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Neurochemical evidence of dopamine release by lateral olivocochlear efferents and its presynaptic modulation in guinea-pig cochlea
Pergamon
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Neuroscience Vol. 90, No. 1, pp. 131–138, 1999 Copyright 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00461-8
NEUROCHEMICAL EVIDENCE OF DOPAMINE RELEASE BY LATERAL OLIVOCOCHLEAR EFFERENTS AND ITS PRESYNAPTIC MODULATION IN GUINEA-PIG COCHLEA A. GA u BORJA u N, B. LENDVAI and E. S. VIZI* Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences and Haynal Imre University of Health Sciences, P.O. Box 67, H-1450, Budapest, Hungary Abstract––In this study, using an in vitro superfusion technique for the first time, we provide direct neurochemical evidence of the transmitter role of dopamine at the level of lateral olivocochlear efferent fibres of the guinea-pig cochlea. Our results revealed that nerve terminals are able to take up and release dopamine upon axonal stimulation. Since dopamine is thought to protect the afferent nerve fibres from damage due to acoustic trauma or ischaemia, enhancement of the release of dopamine, a potential therapeutic site of these injuries, was investigated. Positive modulation of dopamine release has been shown by a D1 dopamine receptor agonist, an antagonist and piribedil. Furthermore, negative feedback on the stimulation-evoked release of dopamine via D2 dopamine receptors has been excluded. Electrical stimulation of the cochlear tissue produced a significant and reproducible release of [3H]dopamine, which could be blocked by tetrodotoxin (1 µM) and cadmium (100 µM), proving that axonal activity releases dopamine and its dependence on Ca2+ influx verifies its neuronal origin. Nomifensine, a high-affinity dopamine uptake blocker, prevented the tissue from taking up [3H]dopamine from the bathing solution, also indicating the neural origin of dopamine released in response to stimulation. SKF-38393 (a selective D1 agonist) increased both the resting and electrically evoked release of dopamine. Piribedil (a D3/D2/D1 agonist), a drug under investigation, known to prevent acoustic trauma or ischaemia-induced hearing loss, had a similar and concentration-dependent increasing effect on both resting and evoked release of dopamine. The effect of both drugs on stimulation-evoked release could be prevented by SKF-83566 (a selective D1 antagonist). However, SKF-83566 alone enhanced the resting and axonal conductionassociated release of dopamine. D2 agonists and antagonists failed to modulate the release of dopamine, indicating the lack of negative feedback modulation of dopamine release. Our results suggest that the release of dopamine was subjected to modulation by a D1 receptor agonist and an antagonist. In addition, it is concluded that D2 receptors are not involved in the modulation of dopamine release. This observation may have clinical relevance in the prevention or therapy of particular types of hearing loss, because enhanced dopaminergic input into the primary auditory neuron may inhibit the (over)excitation of this neuron by glutamatergic input from inner hair cells. 1999 IBRO. Published by Elsevier Science Ltd. Key words: guinea-pig cochlea, in vitro superfusion, lateral olivocochlear efferents, dopamine release, D1 and D2 agonists and antagonists, piribedil.
Two separate systems originating in the superior olivary complex supply the efferent innervation of the cochlea.8,47 The lateral olivocochlear (LOC) bundle (containing approximately 1300 neurons) accounts for 50–60% of the whole efferent supply, and the medial olivocochlear axons, projecting predominantly to the contralateral outer hair cells, form the other 40–50% in the guinea-pig cochlea.1,26,38 The LOC bundle is orientated toward the homolateral cochlea1,45–47 through the tunnel and inner spiral bundle via unmyelinated axons.3,26,46 These axons give rise to numerous en passant swellings and *To whom correspondence should be addressed. Abbreviations: DOPAC, dihydroxyphenylacetic acid; FRR, fractional release at rest; FRS, fractional release in response to stimulation; HEPES, N-2-hydroxyethylpiperazine-N 2-ethanesulphonic acid; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; IHC, inner hair cell; LOC, lateral olivocochlear. 131
short branches ending in terminal boutons.3,38,46 Both types of vesicle-containing boutons form mainly axodendritic synapses with the dendrites of type I ganglion cells beneath the inner hair cells (IHCs).2,25,26 Axosomatic synapses between the lateral efferents and IHCs, and boutons without clear synaptic specialization, were also described.26,38 Several substances, such as acetylcholine, GABA, dopamine, enkephalins, dynorphins, calcitonin generelated peptide and serotonin, have been proposed to have neurotransmitter or neuromodulator functions in these synapses.10,11,15,30,37 The present study was focused on dopaminergic neurotransmission in the guinea-pig cochlea. Dopamine was shown in the rat cochlear homogenates using high-performance liquid chromatography (HPLC) coupled with electrochemical detection.17 The presence of tyrosine hydroxylase immunoreactivity and the absence of dopamine-âhydroxylase in the LOC efferent fibres6,11,37 suggest
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that lateral efferent innervation is dopaminergic. Nevertheless, there is no direct neurochemical evidence of the transmitter role of dopamine at olivocochlear efferent fibres. Therefore, our goal was to add neurochemical evidence to neuroanatomical,11,37 physiological7,17,24 and pathophysiological6,7,36 data indicating that dopamine could act as a neurotransmitter or neuromodulator at the level of lateral efferent fibres. We first used an in vitro superfusion technique on isolated cochlear tissue in order to characterize the release of dopamine and prove its neuronal origin. The function of dopamine in the biochemistry of hearing is still not clear. It is thought to be involved in postsynaptic inhibitory modulation of the glutamatergic synapses between IHCs and the primary auditory nerve.10,14,16,27,28,30,34 Furthermore, dopamine seems to protect the auditory nerve terminal dendrites against glutamate excitotoxicity induced by acoustic trauma or ischaemia.6,14,34–36 There have been no observations about the modulatory possibilities of cochlear dopamine release yet. However, a positive modulation may play a prominent role in clinical therapy of particular types of hearing loss. Since piribedil, a D3/D2/D1 agonist, was described to protect afferent fibres from damage caused by acoustic trauma or ischaemia in animal experiments,6,7,31 in the present study we also made an attempt to characterize the effect of piribedil on cochlear dopamine release. Part of this work has been reported previously in abstract form.13 EXPERIMENTAL PROCEDURES
Animals and tissue preparation Male guinea-pigs (Huma`n, Go¨do¨lloˆ) weighing 250–350 g were decapitated. The osseous bullae tympani were immediately opened. Under a stereomicroscope, the bony capsule of the cochlea and stria vascularis were carefully removed, then the cochlea was dissected out from the bulla. This separated tissue (4–8 mg) contained the efferent fibres without their perikarya, afferent fibres with their nucleus in ganglion spirale, and IHCs and outer hair cells as well. Experiments were performed in accordance with the National Institutes of Health guidelines on the use of experimental animals. Approval of the Animal Use Committee of the Institute of Experimental Medicine, Hungarian Academy of Sciences was obtained prior to initiating the experiments. All efforts were made to avoid animal suffering and to minimize the number of animals used. Microvolume superfusion system; release of [3H]dopamine Experiments were carried out in perilymph-like solution, described by Ikeda et al.,20 containing (mM): NaCl 150, KCl 3.5, CaCl2 1, MgCl2 1, HEPES 2.75 and Tris-OH 2.25, corrigated pH (7.4) and osmolarity (300 mosm/l) according to physiological values by adding HCl and -glucose. This solution was thermoregulated at 37C and continuously saturated with 100% O2. Isolated cochleae were incubated for 60 min at 37C in 1 ml of perilymph-like solution containing 10 µCi [7,83 H]dopamine (concentration of labelled dopamine was 0.2 µM). After incubation, each cochlea was transferred to
