazals et al., 1998; Dolan et al., 1990; McMahon and Patuzzi, 2002; Patuzzi et al., 2004). The result seen with kainic acid perfusion clearly con¢rms previous observations that the SNN is neural in origin and is sensitive to pharmacological agents that cause a cessation of neural ¢ring (Dolan et al., 1990; Zheng et al., 1996; McMahon and Patuzzi, 2002). The present results on the SNN, which is measured in the absence of overt acoustic stimulation, are therefore consistent with previous ¢ndings from single neuron recordings (Robertson and Paki, 2002) showing that spontaneous as well as sound-evoked activities are depressed by intracochlear perfusion of nimodipine. Indeed, in combination with the results of earlier studies, the present ¢ndings provide support for the notion that the amplitude of the SNN may be able to be used as a measure of the ensemble spontaneous ¢ring rate of auditory a¡erents (but see below). Taken together these results imply that the spontaneous ¢ring rate of auditory a¡erents, as well as that evoked by sound, is regulated by the tonic level of intracellular calcium at transmitter release sites in the hair cells and that this is in turn determined by the number of open L-type channels. Unlike earlier single neuron experiments (Robertson and Paki, 2002), the present technique allowed for perfusion of the entire scala tympani perilymphatic compartment and did not su¡er from the mechanical stability limitations of single cell microelectrode recordings. Hence a more complete dose^response relationship could be produced for the SNN than for single auditory a¡erents. These data (Figs. 4 and 6) show that there is rough correspondence between the dose^response relationship of e¡ects on CAP and SNN. However, there was a suggestion in the mean data that the SNN was a¡ected at somewhat lower nimodipine concentrations than the CAP threshold and this was borne out in individual perfusions, in which clear examples were found of changes in SNN without changes in the CAP threshold for nimodipine concentrations of 1 WM. It is possible that this dissociation is not real, because the SNN may come from more widely distributed cochlear regions than the highly localized 16 kHz CAP threshold measure. However, in a number of animals we also monitored CAP threshold changes at 4 and 10 kHz and these were similar to those seen for 16 kHz tones. Hence the dissociation between CAP and SNN changes is probably genuine. The ¢nding supports results of Sewell (1984) who
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showed that the spontaneous ¢ring rate of single a¡erents is more sensitive than acoustic sensitivity to a drop in the electrical driving force between the apical surface and interior of the hair cells. Presumably the explanation for the present results and those of Sewell lies in the sensitivity of the spontaneous rate to small changes in inner hair cell calcium level, determined by their voltage-gated Ca2þ channels. It is of interest that the reduction caused by 10 WM nimodipine in the peak amplitude of the SNN was comparable to that produced by 500 WM kainic acid. The latter drug, at such a high concentration, causes a complete postsynaptic blockade of glutamatergic a¡erent transmission, as evidenced by the total and irreversible abolition of all neural components of the sound-evoked response (see Sellick et al., 2003; Bledsoe et al., 1981), and hence it appears that nimodipine too, may be able to produce a su⁄cient block of L-type channels to totally suppress all spontaneous a¡erent ¢ring. Again this was suggested by previous data in which a small number of examples were obtained of complete suppression by nimodipine of spontaneous ¢ring of single a¡erent neurons (Robertson and Paki, 2002). The above data are all consistent with the notion that nimodipine exerts its e¡ects on the presynaptic calcium channels of the inner hair cells from which the type I primary a¡erents receive their excitation. Such in vivo observations are in broad agreement with demonstrations of dihydropyridine-sensitive calcium currents in isolated hair cells, although there is an interesting point of di¡erence between the in vivo data and that from isolated hair cells. Whole-cell Ca2þ currents in isolated hair cells, although they are greatly reduced by dihydropyridines, are reportedly resistant to complete block. This property is believed to be characteristic of the subtype of L-type calcium channels expressed by cochlear inner hair cells. However the fact that the spontaneous ¢ring of neurons can be completely suppressed by nimodipine raises the intriguing possibility that whole-cell calcium current measurements may not necessarily faithfully re£ect all of the synaptically relevant component of calcium currents. The body of data from this and previous studies constitutes strong circumstantial evidence for a presynaptic site of action of nimodipine. However, neither the fact that nimodipine reduces the SNN amplitude, nor indeed any of the previous single neuron and gross potential results can exclude the possibility that nimodipine might a¡ect primary a¡erent ¢ring through some postsynaptic mechanism. However, we believe that key aspects of the present SNN and CAP data can e¡ectively rule out this as a complicating mechanism. McMahon and Patuzzi (2002) and Patuzzi et al. (2004) have provided compelling evidence that both the spectral peak of the SNN (centered at approxi-
mately 900 Hz), and the characteristic waveform of the CAP complex originate from an intrinsic electrical resonance of the postsynaptic a¡erent dendrite membrane. Based on this notion, a drug acting on the postsynaptic dendrite properties might be expected to alter the features of this resonance, perhaps changing the position of the peak in the SNN spectrum (Patuzzi et al., 2004). A purely presynaptic e¡ect on transmitter release rate, on the other hand, would cause a drop in the peak amplitude but no change in peak frequency of the SNN spectrum. In the present data, the position of the spectral peak in the SNN was not changed by nimodipine perfusion (Figs. 2, 7) despite an evident fall in the peak amplitude, and this is therefore consistent with a presynaptic, not a postsynaptic action. However, a lack of shift in the peak of the SNN spectrum, while it is consistent with a presynaptic e¡ect, cannot be used to totally rule out a postsynaptic component, because a simple reduction in SNN amplitude could conceivably be due either to a change in overall spontaneous neural ¢ring rate, or to a change in the amplitude of the postsynaptic membrane resonance without a change in its basic frequency. However, the present CAP waveform data e¡ectively rule out this possibility. When the CAP after nimodipine perfusion was appropriately scaled by increasing the sound pressure level to a similar level above threshold to that before nimodipine perfusion, the CAP waveforms after perfusion were not discernibly di¡erent from the waveforms before perfusion (Fig. 8). This would not be the case if there had been a change in the relative contribution of a postsynaptic membrane resonance to some components of the CAP response. By providing evidence for an exclusively presynaptic action of nimodipine in the intact cochlea, the present in vivo data both con¢rm and extend the conclusions of previous studies, indicating that both spontaneous and evoked releases of neurotransmitter at the inner hair cell-a¡erent neuron synapse are governed by the functioning of voltage-gated calcium channels of the L-type. The study also supports other work (McMahon and Patuzzi, 2002; Patuzzi et al., 2004) showing how knowledge of the fundamental origins of the SNN can make it a useful, easily measured tool in dissecting basic cellular processes in the inner ear.
