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, 8 (6), e64726

Tinnitus, Unipolar Brush Cells, and Cerebellar Glutamatergic Function in an Animal Model


Tinnitus, Unipolar Brush Cells, and Cerebellar Glutamatergic Function in an Animal Model

Carol A Bauer et al. PLoS One.


Unipolar brush cells (UBCs) are excitatory interneurons found in the dorsal cochlear nucleus (DCN) and the granule cell layer of cerebellar cortex, being particularly evident in the paraflocculus (PFL) and flocculus (FL). UBCs receive glutamatergic inputs and make glutamatergic synapses with granule cells and other UBCs. It has been hypothesized that UBCs comprise local networks of tunable feed-forward amplifiers. In the DCN they might also participate in feed-back amplification of signals from higher auditory centers. Recently it has been shown that UBCs, in the vestibulocerebellum and DCN of adult rats, express doublecortin (DCX), previously considered a marker of newborn and migrating neurons. In an animal model, both the DCN, and more recently the PFL, have been implicated in contributing to the sensation of acoustic-exposure-induced tinnitus. These studies support the working hypothesis that tinnitus emerges after loss of peripheral sensitivity because inhibitory processes homeostatically down regulate, and excitatory processes up regulate. Here we report the results of two sequential experiments that examine the potential role of DCN and cerebellar UBCs in tinnitus, and the contribution of glutamatergic transmission in the PFL. In Experiment 1 it was shown that adult rats with psychophysical evidence of tinnitus induced by a single unilateral high-level noise exposure, had elevated DCX in the DCN and ventral PFL. In Experiment 2 it was shown that micro-quantities of glutamatergic antagonists, delivered directly to the PFL, reversibly reduced chronically established tinnitus, while similarly applied glutamatergic agonists induced tinnitus-like behavior in non-tinnitus controls. These results are consistent with the hypothesis that UBC up regulation and enhanced glutamatergic transmission in the cerebellum contribute to the pathophysiology of tinnitus.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Doublecortin (DCX) immunoreactivity (IR) in UBCs of the DCN and cerebellum ipsilateral to the exposure.
A. Photomicrograph through the brainstem and cerebellum showing DCX-IR neurons. The rectangle shows the location of the higher magnification photomicrograph in B. B. DCX-IR in UBCs of the vPFL. C. DCX-IR neurons in the vPFL, FL and DCN on the side contralateral to the exposure. The rectangle shows the location of the photomicrograph in D. Scale bar 500 µm. D. DCX-IR neurons are UBCs, example at arrow. Scale bar 50 µm. Abbreviations: DCN, dorsal cochlear nucleus; FL, flocculus; UBC, unipolar brush cell vPFL. ventral paraflocculus.
Figure 2
Figure 2. Auditory brainstem response (ABR) group-average thresholds before and after high-level sound exposure.
Unexposed rats are included for comparison. Post-exposure thresholds, obtained immediately after right ear exposure, show temporary right (exposed) ear elevation. End-of-study thresholds, obtained at the conclusion of psychophysical testing, show a recovery of exposed animals to normal threshold levels. Error bars in this and other figures indicates the standard error of the mean.
Figure 3
Figure 3. Group average psychophysical discrimination functions of exposed and unexposed animals in Experiment 1.
The suppression ratio, plotted on the y-axis, indicates relative lever press rate. Sound level, plotted on the x-axis, indicates the ambient level in the 1-min test periods that substitute for background noise. The functions slope upward from speaker OFF because food is obtained for lever pressing when the sound is on, but a foot shock is delivered at the end of speaker OFF periods when speaker-off lever pressing is above a criterion level. Panel A shows the results when tested with the diagnostic stimulus of 20 kHz tones. Panel B shows the results when tested with the non-diagnostic stimulus of broad-band noise (BBN). Hearing level is comparable in both exposed (triangular data points) and unexposed (circular data points) animals, as indicated by their BBN performance. The down-shift of the exposed rats' function when tested with 20 kHz shows greater suppression to sound resembling their sensation during speaker off periods, i.e., tinnitus. Statistically significant separation of the functions above speaker-off is indicated by repeated-measures ANOVA in Panel A.
Figure 4
Figure 4. Doublecortin (DCX) immunoreactivity in the ipsilateral and contralateral DCN and PFL of exposed rats, and unexposed rats.
Both Ipsilateral and contralateral DCN and PFL showed significant elevation (independent t tests) above that of unexposed control animals, with higher ipsilateral levels.
Figure 5
Figure 5. Schematic of the osmotic pump delivery of drugs in Experiment 2.
The pump body was subcutaneously implanted in the dorso-caudal neck region. The catheter terminated dorsal to the ipsilateral PFL, anchored to a 1 mm diameter skull opening. Infusion volume and rate are shown.
Figure 6
Figure 6. Drug diffusion as indicated by methylene blue.
Two rats were implanted with pumps filled with 0.25% methylene blue to indicate the diffusion field of pump-delivered drugs. The diffusion coefficient of methylene blue was 85% that of the drug cocktail average diffusion coefficient. The top panel shows the macroscopic diffusion of methylene blue over the surface of the PFL. The lower panel shows deep tissue penetration in a coronal section of the PFL. Methylene blue infused and penetrated the PFL up to its stalk, but did not extend into brainstem areas encompassing the cochlear nucleus.
Figure 7
Figure 7. Experiment 2, glutamatergic antagonist effects on exposed rats with tinnitus.
Glutamatergic antagonists were delivered to the ipsilateral PFL of exposed rats with significant evidence of tinnitus (triangular data points): Unexposed group results are depicted as circular data points and exposed non-drug animals as square data points. Pre-drug data are shown in the upper left panel. Exposed pre-drug animals were significantly downshifted from unexposed controls and were no different than exposed no-drug animals with tinnitus (repeated-measures F statistics shown in panel). After 72 hrs of antagonist cocktail, the treated animals were significantly up-shifted from the untreated exposed animals, therefore showing attenuation of tinnitus. Antagonist amelioration of tinnitus gradually waned over subsequent treatment days (bottom panels, left to right), returning to pre-treatment strength at 192 hrs.
Figure 8
Figure 8. Experiment 2 glutamatergic agonist effect on exposed rats with no evidence of tinnitus (triangular data points).
The results of unexposed and exposed control groups are depicted as in Figure 7. Pre-drug data are shown in the upper left panel. The agonist cocktail induced significant tinnitus in the exposed no-tinnitus animals, indicated by a function down-shift (upper right panel) after 300 hrs of treatment, but their tinnitus did not attain the same level as the exposed group with tinnitus (F statistics in panel). Nevertheless, the agonist-induced tinnitus persisted for at least 30 days post drug (lower right panel), at which time testing was discontinued.
Figure 9
Figure 9. Experiment 2 glutamatergic agonist effect on unexposed rats with no evidence of tinnitus.
The glutamatergic agonist cocktail was delivered to the right PFL of unexposed rats with no evidence of tinnitus (open circular data points). Unexposed and exposed control groups are depicted as in Figure 7. Pre-drug data are shown in the upper left panel. The agonist cocktail induced a significant tinnitus-like downshift in the function of unexposed animals (upper right panel) after 72 hrs, although, as before, their tinnitus did not attain the same level as the exposed group with tinnitus (F statistics in panel). This tinnitus-like induction gradually washed out over the course of treatment (lower panels, left to right).

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