Hearing Research, 55 (1991) 133-142 0 1991 Elsevier Science Publishers B.V. All rights reserved

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133 0378-5955/91/$03.50

01611

Effects of opioid be drugs on auditory evoked potentials suggest a role of lateral olivocochlear dynorphins in auditory function Tony L. Sahley

‘y3,

Robin B. Kalis h ‘, Frank E. Musiek * and Douglas W. Hoffman



’ Neurochemistry Laboratory, Departments of Psychiatry and Pharmacology, and ’ Division of Audiology, Department of Surgery, Dartmouth Medical School, Hanocer, New Hampshire; and 3 Department of Otolaryngology, University of California, San Francisco, California, U.S.A. (Received

20 December

1990; accepted

2 March

1991)

Multiple gene products of opioid peptide families (e.g., enkephalins, dynorphins) with differing opioid receptor specificities are present within olivocochlear efferent terminals. Enkephalins activate CL- and d-opioid receptors, and are generally inhibitory in the nervous system, and dynorphins are k-receptor agonists, which may be excitatory to postsynaptic neurons. We have examined the effects of intravenously administered opioid agonists and antagonists on click-evoked N, and Nz amplitudes and latencies of the compound action potential in the chinchilla recorded at the round window. Parenteral administration of the opioid receptor antagonist naloxone or the potent p-receptor agonist fentanyl did not alter N, and Nz amplitudes or latencies. The K-receptor agonist, p-receptor antagonist pentazocine caused marked increases in N, and N, amplitudes over baseline values at threshold intensities. These effects were not abolished by naloxone. No effects were seen on the cochlear microphonic, supporting a site of action of these effects at the lateral olivocochlear efferent terminals on auditory nerve dendrites under inner hair cells. Similar results were obtained when far field auditory evoked responses were recorded. Results were obtained under ketamine/ pentobarbital anesthesia, which provided stable recording baselines in contrast to tiletamine/zolezepam/pentobarbital, with which an upward drift in auditory potentials was observed.This stimulatory action of K-agonists on auditory-evoked potential amplitudes appears to represent a physiological role of the lateral olivocochlear efferent innervation. The different neurotransmitters of the olivocochlear efferents (e.g. enkephalins, dynorphins, acetylcholine) may have antagonistic actions on auditory potentials, as may the lateral and medial systems themselves.

Opioid;

Opioid

peptides;

Dynorphin;

Olivocochlear

efferent

bundle;

Introduction

Research on opioid peptides was given a great impetus by the discovery of stereospecific opiate drug binding in the vertebrate central nervous system (Pert and Snyder, 1973; Simon et al., 1973; Terenius, 1973). This discovery initiated the active search for an endogenous opioid-like ligand for the receptor, which led to the isolation and identification of the naturally occurring opioid pentapeptides, methionine and leucine enkephalin (Hughes et al., 1975). With the application in 1982 of recombinant DNA biochemistry (Comb et al., 1982; Gubler et al., 1982; Kakidani et al., 1982; Noda et al., 1982), three genetically distinct opioid peptide families have been identified. Proopiomelanocortin (POMC) contains one potent opioid peptide, p-endorphin, which is the ligand for the E-opioid receptor (Chang, 1984). Proenkephalin (proenkephalin A) is precursor to the enkephalins, all of which are &receptor agonists. Prodynorphin (proenkephalin B) gives rise to the K-WXptOr ligands, the neoendorphins and

Correspondence to: Douglas W. Hoffman, Department of PsychiatryHB7770, Dartmouth Medical School, Hanover, NH 03756, U.S.A. Fax: (603) 646-6 129.

Cochlea;

Auditory

evoked

potential;

Pentazocine;

U50-488.