Acknowledgements Supported by grants from the National Health and Medical Research Council and the Medical Health and Research Infrastructure Fund (WA). S.Y.Z. was the recipient of an AusAid scholarship. The authors are indebted to G. O’Beirne for generous provision of Labview programs.
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References Bledsoe, S.C., Jr., Bobbin, R.P., Chihal, D.M., 1981. Kainic acid: an evaluation of its action on cochlear potentials. Hear. Res. 4, 109^ 120. Bobbin, R.P., Jastrebo¡, P.J., Fallon, M., Littman, T., 1990. Nimodipine, an L-channel Ca2þ antagonist, reverses the negative summating potential recorded from the guinea pig cochlear. Hear. Res. 46, 277^287. Cazals, Y., Huang, Z.W., 1996. Average spectrum of cochlear activity: a possible synchronized ¢ring, its olivo-cochlear feedback and alterations under anesthesia. Hear. Res. 101, 81^92. Cazals, Y., Horner, K.C., Huang, Z.W., 1998. Alterations in average spectrum of cochleoneural activity by long-term salicylate treatment in the guinea pig: a plausible index of tinnitus. J. Neurophysiol. 80, 2113^2120. Cheatham, M.A., Dallos, P., 1984. Summating potential (SP) tuning curves. Hear. Res. 16, 189^200. Dolan, D.F., Nuttall, A.L., Avinash, G., 1990. Asynchronous neural activity recorded from the round window. J. Acoust. Soc. Am. 87, 2621^2627. Engel, J., Michna, M., Platzer, J., Striessnig, J., 2002. Calcium channels in mouse hair cells: function, properties and pharmacology. Adv. Otorhinolaryngol. 59, 35^41. Evans, E.F., Borerwe, T.A., 1982. Ototoxic e¡ects of salicylates on the responses of single cochlear nerve ¢bres and on cochlear potentials. Br. J. Audiol. 16, 101^108. Evans, E.F., Wilson, J.P., Borerwe, T.A., 1981. Animal models of tinnitus. Ciba Found. Symp. 85, 108^138. Hisashi, K., Nakagawa, T., Yasuda, T., Kimitsuki, T., Komune, S., Komiyama, S., 1995. Voltage-dependent Ca2þ channels in the spiral ganglion cells of guinea pig cochlea. Hear. Res. 91, 196^201. Issa, N.P., Hudspeth, A.J., 1994. Clustering of Ca2þ channels and Ca2þ -activated Kþ channels at £uorescently labeled presynaptic active zones of hair cells. Proc. Natl. Acad. Sci. USA 91, 7578^ 7582. Jastrebo¡, P.J., Sasaki, C.T., 1986. Salicylate induced changes in spontaneous activity of single units in the inferior colliculus of the guinea pig. J. Acoust. Soc. Am. 80, 1384^1391. Johnstone, J.R., Alder, V.A., Johnstone, B.M., Robertson, D., Yates, G.K., 1979. Cochlear action potential threshold and single unit thresholds. J. Acoust. Soc. Am. 65, 254^257. Kollmar, R., Montgomery, L.G., Fak, J., Henry, L.J., Hudspeth, A.J., 1997a. Predominance of the K1D subunit in L-type voltage-gated Ca2þ channels of hair cells in the chicken’s cochlea. Proc. Natl. Acad. Sci. USA 94, 14883^14888.
