Effects of repeated cocaine injections on cochlear function

BRAIN RESEARCH ELSEVIER

Brain Research 668 (1994) 230-238

Research report

Effects of repeated cocaine injections on cochlear function Bhagyalakshmi G. Shivapuja a , , Zhi-Ping Gu b, Shi Yuan Liu a, Samuel S. Saunders Eugene P. Schoener c

a,

a Department of Otolaryngology, Henry Ford Hospital, Detroit, MI, USA b Department of Otolaryngology, First Hospital, Beijing Medical University, Beijing, People's Republic of China c Addiction Research Institute, Wayne State University, Detroit, MI, USA Accepted 27 September 1994

Abstract

The effects of repeated cocaine administration on cochlear function were evaluated by measuring amplitude-intensity and latency-intensity functions of the whole-nerve action potential of the auditory nerve. Whole-nerve action potential input/output functions obtained using tone-pips of 0.5, 1, 2, 4 and 8 kHz in a group of cocaine-treated subjects were compared with those obtained in saline-treated animals. All measurements were made 24 h after the last treatment. Amplitudes of whole-nerve action potentials were enhanced in the cocaine-treated animals compared to the control group. No statistically significant differences in latency-intensity functions were seen after cocaine treatment. The effect of chronic cocaine exposure also was examined on catecholamine innervation in the cochlea using immunohistochemical techniques. The density of adrenergic innervation was reduced in the cocaine-treated animals.

Keywords: Chronic cocaine; N 1 amplitude-intensity functions; N 1 latency-intensity functions; Tyrosine hydroxylase

1. Introduction

The addictive nature of cocaine is well known. Effects of cocaine on the central nervous system and the vascular system have been studied extensively [19,12,20]. Acutely, cocaine manifests a local anesthetic property at high doses and psychomotor stimulation at low doses. Since cocaine targets catecholaminergic mechanisms, it is also a potent sympathomimetic agent [27]. The euphoria caused by cocaine has been associated with the activation of dopaminergic pathway in the meso-limbic system [15,4]. Although euphoria due to cocaine is rapid and brief, addiction to the drug seems to be associated with long-term changes in brain function after repeated drug exposure [24,19]. Chronic cocaine use is associated with behavioral changes characterized by tolerance [21] or by sensitization [15]. Changes in neural activity with repeated cocaine exposure have also been described in the central nervous system [23].

The effects of cocaine on sensory systems are not well understood. However, direct action of cocaine on the cochlea has been implied by several investigators [5,6,11,10]. Acute effects of cocaine on the cochlear function were described in an earlier study [30], which showed a reduction in the auditory-nerve compound action potential amplitude after a single injection of cocaine at low to moderate dose. Furthermore, changes in the micro-circulation of the cochlea occurred after a single low-dose injection of cocaine [30]. These findings raise concern about the impact of chronic cocaine exposure on the cochlea. The present study was designed to evaluate the direct action of repeated administrations of cocaine on the whole nerve action potential of the auditory nerve and the catecholaminergic innervation of the cochlea.

2. Materials and methods 2.1. Experiment 1: electrophysiology

* Corresponding author. Otolaryngology Research Labs., E&R 7034, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI-48202, USA. Fax: (1) (313) 556 8110. 0006-8993/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 4 ) 0 1 1 8 8 - 5

2.1.1. Subjects and surgery Ten healthy chinchillas (Chinchilla Lanigera) weighing between 500-600 g were randomly assigned to the control or experimental