dynorphins (Akil et al., 1984; Khachaturian et al., 1985; Watson et al., 1982, 1984). Opioid alkaloid drugs such as morphine play an important p-receptor mediated role in antinociception (Akil et al, 1984; Basbaum, 1984; Basbaum and Fields, 1984). It has become increasingly clear, however, that nociception is a part of the larger activity endogenous opioid peptides have in the modulation of sensory transduction and neuronal transmission. Neuroactive opioid peptides and their receptors are widely distributed within mammalian forebrain, midbrain, limbic system and brainstem structures, including primary sensory nuclei of the visual, auditory, olfactory and somatosensory systems (Akil et al., 1984; Mansour et al., 1987; Khachaturian et al., 1985; Watson et al., 1982; 1984). Their presence in these structures suggests that these substances perform complex and integrated functions in the neuromodulation of biologic systems that process information and regulate attention (Akil et al., 1984; Lewis et al., 1981; Miller and Pickel, 1980). The opioid peptides are also found in the auditory system, most prominently in the terminals of the lateral olivocochlear (OC) system innervating eighth nerve dendrites under inner hair cells (Altschuler et al., 1984, 1985; Drescher and Drescher, 1985; Eybalin et al., 1984; Fex and Altschuler, 1981; Hoffman, 1986; Hoff-

man et al., 1983, 1984, 1985). Efferent cell bodies in the lateral superior olivary region (the ‘lateral’ efferent system) synapse directly on auditory nerve dendrites under inner hair cells, while medial periolivary nuclei send terminal fibers (the ‘medial’ efferents) to the basal and circumnuclear regions of outer hair cells (Warr et al, 1986). Both proenkephalin and prodynorphin gene products are found in lateral efferent OC cell bodies, fibers and terminals (Altschuler and Fex, 1986; Altschuler et al., 1984, 1985; Eybalin et al, 1984; Fex and Altschuler, 1981; 1986; Hoffman et al., 1983, 1985; Ryan et al., 1988). Specific opioid receptor subtypes (e.g. ~,c~,K,E,) in the olivocochlear efferent system have not yet been identified. Opioid receptor presence in the cochlea has been shown by stereospecific binding (Hoffman, 19861, and has gained additional support by the demonstration (Eybalin et al., 1987) of naloxone-reversible inhibition of receptor-linked adenylate cyclase in guinea pig homogenates. Both p-and &opioid receptors in cochlear tissues may be coupled to the same adenylate cyclase (Eybalin et al., 1987). It would seem equally likely that K-receptors are also present in cochlea, based on the known presence of prodynorphin gene products in efferent terminals (Altschuler et al., 1985; Hoffman et al., 1985). The goal of these studies has been to identify a specific receptor-mediated role of the opioid peptides found in lateral olivocochlear efferents. Such an effect must represent a role of the lateral olivocochlear efferent system in auditory processing.

Materials

and Methods

Chinchillas (chinchilla faniger, 450-600 g> were anesthetized with ketamine hydrochloride (50 mg/kg) or Telazol” (30 mg/kg) administered intramuscularly, and subsequently maintained on pentobarbital. Telazol is a nonnarcotic, nonbarbituate anesthetic agent consisting of equal parts by weight of the dissociative anesthetic agent tiletamine HCl, and the diazepinone sedative muscle relaxant zolazepam HCl (Hrapkiewicz et al., 1989). A tracheostomy was performed in order to assist breathing. Body temperature was monitored with a rectal thermistor probe and maintained at a constant level (35-35.5”C) throughout testing with a heating blanket. The internal jugular vein was cannulated with PE-10 polyethylene tubing. Additional ketamine (5 10 mg/kg, im) was given as needed. Insertion of the tubing into the right internal jugular vein never exceeded 2 cm. The distal end of the tube fit onto a 30G needle, inserted into a zero dead volume three-way valve. An additional length of PE-10 tubing extended from the stopcock to a 10 ml syringe mounted on a perfusion

pump system located outside of the test chamber. Following cannulation, anesthesia was maintained with sodium pentobarbital (6.5 mg/ml sterile lactated Ringer’s solution), delivered at a constant flow rate of 5 pl/min, as controlled by the syringe perfusion pump. The open port on the three-way valve was fitted with a Luer-lock hub and used to administer pharmacological agents directly into the jugular vein. To expose the cochlear base for N,/N, recording, the skin, muscle and connective tissue overlying the left auditory bulla in the region just caudal and slightly ventral to the osseous external auditory canal were removed. The soft bone of the auditory bulla was carefully excised with a small rongeur, exposing the tympanic annulus, cochlear base, round window, and the incudostapedial joint. This region corresponded to the mastoid zone previously detailed by Browning and Granich (1978) in this species. The pinna was retracted anteriorward exposing the osseous canal entrance, and an opening into the canal was made. The postauricular venous system was surgically ligated when necessary, and the distal portion of the facial nerve as it exits from its foramen located on the lateral posterioventral surface of the external osseous auditory canal was also sacrificed. Auditory testing and stimulus generation