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Kollmar, R., Fak, J., Montgomery, L.G., Hudspeth, A.J., 1997b. Hair cell-speci¢c splicing of mRNA for the K1D subunit of voltagegated Ca2þ channels in the chicken’s cochlear. Proc. Natl. Acad. Sci. USA 94, 14889^14893. Lowe, M., Robertson, D., 1995. The behaviour of the f2^f1 acoustic distortion product: lack of e¡ect of brainstem lesions in anesthetized guinea pigs. Hear. Res. 83, 133^141. McMahon, C.M., Patuzzi, R.B., 2002. The origin of the 900 Hz spectral peak in spontaneous and sound-evoked round-window electrical activity. Hear. Res. 173, 134^152. Patuzzi, R., Brown, D.J., McMahon, C.M., Halliday, A.F., 2004. Determinants of the spectrum of the neural activity at the round window: transmitter release and neural depolarization. Hear Res. in press. Platzer, J., Engel, J., Schrott-Fischer, A., Stephan, K., Boya, S., Chen, H., Zheng, H., Striessing, J., 2000. Congential deafness and sinoatrial node dysfunction in mice lacking class D L-type Ca2þ channels. Cell 102, 88^97. Robertson, D., Johnstone, B.M., 1979. E¡ects of divalent cations on spontaneous and evoked activity of single mammalian auditory neurons. P£ugers Arch. 380, 1^12. Robertson, D., Paki, B., 2002. Role of L-type Ca2þ channels in transmitter release from mammalian inner hair cells. II. Single-neuron activity. J. Neurophysiol. 87, 2734^2740. Sellick, P.M., Patuzzi, R., Robertson, D., 2003. Primary a¡erent and cochlear nucleus contributions to extracellular potentials during tone-bursts. Hear. Res. 176, 42^58. Sewell, W.F., 1984. The e¡ects of furosemide on the endocochlear potential and auditory-nerve ¢ber tuning curves in cats. Hear. Res. 14, 305^314. Shivashankar, N., Satishchandra, P., Shashikala, H.R., Gore, M., 2003. Primary auditory neuropathy ^ an enigma. Acta Neurol. Scand. 108, 130^135. Siegel, J.H., Relkin, E.M., 1987. Antagonistic e¡ects of perilymphatic calcium and magnesium on the activity of single cochlear a¡erent neurons. Hear. Res. 28, 131^147. Zhang, S.Y., Robertson, D., Yates, G., Everett, A., 1999. Role of L-type Ca2þ channels in transmitter release from mammalian inner hair cells I. Gross sound-evoked potentials. J. Neurophysiol. 82, 3307^3315. Zheng, X.Y., Wang, J., Salvi, R., Henderson, D., 1996. E¡ects of kainic acid on the cochlear potentials and distortion product oto-acoustic emissions in chinchilla. Hear. Res. 95, 161^167. Zidanic, M., Fuchs, P.A., 1995. Kinetic analysis of barium currents in chick cochlear hair cells. Biophys. J. 68, 1323^1336.
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E¡ects of a calcium channel blocker on spontaneous neural noise and gross action potential waveforms in the guinea pig cochlea T. Sueta b
a;b
, S.Y. Zhang c , P.M. Sellick b , R. Patuzzi b , D. Robertson
b;
a Department of Otolaryngology, Fukuoka University, Fukuoka, Japan The Auditory Laboratory, Physiology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Hwy, Crawley, WA 6009, Australia c Department of Otolaryngology, Guangdong Provincial People’s Hospital, Guanzhou, PR China
Received 8 September 2003; accepted 11 November 2003
Abstract The effects of the L-type Ca2þ channel blocker nimodipine on the spectrum of the spontaneous neural noise (SNN) and the waveform of the gross sound-evoked compound action potential (CAP) were investigated by perilymphatic perfusion in the guinea pig cochlea. Both the SNN and the CAP were reversibly suppressed by nimodipine. The percentage reduction in SNN was dosedependent in a manner very similar to the results obtained with the measures of CAP threshold changes. The reduction in the peak SNN caused by 10 WM nimodipine was the same as that caused by 500 WM kainic acid, which totally eliminated any neural responses. For 1 WM nimodipine there was an apparent dissociation between the SNN and CAP changes such that the SNN could be markedly suppressed with only very small changes in CAP thresholds. These results imply that spontaneous release of neurotransmitter from the inner hair cell is more sensitive to block of calcium channels than evoked release. There was no evidence for any marked shift caused by nimodipine, in the position of the main (900 Hz) spectral peak in the SNN. Comparison of the CAP waveform before and after nimodipine perfusion showed that the CAP waveforms were unchanged despite the change in sensitivity. These data do not support the notion of any significant postsynaptic site of action of nimodipine. The data hence provide further support for an exclusively presynaptic role for L-type Ca2þ channels in the regulation of both evoked and spontaneous neurotransmitter release from inner hair cells. 7 2003 Elsevier B.V. All rights reserved. Key words: Cochlea; Transmitter; Calcium channel; Neural noise; Compound action potential; Nimodipine
1. Introduction The excitation of auditory nerve ¢bers in mammals is achieved by release of excitatory neurotransmitter from the inner hair cells of the cochlea. It has been shown that this neurotransmitter release, in conformity with other chemical synapses, is a calcium-dependent process
* Corresponding author. Tel.: +61 (8) 9380 3291; Fax: +61 (8) 9380 1025. E-mail address: [email protected] (D. Robertson). Abbreviations: CAP, compound action potential; SP, summating potential; DC, direct current; SNN, spectrum of the spontaneous neural noise; DMSO, dimethylsulfoxide; N1 , ¢rst negative peak of CAP; N2 , second negative peak of CAP; P1 , ¢rst positive peak of CAP
governed by the operation of voltage-gated calcium channels in the basolateral walls of the hair cells (Robertson and Johnstone, 1979; Siegel and Relkin, 1987; Issa and Hudspeth, 1994). There is now good evidence from a variety of experimental models that these calcium channels belong to the major group of dihydropyridine-sensitive channels known as ‘L-type’ (Bobbin et al., 1990; Zhang et al., 1999; Robertson and Paki, 2002; Platzer et al., 2000; Engel et al., 2002; Kollmar et al., 1997a,b; Zidanic and Fuchs, 1995). The role of these hair cell calcium channels in exerting presynaptic control over both the acoustically driven and the spontaneous ¢ring of auditory nerve ¢bers is of considerable interest because their abnormal function could be a basis for conditions such as tinnitus (see for example Evans et al., 1981; Evans and Borerwe, 1982;