B.G. Shivapuja et al. / Brain Research 668 (1994) 230-238 group. All animals were treated daily with an injection of either cocaine (15 mg/kg, I.P.) or saline (0.15 M NaCI, I.P.) of equivalent volume for 90 days. Recordings were obtained 24 h after the last treatment. All measurements were made with the animals under anesthesia. Anesthesia was induced with 10-13 mg/kg Ketamine (S.C.) and subsequently maintained with Nembutal (35 mg/kg, I.P.). Body temperature was monitored with a rectal probe and maintained at 37°C with a heating blanket. The animal was hydrated with 0.5 cc of saline periodically. The right pinna was removed to place the sound source in close proximity to the tympanic membrane. A silver ball electrode was placed on the round window and cemented into place. 2.1.2. Stimuli Acoustic stimuli consisting of tone bursts (10 ms, 1 ms rise-fall) ranging in frequency from 0.5 to 8 kHz were generated using a signal processing system board (Spectrum, Model TMS320C25) and a 386PC computer. The output of this board was fed into a programmable mixer/switch, programmable attenuator (Wavetek, Series P557TTL), impedance matching network, and an earphone (Bayer Dynamic, DT48). The stimulus levels were calibrated before each experiment using a probe microphone (Etymotic, ER7C) and a sound pressure level meter (Larsen-Davies, Model #800B). The starting phase of the stimulus was randomized to minimize cochlear microphonics.

2.1.3. Data collection: recording The output from the round window electrode was fed to an amplifier (GRASS, Model P511H, 50 X gain, 30 Hz-30 kHz bandpass), and then to a signal processing board (Spectrum, Model TMS320C25) to digitize (sampling frequency 17 kHz) and average the N 1 response. The output of the amplifier was viewed on an oscilloscope (Tektronix, Model #5112). The averaged waveforms were stored on a 386-PC computer for off-line analysis. A total of 256 sweeps were averaged. Each sweep represented 15 ms of neural response starting at onset of the stimulus. Amplitude-intensity and latency-intensity functions (20-80 dB SPL) were obtained for tone-pips of 0.5, 1, 2, 4, and 8 kHz. Two measurements at each stimulus condition were obtained for each animal.

2.1.4. Data analysis Separate analyses were performed on amplitude and latency measures. Average amplitude-intensity functions or latency-intensity functions were compared across the two groups. Analysis of variance (ANOVA) was used to compare the CAP amplitude and latency as a function of stimulus frequency and stimulus intensity for the two groups.

2.2. Experiment 2: immunohistochemistry 2.2.1. Subjects Ten additional healthy chinchillas (Chinchilla lanigera) weighing between 500-600 g were randomly assigned to either control or experimental group. All animals were treated daily with an injection of either cocaine (15 mg/kg, I.P.) or saline (0.15 M NaCI, I.P.) of equivalent volume for 42 days. The animals were sacrificed 24 h after the last cocaine or saline injection. Z2.2. Immunostaining Pilot experiments were conducted to evaluate the feasibility of using commercially available antibodies to tyrosine hydroxylase (TH) or to dopamine-/3-hydroxylase (DBH) for identifying the presence of adrenergic fibers in the chinchilla cochlea. Based on the reactivity of the above antibodies to chinchilla tissue, a decision was made to use TH antibody to evaluate differences between control and cocainetreated animals.

231

Tyrosine hydroxylase presence in the cochlea was assessed using a modified immunostaining technique described previously [2]. The cochleas were perfused in vivo with 4% paraformaldehyde in phosphate buffer (pH 7.4) removed and post-fixed with the same fixative for 3 h at room temperature. Then the cochleas were decalcified overnight at room temperature in 0.35 M EDTA (pH 7.4). The organ of Corti (PC) was dissected into individual turns and the lateral wall was removed. The individual P C turns were placed in 1% Triton X-100 in PBS for 30 min. The turns were then rinsed twice with phosphate-buffered saline (PBS) containing 0.1% Triton X-100 followed by permeation in graded alcohol (30%, 50%, 70%, 50%, 30%) for 10 min each. After three additional rinses in PBS with 0.1% Triton X-100, the tissue was placed in 0.15% hydrogen peroxide in methanol for 25 min. Then, the tissue was placed in 10% normal goat serum for 2 h at room temperature. Next the tissue was incubated for 68 h at 4°C in a solution containing primary antibody to TH (Chemicon, Temecula, CA) diluted 1:500, 4% normal goat serum and 0.1% Triton X-100. The tissue was rinsed 3 times with PBS containing 0.1% Triton X-100 before being incubated in the secondary antibody, biotinylated goat anti-rabbit IgG (Vector Labs, Inc., Burlingame, CA) at room temperature for 3 h. The tissue was rinsed again in PBS containing 0.1% Triton X-100 and was incubated for two hours at room temperature in the Vectastain ABC reagent (Vector Labs, Inc., Burlingame, CA). This was followed by another rinse with PBS and an 8 min incubation in 0.075% diaminobenzidine tetrahydrochloride (DAB), 0.005% hydrogen peroxide and 0.5% solution of gelatin. The tissue was rinsed in PBS before being mounted on slides with glycerol for viewing under light microscope. With each series of cochleas (8 to 10), one normal cochlea was used for negative control (primary antibody was replaced by buffer) and one was used for a positive control to assess signal to noise ratio of TH staining. Changes in TH staining of the cochleas between the two groups was determined by an observer familiar with the appearance of normal cochlear tissue. This observer was asked to judge randomly selected samples from both groups for a visible reduction in density of innervation.