During all phases of auditory testing, animals were maintained in a prone position on a surgical table and were secured by the head. The adjustable head-holding device allowed for easy access to the left auditory canal and opened bulla, and was mounted on the surgical table housed within a sound attenuating chamber. The sound attenuating chamber was located within a quiet isolated room (background 51 dBA). All auditory potentials were elicited by 100 ps rectangular electric waves generated by a Nicolet 1007A click generator. Clicks were delivered through an Etymotic ER-3A transducer, which has a flat frequency response from 250-4000 Hz with a rapid attenuation at higher frequencies (Wilber et al., 1988). The ER-3A sound tube introduces a 0.9 ms delay in the delivery of the acoustic stimulus. The click generator was capable of delivering stimuli ranging from 22 to 135 dB peak sound pressure level (SPL) (re: 20 PPa). The acoustic stimuli were delivered from the transducer through a foam earplug shaped to fit snugly into the left osseous ear canal. Electrophysiologically obtained auditory thresholds were defined as the lowest intensity producing at least one visually identifiable and replicable action potential. Thresholds were established by a tracking procedure whereby the intensity was reduced in 2 dB steps from 40 dB SPL, until no response could be observed. The intensity level was then increased in 2 dB steps, and threshold was taken as the intensity at which waveform peaks of interest (near field, N, and N2; far field,

135

waves 1-3) could be replicated. In each animal, the stimulus intensity defined as threshold during the predrug baseline recordings was used as threshold throughout subsequent recordings from that animal. Neuroelectric activity was amplified 100,000 times (Nicolet HGA-200A) filtered (Nicolet 5OlA) and averaged on-line with a Nicolet Clinical Averaging System (CAlOOO). The data channel was digitized with an g-bit A/D converter every 100 ~LS(10 kHz sampling rate). Electrocochleography N, and N2 components

of the compound action potential (CAP) were elicited by 300 alternating polarity clicks, delivered at a rate of 18.3/s. N, and N2 potentials were recorded using standard platinum-alloy subdermal needle electrodes. The active, noninverting (+I electrode was positioned at the scalp vertex, and a common (ground) electrode was inserted over the neck musculature. The inverting ( - ) reference electrode was placed near the round window of the cochlea, and consisted of a subdermal platinum-alloy needle electrode wrapped with a small section of fine diameter silver wire flamed at the tip to form a 0.5 mm diameter ball. The impedance of the round window electrode was maintained from 4500-85000 by the application of a minute amount of water soluble transmission gel to the electrode tip. The spherical electrode tip was placed onto the round window niche and held in place over the mastoid opening with a custom designed electrode clamp. The electrode clamp was adjustably secured to the head holder, allowing for placement or removal of the electrode. Neuroelectrical activity was amplified and band pass filtered from 150 Hz to 3000 Hz. Three hundred sweeps, each having a 5 ms epoch were averaged on line at each of four stimulus intensities. The four intensity levels were threshold, and 10, 30, and 50 dB above threshold (SL). All animals used in the present investigation had N,/N, thresholds ranging from 22 to 40 dB SPL, with an average threshold of 29 dB SPL. N, and N2 potentials were obtained at the four intensities using a counter-balanced intensity order across animals. Two N, and N2 potentials were obtained at each intensity, and absolute latency and peak-to-trough amplitude of N, and N2 were evaluated on-line at each of the four stimulus intensity levels. Six N, and N2 amplitudes were recorded from each animal at each intensity during each of three 30 min baseline periods (which included a 30 min Ringer’s period), and during three 30 min periods following either drug or Ringer’s administration. Baseline was defined as the average of the mean amplitude values obtained during the three predrug (Bl-B3) testing periods. The values of the individual baseline periods (BlB3) relative to the overall average are shown in Figs. 2 and 3 in order to represent the variability we routinely