0378-5955 / 03 / $ ^ see front matter 7 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0378-5955(03)00374-5
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Jastrebo¡ and Sasaki, 1986) and some forms of auditory dysfunction in which hair cell responses appear normal but neural responses are a¡ected (Shivashankar et al., 2003). However, previous studies investigating this question had limitations. For example, isolated hair cell experiments cannot directly test the functional role of calcium channels in determining neural output. On the other hand, in vivo experiments measuring the e¡ects of dihydropyridines on single or gross neural potentials in the cochlea, while they have been used successfully to localize the site of action to the inner hair cell-a¡erent neuron synapse, cannot distinguish between presynaptic and postsynaptic disturbances of neurotransmission. The possibility of a postsynaptic action of nimodipine has to be considered in view of the demonstration that spiral ganglion neuron somata possess voltage-gated calcium channels (Hisashi et al., 1995). In this paper measurements of the electrical noise recorded from the unstimulated cochlea (Cazals and Huang, 1996; Cazals et al., 1998; Dolan et al., 1990; McMahon and Patuzzi, 2002; Patuzzi et al., 2004) were used as an index of spontaneous ¢ring of auditory nerve ¢bers in the guinea pig during intracochlear perfusion with the calcium channel blocker nimodipine. At the same time, detailed measurements were made of the waveform of the compound action potential (CAP) response to tone bursts. The results indicate that nimodipine can suppress all ¢ring of auditory neurons by an action that is most likely to be purely presynaptic in nature.
2. Materials and methods Experiments were performed on 46 young pigmented guinea pigs (207^430 g) of either sex. They were anesthetized with a combination of Nembutal and Hypnorm (fentanyl/£uanisone). Subsequently, the depth of anesthesia was maintained by hourly administration of Hypnorm and half doses of Nembutal every 2 h (Lowe and Robertson, 1995; Zhang et al., 1999). All procedures conformed to the Code of Practice of the National Health and Medical Research Council of Australia and were approved by the Animal Ethics Committee of The University of Western Australia. For initial evaluation of the cochlear condition, the tympanic bulla was opened via a dorsolateral approach and a silver wire hook electrode was placed on the bony shelf near the round window. A chloride silver wire reference electrode was inserted into the animal’s neck muscles. The threshold of the auditory nerve CAP was measured at tone burst frequencies ranging from 2 to 20 kHz. If the CAP thresholds determined by this initial screening were within normal limits as determined by previous work in the laboratory, preparations were
made for intracochlear perfusion and recording from within scala tympani, as detailed below. The whole cochlear perfusion method was employed. A small hole was made in the basal turn scala tympani and the outlet hole was drilled in the apex. Care must be taken to avoid blockage of the perfusion holes by blood clots. A perfusion pipette (V80 Wm tip diameter) was sealed into the basal turn hole with a small bead of silicone ¢xed to the shank of the pipette. Perfusion was performed at a rate of 3 Wl/min for 10 min. Control arti¢cial perilymph consisted of (in mM) 137 NaCl, 5 KCl, 2 CaCl2 , 1 MgCl2 , 1 NaH2 PO4 , 12 NaHCO3 , and 11 glucose (Zhang et al., 1999). The solution pH was adjusted to 7.4 at 37‡C. Nimodipine was mixed with arti¢cial perilymph after being dissolved in dimethylsulfoxide (DMSO). The solutions containing nimodipine at concentrations ranging from 0.5 to 10 WM contained DMSO concentrations ranging from 0.005 to 0.1%. We have previously shown that 0.1% DMSO in arti¢cial perilymph does not a¡ect cochlear neural threshold (Zhang et al., 1999). Kainic acid was used at 500 WM only. All salts and chemicals were obtained either from Sigma or BDH chemicals. Measurements of the CAP, the direct current (DC) receptor potential, or summating potential (SP) and the spectrum of the spontaneous neural noise (SNN), before, during and after perfusion, were made using a ¢ne etched tungsten wire inserted into the shank of the perfusion pipette. Acoustic stimuli consisted of computergenerated gated tone bursts (10 ms duration with 1 ms rise^fall times, repetition period 201 ms) delivered in a calibrated closed sound delivery system. Potentials were ampli¢ed 100 times and ¢ltered (low- and high-frequency cuto¡s 1 Hz and 3 kHz respectively) before display and storage in computer memory. All acoustic stimuli were generated using custom software with a PC and stereo sound card (DigitalAudio, CardDeluxe, 24 bits, 96 kHz). One channel of the sound card was used for recording electrical activity. The CAP and the DC receptor potential (SP) were recorded by averaging the responses to 20 tone burst presentations and the visual detection thresholds of both the CAP and the SP were estimated from these recordings at 1 min intervals during the experiment. The rationale for recording both CAP and SP thresholds is that the SP threshold is a reasonable measure of the sensitivity of the outer hair cells, whereas the CAP threshold is a measure of the sensitivity of the excitatory synaptic drive from the inner hair cells to the afferent neurons. Hence it has been shown that these two measures covary in response to treatments that a¡ect the outer hair cell cochlear ampli¢er, whereas treatments that a¡ect the inner hair cell synapse cause larger changes in CAP than SP thresholds (Zhang et al., 1999).
