Chronic tinnitus and unipolar brush cell alterations in the cerebellum and dorsal cochlear nucleus

Abstract

Animal model research has shown that the central features of tinnitus, the perception of sound without an acoustic correlate, include elevated spontaneous and stimulus-driven activity, enhanced burst-mode firing, decreased variance of inter-spike intervals, and distortion of tonotopic frequency representation. Less well documented are cell-specific correlates of tinnitus. Unipolar brush cell (UBC) alterations in animals with psychophysical evidence of tinnitus has recently been reported. UBCs are glutamatergic interneurons that appear to function as local-circuit signal amplifiers. UBCs are abundant in the dorsal cochlear nucleus (DCN) and very abundant in the flocculus (FL) and paraflocculus (PFL) of the cerebellum. In the present research, two indicators of UBC structure and function were examined: Doublecortin (DCX) and epidermal growth factor receptor substrate 8 (Eps8). DCX is a protein that binds to microtubules where it can modify their assembly and growth. Eps8 is a cell-surface tyrosine kinase receptor mediating the response to epidermal growth factor; it appears to have a role in actin polymerization as well as cytoskeletal protein interactions. Both functions could contribute to synaptic remodeling. In the present research UBC Eps8 and DCX immunoreactivity (IR) were determined in 4 groups of rats distinguished by their exposure to high-level sound and psychophysical performance: Unexposed, exposed to high-level sound with behavioral evidence of tinnitus, and two exposed groups without behavioral evidence of tinnitus. Compared to unexposed controls, exposed animals with tinnitus had Eps8 IR elevated in their PFL; other structures were not affected, nor was DCX IR affected. This was interpreted as UBC upregulation in animals with tinnitus. Exposure that failed to produce tinnitus did not increase either Eps8 or DCX IR. Rather Eps8 IR was decreased in the FL and DCN of one subgroup (Least-Tinnitus), while DCX IR decreased in the FL of the other subgroup (No-Tinnitus). Neuron degeneration was also documented in the cochlear nucleus and PFL of exposed animals, both with and without tinnitus. Degeneration was not found in unexposed animals. Implications for tinnitus neuropathy are discussed in the context of synaptic remodeling and cerebellar sensory modulation.

Keywords: Animal model; Auditory afferent degeneration; Cerebellum; Doublecortin; Eps8; Tinnitus; Unipolar brush cell.

Figure 1

Psychophysical performance of animals (n = 4 / group) selected for immunoreactive (IR) quantification. Top panel: 20 kHz tone presentations, used as the tinnitus diagnostic. The Exposed Tinnitus group function with dark triangular data points, was significantly down shifted (p = 0.021) from the Unexposed Control group, dark function with round data points. The Exposed No-Tinnitus group, unfilled square data points, was not significantly different than Unexposed (p = 0.482), and the Least-Tinnitus group, shaded square data points, was significantly up shifted from the Unexposed (p = 0.009). Bottom panel: Broad-band noise (BBN) presentations, used as a diagnostic for hearing loss; BBN testing showed no significant difference (p = 0.479) between groups. In both panels the X-axis shows the level of test sound presentations and the Y-axis (Suppression Ratio) shows bar pressing rate relative to background-sound bar pressing. Error bars show the standard error of the mean.

Figure 2

Auditory brainstem response (ABR) pure-tone thresholds (8 – 32 kHz) of the four experimental groups (same animals, as depicted in Figure 1). Top panel: pre-exposure thresholds; mid panel: immediate post-exposure thresholds; lower panel: end-of-behavioral testing thresholds. Only the right ear was exposed for 1 hr; n = 4/group; error bars show the standard error of the mean.

Figure 3

Representative epidermal growth factor 8 (Eps8) IR in the paraflocculus (PFL) of an exposed animal with tinnitus. Panes A – C show successively higher magnified views of a unipolar brush cell (UBC) configuration with brush contact between two adjacent UBCs (arrows in C). Calibration bar = 50 μm.

Figure 4

Representative doublecortin (DCX) IR in the PFL of an exposed animal with tinnitus. Panels A – C show successively higher magnified views of a typical UBC region. Arrows in C indicate a cell body and unipolar brush. Calibration bar = 50 μm.

Figure 5

Macroscopic bilateral view of Eps8 (top panes) and DCX (lower panes) IR in the animal depicted in Fig. 3. DCN, dorsal cochlear nucleus; FL, flocculus; PFL, paraflocculus. Eps8 and DCX IR puncta are evident the PFL, FL, and DCN.

Figure 6

Graphic summary of significant (p < 0.05) group IR differences between Unexposed Controls and Exposed Tinnitus, and Exposed No-Tinnitus. In the Tinnitus group (top) Eps8 was increased in the PFL. In the No-Tinnitus group (bottom) both EPS8 and DCX were decreased in the FL.

Figure 7

DCN Rostro-caudal DCX and Eps8 IR gradients in the four treatment groups (Unexposed, Exposed Tinnitus, Exposed No-Tinnitus, Exposed Least-Tinnitus). DCX IR (right panels) was higher in rostral sections than in caudal (F_3,96 = 17.14; p < 0.00001), with no significant difference between groups (F_7,96 = 0.83; p = 0.686). Eps8 also showed a high-rostral low-caudal gradient, but it was significant only in the three exposed groups (F_3,84 = 18.91; p < 0.0001), and not in the Unexposed group (F_3,28 = 1.10; p = 0.366).

Figure 8

Cochlear nucleus degeneration volume evident in Exposed Tinnitus, Exposed No-Tinnitus, and Unexposed groups. Error bars indicate total range and data points the group mean.

Figure 9

Representative cochlear nucleus (A & B) and PFL (C) degeneration in an Exposed Tinnitus animal. Diffuse degeneration was evident in the dorsal and intermediate acoustic stria as well as in the ventral cochlear nucleus (A. arrow and B. inset) medial to the DCN. Fiber tract degeneration (C. arrows) was also evident in the PFL.