Salicylate induces tinnitus through activation of cochlear NMDA receptors

Abstract

Salicylate, the active component of aspirin, is known to induce tinnitus. However, the site and the mechanism of generation of tinnitus induced by salicylate remains unclear. Here, we developed a behavioral procedure to measure tinnitus in rats. The behavioral model was based on an active avoidance paradigm in which rats had to display a motor task (i.e., to jump on a climbing pole when hearing a sound). Giving salicylate led to a decrease in the percentage of correct responses (score) and a drastic increase in the number of false positive responses (i.e., animals execute the motor task during a silent period). Presentation of the sound at a constant perceptive level prevents decrease of the score, leading to the proposal that score is related to hearing performance. In contrast, the increase of false positive responses remained unchanged. In fact, animals behaved as if they hear a sound, indicating that they are experiencing tinnitus. Mefenamate in place of salicylate also increased the number of false positive responses, suggesting that salicylate-induced tinnitus is related to an inhibition of cyclooxygenase. One physiological basis of salicylate ototoxicity is likely to originate from altered arachidonic acid metabolism. Because arachidonic acid potentiates NMDA receptor currents, we tested the involvement of cochlear NMDA receptors in the occurrence of tinnitus. Application of NMDA antagonists into the perilymphatic fluids of the cochlea blocked the increase in pole-jumping behavior induced by salicylate, suggesting that salicylate induces tinnitus through activation of cochlear NMDA receptors.

Fig. 1.

Schematic representation of the behavioral protocol. Animals were conditioned to jump on a climbing pole in response to a sound stimulation. Each session included 10 trials. The conditioning procedure requires up to seven sessions lasting 15–20 min (i.e., 2 or 3 d). When conditioned (criterion, 3 consecutive sessions with a score ≥80%), animals were included in the experiments. The behavioral testing protocol (9 d) consisted of a daily measurement of the correct responses to sound (score) and climbings during intertrial periods (false positives or responses during silent periods) in the same 10 min session. Saline, salicylate, or mefenamate were injected daily 2 hr before the testing session.

Fig. 2.

Measurement of the score and false positive responses in salicylate-treated animals. Animals were conditioned to display a motor task (i.e., to jump on a climbing pole) in response to the presentation of a sound (10 kHz, 3 sec duration). Arepresents the percentage of correct responses to sound (score) measured before (day 0), during (days 1–4; gray area), and after intraperitoneal injections of saline or salicylate (300 mg/kg).B represents the number of abnormal jumps during silent periods (false positives). The score remained stable (∼90%) during the time course of the experiment for the saline group (filled circles;n = 10), even during the intraperitoneal injections of saline. Note the absence of false positive responses (i.e., animals did not execute the task during silent periods). Injections of salicylate (open circles; n = 10) reduced the score (p < 0.05 at days 3 and 4) and significantly (p < 0.001) increased the number of false positives as soon as the first day of treatment. A complete recovery was seen when the treatment was stopped. Note the different time pattern of change induced by salicylate on the score and the false positives.

Fig. 3.

CAP and DPOAE measurements. Hearing loss induced by salicylate was assessed by recording CAP and DPOAE measurements. A, Shown are the CAP audiograms for animals (n = 6) before salicylate treatment (day 0; circles), after the second injection of salicylate (day 2; squares), after the third injection of salicylate (day 3; upward triangles), and the first day of recovery (day 5; inverted triangles). The broken line represents 10 kHz. B, CAP threshold shift for 10 kHz before, during, and after salicylate treatment (gray area). CAP threshold shifts at 10 kHz were calculated as the difference in decibels between the auditory threshold recorded at day 0 and those recorded on following days. C, Shown are the DPOAE amplitudes as a function of f₂ frequency for animals (n = 6) before salicylate treatment (day 0; circles), after the second injection of salicylate (day 2; squares), after the third injection of salicylate (day 3; upward triangles), and the first day of recovery (day 5; inverted triangles). The broken line represents 10 kHz. After the third injection of salicylate (day 3), DPOAEs disappeared into the noise floor. A complete recovery to pretreatment amplitudes was seen 1 d after the end of salicylate treatment. D, Changes in DPOAE amplitudes forf₂ = 10 kHz before, during, and after salicylate treatment (gray area). Note the similarity of the time course of hearing loss assessed by CAP and DPOAE measurements and the reduction of the score shown in Fig. 2A. The first significant change in CAP and DPOAE was observed at day 2 (p < 0.001).

Fig. 4.

Measurement of score and false positive responses at constant perceptive level. Hearing loss induced by salicylate was compensated by adjusting the intensity of sound eliciting behavioral responses as a function of threshold shift (gray area;n = 10). A, A slight, but not significant (p = 0.287), increase of the score was observed in animals in which sound was presented at constant perceptive level. B, Presentation of sound at a constant perceptive level did not affect the increase of false positive responses induced by salicylate treatment, the first significant increase being observed at day 1 (p < 0.001).

Fig. 5.

Measurement of the score and false positive responses in mefenamate-treated animals. To determine whether changes of behavioral responses induced by salicylate were linked to cyclooxygenase inhibition, we investigated the effect of intraperitoneal injection of mefenamate. The mefenamate treatment (35 mg/kg) lasted 4 d (from day 1 to day 4; gray area).A represents the percentage of correct responses to sound (score). B represents the number of abnormal jumping during silent periods (false positives). Whereas mefenamate did not significantly change the score, it did significantly increase in the number of false positive responses (n = 10), the first significant increase being observed at day 2 (p < 0.001).

Fig. 6.

Score and false positive responses after cochlear NMDA receptor blockade. NMDA antagonists were applied into the perilymphatic fluids using Gelfoam placed on the round window of both ears. Surgery to place Gelfoam on the round window was done immediately after the first behavioral measurement (day 0). A, Gelfoam bathed with control artificial perilymph alone (AP;n = 10; filled circles) or containing 50 μm 7-CK (n = 10; open circles) did not change the degree and the time course of the reduction of the score. B, In contrast, application of 7-CK into the perilymphatic fluids significantly reduced the number of false positive responses. C, Shown are the number of false positive responses measured at day 4 in animals injected with saline solution (saline) and in animals injected with salicylate plus Gelfoam bathed with artificial perilymph alone (AP; n = 10) or MK-801 (10 μm; n = 10), 7-CK (50 μm; n = 10), or gacyclidine (50 μm; n = 10). When compared with artificial perilymph alone, local application of MK-801, 7-CK, or gacyclidine significantly (p < 0.001) reduced the occurrence of the false positive responses. When compared with animals injected with mefenamate alone (control), application of Gelfoam containing 50 μm 7-CK significantly reduced the occurrence of the false positive responses.