observed during the baseline periods. Control animals were given equivalent volumes of drug vehicle (Ringer’s solution) in place of drug solutions, and recordings were obtained from these animals in the same manner as experimentals. Potentials were displayed negative up at each intensity for online analysis. Amplitudes were measured from the point of maximum negativity of N, or N2 to the point of maximum positivity of the following trough (Pi or P,, respectively). To investigate possible opioid effects at the outer hair cells, the cochlear microphonic (CM) was obtained in three animals. Microphonic activity was band pass filtered from 150 Hz to 8 kHz and recorded using the same electrode montage. CM potentials were elicited by 300 negative polarity clicks (18.3/s) and were obtained at the single intensity level of 10 dB (SL) above the threshold for the CAP, at which level stable CM recordings could be obtained. Unlike efferent effects on N,, medial efferent effects on the CM are independent of the intensity of the stimulus eliciting the CM (Wiederhold, 1986). Microphonic potentials were obtained over the same length of time as N, and N, recordings. At least six CM potentials were recorded in each successive 30 min test period. Auditory brainstem response recording

Absolute latency and peak-to-trough amplitudes of the separate wave components (waves l-4) of the auditory brainstem response (ABR) were also obtained in three animals. Far field recorded ABRs were elicited by 1200 alternating polarity clicks delivered at a rate of 68.3/s. ABRs were recorded using an additional, independent set of platinum-alloy subdermal needle electrodes. The active ( + ) electrode was positioned at the scalp vertex with an ipsilateral reference ( - ) electrode at the mastoid. A ground electrode was inserted over the contralateral mastoid. Electrode impedance in all animals was < 3000 0. Neuroelectrical activity was amplified and band pass filtered from 150 to 1500 Hz. Twelve hundred sweeps, each having a 10 ms epoch were averaged online at the four intensity levels indicated. Acoustic reflex threshold

The intensity level required to elicit an acoustic reflex threshold in this species was determined in 3 additional animals. The contralateral middle ear muscle reflex was elicited by pure tones (1 s duration; 2 ms onset/offset) from 500 Hz to 3 kHz. Reflex thresholds were measured using a probe tone frequency of 660 Hz, and were detected with a Grason Stadler 1720B otoadmittance meter. Thresholds were obtained with a descending-ascending method of limits, and were bracketed in 2 dB increments over 3 consecutive sweeps, beginning at 100 dB SPL. ARTS were accepted as a visually monitored 0.2 mmho compliance change.

136

Drug administration

16 mg/kgI, and two 7 X 6 (mean) data matrices (pentazocine 8 mg/kg). In each of the four analyses, control vs experimental comparisons were made across three 30 min baseline and three 30 min postbaseline periods. Statistical significance was defined by an alpha level of < 0.05.

All drugs were dissolved in lactated Ringer’s solution. All solutions were administered iv at a constant volume (1 p1/5g body weight) and rate (50 pl/min). The following drugs were tested: (-I-) pentazocine HCl (p-antagonist, K-agonist; 8 mg/kg and 16 mg/kg) (Sigma); U-50,488H (trans-3,4-dichloro-N-methyl-N-[2(l-pyrrolidinylj-cyclohexyll-benzeneacetamide} (K-agonist; 6 mg/kg and 20 mg/kg) (Research Technology Branch, NIDA), the potent p-agonist fentanyl citrate (2 mg/kgI (Sigma), and the strong p-antagonist, weakly K-antagonistic, naloxone HCl (1.1 mg/kg) (Sigma). Doses represent total amounts infused over the course of 30-60 min. Absolute latency and peak-to-trough amplitudes of N, and NZ were evaluated online after drug administration at each of the four stimulus intensity levels determined during the predrug baseline recording periods. Control animals were given equivalent volumes of drug vehicle (Ringer’s solution) in place of drug solutions, delivered at the same flow rate. Dosages were based on achieving approximate equal potencies across the drugs used.