a thermoregulated (37C) Plexiglas microvolume chamber (inside volume: 100 µl).43 The tissue was superfused at a rate of 0.3 ml/min. After 60-min preperfusion, the outflow was collected in 0.9-ml (3-min) fractions for an additional 60 min. Tissues were stimulated at 60 V, 2 Hz, 0.5 ms impulse duration for 3 min (360 pulses) via platinum electrodes inserted from the top and bottom of the chamber using a stimulator (Eltron) during the third and 13th collection periods (S1 and S2). Drugs were added at the sixth fraction to the perfusion fluid and they were maintained until the end of the experiment. At the end of the perfusion period, the tissue was removed from the perfusion chamber, suspended in 500 µl of 10% trichloroacetic acid and sonicated. An aliquot (100 µl) was assayed for tissue radioactivity. To determine radioactivity released from the tissue, 0.5-ml aliquots of the collected fractions were also assayed for radioactivity by a liquid scintillation counter (Packard TR 1900) and expressed in Bq/g. The samples were analysed by HPLC with electrochemical detection, as described previously.19 Data analysis The outflow of tritium, expressed as a fractional rate, i.e. as a percentage of the amount of radioactivity in the tissue at the time of the release, was determined. To calculate the electrical field stimulation-induced overflow, the mean of the basal release determined before and after stimulation was subtracted from the total efflux of radioactivity from the tissue in response to electrical stimulation. The effects of drugs on the electrical stimulation-induced outflow were expressed as the calculated ratio of fractional release in response to the second stimulation (FRS2) over the fractional release induced by the first, control stimulus (FRS1). The effect induced on basal release was expressed as the ratio of the average of 11th and 12th over the average of first and second fractional release (FRR2/FRR1). All data are presented as meansS.E.M. Statistical analysis Student’s test and one-way ANOVA with Tukey’s post hoc test were used to determine the significance of data (*P<0.05, **P<0.01 or ***P<0.001). Materials [7,8-3H]Dopamine (specific activity 1.81 TBq/mmol, 49.0 Ci/mmol) was purchased from Amersham. Tetrodotoxin (Sigma, St Louis, MO, U.S.A.), cadmium chloride 2,5-hydrate (Reanal, Hungary), nomifensine maleate (Sigma), sulpiride (Sigma), piribedil (Servier, France), R(+)SKF-38393 hydrochloride (RBI, U.S.A.), ()-SKF-83566 hydrochloride (RBI, U.S.A.), quinpirole (RBI, U.S.A.) and bromocriptine mesilate (Sandoz, Switzerland) were used. RESULTS
Release of [3H]dopamine Release at rest. After 60 min loading with [3H]dopamine, followed by a 60-min washout, the cochlea contained 825105 kBq/mg radioactivity. At rest 1.700.13% (average of fractional release from the first and second samples) of the total radioactive content was released during the 3-min collection periods. Change in 30 min in resting release was compared to control (see Table 1, FRR2/FRR1). Release in response to stimulation. In response to electrical field stimulation (2 Hz, 0.5 ms, 360 shocks), 2.790.26% of the radioactivity present in the tissue
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Table 1. Effect of different drugs on [3H]dopamine release at rest and in response to stimulation (2 Hz, 360 shocks) from superfused lateral olivocochlear efferent fibres of the guinea-pig cochlea Drugs Control Tetrodotoxin Cadmium chloride Nomifensine SKF-38393 SKF-83566 Piribedil Bromocriptine Quinpirole Sulpiride
Concentration
n
FRS2/FRS1
P
FRR2/FRR1
P
1 µM 100 µM 20 µM 10 µM 100 µM 100 µM 500 µM 1 mM 20 µM 20 µM 100 µM 500 µM 100 µM 500 µM
8 6 7 6 4 8 8 8 6 4 6 5 2 7 4
0.970.09 0.190.04 0.190.05 1.310.09 1.650.13 1.680.24 1.340.09 1.610.09 2.120.45 0.940.11 1.190.10 1.240.11 1.000.00 1.030.07 0.940.06
0.000031 0.000010 0.024 0.0052 0.016 0.013 0.00021 0.013 0.34 0.14 0.10 0.88 0.64 0.84
0.900.03 1.030.07 0.840.03 0.970.04 1.530.17 1.120.08 0.970.04 1.170.06 1.830.36 1.000.04 0.880.05 0.880.03 0.980.01 0.910.04 0.910.04
0.18 0.19 0.13 0.022 0.015 0.14 0.0014 0.011 0.92 0.75 0.69 0.16 0.83 0.82
For stimulation, see Experimental Procedures. Drugs were added at the sixth fraction and maintained throughout the experiments. Note that FRR2/FRR1 or FRS2/FRS1 ratios less than the control indicate reduction, and values higher than the control indicate an increase of dopamine release.
at the time of the stimulation was released over the basal release (FRS1). When the stimulation was repeated after 30 min, the amount of radioactivity released above the resting release was 2.660.28% (FRS2; Fig. 1A), indicating that the stimulation-evoked release was fairly constant: the ratio between the amount of radioactivity released by consecutive stimulations (FRS2/FRS1) was 0.969 0.095 (n=8). HPLC data showed that 91–95% of the released tritiated activity was dopamine and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The other 5–9% was norepinephrine and epinephrine. The question arises whether the radioactivity determined in the tissue and released in response to field stimulation is of neuronal origin or whether it was taken up by and released from non-neuronal elements of the preparation. Evidence of neuronal origin of dopamine When the voltage-dependent Na+ influx, i.e. the axonal conduction, was inhibited by the application of tetrodotoxin (1 µM), the evoked release of [3H]dopamine in response to field stimulation was blocked (Fig. 1B), showing the Na+ influx dependence of electrically evoked release (Table 1). The inhibition of voltage-dependent Ca2+ channels by cadmium (100 µM), a non-selective voltagedependent Ca2+ channel blocker, prevented the effect of electrical stimulation on the release of [3H]dopamine (Fig. 1C), indicating the role of voltage-dependent Ca2+ channels in electrically evoked release (Table 1). In four experiments, a high-affinity dopamine uptake blocker, nomifensine12 (20 µM), was added into the fluid 15 min before the preparation was loaded for 60 min with [3H]dopamine, and it was kept in the solution throughout the loading time. If
[3H]dopamine was taken up and stored by dopaminergic terminals, the neuronal uptake of [3H]dopamine should have been blocked by nomifensine, and field stimulation should have failed to release radioactivity. This was indeed the case; the fractional release of [3H]dopamine from nomifensine-pretreated preparations in response to field stimulation (FRS1) was 0.670.02%, significantly lower (P=0.0002) than in control cases. Application of nomifensine from the sixth fraction to the perfusing solution increased the evoked release of dopamine, proving the blocked elimination of released dopamine (data are shown in Table 1). Presynaptic modulation of dopamine release After perfusing 10 µM SKF-38393, a D1 dopamine receptor subtype-specific agonist, through the cochlear tissue, basal and stimulation-evoked release of dopamine was increased. Figure 2 shows the highly significant increase in resting and in electrically evoked release in the presence of SKF-38393 (data are shown in Table 1). SKF-83566 (100 µM), a D1selective antagonist, caused an increase in both resting and evoked release of dopamine (Table 1). SKF-83566 applied before the agonist and kept in the solution throughout the experiments prevented the D1 receptor agonist-induced increase in electrically evoked release. The D1 antagonist failed to decrease the D1 agonist-increased resting release (Fig. 2). Effect of piribedil on dopamine release Piribedil was used at concentrations of 100 µM, 500 µM and 1 mM, as applied by others.6,7,31 The resting and electrically evoked release of [3H]dopamine was significantly increased in a
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A. Ga´borja´n et al. DISCUSSION
Neurochemical evidence of dopaminergic transmission in the cochlea
Fig. 1. Fractional release of radioactivity ([3H]dopamine) in response to electrical field stimulations (2 Hz, 360 stimuli, 0.5 ms; S1, S2) from isolated cochlea preloaded with [3H]dopamine. (A) In control experiments (n=8), the consecutive stimulation-evoked release remained constant. (B, C) Tetrodotoxin (TTX, 1 µM; n=6; B) and cadmium chloride (CdCl2, 100 µM; n=7; C) applied from the sixth fraction prevented the electrically (S2) evoked release. S1 was used as a control stimulation. The symbols present the mean values and the vertical bars show the S.E.M.