3. Results

3.1. Experiment 1: electrophysiology The whole-nerve or compound action potential of the auditory nerve can be defined as a synchronized response by a subset of fibers in the auditory nerve to the onset of a discrete auditory stimulus. This response is characterized by a negative peak known as the N~ potential. Fig. 1 shows representative waveforms of N t potential in response to a 2,000 Hz tone at 50 dB SPL for a control (solid line) and an experimental animal (dotted line). The responses shown were obtained by averaging two measurements for each animal. N 1 arises from the initial synchronized firing of a defined group of auditory-nerve fibers. The source for the N 2 potential is not well understood. In the cocaine-treated animals, the N 1 and the N 2 amplitudes increase and their latencies decrease.

3.1.1. Amplitude-intensity functions Amplitude-intensity functions for 0.5, 1, 2, 4 and 8 kHz are shown in Fig. 2 A - E , respectively. In each

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TIME (ms) Fig. 1. Averaged waveforms of compound action potential recorded from the round window. Solid line represents the response obtained from a control animal for a tone-burst of 2 kHz at 50 dB SPL. Dotted line represents the response obtained from a cocaine-treated animal (15 mg/kg daily for 90 days) for the same 2 kHz at 50 dB SPL tone-burst. Notice the enhancement in N1 and N2 amplitudes and a decrease in the latency of N l and N 2 response for the cocaine-treated animal. panel, the hollow symbols represent the m e a n N 1 amplitude obtained from the five animals in the control group and the solid symbols represent the m e a n N L amplitude from the five cocaine-treated animals. T h e leftward shift of this function for the cocaine-treated group demonstrates an increase in the m e a n N t amplitudes obtained from these animals. This increase seems to be constant with stimulus intensity > 30 dB for frequencies below 4 kHz. At 4 and 8 kHz, the increase in N 1 amplitude seems to be mainly in the mid-intensity range. To preclude any bias, responses that were saturated or close to saturation were not included in the statistical analysis. Saturation of responses was d e t e r m i n e d as follows: for each animal, condition, and frequency, a line of best fit between stimulus level in dB sound pressure level and logarithm of response amplitude was determined by linear regression. Residuals from the regression were examined, and points were eliminated when all determinations for a given level fell below the predicted amplitude based on regression for the range of stimuli tested. Regression was then recomputed, and the residuals examined again, until no