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Previous studies have shown that CAP threshold measurements for di¡erent tone burst frequencies are relatively unprejudiced by electrode position (Johnstone et al., 1979), but it is important that SP recordings are obtained at a frequency appropriate for the position of the recording electrode (Cheatham and Dallos, 1984). In this experiment, our recording electrode was placed near the high-frequency end of the cochlea in the basal turn, and a 16 kHz tone burst was therefore employed to measure both neural and hair cell function at the characteristic frequency of the recording electrode position. On several occasions, we also measured the CAP thresholds at 4 and 10 kHz in order to check for e¡ects ‘downstream’ from the site of the cochlear perfusion pipette. These CAP thresholds to lower frequencies behaved similarly to the 16 kHz thresholds for all perfusions in which they were investigated, and hence they were not recorded routinely. In addition to CAP and SP measurement, the SNN was also recorded before and after perfusion with control arti¢cial perilymph and with perilymph containing kainic acid and nimodipine. The SNN was averaged with a Blackman^Harris window using a Labview program. In most experiments, the spectrum was obtained from the average of 30 separate 1 s samples, using a sampling rate of 22 050 Hz. In a small number of early experiments a di¡erent sound card (Crystal semiconductor corporation, 16 bits, 44 kHz) and an earlier version of the Labview program were used for SNN data acquisition. Although this resulted in slightly altered appearance of the SNN recordings, these di¡erences did not a¡ect the interpretation of the results obtained.
3. Results 3.1. Control perfusions and SNN We have previously shown that perfusion of the scala tympani with arti¢cial perilymph caused no signi¢cant
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changes in the CAP and SP thresholds (Zhang et al., 1999). Fig. 1A shows an example of the spectrum of the SNN before and at the end of a 10 min perfusion with arti¢cial perilymph. It is clear from this example that arti¢cial perilymph caused insigni¢cant changes in the amplitude and shape of the SNN. In contrast to the results obtained with arti¢cial perilymph, perfusion with arti¢cial perilymph containing 500 WM kainic acid caused a large change in the SNN (Fig. 1B). This concentration of kainic acid rapidly eliminated all neural responses to tone burst stimuli, as assessed by the CAP response. Changes were seen in di¡erent regions of the SNN, but for the remainder of this paper, attention is focused on those changes occurring between about 300 and 2000 Hz, because this region of the spectrum has been shown to be most closely associated with primary auditory a¡erent discharge (McMahon and Patuzzi, 2002). Changes at very low frequencies are hard to interpret, especially in view of the fact that it has been shown that at least some of this part of the SNN emanates from extracochlear, and probably non-neural sources such as EMG and electrical contamination. 3.2. Nimodipine perfusion and SNN Perfusion of the scala tympani with arti¢cial perilymph containing nimodipine at concentrations ranging from 0.5 to 10 WM caused varying amounts of alteration of the SNN (Fig. 2). For a concentration of nimodipine of 0.5 WM, the e¡ects of perfusion did not di¡er signi¢cantly from control perfusions (Fig. 2A). At a concentration of 1 WM, the peak of the SNN centered at around 700^1000 Hz was substantially reduced in amplitude after perfusion, but the peak of the spectrum was still visible and remained at the same frequency (Fig. 2B). 5 and 10 WM perfusions (Fig. 2C, D) further reduced the SNN amplitude, with e¡ects mostly evident in the 700^1000 Hz region of the SNN spectrum. 10 WM nimodipine in fact, produced a reduction similar to that seen after 500 WM kainic acid perfusion. We
Fig. 1. Examples from one animal showing the e¡ects of perfusion on the SNN recorded from basal turn scala tympani. Solid lines, before perfusion, gray lines, after 10 min perfusion with A: arti¢cial perilymph, B: 500 WM kainic acid.