concentration-dependent manner (Table 1). This effect could not be blocked by the D2 antagonist, sulpiride, but the D1-selective antagonist, SKF83566, prevented the effect of piribedil (Fig. 3). D2 receptor-specific effects Neither D2 dopamine receptor subtype-selective agonists, such as bromocriptine (at a concentration of 20 µM) and quinpirole (at concentrations of 20, 100 and 500 µM), nor selective antagonists, such as sulpiride (at concentrations of 100 and 500 µM), influenced the stimulation-evoked release of dopamine (Table 1), suggesting the absence of presynaptic D2 receptors on dopaminergic terminals.
In this work, we provide for the first time neurochemical evidence of the neurotransmitter role of dopamine in the mammalian auditory organ. To establish the identity of a neurotransmitter, an important criterion is the ability of nerve endings to take up and release the candidate substance upon electrical stimulation of the nerve. However, there are some indirect conclusions relying upon HPLC studies under noise conditions, which showed decreased dopamine content suggesting noise-evoked release of dopamine;16,18,40 until now, there has not been a reliable method to obtain direct evidence of the release of dopamine. We first used an in vitro superfusion technique on guinea-pig cochleae to confirm the release of dopamine from nerve terminals of LOC neurons in response to stimulation. Electrical depolarization of the cochlear tissue produced a significant and reproducible increase in [3H]dopamine release. The tritiated outflow we measured was due to release from dopaminergic fibres, as shown by HPLC combined with electrochemical detection. The majority of radioactivity released at rest and in response to stimulation (91% and 95%, respectively) was dopamine and its metabolites (DOPAC, HVA); only small amounts were epinephrine and norepinephrine, as described previously.13 We can assume that dopamine was released from lateral efferent fibres, because no other dopaminergic innervation has been shown in the cochlea.11,14,24,40 It is very important to confirm the neuronal origin of dopamine released by stimulation used in our experiments. The electrical stimuli activate the voltage-dependent Na+ channels present in the axons, imitating the physiological discharging action. The specific block of these channels with 1 µM tetrodotoxin inhibited the axonal conduction and prevented the electrically evoked release of dopamine. If the depolarization can propagate along the axon, voltage-dependent Ca2+ channels are activated, followed by an increase in intracellular Ca2+ concentration, postulated to cause exocytotic release. Blocking these voltage-dependent Ca2+ channels with cadmium, electrical stimulation also failed to release dopamine (Fig. 4). These results show that dopamine can be released from efferent fibres, and the fast Na+ channel and voltage-dependent Ca2+ channel dependence of the release proves its neuronal origin. Another important criterion for the role of neurotransmitter in the case of dopaminergic transmission is that neuronal nerve endings must be able to take up the released dopamine by their membrane uptake carriers and terminate the action of the transmitter in the extracellular space. In this study, the special inhibition of the dopaminergic uptake carrier by nomifensine12 during incubation with [3H]dopamine
Cochlear dopaminergic neurotransmission
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Fig. 2. Effect of the D1 agonist, SKF-38393, at a concentration of 10 µM on fractional release of [3H]dopamine (n=4). Drugs were added as indicated. The D1 antagonist, SKF-83566, at a concentration of 100 µM was applied 20 min before fraction collection (35 min before agonist administration) and kept in the solution throughout the experiments (n=4).
Fig. 3. Effect of 500 µM piribedil on [3H]dopamine release. The D2 antagonist, sulpiride, at a concentration of 100 µM, was applied 20 min before fraction collection and kept in the solution throughout the experiments (n=6). The D1 antagonist, SKF-83566, at a concentration of 100 µM, was applied similarly, as indicated (n=5).
decreased the releasable amount of dopamine at the release site, supporting its neuronal origin. Blocking the uptake mechanism by nomifensine after control stimulation caused an increase in the evoked release of dopamine, because of the failure of elimination. Our results provide neurochemical evidence of dopaminergic neurotransmission in the cochlea between the LOC efferents and type I afferent fibres (Fig. 4), and prove the neuronal origin of dopamine released by electrical stimulation.
There is some additional, indirect evidence that fulfils the criteria of dopamine-mediated chemical transmission of LOC efferent innervation. The existence of tyrosine hydroxylase and aromatic amino acid decarboxylase immunoreactivity, and lack of the enzymes dopamine-â-hydroxylase and phenylethanolamine-N-methyltransferase, suggest that the innervation from the lateral nuclei of the superior olive is dopaminergic.6,11,22,37,39 Depletion studies after 6-hydroxydopamine administration also
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Fig. 4. Scheme of synaptic interactions in the cochlea and the role of dopamine released from the axon terminals of LOC efferent fibres. The release of dopamine associated with axonal firing is subject to modulation by different drugs. Therefore, any drug able to release or potentiate dopamine release in fact inhibits the (over)excitation of primary auditory neurons caused by glutamatergic input from IHCs via D2 receptors present on dendrites of afferent fibres, as shown by others (see text). The clinical effect of piribedil is partly mediated via a presynaptic effect potentiating the release of dopamine and partly via stimulation of D2 receptors located on the afferent fibres.
support the notion that LOC fibres are able to synthesize dopamine.6,11 In accordance with data indicating dopamine-containing nerves, endogenous dopamine could also be detected using HPLC in rat cochlear homogenates,17 and under noise activation the level of dopamine metabolites, DOPAC and HVA, increased.16,18,40 It has also been shown that the interaction of dopamine with its specific receptors leads to changes in postsynaptic activity. The compound action potential of the auditory nerve was measured by noise exposure and showed a significantly reduced amplitude on adding dopamine and its analogues. This effect is thought to be mediated via the D2 or D3 subtypes of dopamine receptors.14,30 In addition, polymerase chain reaction analysis and nucleotide sequencing identified D2(long) and D3 receptor messages for dopamine receptor subtypes in the mammalian cochlea.24 Our results, taken together with previous findings, prove the neurotransmitter role of dopamine at the level of LOC efferent terminals.
