Identification and quantification of GABAA R-α1-positive cells in the DCN of rats with behavioral evidence of noise-induced tinnitus

Abstract

Tinnitus, the perception of a phantom sound, often occurs as a clinical sequela of auditory traumas. However, the underlying mechanisms of tinnitus are largely unknown. In our previous studies, we found more gamma-aminobutyric acid A receptor alpha 1 subunit (GABA_AR-α1)-positive cells in the dorsal cochlear nucleus (DCN) after noise exposure, however, we were not able to identify the specific types of DCN cells that up-regulated GABA_AR-α1 after the insult. In the current study, we used Nissl staining, Purkinje cell protein 4 (PCP4) and glutamic acid decarboxylase 67 (GAD67) immunolabeling to identify GABA_AR-α1-positive cells in the DCN. Each type of GABA_AR-α1-positive cells was quantified and statistically analyzed using immunostaining and Nissl staining according to their morphology, size and location in the DCN. GABA_AR-α1-positive cartwheel cells, Golgi cells, as well as ML-stellate and vertical cells were confirmed by dual immunolabeling. In the DCN, the most common GABA_AR-α1-positive cells were Golgi cells followed by vertical cells and cartwheel cells while very few other cells were GABA_AR-α1-positive in all conditions. We found significantly more GABA_AR-α1-positive Golgi cells in the DCN of noise-exposed rats without behavioral evidence of tinnitus compared to normal controls and noise-exposed rats with behavioral evidence of tinnitus. This heightened "context-dependent" inhibition may help to maintain a balanced neuronal network, preventing the potential for tinnitus-related hyperactivity in the auditory pathways.

Keywords: Acoustic trauma; Central auditory system; GABA receptor; Neurons; Tinnitus.

Fig. 1

Noise exposure caused a transient threshold elevation at 24 h post-exposure, with near-complete recovery by 4 weeks post-exposure. A. ABR thresholds were temporarily elevated by approximately 27 dB relative to baseline (pre-exposure) levels (n = 5; two-way repeated-measures ANOVA, p < 0.001). B. A slight but statistically significant permanent threshold elevation of approximately 4 dB remained (n = 30; two-way repeated-measures ANOVA, p < 0.001). C. Inset displays the same ABR thresholds from the cohort of noise-exposed animals shown in panel B, separated into two cohorts based on the presence (N/T+ ) or absence (N/T− ) of noise-induced tinnitus (n = 20 for N/T+ , n = 10 for N/T− ; two-way mixed ANOVA, p > 0.05). Mean ABR thresholds (±SEMs) are shown at each test frequency. P-values represent the main effect of time or group, as applicable, from global ANOVA tests. Color key in A applies to both panels A and B. Auditory brainstem responses: ABRs. Re-graphed and adapted with permission from the International Tinnitus Journal (Du et al., 2024).

Fig. 2

Examples of GABA_AR-α1-positive cells in the DCN of NC (A) and N/T- (B) rats. A. The three layers of the DCN, the molecular layer (ML), the fusiform cell layer (FCL) and the deep layer (DL), are shown at low magnification. B. GABA_AR-α1-positive cells varied in size and shape. The scale bars = 50 μM in A and 20 μM in B.

Fig. 3

Examples of GABA_AR-α1-positive ML-stellate and cartwheel cells in the ML of the DCN. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A-C. GABA_AR-α1-positive ML-stellate cells (arrows in D-F) and cartwheel cells (arrowheads in D and F) are shown in the ML in NCs (A), noise-exposed rats without tinnitus (N/T-, B), and noise-exposed rats with tinnitus (N/T+, C). The squares in A-C indicate where images in D-F were collected in the DCN, respectively. The scale bar in C = 50 μM for A-C, while the scale bar in F = 20 μM for D-F.

Fig. 4

Examples of GABA_AR-α1-positive cartwheel cells in the FCL of the DCN. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A-C. GABA_AR-α1-positive (arrows in D-F) and negative (arrowheads in D-F) cartwheel cells are shown in the FCL among NCs (A and D), N/T- (B and E), and N/T+ (C and F) rats. The squares in A-C indicate where images in D-F were collected in the DCN, respectively. The scale bar in C = 50 μM for A-C, while the scale bar in F = 20 μM for D-F.

Fig. 5

Examples of GABA_AR-α1-positive Golgi cells in the FCL of the DCN. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A-C. GABA_AR-α1-positive Golgi cell (arrows in D-F) are shown in the FCL among NCs (A and D), N/T- (B and E), and N/T+ (C and F) rats. The squares in A-C indicate where images in D-F were collected in the DCN, respectively. The scale bar in C = 50 μM for A-C, while the scale bar in F = 20 μM for D-F.

Fig. 6

Examples of GABA_AR-α1-positive fusiform cells in the FCL of the DCN. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A-C. GABA_AR-α1-positive (arrows in D-F) and negative (arrowheads in D-F) fusiform cell are shown in the FCL among NCs (A and D), N/T- (B and E), and N/T+ (C and F) rats. The squares in A-C indicate where images in D-F were collected in the DCN, respectively. The scale bar in C = 50 μM for A-C, while the scale bar in F = 20 μM for D-F.

Fig. 7

Examples of GABA_AR-α1-positive granule and unipolar-brush cells in the DCN of N/T+ (A) and NC (B) rats. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A and B. Arrows denote a GABA_AR-α1-positive granule cell in the FCL (C) and a unipolar brush cell (D) in the DL of the DCN. Arrowheads denote GABA_AR-α1-positive Golgi cells in these two layers (C and D). The squares in A and B indicate where images in C and D were collected in the DCN, respectively. The scale bar in B = 50 μM for A and B, while the scale bar in D = 20 μM for C and D.