further pruning was indicated. With this process, it was d e t e r m i n e d that responses in the range of 3 0 - 5 5 dB SPL were not saturated. Therefore, a r e p e a t e d measure analysis of variance with two repeating factors (stimulus frequency (5 levels) and stimulus intensity (6 levels)) and 1 grouping factor ( c o n t r o l / e x p e r i m e n t a l ) was p e r f o r m e d on this r e d u c e d data set. T h e amplitude clearly increases with stimulus level for both groups and all frequencies. This increase is confirmed by the significant effect of intensity ( F = 109.46, d f = 5,329, P < 0.0001). T h e growth of amplitude with intensity is essentially linear in dB versus log amplitude coordinates at the lower 3 frequencies, while there is clear curvature at the 2 higher frequencies. This difference in functional form is reflected in a significant frequency by intensity interaction ( F = 2.59, df = 20,329, P < 0.0003). At m o d e r a t e to loud stimulus levels ( > 35 dB SPL), the amplitude is generally higher for subjects receiving cocaine; this is reflected in a significant group by intensity interaction ( F = 1.80, d f = 5 , 3 2 9 , P < 0.0001). • 3.1.2. Latency-intensity functions Statistical analysis on latency measures on the above data set were p e r f o r m e d as described above. While latency appears to be decreased in the cocaine-treated group in Fig. 1, analysis revealed no statistical difference between the two groups. However, latency did decrease as a function of stimulus frequency ( F = 73.61, df = 4,329, P < 0.0001) and intensity ( F = 44.25, df = 5,329, P < 0.001) as r e p o r t e d previously [26]. 3.2. Experiment 2." immunohistochemistry Figs. 3 and 4 illustrate nerve terminals stained in the cochlea using an antibody to TH. T h e sections shown are longitudinal sections focused on the plane below the hair cells in the region of the habenula perforata. T h e dark labelled nerve terminals are consistent with those described as adrenergic nerve terminals in cats [31], gerbils [2] and guinea pigs [14]. T h e b e a d e d nature of these terminals is due to the varicosities. Fig. 3A. shows a section from the basal turn in the region of 8 kHz of a normal cochlea. T h e light area just medial to the habenula perforata is the unmyelinated fiber region. T h e r e is rich arborization of the fibers in the region of the habenula perforata. Similar arborization has b e e n described in the cat cochlea [31]. Fig. 3B illustrates a similar section in the 8 k H z region of the

Fig. 2. Mean amplitude-intensity functions obtained in response to tone-bursts of 500 Hz (Panel A), 1 kHz (Panel B), 2 kHz (Panel C), 4 kHz (Panel D) and 8 kHz (Panel E) are shown. Open symbols show the mean amplitudes obtained from a group of control animals. Solid symbols show the mean amplitude obtained from the cocaine-treated animals. Error bars indicate + 1 standard error of the mean (S.E.M.). Intensity levels in the range 30-55 dB SPL show a significant difference between the two groups. These animals were administered 15 mg/kg of cocaine daily I.P. for 90 days.

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Fig. 3. A: longitudinal section near the 8 kHz region of the basal turn of a normal cochlea. Large arrowheads point to the beaded adrenergic nerve terminals running radially from the modiolus (not seen) to the periphery. Notice the rich arborization in the habenula perforata (HP). 'T' denotes the tunnel of Corti. B: longitudinal section near the 8 kHz region of the basal turn of a cochlea in a cocaine-treated animal. Large arrowheads point to the beaded adrenergic nerve terminals running radially from the modiolus (not seen) to the periphery. 'H' denotes HP. Notice the lack of arborization of the nerve terminals near HP and the reduction in the number of nerve terminals. Stars indicate regions with no nerve terminals. basal turn in a cocaine-treated animal. Note the reduction in density of nerve terminals. This was a typical finding in the base and hook regions of the cochlea in cocaine-treated animals. Changes were also noted in other regions of the organ of Corti. A longitudinal section of this structure in the apical/middle turn (500 Hz region) of a normal chinchilla (Fig. 4A) shows similar beaded fibers running radially from the modiolus to the periphery (large arrow-heads). Radiating capillaries are visible in this section. The number of arborizations near the habenula perorata region (labelled ' H ' ) in the middle and apical turns of the cochlea is somewhat limited. This distinction is seen in normal and cocaine-treated animals. Fig. 4B shows the reduction of labelled terminals in a similar section through the 500 Hz region of the apical/middle turn in a cocaine-treated animal.