The methods used in these experiments are suitable for investigating the presynaptic effect of different dopamine receptor-specific drugs. In our experiments, both the D1-selective agonist,23,48 SKF38393, and the antagonist,5 SKF-83566, caused an increase in basal and evoked release of dopamine. The D1 antagonist prevented the effect of the agonist. D2 receptor-specific agonists (bromocriptine, quinpirole) and antagonist (sulpiride) had no effect on dopamine release, suggesting the lack of this receptor subtype at the presynaptic site. It is concluded that the resting and stimulationevoked release of dopamine from the olivocochlear efferent fibres can be increased by different drugs. Since it has been observed that dopamine may have some sort of protective action against damage due to intense sound,6,7,31,36 the possibility arises that the enhancement of dopamine release may be clinically useful to diminish the damage in the cochlea caused by acoustic trauma or ischaemia. The lack of negative feedback suggests a crucial role of dopamine release.
Presynaptic modulation of dopamine release Modulation of dopamine release in the auditory system could be envisaged through a neuronal circuit: the activation of afferent fibres causes synaptic activity in the cochlear nuclei, which are connected to LOC neurons. In this way, the release of dopamine and other neurotransmitters from LOC efferents could be modified.16,18,32 The other possibility is a local modulation via a presynaptic modulatory site.41
Effect of piribedil on dopaminergic neurotransmission Piribedil has been described as a D2-specific agonist, but a novel radioligand binding study showed its 20-fold higher affinity for the D3 than for the D2 type of dopamine receptor, and little affinity for the D1 receptor subtype.4 Piribedil (Trivastal) is used clinically for the treatment of dopamine system dysfunction.9,21,29 Other clinical studies with piribedil
Cochlear dopaminergic neurotransmission
showed an improvement of symptoms (vertigo, tinnitus, sudden hearing loss) associated with cochleo-vestibular diseases. Piribedil has also been a popular drug to characterize the function of dopamine in the lateral efferent neurotransmission and to analyse dopamine receptor subtypes in these synapses.6,7,18,31,40 Intracochlear perfusion of 0.1– 1 mM of piribedil induced a dose-dependent decrease in the amplitude of the compound action potential and a concomitant increase in N1 latency of the auditory nerve measured by high-intensity toneburst stimulation.6,7,31 Piribedil also prevented the ischaemia-induced6,7,36 and acoustic traumainduced7,31 swelling of the radial dendrites. We found that dopamine release from isolated cochleae was enhanced by piribedil in a manner resembling that observed with other compounds (Figs 2, 3). Furthermore, the dopamine-releasing effect of piribedil could be blocked by a D1 antagonist. The presynaptic effect of piribedil to increase the resting release of dopamine and potentiate the release associated with axonal activity does not exclude its action on postsynaptic inhibitory D2 receptors (see Fig. 4). It seems very likely that its effect is partly indirect, via release of dopamine, and partly direct, via D2 receptors expressed on afferent fibres, as suggested by others.6,7,27 According to this model, the potentiation of dopamine release could lead to a more effective block of postsynaptic activity. It is worth mentioning that not all efferent en passant boutons make synaptic contacts with afferent
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fibres.26,38,46 Therefore, it is possible that dopamine or other transmitters released from non-synaptic varicosities diffuse far away from the release site and may have modulatory actions in a more diffuse fashion, as suggested similarly for other regions of the brain.42,44
CONCLUSIONS
For the first time, neurochemical evidence has been obtained for the transmitter role of dopamine at LOC efferent innervation of the cochlea. Dopamine—released into the vicinity and/or axodendritic synapses between LOC efferent fibres and dendrites of afferent fibres—may be involved in an inhibitory modulation of the effect of extreme synaptic activity of IHCs on primary auditory neurons,33 by this means protecting the auditory nerve from glutamate excitotoxicity (Fig. 4). It could also play a role in repair of the consequences of cellular excitotoxicity.30,34,35 Interventions to increase dopamine release in the cochlea could be a therapeutic target for the future and our results could contribute to progress in therapy or in prevention of particular types of hearing loss. Acknowledgements—This study was supported by a Hungarian Higher Education Research and Development Grant (FKFP 1343/1997), a Hungarian Research Grant (OTKA) and a Hungarian Medical Research Grant (ETT).
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(1987) Tyrosine hydroxylase immunoreactivity identifies possible catecholaminergic fibers in the organ of Corti. Hearing Res. 30, 33–38. Kaiser C., Dandridge P. A., Garvey E., Hahn R. A., Sarau H. M., Setler P. E., Bass L. S. and Clardy J. (1982) Absolute stereochemistry and dopaminergic activity of enantiomers of 2,3,4,5-tetrahydro-7,8-dihydroxy-1-phenyl-1H3-benzazepine. J. med. Chem. 25, 697–703. Karadaghy A. A., Lasak J. M., Chomchai J. S., Khan K. M., Drescher M. J. and Drescher D. G. (1997) Quantitative analysis of dopamine receptor messages in the mouse cochlea. Molec. Brain Res. 44, 151–156. Liberman M. C. (1980) Efferent synapses in the inner hair cell area of the cat cochlea: an electron microscopic study of serial sections. Hearing Res. 3, 189–204. Liberman M. C., Dodds L. W. and Pierce S. (1990) Afferent and efferent innervation of the cat cochlea: quantitative analysis with light and electron microscopy. J. comp. Neurol. 301, 443–460. Oestreicher E., Arnold W., Ehrenberger K. and Felix D. (1997) Dopamine regulates the glutamatergic inner hair cell activity in guinea pigs. Hearing Res. 107, 46–52. Ohmori H. (1996) Afferent and efferent synaptic transmission in hair cells. News physiol. Sci. 11, 161–166. Ollat H. (1992) Dopaminergic insufficiency reflecting cerebral ageing: value of a dopaminergic agonist, piribedil. J. Neurol., Germany, Suppl. 239, S13–S16. Puel J. L. (1995) Chemical synaptic transmission in the cochlea. Prog. Neurobiol. 