Fig. 8

Examples of GABA_AR-α1-positive vertical and Golgi cells in the DL of the DCN. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A-C. GABA_AR-α1-positive vertical (arrows in D-F) and Golgi (arrowhead in E) cells are shown in the DL among NCs (A and D), N/T- (B and E), and N/T+ (C and F) rats. The squares in A-C indicate where images in D-F were collected in the DCN, respectively. The scale bar in C = 50 μM for A-C, while the scale bar in F = 20 μM for D-F.

Fig. 9

Examples of GABA_AR-α1-positive giant cells in the DL of the DCN. The three layers of the DCN (ML, FCL and DL) are shown at low magnification in A-C. GABA_AR-α1-positive (arrows in D-F) and negative (arrowheads in D-F) giant cells are also denoted. The squares in A-C indicate where images in D-F were collected in the DCN, respectively. The scale bar in C = 50 μM for A-C, while the scale bar in F = 20 μM for D-F.

Fig. 10

Quantification and statistical analysis of GABA_AR-α1-positive Golgi cells in the DCN. Significantly more GABA_AR-α1-positive Golgi cells were measured in the DCN of noise-exposed rats without tinnitus (N/T-) compared to the NC and noise-exposed with tinnitus (N/T+) groups. Data are expressed as mean ± SEM. The statistical significance between groups is shown by asterisks: ***p < 0.001.

Fig. 11

Quantification and statistical analysis of GABA_AR-α1-positive cartwheel cells in the DCN. Significantly more GABA_AR-α1-positive cartwheel cells were measured in the DCN of noise-exposed rats without tinnitus (N/T-) compared to the NC group. No such statistical significance was measured when N/T+ cell densities were compared to NCs or the N/T- group (p > 0.05). Data are expressed as mean ± SEM. The statistical significance between groups is shown by asterisks: *p < 0.05.

Fig. 12

Quantification and statistical analysis of GABA_AR-α1-positive vertical cells in the DCN. Significantly more GABA_AR-α1-positive vertical cells were measured in the DCN of noise-exposed rats without tinnitus (N/T-) compared to the NC group. No such statistical significance was measured when N/T+ cell densities were compared to NCs or the N/T- group (p > 0.05). Data are expressed as mean ± SEM. The statistical significance between groups is shown by asterisks: *p < 0.05.

Fig. 13

Quantification and statistical analysis of GABA_AR-α1-positive fusiform, giant, granule, ML-stellate and unipolar-brush cells in the DCN. No significant difference was found among the three groups in the densities of GABA_AR-α1-positive fusiform, giant, granule, ML-stellate and unipolar-brush cells. Data are expressed as mean ± SEM.

Fig. 14

Confirmation of GABA_AR-α1-positive cartwheel cells in the DCN by GABA_AR-α1 and PCP4 dual immunolabelling. GABA_AR-α1 and PCP4 dual immuno-positive cartwheel cells were identified in the ML of the DCN in both the N/T- and N/T+ groups (arrows in B and C). PCP4-positive but GABA_AR-α1-negative cartwheel cells were also observed in the ML (arrowheads in A and C). GABA_AR-α1 and PCP4 dual immune-positive cartwheel cells were also identified in the FCL of the DCN in the three evaluation groups (arrows in G-I), and PCP4-positive but GABA_AR-α1-negative cartwheel cells were also observed in the FCL (arrowheads in D-I). The dashed lines in A-E indicate the ependymal layer of the DCN. The squares in D-F indicate where images in G-I were collected in the DCN, respectively. The scale bar in I = 10 μM for A-C and G-I, while the scale bar in F = 10 μM for D-F.

Fig. 15

Confirmation of GABA_AR-α1-positive cells in the DCN by GABA_AR-α1 and GAD67 dual immunolabelling. GABA_AR-α1 and GAD67 dual immuno-positive cartwheel (arrowheads in A and C) and ML-Stellate (arrows in A-C) cells were identified in the ML of the DCN of NCs (A), the N/T- (B) and N/T+ (C) groups. GABA_AR-α1 and GAD67 dual immuno-positive Golgi (arrows in D-F) and cartwheel cells (arrowheads in D-F) were also observed in the FCL of the DCN of the three evaluation conditions. GABA_AR-α1 and GAD67 dual immuno-positive vertical cells were identified in the DL of the DCN of the three evaluation conditions (arrows in G-I). The dashed lines in A-C indicate the ependymal layer of the DCN. The scale bar in I = 10 μM for A-I.

Fig. 16

Schematic diagram of cell interactions in the DCN. The DCN receives its primary inputs from the auditory nerve (AN) and sends its primary outputs to the inferior colliculus (IC). The principal cells [Fusiform cells (FC) and giant cells (GiC)] receive GABAergic and glycinergic inhibition directly from inhibitory interneurons [cartwheel cells (CW), ML-stellate cells (SC) and vertical cells (VC)]. Golgi cells (GoC) deliver GABAergic inhibitory signals to cartwheel cells, other Golgi cells and granule cells (GrC). The potential consequences of GABA_AR-α1 up-regulation on inhibitory neurons in the DCN depends on the functional status of inhibitory neurons that directly connected to the principal neurons (detailed in the Discussion). PF stands for parallel fibers, MF for Mossy fibers. Cells in blue indicate inhibitory neurons, and in red indicate excitatory neurons. Red lines and arrows mean excitation and blue lines mean inhibition.