Results

A consistent upward drift in N, amplitudes was observed in animals anesthetized with Telazol@ (tiletamine/ zolezepam) and pentobarbital, which was not seen with ketamine/pentobarbital. Amplitudes at all stimulus intensities continued to drift upward over all recording periods. This difference between the drugs is illustrated in Fig. 1, at 50 dB SL. The use of tiletamine/zolezepam was therefore discontinued, and results shown in Figs. 2-5 are from ketamine/pentobarbital anesthetized animals only. The threshold for activation of the middle ear reflex in ketamine/pentobarbital anesthetized chinchillas was found to be 82 f 0.4 and 90 + 3.9 dB SPL (mean + SEMI at 500 Hz and 1 kHz, respectively (N = 3). Threshold for activation increased with increasing frequencies over 1 kHz. Mu-opioid receptor-preferring drugs did not affect either N, or N2 amplitudes. The p-opioid receptor antagonist naloxone (1.1 mg/kg; N = 21, and the potent k-receptor agonist fentanyl(2 mg/kg; N = 2) both failed to produce postbaseline amplitude changes in potentials from the cochlea and eighth nerve (Fig. 2) at any intensity tested. In contrast, the K-opioid receptor agonist, p-receptor antagonist pentazocine increased N, and N2 amplitudes markedly. Pentazocine (16 mg/kg; N = 5) significantly increased N, amplitudes [F(1,8) = 18.29; P < 0.0051 when compared to postbaseline control values (Fig 2a). Peak N, amplitude effects ranged up to 263% over baseline (mean = 133% in-

Data analysis

Pentazocine effects on the N, and N2 amplitudes at threshold were statistically evaluated in a total of 14 animals, using multivariate analyses of variance. Relative changes in N1/N2 amplitude from baseline N,/N, amplitude were assessed in ketamine/pentobarbitol anesthetised animals following the administration of pentazocine at two doses (16 mg/kg and 8 mg/kg), using four separate analyses. Pentazocine (16 mg/kg) effects were evaluated in five experimental and five matched control animals. Effects of 8 mg/kg pentazocine were evaluated in 4 animals. For statistical analysis, N, and N2 amplitudes recorded during each of the six 30 min test periods were averaged, creating two 10 x 6 data matrices of mean values (pentazocine

E 0

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Fig. I. Preanesthesia N = 2), or ketamine

effects on auditory (50 mg/kg;

evoked potentials.

N = 2) preanesthesia

60

45

Time

75

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(min) Effects of iv pentobarbital

on baseline N, amplitudes

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keiamine

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137

67% for N,, and 47% for Nz. Following the second 4 mg/kg delivery, average peak elevations reached as high as 97% for N,, and 103% for Nz over baseline values. While N, and N2 baseline amplitudes were stable across all animals [F(V) = 0.67; pns. and F(1,5) = 0.27; pns., respectively], pentazocine (8 mg/kg) effects on N, and N2 amplitudes nevertheless failed to reach levels of statistical significance [F(1,5) = 3.35; pns. and F(1,5) = 2.30; pns., respectively]. All four wave components of the ABR recorded at threshold (with surface electrodes) demonstrated amplitude changes following either 8 mg/kg (N = 2) or 16 mg/kg (N = 1) pentazocine. Representative waveforms of both near field and far field potentials in the same animal obtained following a single dose of pentazocine (16 mg/kg) are shown in Fig. 4. In general, drug-induced latency changes were not observed in any animal at any of the four intensity levels tested. A similar (197%) N, amplitude increase was observed in one animal tested with the selective K-reCeptor agonist U-50,488H (20 mg/kg), while no effects were seen in three animals tested at a lower dose (6

crease over baseline). Peak Nz amplitudes after pentazocine increased up to 230% over baseline (mean = 122%). Pentazocine (16 mg/kg) effects on the N2 (Fig 2b) however, only approached a level of significance [F(1,8) = 4.25; P = 0.071. Amplitude increases were observed exclusively at threshold intensities (Fig. 3). During baseline testing, threshold N, and N2 amplitudes in the experimental animals did not differ significantly from those in the 5 controls [F(1,8) = 0.74; pns]. Maximum effects in both waves were observed within 60 min post administration, returning to baseline values by 135 min. Peak effects of pentazocine on wave amplitudes were not affected by co-adminstration of the opioid receptor antagonist naloxone (1 mg/kg) (N = 1; not shown). In 4 other animals, the effect of a total dose of 8 mg/kg pentazocine on N, and Nz amplitudes, given in two 4 mg/kg injections 30 min apart, was investigated. Pentazocine at this lower dose produced similar amplitude increases of N, and N, compared to postbaseline values in controls (Fig 2). Following the first iv dose, pentazocine led to average peak elevations as high as

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'(16mglkg) Fmtazocin,

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Dl

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D3

Treatment

‘(16mglkg)

pmtazocins (8mnlkn) fentanyl (Zmnlkn)

naloxono (l.lmg/kg)

Treatment Fig. 2. Effects of opiate drugs on N, and N2 amplitudes. Six amplitude measurements were made in each 30 min period (Bl-D3) at threshold following pentazocine at 16 mg/kg (N = 5) or 8 mg/kg (N = 4), fentanyl at 2 mg/kg (N = 2), Ringer’s (N = 5), or naloxone at 1.1 mg/kg (N = 2). Data points represent the percent change of the mean amplitude from baseline (average of the amplitude values obtained in that animal prior to drug administration). Bl-B3, baseline recording periods; Dl-D3, postdrug recording periods. (A) N,; (B) Na.