47, 449–476. Puel J. L., d’Aldin C. G., Saffiedine S., Eybalin M. and Pujol R. (1996) Excitotoxicity and plasticity of IHC-auditory nerve contributes to both temporary and permanent threshold shift. In Scientific Basis of Noise-induced Hearing Loss (eds Axelsson A., Hamernik R. P. and Salvi R. J.), pp. 36–42. Thieme Medical, New York. Puel J. L., Bobbin R. P. and Fallon M. (1988) An ipsilateral cochlear efferent loop protects the cochlea during intense sound exposure. Hearing Res. 37, 65–70. Puel J. L., Pujol R., Tribillac F., Ladrech S. and Eybalin M. (1994) Excitatory amino acid antagonists protect cochlear auditory neurons from excitotoxicity. J. comp. Neurol. 341, 241–256. Pujol R. (1994) Lateral and medial efferents: a double neurochemical mechanism to protect and regulate inner and outer hair cell function in the cochlea. Br. J. Audiol. 28, 185–191. Pujol R., Eybalin M. and Puel J. L. (1995) Recent advances in cochlear neurotransmission: physiology and pathophysiology. News physiol. Sci. 10, 178–183. Pujol R., Puel J. L., d’Aldin C. G. and Eybalin M. (1993) Pathophysiology of the glutamatergic synapses in the cochlea. Acta otolar., Stockh. 113, 330–334. Safieddine S., Prior A. M. S. and Eybalin M. (1997) Choline acetyltransferase, glutamate decarboxylase, tyrosine hydroxylase, calcitonin gene-related peptide and opioid peptides coexist in lateral efferent neurons of rat and guinea-pig. Eur. J. Neurosci. 9, 356–367. Satake M. and Liberman M. C. (1996) Morphological subclasses of lateral olivocochlear terminals? Ultrastructural analysis of inner spiral bundle in cat and guinea pig. J. comp. Neurol. 371, 621–632. Usami S., Hozawa J., Tazawa M., Yoshihara T., Igarashi M. and Thompson G. C. (1988) Immunocytochemical study of catecholaminergic innervation in the guinea pig cochlea. Acta otolar., Stockh., Suppl. 447, 36–45. Vicente-Torres M. A., Gil-Loyzaga P., Ferna´ndez P., Arce A. and Esquifino A. (1993) Piribedil could modify dopamine turnover in cochleas under noise stimulation. J. Oto-Rhino-Laryngol. 55, 322–324. Vizi E. S. (1979) Presynaptic modulation of neurochemical transmission. Prog. Neurobiol. 12, 181–290. Vizi E. S. (1984) Non-synaptic Interaction Between Neurons. Wiley, Oxford. Vizi E. S., Ha´rsing L. G., Zima´nyi I. and Gaa´l G. (1985) Release and turnover of noradrenaline in isolated median eminence: lack of negative feedback modulation. Neuroscience 16, 907–916. Vizi E. S. and La´bos E. (1991) Non-synaptic interactions at presynaptic level. Prog. Neurobiol. 37, 145–163. Warr W. B. (1975) Olivocochlear and vestibular efferent neurons of the feline brain stem: their location, morphology and number determined by retrograde axonal transport and acetylcholinesterase histochemistry. J. comp. Neurol. 161, 159–182. Warr W. B., Boche J. B. and Neely S. T. (1997) Efferent innervation of the inner hair cell region: origins and terminations of two lateral olivocochlear systems. Hearing Res. 108, 89–111. Warr W. B. and Guinan J. J. (1979) Efferent innervation of the organ of Corti: two separate systems. Brain Res. 173, 152–155. Zhou Q. Y., Grandy D. K., Thambi L., Kushner J. A., Van Tol H. H. M., Cone R., Pribnow D., Salon J., Bunzow J. R. and Civelli O. (1990) Cloning and expression of human and rat D1 dopamine receptors. Nature 347, 76–80. (Accepted 4 August 1998)
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Neuroscience Vol. 90, No. 1, pp. 131–138, 1999 Copyright 1999 IBRO. Published by Elsevier Science Ltd Printed in Great Britain. All rights reserved 0306–4522/99 $19.00+0.00 S0306-4522(98)00461-8
NEUROCHEMICAL EVIDENCE OF DOPAMINE RELEASE BY LATERAL OLIVOCOCHLEAR EFFERENTS AND ITS PRESYNAPTIC MODULATION IN GUINEA-PIG COCHLEA A. GA u BORJA u N, B. LENDVAI and E. S. VIZI* Department of Pharmacology, Institute of Experimental Medicine, Hungarian Academy of Sciences and Haynal Imre University of Health Sciences, P.O. Box 67, H-1450, Budapest, Hungary Abstract––In this study, using an in vitro superfusion technique for the first time, we provide direct neurochemical evidence of the transmitter role of dopamine at the level of lateral olivocochlear efferent fibres of the guinea-pig cochlea. Our results revealed that nerve terminals are able to take up and release dopamine upon axonal stimulation. Since dopamine is thought to protect the afferent nerve fibres from damage due to acoustic trauma or ischaemia, enhancement of the release of dopamine, a potential therapeutic site of these injuries, was investigated. Positive modulation of dopamine release has been shown by a D1 dopamine receptor agonist, an antagonist and piribedil. Furthermore, negative feedback on the stimulation-evoked release of dopamine via D2 dopamine receptors has been excluded. Electrical stimulation of the cochlear tissue produced a significant and reproducible release of [3H]dopamine, which could be blocked by tetrodotoxin (1 µM) and cadmium (100 µM), proving that axonal activity releases dopamine and its dependence on Ca2+ influx verifies its neuronal origin. Nomifensine, a high-affinity dopamine uptake blocker, prevented the tissue from taking up [3H]dopamine from the bathing solution, also indicating the neural origin of dopamine released in response to stimulation. SKF-38393 (a selective D1 agonist) increased both the resting and electrically evoked release of dopamine. Piribedil (a D3/D2/D1 agonist), a drug under investigation, known to prevent acoustic trauma or ischaemia-induced hearing loss, had a similar and concentration-dependent increasing effect on both resting and evoked release of dopamine. The effect of both drugs on stimulation-evoked release could be prevented by SKF-83566 (a selective D1 antagonist). However, SKF-83566 alone enhanced the resting and axonal conductionassociated release of dopamine. D2 agonists and antagonists failed to modulate the release of dopamine, indicating the lack of negative feedback modulation of dopamine release. Our results suggest that the release of dopamine was subjected to modulation by a D1 receptor agonist and an antagonist. In addition, it is concluded that D2 receptors are not involved in the modulation of dopamine release. This observation may have clinical relevance in the prevention or therapy of particular types of hearing loss, because enhanced dopaminergic input into the primary auditory neuron may inhibit the (over)excitation of this neuron by glutamatergic input from inner hair cells. 1999 IBRO. Published by Elsevier Science Ltd. Key words: guinea-pig cochlea, in vitro superfusion, lateral olivocochlear efferents, dopamine release, D1 and D2 agonists and antagonists, piribedil.