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Fig. 2. Examples from individual animals of the e¡ects of nimodipine perfusion on the SNN. Solid lines, spectrum before perfusion, gray lines, after 10 min perfusion with A: 0.5 WM nimodipine, B: 1 WM nimodipine, C: 5 WM nimodipine, D: 10 WM nimodipine.
were able to quantitatively estimate the peak frequency of the SNN for eight separate perfusions with 5 WM nimodipine. The mean peak frequency was 907 Hz (S.E.M. 6.5 Hz) before perfusion and 910 Hz (S.E.M. 7.9 Hz) after perfusion with the peak after perfusion being sampled when there was a substantial reduction in the peak amplitude compared to before perfusion. The di¡erence in mean peak frequency was not signi¢cant. Such estimates were not possible for 10 WM perfusions because of the loss of a discrete peak in the relevant frequency region. The e¡ects of nimodipine perfusion were only slowly reversible if the natural replacement of perilymph was used as the means of wash-
Fig. 3. Example of reversibility of nimodipine e¡ects. Solid black line, before perfusion; gray line, after perfusion with 5 WM nimodipine; broken line, after a 10 min wash with control arti¢cial perilymph.
out of the drug. However, reversibility was rapid if the nimodipine perfusion was followed by a second perfusion with normal arti¢cial perilymph (Fig. 3). To quantify the reduction in SNN, we obtained a single measure of the SNN amplitude, by averaging the amplitude readings at all spectral frequencies between 400 and 1000 Hz. Fig. 4 shows the percent reduction in this measure seen at the end of the various 10 min perfusions. The average reduction in the SNN measure after arti¢cial perilymph was only 2.2%. After 0.5 WM nimodipine, the reduction was only 0.6% and
Fig. 4. Mean SNN reduction at the end of 10 min perfusions with various solutions. Arti¢cial perilymph (n = 11); 0.5 WM nimodipine (n = 9); 1 WM nimodipine (n = 7); 5 WM nimodipine (n = 9); and 10 WM nimodipine (n = 8); KA, 500 WM kainic acid (n = 10). Note absence of signi¢cant di¡erence between arti¢cial perilymph and 0.5 WM nimodipine and between 10 WM nimodipine and 500 WM kainic acid.
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Fig. 5. Example of time course of CAP and SP threshold changes from one animal with a 10 WM nimodipine perfusion. Thick line at top shows perfusion length (10 min). Solid circle: CAP thresholds, open circles: SP thresholds. Note substantially larger e¡ect on CAP threshold 60 dB change maximum possible limit of sound delivery system.
this was not signi¢cantly di¡erent from the result with arti¢cial perilymph (unpaired t-test, P s 0.9). The reductions in the measure of the SNN peak were 20.5, 30.5, 40.7% in 1, 5, 10 WM nimodipine respectively and these were all signi¢cantly di¡erent from perfusion with arti¢cial perilymph or with 0.5 WM nimodipine. After perfusion with 500 WM kainic acid the mean SNN reduction was 43.3%. The reduction in SNN caused by 10 WM nimodipine was not signi¢cantly di¡erent from that caused by 500 WM kainic acid (unpaired t-test, P s 0.9). 3.3. Correlations between SNN, CAP and SP thresholds For all the above perfusions in which the SNN was
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Fig. 6. Mean maximum CAP threshold changes with arti¢cial perilymph (n = 11), 0.5 WM nimodipine (n = 9), 1 WM nimodipine (n = 7), 5 WM nimodipine (n = 9) and 10 WM nimodipine (n = 8).
measured, we also estimated the CAP and SP thresholds before, during and after perfusion. The mean maximum CAP threshold change after arti¢cial perilymph was 2.7 dB. Typically, arti¢cial perilymph caused very small depression of CAP thresholds in the ¢rst few minutes of perfusion, but this rapidly recovered over the course of the perfusion. In agreement with previous studies, nimodipine perfusions caused marked changes. Fig. 5 shows an example of the e¡ects of 10 WM nimodipine on CAP and SP thresholds, illustrating the classical result of a much greater e¡ect on CAP compared to SP thresholds (see for example Zhang et al., 1999; Bobbin et al., 1990). Fig. 6 shows the average e¡ects on CAP thresholds for the di¡erent perfusions. After ni-
Fig. 7. Example of changes produced by perfusion with 1 WM nimodipine and washout on CAP and SP thresholds (E) (solid circle: CAP, open circle: SP) and SNN (A^D). Arrows indicate time points at which each SNN measurement was obtained. In E, dark horizontal line shows length of nimodipine perfusion, dotted line shows change of perfusion pipette, gray line shows washout. Note signi¢cant reversible change in SNN peak in absence of substantial change in CAP and SP thresholds.