138

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Treatment Fig. 3. Effect of pentazocine (16 mg/kg) on N, and Nz amplitudes at different stimulus intensities in one representative animal. Six amplitude measurements were obtained in each 30 min period (Bl-D3) at each of the four stimulus intensities indicated. Data points represent the percent change from the average of the mean baseline amplitude values obtained during the three 30 min baseline recording periods (Bl-B3). (A) N,; (B) Nz.

mg/kg). Neither pentazocine (16 mg/kg) (Fig. 5) nor U-50,488H (20 mg/kg) (N = 1; not shown) had any measurable effect on CM.

Discussion

Opioid peptides are known to occur in olivocochlear neurons terminating under inner hair cells in the

cochlea. Here they may function as neuromodulators, influencing the activity of co-localized transmitters, or they may cross the synapse and act directly on receptors on eighth nerve dendrites. Either mode of action is likely to result in complex actions on the early components of the auditory evoked response, N, and A$, but not on the CM. The first and second negative peaks (N, and N2) of the auditory nerve compound action potential reflect

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1

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Time

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Fig. 4. Effect of pentazocine (16 mg/kg) on threshold auditory evoked potentials in one animal. Representative near field (A) and far field (B) recorded potentials obtained during baseline and at 30 (near and far field) and 60 min (far field) post administration of pentazocine are presented. Potentials are displayed negative up.

139

determination of the degree of threshold shift awaits further study. Both ketamine and pentobarbital anesthesia have been reported to produce respiratory depression and a subsequent rise in blood pC0, levels (Brown et al., 1989). The respiratory depression produced by morphine and other opiates involves a reduction in the responsiveness of brain stem respiratory neurons to increases in pCO,, which results in increases in blood pC0,. Such effects are mediated in large part by p-opiate receptors (Furst and Weinger, 1990; Jaffe and Martin, 1985). Progressive increases in pC0, (or decreases in arterial ~0,) generally lead to amplitude reductions in the CAP (Hildesheimer et al., 1987). However, fentanyl and morphine have been shown not to alter the amplitudes and latencies of the early peaks of the auditory evoked response (Fig. 2; see also Sahley et al., 1987; Samra et al., 1984; 1985). Fentanyl combined with pentobarbital anesthesia has been reported to elevate pC0, to an even greater level than ketamine plus pentobarbital (Brown et al., 19891, probably due to enhanced suppression of neurogenic respiratory drive. However, in the present investigation, no amplitude changes were observed after prolonged pentobarbital anesthesia, nor in animals treated with fentanyl and pentobarbital. This is in spite of the fact that naloxone-reversible sedation and apparent depression of respiratory rate were observed in fentanyl-treated animals. It is for these reasons unlikely that the effects observed in the present investigation are due to pC0, fluctuations. Ketamine is a dissociative anesthetic related to phencyclidine which may selectively antagonize the activity of excitatory amino acids at the NMDA-type receptor (Fagg et al., 1986). Ketamine administration has been reported to lead to dose and stimulus intensity-dependent changes in both the latency and amplitude of the major components of the ABR, consisting of amplitude increases in waves 1 and 3 with increasing stimulus intensities (Church and Gritzke, 1987). Such effects, however, have been reported at doses consider-