4. Discussion The present study was designed to evaluate the direct effects of repeated cocaine administration on

cochlear function and structure. The electrophysiologic results reveal an enhancement in N 1 amplitude obtained 24 h after the last cocaine injection. The degree of increase seems to be constant throughout the intensity range tested for frequencies of 500-2000 Hz. However, at 4 kHz and 8 kHz, the elevation is seen only in the mid-intensity ranges. Enhancement of the N~ response in the cocaine-treated animals may be due to (i) an increase in spread of excitation over the basilar membrane a n d / o r (ii) an increase in synchronization of single fiber responses per unit area of the basilar membrane when compared to normal animals. No significant difference in latency of the N~ response between control and cocaine-treated subjects suggests that the enhancement of N~ amplitude is due more likely to an increase in synchronization of single fiber responses per unit area of the basilar membrane rather than a change in spread of excitation on the basilar membrane between the two groups. Change in the shape of i n p u t / o u t p u t function seen at 4 kHz and 8 kHz may have occurred for several reasons, the most of which is saturation of the N 1 response at high intensity for the 4kHz and 8 kHz stimuli. If high intensity

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responses were saturated or close to saturation in the normal animals, then elevated responses in the cocaine-treated animals would be subject to a ceiling effect. It is reasonable to expect saturation of the N~ response at high intensity due to the spread of excitation towards the basal regions of the basilar membrane. Anesthetic agents used in these experiments may have influenced the cocaine action seen in the cochlea. Animals were initially anesthetized with ketamine and maintained under anesthesia with sodium pentobarbital. Since Nembutal mainly targets GABA, minimal interaction of this agent with cocaine is expected. Furthermore, given that ketamine is a short acting drug [32] and since surgical preparation and calibration procedures lasted 45-60 min, the influence of ketamine during data collection is expected to be minimal. Nevertheless, interaction of ketamine with cocaine action cannot be ruled out completely. In acute conditions, cocaine can prolong sleep time induced by ketamine [33]. Additionally, ketamine can increase circulating levels of catecholamines [22,28] which, in turn, can potentiate acute action of cocaine. However, in the chronic condition, the enhancement of evoked potential may be due to a reduction in catecholamine release

in the cochlea. Therefore, a temporary increase in circulating catecholamines may result in an underestimation of the chronic action of cocaine in the cochlea. Furthermore, similar enhancement of the N~ response is seen in alert animals monitored daily during cocaine administration of much smaller doses (1-3 mg/kg, I.P.) (unpublished data). The anatomical basis for enhancement of the whole-nerve action potential in cocaine-treated animals was investigated viz. catecholaminergic systems in the cochlea because of the profound action of cocaine on catecholamines. Catecholaminergic systems in the cochlea were studied by assessing the presence of tyrosine hydroxylase (TH), the rate limiting enzyme in the conversion of tyrosine to dopamine. Since dopamine is converted to norepinephrine by the enzyme dopamine-/3-hydroxylase (DBH), the effects of repeated cocaine injections can be evaluated on both the dopaminergic and adrenergic systems in the same specimen via tyrosine hydroxylase. TH activity in the inner spiral bundle (ISB) and tunnel spiral bundle (TB) underneath the inner hair-cells and in the sympathetic system of the guinea pig cochlea has been demonstrated [14]. The sympathetic fibers that revealed TH activity in the guinea pig cochlea showed DBH activity

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as well. In the chinchilla cochlea, no T H was seen in the ISB or TB. T H presence was noted only in fibers corresponding to the sympathetic fibers of the guinea pig cochlea. We note here that D B H was also present in this same population of fibers in the cochleas from two normal chinchillas. The restriction of T H to sympathetic fibers is consistent with the findings in the gerbil [2]. Two adrenergic subsystems, perivascular and nonperivascular have been described previously [31,7,8]. Non-perivascular adrenergic nerve terminals in close proximity to unmyelinated afferent fibers in the habenula perforata have been noted. Arborization in the HP area has also been attributed to non-perivascular terminals [31] (see Fig. 3). Finally, it has been speculated that these adrenergic terminals may be involved in spike generation a n d / o r spike conduction [31]. However, the physiologic relevance of these adrenergic nerve terminals in the HP region has not been determined yet. In the cocaine-treated animals, there is an increase in the whole nerve action potential amplitude