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modipine perfusion, average CAP thresholds were elevated by 4.9, 9.2, 24.6, and 51.3 dB (0.5, 1, 5 and 10 WM nimodipine respectively). All except the change after 0.5 WM were signi¢cantly di¡erent from the e¡ect of arti¢cial perilymph perfusion. The CAP threshold changes were dose-dependent in a manner roughly similar to the results obtained with the measures of SNN. However, for concentrations of nimodipine of 1 WM, we sometimes saw a dissociation between the SNN and CAP results, such that there was only a very small change in CAP thresholds, although SNN was markedly suppressed. This is illustrated in Fig. 7. 3.4. CAP waveforms and nimodipine We also investigated whether the shape of the CAP waveform was altered after nimodipine perfusions that caused CAP threshold elevations and SNN reduction. We did this by matching as closely as possible the ¢rst negative peak of CAP^¢rst positive peak of CAP (N1 ^ P1 ) amplitude at di¡erent sound intensities before and after perfusion. Fig. 8 shows examples of this matching procedure from three di¡erent animals after perfusion
with 5 WM nimodipine. The CAP waveforms after perfusion are seen superimposed on a substantial DC component which is the relatively una¡ected SP generated at the higher sound pressures used. The records after perfusion have been shifted vertically to produce the best visual match of the CAP component of the responses. Fig. 8A^C show examples from three di¡erent animals. The CAP threshold changes were 6, 18 and 18 dB respectively and the level of the tone was 6 dB above threshold in each case. Fig. 8D^G illustrate the results of the matching procedure in a single animal at di¡erent points on the CAP input^output function. It is apparent from these results that the overall CAP waveforms are very similar before and after perfusion. One quantitative measure of features of the CAP waveform is the N1 ^N2 latency di¡erence, and in Fig. 9 this is plotted against N1 ^P1 amplitude for all perfusions. For arti¢cial perilymph perfusion, the data before and after perfusions overlap completely because neither the N1 ^P1 amplitude nor the N1 ^N2 latency di¡erences were signi¢cantly altered by this control perfusion. The data from perfusions with 5 and 10 WM nimodipine show a clear leftward shift in the distribution of data
Fig. 8. Examples of comparison of CAP waveforms before and after 5 WM nimodipine perfusion. A^C were obtained from three di¡erent animals. The CAP threshold changes were 6, 18 and 18 dB respectively and the waveforms shown were all obtained at 6 dB above visual detection threshold. D^G were obtained from one animal at a time after perfusion when the CAP threshold change was 6 dB. Solid lines in all panels, CAP waveforms before perfusion; gray lines, after 10 min perfusion with 5 WM nimodipine.
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Fig. 9. Relation between N1 ^N2 latency di¡erence and N1 ^P1 amplitude before (solid circle) and after (open circle) perfusion. A: Arti¢cial perilymph, n = 5. B: 5 WM nimodipine, n = 4. C: 10 WM nimodipine, n = 4.
points after perfusion, re£ecting the reduced CAP amplitude. There is, however, no systematic vertical displacement of the data points, consistent with the lack of obvious change in the shape of the matched CAP waveforms illustrated in Fig. 8.
4. Discussion In the present study we have shown that intracochlear perfusion of nimodipine, a blocker of L-type voltagegated Ca2þ channels, reduces the amplitude of the SNN in a dose-dependent manner, in addition to its welldocumented e¡ect on the CAP threshold evoked by tone burst stimuli (Bobbin et al., 1990; Zhang et al., 1999). In agreement with previous reports, the threshold of the DC receptor potential (SP) was less a¡ected than the CAP and the SNN, consistent with a rather selective action of nimodipine on the ¢nal stage of auditory transduction, the release of neurotransmitter from the inner hair cells and/or its interaction with receptors on the postsynaptic dendrite membrane. The fact that there is a change in SP threshold, albeit less than on the CAP, is explained by an additional action of nimodipine on the outer hair cells which are responsible for determining the normal sensitive mechanical drive to
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the inner hair cells (Bobbin et al., 1990; Zhang et al., 1999). The mechanism of this outer hair cell component to the e¡ect is unknown. The CAP threshold is a measure of sound-evoked neural responses while the SNN is considered to re£ect the spontaneous ensemble ¢ring of auditory a¡erents (Cazals and Huang, 1996; Cazals et al., 1998; Dolan et al., 1990; McMahon and Patuzzi, 2002; Patuzzi et al., 2004). The result seen with kainic acid perfusion clearly con¢rms previous observations that the SNN is neural in origin and is sensitive to pharmacological agents that cause a cessation of neural ¢ring (Dolan et al., 1990; Zheng et al., 1996; McMahon and Patuzzi, 2002). The present results on the SNN, which is measured in the absence of overt acoustic stimulation, are therefore consistent with previous ¢ndings from single neuron recordings (Robertson and Paki, 2002) showing that spontaneous as well as sound-evoked activities are depressed by intracochlear perfusion of nimodipine. Indeed, in combination with the results of earlier studies, the present ¢ndings provide support for the notion that the amplitude of the SNN may be able to be used as a measure of the ensemble spontaneous ¢ring rate of auditory a¡erents (but see below). Taken together these results imply that the spontaneous ¢ring rate of auditory a¡erents, as well as that evoked by sound, is regulated by the tonic level of intracellular calcium at transmitter release sites in the hair cells and that this is in turn determined by the number of open L-type channels. Unlike earlier single neuron experiments (Robertson and Paki, 2002), the present technique allowed for perfusion of the entire scala tympani perilymphatic compartment and did not su¡er from the mechanical stability limitations of single cell microelectrode recordings. Hence a more complete dose^response relationship could be produced for the SNN than for single auditory a¡erents. These data (Figs. 4 and 6) show that there is rough correspondence between the dose^response relationship of e¡ects on CAP and SNN. However, there was a suggestion in the mean data that the SNN was a¡ected at somewhat lower nimodipine concentrations than the CAP threshold and this was borne out in individual perfusions, in which clear examples were found of changes in SNN without changes in the CAP threshold for nimodipine concentrations of 1 WM. It is possible that this dissociation is not real, because the SNN may come from more widely distributed cochlear regions than the highly localized 16 kHz CAP threshold measure. However, in a number of animals we also monitored CAP threshold changes at 4 and 10 kHz and these were similar to those seen for 16 kHz tones. Hence the dissociation between CAP and SNN changes is probably genuine. The ¢nding supports results of Sewell (1984) who