Two separate systems originating in the superior olivary complex supply the efferent innervation of the cochlea.8,47 The lateral olivocochlear (LOC) bundle (containing approximately 1300 neurons) accounts for 50–60% of the whole efferent supply, and the medial olivocochlear axons, projecting predominantly to the contralateral outer hair cells, form the other 40–50% in the guinea-pig cochlea.1,26,38 The LOC bundle is orientated toward the homolateral cochlea1,45–47 through the tunnel and inner spiral bundle via unmyelinated axons.3,26,46 These axons give rise to numerous en passant swellings and *To whom correspondence should be addressed. Abbreviations: DOPAC, dihydroxyphenylacetic acid; FRR, fractional release at rest; FRS, fractional release in response to stimulation; HEPES, N-2-hydroxyethylpiperazine-N 2-ethanesulphonic acid; HPLC, high-performance liquid chromatography; HVA, homovanillic acid; IHC, inner hair cell; LOC, lateral olivocochlear. 131
short branches ending in terminal boutons.3,38,46 Both types of vesicle-containing boutons form mainly axodendritic synapses with the dendrites of type I ganglion cells beneath the inner hair cells (IHCs).2,25,26 Axosomatic synapses between the lateral efferents and IHCs, and boutons without clear synaptic specialization, were also described.26,38 Several substances, such as acetylcholine, GABA, dopamine, enkephalins, dynorphins, calcitonin generelated peptide and serotonin, have been proposed to have neurotransmitter or neuromodulator functions in these synapses.10,11,15,30,37 The present study was focused on dopaminergic neurotransmission in the guinea-pig cochlea. Dopamine was shown in the rat cochlear homogenates using high-performance liquid chromatography (HPLC) coupled with electrochemical detection.17 The presence of tyrosine hydroxylase immunoreactivity and the absence of dopamine-âhydroxylase in the LOC efferent fibres6,11,37 suggest
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that lateral efferent innervation is dopaminergic. Nevertheless, there is no direct neurochemical evidence of the transmitter role of dopamine at olivocochlear efferent fibres. Therefore, our goal was to add neurochemical evidence to neuroanatomical,11,37 physiological7,17,24 and pathophysiological6,7,36 data indicating that dopamine could act as a neurotransmitter or neuromodulator at the level of lateral efferent fibres. We first used an in vitro superfusion technique on isolated cochlear tissue in order to characterize the release of dopamine and prove its neuronal origin. The function of dopamine in the biochemistry of hearing is still not clear. It is thought to be involved in postsynaptic inhibitory modulation of the glutamatergic synapses between IHCs and the primary auditory nerve.10,14,16,27,28,30,34 Furthermore, dopamine seems to protect the auditory nerve terminal dendrites against glutamate excitotoxicity induced by acoustic trauma or ischaemia.6,14,34–36 There have been no observations about the modulatory possibilities of cochlear dopamine release yet. However, a positive modulation may play a prominent role in clinical therapy of particular types of hearing loss. Since piribedil, a D3/D2/D1 agonist, was described to protect afferent fibres from damage caused by acoustic trauma or ischaemia in animal experiments,6,7,31 in the present study we also made an attempt to characterize the effect of piribedil on cochlear dopamine release. Part of this work has been reported previously in abstract form.13 EXPERIMENTAL PROCEDURES
Animals and tissue preparation Male guinea-pigs (Huma`n, Go¨do¨lloˆ) weighing 250–350 g were decapitated. The osseous bullae tympani were immediately opened. Under a stereomicroscope, the bony capsule of the cochlea and stria vascularis were carefully removed, then the cochlea was dissected out from the bulla. This separated tissue (4–8 mg) contained the efferent fibres without their perikarya, afferent fibres with their nucleus in ganglion spirale, and IHCs and outer hair cells as well. Experiments were performed in accordance with the National Institutes of Health guidelines on the use of experimental animals. Approval of the Animal Use Committee of the Institute of Experimental Medicine, Hungarian Academy of Sciences was obtained prior to initiating the experiments. All efforts were made to avoid animal suffering and to minimize the number of animals used. Microvolume superfusion system; release of [3H]dopamine Experiments were carried out in perilymph-like solution, described by Ikeda et al.,20 containing (mM): NaCl 150, KCl 3.5, CaCl2 1, MgCl2 1, HEPES 2.75 and Tris-OH 2.25, corrigated pH (7.4) and osmolarity (300 mosm/l) according to physiological values by adding HCl and -glucose. This solution was thermoregulated at 37C and continuously saturated with 100% O2. Isolated cochleae were incubated for 60 min at 37C in 1 ml of perilymph-like solution containing 10 µCi [7,83 H]dopamine (concentration of labelled dopamine was 0.2 µM). After incubation, each cochlea was transferred to
a thermoregulated (37C) Plexiglas microvolume chamber (inside volume: 100 µl).43 The tissue was superfused at a rate of 0.3 ml/min. After 60-min preperfusion, the outflow was collected in 0.9-ml (3-min) fractions for an additional 60 min. Tissues were stimulated at 60 V, 2 Hz, 0.5 ms impulse duration for 3 min (360 pulses) via platinum electrodes inserted from the top and bottom of the chamber using a stimulator (Eltron) during the third and 13th collection periods (S1 and S2). Drugs were added at the sixth fraction to the perfusion fluid and they were maintained until the end of the experiment. At the end of the perfusion period, the tissue was removed from the perfusion chamber, suspended in 500 µl of 10% trichloroacetic acid and sonicated. An aliquot (100 µl) was assayed for tissue radioactivity. To determine radioactivity released from the tissue, 0.5-ml aliquots of the collected fractions were also assayed for radioactivity by a liquid scintillation counter (Packard TR 1900) and expressed in Bq/g. The samples were analysed by HPLC with electrochemical detection, as described previously.19 Data analysis The outflow of tritium, expressed as a fractional rate, i.e. as a percentage of the amount of radioactivity in the tissue at the time of the release, was determined. To calculate the electrical field stimulation-induced overflow, the mean of the basal release determined before and after stimulation was subtracted from the total efflux of radioactivity from the tissue in response to electrical stimulation. The effects of drugs on the electrical stimulation-induced outflow were expressed as the calculated ratio of fractional release in response to the second stimulation (FRS2) over the fractional release induced by the first, control stimulus (FRS1). The effect induced on basal release was expressed as the ratio of the average of 11th and 12th over the average of first and second fractional release (FRR2/FRR1). All data are presented as meansS.E.M. Statistical analysis Student’s test and one-way ANOVA with Tukey’s post hoc test were used to determine the significance of data (*P<0.05, **P<0.01 or ***P<0.001). Materials [7,8-3H]Dopamine (specific activity 1.81 TBq/mmol, 49.0 Ci/mmol) was purchased from Amersham. Tetrodotoxin (Sigma, St Louis, MO, U.S.A.), cadmium chloride 2,5-hydrate (Reanal, Hungary), nomifensine maleate (Sigma), sulpiride (Sigma), piribedil (Servier, France), R(+)SKF-38393 hydrochloride (RBI, U.S.A.), ()-SKF-83566 hydrochloride (RBI, U.S.A.), quinpirole (RBI, U.S.A.) and bromocriptine mesilate (Sandoz, Switzerland) were used. RESULTS
Release of [3H]dopamine Release at rest. After 60 min loading with [3H]dopamine, followed by a 60-min washout, the cochlea contained 825105 kBq/mg radioactivity. At rest 1.700.13% (average of fractional release from the first and second samples) of the total radioactive content was released during the 3-min collection periods. Change in 30 min in resting release was compared to control (see Table 1, FRR2/FRR1). Release in response to stimulation. In response to electrical field stimulation (2 Hz, 0.5 ms, 360 shocks), 2.790.26% of the radioactivity present in the tissue
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Table 1. Effect of different drugs on [3H]dopamine release at rest and in response to stimulation (2 Hz, 360 shocks) from superfused lateral olivocochlear efferent fibres of the guinea-pig cochlea Drugs Control Tetrodotoxin Cadmium chloride Nomifensine SKF-38393 SKF-83566 Piribedil Bromocriptine Quinpirole Sulpiride
Concentration
n
FRS2/FRS1
P
FRR2/FRR1
P
1 µM 100 µM 20 µM 10 µM 100 µM 100 µM 500 µM 1 mM 20 µM 20 µM 100 µM 500 µM 100 µM 500 µM
8 6 7 6 4 8 8 8 6 4 6 5 2 7 4
0.970.09 0.190.04 0.190.05 1.310.09 1.650.13 1.680.24 1.340.09 1.610.09 2.120.45 0.940.11 1.190.10 1.240.11 1.000.00 1.030.07 0.940.06
0.000031 0.000010 0.024 0.0052 0.016 0.013 0.00021 0.013 0.34 0.14 0.10 0.88 0.64 0.84
0.900.03 1.030.07 0.840.03 0.970.04 1.530.17 1.120.08 0.970.04 1.170.06 1.830.36 1.000.04 0.880.05 0.880.03 0.980.01 0.910.04 0.910.04
0.18 0.19 0.13 0.022 0.015 0.14 0.0014 0.011 0.92 0.75 0.69 0.16 0.83 0.82
For stimulation, see Experimental Procedures. Drugs were added at the sixth fraction and maintained throughout the experiments. Note that FRR2/FRR1 or FRS2/FRS1 ratios less than the control indicate reduction, and values higher than the control indicate an increase of dopamine release.