neural activity of the auditory periphery and lower brainstem, respectively, in response to an auditory stimulus. In most mammalian species, the amplitude of N, represents neural activity and synchronization coincident with the onset discharges produced by single units innervating the basal turn of the organ of Corti (Moller and Jannetta, 1985). The magnitude of N2 in rodents coincides with the discharge level of neurons located within the region nearer to the ipsilateral cochlear nucleus and contralateral superior olivary complex (Wada and Starr, 1983a; 1983b). The CM potential is an extracellular frequency- and intensitydependent response to sound which is generated predominantly by the cochlear outer hair cells (Dallas, 1984; Wang and Dallos, 1972). Effects occurring at outer hair cells are reflected in CM and in N, and N2, while more proximally occurring actions at inner ear cells only affect Nr and N2, and possibly subsequent waves of the ABR. Pentazocine amplification of N, and N2 potentials suggests a K-opioid receptor mediated role of the endogenous K-agonists, the dynorphins, which is further supported by the preliminary results with U50,488H. This may reflect K-receptor effects on N, which are cochlear in origin. The lack of effect of a p-agonist and antagonist (fentanyl and naloxone, respectively) in the present study corroborates previous research which failed to identify a F-mediated effect on electrophysiological potentials arising from the auditory periphery and lower brainstem (Sahley et al., 1987; Samra et al., 1984; Velasco et al., 1984). These data were recorded as amplitude increases at the stimulus intensity determined to be the auditory threshold in each animal prior to drug treatment. These were the best quantitative data obtainable at the very low stimulus intensities at which these drug effects were seen. It may reasonably be inferred that thresholds shift to a lower value in dB SPL as a consequence of these drug-induced amplitude increases. These could not be quantified as reliably with our present equipment as could the amplitude increases. Quantitative 200 t I

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Treatment Fig. 5. Effects were averaged Baseline

of pentazocine (16 mg/kg) on N, and CM amplitudes in the same animal. Threshold N, and CM (10 dBSL) amplitude values over each of the six 30 min test periods (N = 2). Data points represent the percent change of each mean amplitude from baseline. is defined as the average of the mean amplitude values obtained during the three 30 min baseline recording periods (Bl-B3).

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ably higher (i.e., 200 mg/kg) than the 50 mg/kg dose used in the present investigation. Ketamine at 100 mg/kg does not affect amplitudes or latencies of early components (N, and N,) of the ABR (Bobbin et al., 1979). Ketamine plus pentobarbital has been reported to increase 2-deoxyglucose uptake in cochlear nucleus and superior olive, among other auditory nuclei (Wang et al., 1987). This increase in 2-deoxyglucose uptake does not appear to be reflected in electrophysiological activity of the auditory nerve in our study, and may occur more centrally. Telazol is a nonnarcotic, nonbarbituate anesthetic agent consisting of equal parts by weight of tiletamine HCl and zolazepam HCI. Tiletamine is a dissociative anesthetic agent similar to ketamine, whose pharmacologic action is characterized by rapid induction, profound analgesia, normal pharyngeal and laryngeal reflexes, and cataleptoid anesthesia. Zolazepam is pharmacologically similar to diazepam (Hrapkiewicz et al., 19891, and so is likely to modulate GABA receptor activity. The changing baseline activity in animals maintained on this combination may reflect underlying metabolic activity or changes in depth of anesthesia, as well as synaptic activity-mediated changes. In spite of its advantages as an anesthetic agent, it appears to have limited utility for electrophysiological studies of evoked potentials. It cannot be clearly discerned from the effects of anesthetic agents on our recordings whether we do observe any drug effects on auditory evoked potentials which may be ascribed to actions at GABA or NMDA-type receptors. However, in a limited number of preliminary experiments completed to date, (- 1 pentazocine, the active K-agonist, had all the activity of the racemic pair, while the (+) enantiomer of pentazocine, which is a phencyclidine congener active at NMDA-type receptors, had no activity whatsoever on N, and Nz. This would tend to argue against the presence of peripheral NMDA-type receptor affecting auditory potentials. Effects of hypothermia on the ABR are well documented, and are seen primarily as a 2% cumulative prolongation of the I-V interpeak latency for every 1°C decline in body temperature (Kileny and McIntyre, 1985; Sohmer, 1989). Amplitude fluctuations in ABR waves do not result from body temperature changes (Sohmer et al., 1989). Temperature effects on the latencies and amplitudes of near field cochlear potentials have also been reported in pentobarbital anesthetised rodents. N, latencies can increase as much as 0.04 ms for every 1°C fall (from 35 to 30°C) in intracranial temperature, but N, amplitudes are generally unaffected by temperature fluctuations (Inamura et al., 1987). In our studies, changes in latencies of ABR waves were not observed with any drug tested. Therefore, the pentazocine induced amplitude effects pre-