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showed that the spontaneous ¢ring rate of single a¡erents is more sensitive than acoustic sensitivity to a drop in the electrical driving force between the apical surface and interior of the hair cells. Presumably the explanation for the present results and those of Sewell lies in the sensitivity of the spontaneous rate to small changes in inner hair cell calcium level, determined by their voltage-gated Ca2þ channels. It is of interest that the reduction caused by 10 WM nimodipine in the peak amplitude of the SNN was comparable to that produced by 500 WM kainic acid. The latter drug, at such a high concentration, causes a complete postsynaptic blockade of glutamatergic a¡erent transmission, as evidenced by the total and irreversible abolition of all neural components of the sound-evoked response (see Sellick et al., 2003; Bledsoe et al., 1981), and hence it appears that nimodipine too, may be able to produce a su⁄cient block of L-type channels to totally suppress all spontaneous a¡erent ¢ring. Again this was suggested by previous data in which a small number of examples were obtained of complete suppression by nimodipine of spontaneous ¢ring of single a¡erent neurons (Robertson and Paki, 2002). The above data are all consistent with the notion that nimodipine exerts its e¡ects on the presynaptic calcium channels of the inner hair cells from which the type I primary a¡erents receive their excitation. Such in vivo observations are in broad agreement with demonstrations of dihydropyridine-sensitive calcium currents in isolated hair cells, although there is an interesting point of di¡erence between the in vivo data and that from isolated hair cells. Whole-cell Ca2þ currents in isolated hair cells, although they are greatly reduced by dihydropyridines, are reportedly resistant to complete block. This property is believed to be characteristic of the subtype of L-type calcium channels expressed by cochlear inner hair cells. However the fact that the spontaneous ¢ring of neurons can be completely suppressed by nimodipine raises the intriguing possibility that whole-cell calcium current measurements may not necessarily faithfully re£ect all of the synaptically relevant component of calcium currents. The body of data from this and previous studies constitutes strong circumstantial evidence for a presynaptic site of action of nimodipine. However, neither the fact that nimodipine reduces the SNN amplitude, nor indeed any of the previous single neuron and gross potential results can exclude the possibility that nimodipine might a¡ect primary a¡erent ¢ring through some postsynaptic mechanism. However, we believe that key aspects of the present SNN and CAP data can e¡ectively rule out this as a complicating mechanism. McMahon and Patuzzi (2002) and Patuzzi et al. (2004) have provided compelling evidence that both the spectral peak of the SNN (centered at approxi-
mately 900 Hz), and the characteristic waveform of the CAP complex originate from an intrinsic electrical resonance of the postsynaptic a¡erent dendrite membrane. Based on this notion, a drug acting on the postsynaptic dendrite properties might be expected to alter the features of this resonance, perhaps changing the position of the peak in the SNN spectrum (Patuzzi et al., 2004). A purely presynaptic e¡ect on transmitter release rate, on the other hand, would cause a drop in the peak amplitude but no change in peak frequency of the SNN spectrum. In the present data, the position of the spectral peak in the SNN was not changed by nimodipine perfusion (Figs. 2, 7) despite an evident fall in the peak amplitude, and this is therefore consistent with a presynaptic, not a postsynaptic action. However, a lack of shift in the peak of the SNN spectrum, while it is consistent with a presynaptic e¡ect, cannot be used to totally rule out a postsynaptic component, because a simple reduction in SNN amplitude could conceivably be due either to a change in overall spontaneous neural ¢ring rate, or to a change in the amplitude of the postsynaptic membrane resonance without a change in its basic frequency. However, the present CAP waveform data e¡ectively rule out this possibility. When the CAP after nimodipine perfusion was appropriately scaled by increasing the sound pressure level to a similar level above threshold to that before nimodipine perfusion, the CAP waveforms after perfusion were not discernibly di¡erent from the waveforms before perfusion (Fig. 8). This would not be the case if there had been a change in the relative contribution of a postsynaptic membrane resonance to some components of the CAP response. By providing evidence for an exclusively presynaptic action of nimodipine in the intact cochlea, the present in vivo data both con¢rm and extend the conclusions of previous studies, indicating that both spontaneous and evoked releases of neurotransmitter at the inner hair cell-a¡erent neuron synapse are governed by the functioning of voltage-gated calcium channels of the L-type. The study also supports other work (McMahon and Patuzzi, 2002; Patuzzi et al., 2004) showing how knowledge of the fundamental origins of the SNN can make it a useful, easily measured tool in dissecting basic cellular processes in the inner ear.
Acknowledgements Supported by grants from the National Health and Medical Research Council and the Medical Health and Research Infrastructure Fund (WA). S.Y.Z. was the recipient of an AusAid scholarship. The authors are indebted to G. O’Beirne for generous provision of Labview programs.
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