at the time of the stimulation was released over the basal release (FRS1). When the stimulation was repeated after 30 min, the amount of radioactivity released above the resting release was 2.660.28% (FRS2; Fig. 1A), indicating that the stimulation-evoked release was fairly constant: the ratio between the amount of radioactivity released by consecutive stimulations (FRS2/FRS1) was 0.969 0.095 (n=8). HPLC data showed that 91–95% of the released tritiated activity was dopamine and its metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA). The other 5–9% was norepinephrine and epinephrine. The question arises whether the radioactivity determined in the tissue and released in response to field stimulation is of neuronal origin or whether it was taken up by and released from non-neuronal elements of the preparation. Evidence of neuronal origin of dopamine When the voltage-dependent Na+ influx, i.e. the axonal conduction, was inhibited by the application of tetrodotoxin (1 µM), the evoked release of [3H]dopamine in response to field stimulation was blocked (Fig. 1B), showing the Na+ influx dependence of electrically evoked release (Table 1). The inhibition of voltage-dependent Ca2+ channels by cadmium (100 µM), a non-selective voltagedependent Ca2+ channel blocker, prevented the effect of electrical stimulation on the release of [3H]dopamine (Fig. 1C), indicating the role of voltage-dependent Ca2+ channels in electrically evoked release (Table 1). In four experiments, a high-affinity dopamine uptake blocker, nomifensine12 (20 µM), was added into the fluid 15 min before the preparation was loaded for 60 min with [3H]dopamine, and it was kept in the solution throughout the loading time. If
[3H]dopamine was taken up and stored by dopaminergic terminals, the neuronal uptake of [3H]dopamine should have been blocked by nomifensine, and field stimulation should have failed to release radioactivity. This was indeed the case; the fractional release of [3H]dopamine from nomifensine-pretreated preparations in response to field stimulation (FRS1) was 0.670.02%, significantly lower (P=0.0002) than in control cases. Application of nomifensine from the sixth fraction to the perfusing solution increased the evoked release of dopamine, proving the blocked elimination of released dopamine (data are shown in Table 1). Presynaptic modulation of dopamine release After perfusing 10 µM SKF-38393, a D1 dopamine receptor subtype-specific agonist, through the cochlear tissue, basal and stimulation-evoked release of dopamine was increased. Figure 2 shows the highly significant increase in resting and in electrically evoked release in the presence of SKF-38393 (data are shown in Table 1). SKF-83566 (100 µM), a D1selective antagonist, caused an increase in both resting and evoked release of dopamine (Table 1). SKF-83566 applied before the agonist and kept in the solution throughout the experiments prevented the D1 receptor agonist-induced increase in electrically evoked release. The D1 antagonist failed to decrease the D1 agonist-increased resting release (Fig. 2). Effect of piribedil on dopamine release Piribedil was used at concentrations of 100 µM, 500 µM and 1 mM, as applied by others.6,7,31 The resting and electrically evoked release of [3H]dopamine was significantly increased in a
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Neurochemical evidence of dopaminergic transmission in the cochlea
Fig. 1. Fractional release of radioactivity ([3H]dopamine) in response to electrical field stimulations (2 Hz, 360 stimuli, 0.5 ms; S1, S2) from isolated cochlea preloaded with [3H]dopamine. (A) In control experiments (n=8), the consecutive stimulation-evoked release remained constant. (B, C) Tetrodotoxin (TTX, 1 µM; n=6; B) and cadmium chloride (CdCl2, 100 µM; n=7; C) applied from the sixth fraction prevented the electrically (S2) evoked release. S1 was used as a control stimulation. The symbols present the mean values and the vertical bars show the S.E.M.
concentration-dependent manner (Table 1). This effect could not be blocked by the D2 antagonist, sulpiride, but the D1-selective antagonist, SKF83566, prevented the effect of piribedil (Fig. 3). D2 receptor-specific effects Neither D2 dopamine receptor subtype-selective agonists, such as bromocriptine (at a concentration of 20 µM) and quinpirole (at concentrations of 20, 100 and 500 µM), nor selective antagonists, such as sulpiride (at concentrations of 100 and 500 µM), influenced the stimulation-evoked release of dopamine (Table 1), suggesting the absence of presynaptic D2 receptors on dopaminergic terminals.
In this work, we provide for the first time neurochemical evidence of the neurotransmitter role of dopamine in the mammalian auditory organ. To establish the identity of a neurotransmitter, an important criterion is the ability of nerve endings to take up and release the candidate substance upon electrical stimulation of the nerve. However, there are some indirect conclusions relying upon HPLC studies under noise conditions, which showed decreased dopamine content suggesting noise-evoked release of dopamine;16,18,40 until now, there has not been a reliable method to obtain direct evidence of the release of dopamine. We first used an in vitro superfusion technique on guinea-pig cochleae to confirm the release of dopamine from nerve terminals of LOC neurons in response to stimulation. Electrical depolarization of the cochlear tissue produced a significant and reproducible increase in [3H]dopamine release. The tritiated outflow we measured was due to release from dopaminergic fibres, as shown by HPLC combined with electrochemical detection. The majority of radioactivity released at rest and in response to stimulation (91% and 95%, respectively) was dopamine and its metabolites (DOPAC, HVA); only small amounts were epinephrine and norepinephrine, as described previously.13 We can assume that dopamine was released from lateral efferent fibres, because no other dopaminergic innervation has been shown in the cochlea.11,14,24,40 It is very important to confirm the neuronal origin of dopamine released by stimulation used in our experiments. The electrical stimuli activate the voltage-dependent Na+ channels present in the axons, imitating the physiological discharging action. The specific block of these channels with 1 µM tetrodotoxin inhibited the axonal conduction and prevented the electrically evoked release of dopamine. If the depolarization can propagate along the axon, voltage-dependent Ca2+ channels are activated, followed by an increase in intracellular Ca2+ concentration, postulated to cause exocytotic release. Blocking these voltage-dependent Ca2+ channels with cadmium, electrical stimulation also failed to release dopamine (Fig. 4). These results show that dopamine can be released from efferent fibres, and the fast Na+ channel and voltage-dependent Ca2+ channel dependence of the release proves its neuronal origin. Another important criterion for the role of neurotransmitter in the case of dopaminergic transmission is that neuronal nerve endings must be able to take up the released dopamine by their membrane uptake carriers and terminate the action of the transmitter in the extracellular space. In this study, the special inhibition of the dopaminergic uptake carrier by nomifensine12 during incubation with [3H]dopamine
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Fig. 2. Effect of the D1 agonist, SKF-38393, at a concentration of 10 µM on fractional release of [3H]dopamine (n=4). Drugs were added as indicated. The D1 antagonist, SKF-83566, at a concentration of 100 µM was applied 20 min before fraction collection (35 min before agonist administration) and kept in the solution throughout the experiments (n=4).