(N t amplitude) generated by the afferent fibers. Since N 1 responses arise from the synchronized firing of a subset of fibers, this observation implies an influence of the adrenergic nerve terminals on the synchronization of the afferent spike activity needed to generate the N t response. A reduction in the number of adrenergic terminals stained, arborizations in the HP region, and varicosities also suggests a direct action of cocaine on these terminals. Furthermore, we have demonstrated a reduction in N 1 amplitude and cochlear blood flow for low and moderate doses of cocaine in the acute experiments [30]. A reduction in cochlear blood flow implies vasoconstriction. The inhibition of re-uptake of NE at the synaptic cleft by cocaine can lead to increased levels of the neurotransmitter in the synaptic cleft resulting in prolonged post-synaptic excitation. This prolonged excitation can lead to prolonged vasoconstriction and eventually decreased cochlear blood flow. With chronic cocaine use, one would expect reduced post-synaptic sensitivity to the neurotransmitter a n d / o r reduced presynaptic release of the neurotrans-

Fig. 4. A: longitudinal section near the 500 Hz region of the apical/middle turn of a normal cochlea. Large arrowheads point to the beaded adrenergic nerve terminals running radially from the modiolus (not seen) to the periphery. 'H' denotes the HP region. 'C' denotes blood vessels. The small arrows indicate perivascular terminals. The number of arborizations in the apical region is less than in the basal end of the cochlea. B: longitudinal section near the 500 Hz region of the apical/middle turn in a cocaine-treated animal. Large arrowheads points to the beaded adrenergic nerve terminals running radially from the modiolus (not seen) to the periphery. Stars point to regions with no adrenergic terminals. 'T' denotes tunnel of Corti. Notice the marked reduction of nerve terminals after cocaine treatment.

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237

Fig. 4 (continued).

mitter. Given the magnitude of cocaine action on the catelcholaminergic systems, it is reasonable to suggest that cocaine can act directly on the cochlea via the sympathetic fibers. Since catecholamines seem to be present only in the sympathetic system of the chinchilla, the main route of action of cocaine in the cochlea would appear to be via these fibers. The effects of repeated injections of cocaine on the central nervous system (CNS) have been investigated extensively. Increased motor activity is observed in animals treated with cocaine daily [9,25,15]. Alterations in the central dopamine (DA) transmission has been implicated for this sensitization of the system. Using in vivo microdialysis techniques, Kalivas and his colleagues have shown increased extracellular dopamine levels in the nucleus accumbens and striatum after repeated cocaine exposure. However, there have been reports of either no change [29] or decreased DA levels in the nucleus accumbens in response to a challenge cocaine injection after repeated exposure to cocaine [13]. These contradictory results appear to be due to the dosage of cocaine used a n d / o r the withdrawal time allowed before measurement. It has been reported that measurement of DA levels on days 1 to 4 after the cessation of daily cocaine treatment does not show an

increase in response to a challenge injection [17]. A number of mechanisms have been proposed to explain this apparent tolerance. A reduction in dopamine levels at the synaptic level with chronic cocaine use has been suggested [4]. However, this hypothesis has not been substantiated by studies that have examined extracellular DA levels after repeated cocaine use [15,18] or basal extracellular DA levels in early withdrawal [16]. Nevertheless, reduced synthesis of DA during early withdrawal has been reported [15]. Therefore, it is possible that the availability of DA at the terminals in the CNS may be reduced during early withdrawal. The effects of repeated cocaine use in the CNS and in the peripheral sensory end-organ, the cochlea, appear to be similar in that both show increased activity. Nonetheless, the neurotransmitter systems involved are different. While DA seems to be implicated in the CNS, norepinephrine seems to play a key role in influencing the regular sensory transduction in the cochlea. It should be pointed out that the role of the cochlear non-perivascular adrenergic system on sensory function is not known and that the effects of repeated cocaine use on CNS noradrenergic systems is not completely understood. Furthermore, involvement of noradrenergic (NE) systems in 'kindling' has been substantiated

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(for review see [3]), and, depletion of NE facilitates hippocampal kindling [1]. In the case of diminished NE in the cochlea as suggested by the above results, it is reasonable to speculate that attenuation of the NE influence on afferent activity would manifest as increased activity in the auditory nerve.

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