sented are unlikely to result from the 0.5”C fluctuations in core temperature which we observed. It must be considered whether tonic or dynamic changes in middle ear or other muscle activity play a role in the observed drug-induced amplitude changes. Middle ear muscle activity has been shown to have little or no influence on auditory responses at threshold intensity (Guinan and McCue, 1987), high frequency (Zakrisson et al., 1975) or impulse sounds (Solomon and Starr, 1963), such as the click stimuli used in the present investigation. Ketamine/pentobarbital anesthetised animals exhibit an apparent lack of tonic EMG activity within the tensor tympani and stapedius muscles, and EMG thresholds for both muscles have been reported at 90 dB SPL, using 1 kHz tone bursts (Guinan and McCue, 1987; McCue and Guinan, 1988). We also directly measured the acoustic reflex threshold in 3 animals used in the present study, and found the threshold for activation of the middle ear reflex to be far above the intensity of the stimuli at which we observed auditory potential amplitude effects. Similarly, it is unlikely that a reduction in electrical noise resulting from systemic effects of the drug on spontaneous muscle activity or muscle tone is the basis for the observed amplitude increases. Fentanyl has far more pronounced muscle relaxant and respiratory depressant effects in our animals than pentazocine. There were no measurable effects of fentanyl on auditory evoked potentials at dosages ultimately resulting in complete respiratory suppression, even in animals in which reductions in muscle artifact in the ABR recordings could be observed. Recent evidence suggests that the crossed (medial) efferents do not act tonically in determining auditory nerve sensitivity and evoked potential amplitudes (Rajan et al., 1990). Results obtained in the present investigation tend to support such a role for the uncrossed (lateral) efferent system. The lateral efferent terminals on auditory nerve dendrites are anatomically well situated to modulate sensitivity and spontaneous discharge in primary afferents (Liberman, 1980a; 1980b; 1988). One possible mechanism for the threshold effect we observe is a lateral efferent role in increasing the contribution of primary afferent fibers to the threshold response, resulting in greater amplitude of the N, and N, at threshold intensities. Presynaptic interactions of the multiple colocalized transmitters in lateral olivocochlear efferents may also be occurring. Other possibilities include direct excitatory actions of dynorphins on auditory nerve dendrites, perhaps serving to disinhibit them from cholinergic suppression or adaptation phenomena. Earlier work (Bobbin and Konishi, 1974) has defined an inhibitory effect of cholinergic agonists on auditory-evoked potentials, and suppression of auditory-evoked potentials has been accepted as the pri-

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mary action of the olivocochlear efferents. These studies have really been confined to the medial olivocochlear innervation. The stimulatory action of Kagonists on auditory-evoked potential amplitudes may represent a physiological role of the lateral efferents. This action is likely to be mediated by the dynorphinrelated neuropeptides contained in these fibers, which are excitatory to postsynaptic neurons in other parts of the CNS. It would appear then, that the different neurotransmitters of the olivocochlear efferents may have antagonistic actions, as may the lateral and medial systems themselves. The role of olivocochlear efferents in audition is not well defined at this time, in spite of much research. The understanding of their functional significance requires a better understanding of the neurochemical substrate which mediates these actions; i.e., neurotransmitters and their receptors. The complex interactions of multiple neurotransmitters and modulators at the olivocochlear/eighth nerve/hair cell junction may define many characteristics of peripheral auditory processing, and so point the way to understanding the functional role of the olivocochlear innervation. Such an understanding has both neurophysiological and therapeutic significance.

Acknowledgements

This work was supported in part by the National Science Foundation (BNS 8646563; DWHI, the Henry Hey1 Fund of the Hitchcock Foundation (DWH) and the Deafness Research Foundation (FEM & DWH). The authors thank Drs. Alan Basbaum, Christoph Schreiner and Michael Merzenich for valuable discussions of this work, and Dr. Therese Stukel for statistical support. We acknowledge with thanks the gift of the thermistor monitor from the John Fluke Co, and pentazocine and U50,488H from the Research Technology Branch, NIDA.

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Effects of opioid be drugs on auditory evoked potentials suggest a role of lateral olivocochlear dynorphins in auditory function.

Multiple gene products of opioid peptide families (e.g., enkephalins, dynorphins) with differing opioid receptor specificities are present within oliv...
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