Fig. 3. Effect of 500 µM piribedil on [3H]dopamine release. The D2 antagonist, sulpiride, at a concentration of 100 µM, was applied 20 min before fraction collection and kept in the solution throughout the experiments (n=6). The D1 antagonist, SKF-83566, at a concentration of 100 µM, was applied similarly, as indicated (n=5).
decreased the releasable amount of dopamine at the release site, supporting its neuronal origin. Blocking the uptake mechanism by nomifensine after control stimulation caused an increase in the evoked release of dopamine, because of the failure of elimination. Our results provide neurochemical evidence of dopaminergic neurotransmission in the cochlea between the LOC efferents and type I afferent fibres (Fig. 4), and prove the neuronal origin of dopamine released by electrical stimulation.
There is some additional, indirect evidence that fulfils the criteria of dopamine-mediated chemical transmission of LOC efferent innervation. The existence of tyrosine hydroxylase and aromatic amino acid decarboxylase immunoreactivity, and lack of the enzymes dopamine-â-hydroxylase and phenylethanolamine-N-methyltransferase, suggest that the innervation from the lateral nuclei of the superior olive is dopaminergic.6,11,22,37,39 Depletion studies after 6-hydroxydopamine administration also
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Fig. 4. Scheme of synaptic interactions in the cochlea and the role of dopamine released from the axon terminals of LOC efferent fibres. The release of dopamine associated with axonal firing is subject to modulation by different drugs. Therefore, any drug able to release or potentiate dopamine release in fact inhibits the (over)excitation of primary auditory neurons caused by glutamatergic input from IHCs via D2 receptors present on dendrites of afferent fibres, as shown by others (see text). The clinical effect of piribedil is partly mediated via a presynaptic effect potentiating the release of dopamine and partly via stimulation of D2 receptors located on the afferent fibres.
support the notion that LOC fibres are able to synthesize dopamine.6,11 In accordance with data indicating dopamine-containing nerves, endogenous dopamine could also be detected using HPLC in rat cochlear homogenates,17 and under noise activation the level of dopamine metabolites, DOPAC and HVA, increased.16,18,40 It has also been shown that the interaction of dopamine with its specific receptors leads to changes in postsynaptic activity. The compound action potential of the auditory nerve was measured by noise exposure and showed a significantly reduced amplitude on adding dopamine and its analogues. This effect is thought to be mediated via the D2 or D3 subtypes of dopamine receptors.14,30 In addition, polymerase chain reaction analysis and nucleotide sequencing identified D2(long) and D3 receptor messages for dopamine receptor subtypes in the mammalian cochlea.24 Our results, taken together with previous findings, prove the neurotransmitter role of dopamine at the level of LOC efferent terminals.
The methods used in these experiments are suitable for investigating the presynaptic effect of different dopamine receptor-specific drugs. In our experiments, both the D1-selective agonist,23,48 SKF38393, and the antagonist,5 SKF-83566, caused an increase in basal and evoked release of dopamine. The D1 antagonist prevented the effect of the agonist. D2 receptor-specific agonists (bromocriptine, quinpirole) and antagonist (sulpiride) had no effect on dopamine release, suggesting the lack of this receptor subtype at the presynaptic site. It is concluded that the resting and stimulationevoked release of dopamine from the olivocochlear efferent fibres can be increased by different drugs. Since it has been observed that dopamine may have some sort of protective action against damage due to intense sound,6,7,31,36 the possibility arises that the enhancement of dopamine release may be clinically useful to diminish the damage in the cochlea caused by acoustic trauma or ischaemia. The lack of negative feedback suggests a crucial role of dopamine release.
Presynaptic modulation of dopamine release Modulation of dopamine release in the auditory system could be envisaged through a neuronal circuit: the activation of afferent fibres causes synaptic activity in the cochlear nuclei, which are connected to LOC neurons. In this way, the release of dopamine and other neurotransmitters from LOC efferents could be modified.16,18,32 The other possibility is a local modulation via a presynaptic modulatory site.41
Effect of piribedil on dopaminergic neurotransmission Piribedil has been described as a D2-specific agonist, but a novel radioligand binding study showed its 20-fold higher affinity for the D3 than for the D2 type of dopamine receptor, and little affinity for the D1 receptor subtype.4 Piribedil (Trivastal) is used clinically for the treatment of dopamine system dysfunction.9,21,29 Other clinical studies with piribedil
Cochlear dopaminergic neurotransmission
showed an improvement of symptoms (vertigo, tinnitus, sudden hearing loss) associated with cochleo-vestibular diseases. Piribedil has also been a popular drug to characterize the function of dopamine in the lateral efferent neurotransmission and to analyse dopamine receptor subtypes in these synapses.6,7,18,31,40 Intracochlear perfusion of 0.1– 1 mM of piribedil induced a dose-dependent decrease in the amplitude of the compound action potential and a concomitant increase in N1 latency of the auditory nerve measured by high-intensity toneburst stimulation.6,7,31 Piribedil also prevented the ischaemia-induced6,7,36 and acoustic traumainduced7,31 swelling of the radial dendrites. We found that dopamine release from isolated cochleae was enhanced by piribedil in a manner resembling that observed with other compounds (Figs 2, 3). Furthermore, the dopamine-releasing effect of piribedil could be blocked by a D1 antagonist. The presynaptic effect of piribedil to increase the resting release of dopamine and potentiate the release associated with axonal activity does not exclude its action on postsynaptic inhibitory D2 receptors (see Fig. 4). It seems very likely that its effect is partly indirect, via release of dopamine, and partly direct, via D2 receptors expressed on afferent fibres, as suggested by others.6,7,27 According to this model, the potentiation of dopamine release could lead to a more effective block of postsynaptic activity. It is worth mentioning that not all efferent en passant boutons make synaptic contacts with afferent
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fibres.26,38,46 Therefore, it is possible that dopamine or other transmitters released from non-synaptic varicosities diffuse far away from the release site and may have modulatory actions in a more diffuse fashion, as suggested similarly for other regions of the brain.42,44
CONCLUSIONS
For the first time, neurochemical evidence has been obtained for the transmitter role of dopamine at LOC efferent innervation of the cochlea. Dopamine—released into the vicinity and/or axodendritic synapses between LOC efferent fibres and dendrites of afferent fibres—may be involved in an inhibitory modulation of the effect of extreme synaptic activity of IHCs on primary auditory neurons,33 by this means protecting the auditory nerve from glutamate excitotoxicity (Fig. 4). It could also play a role in repair of the consequences of cellular excitotoxicity.30,34,35 Interventions to increase dopamine release in the cochlea could be a therapeutic target for the future and our results could contribute to progress in therapy or in prevention of particular types of hearing loss. Acknowledgements—This study was supported by a Hungarian Higher Education Research and Development Grant (FKFP 1343/1997), a Hungarian Research Grant (OTKA) and a Hungarian Medical Research Grant (ETT).
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