Synaptic Reorganization Response in the Cochlear Nucleus Following Intense Noise Exposure

Abstract

The cochlear nucleus, located in the brainstem, receives its afferent auditory input exclusively from the auditory nerve fibers of the ipsilateral cochlea. Noise-induced neurodegenerative changes occurring in the auditory nerve stimulate a cascade of neuroplastic changes in the cochlear nucleus resulting in major changes in synaptic structure and function. To identify some of the key molecular mechanisms mediating this synaptic reorganization, we unilaterally exposed rats to a high-intensity noise that caused significant hearing loss and then measured the resulting changes in a synaptic plasticity gene array targeting neurogenesis and synaptic reorganization. We compared the gene expression patterns in the dorsal cochlear nucleus (DCN) and ventral cochlear nucleus (VCN) on the noise-exposed side versus the unexposed side using a PCR gene array at 2 d (early) and 28 d (late) post-exposure. We discovered a number of differentially expressed genes, particularly those related to synaptogenesis and regeneration. Significant gene expression changes occurred more frequently in the VCN than the DCN and more changes were seen at 28 d versus 2 d post-exposure. We confirmed the PCR findings by in situ hybridization for Brain-derived neurotrophic factor (Bdnf), Homer-1, as well as the glutamate NMDA receptor Grin1, all involved in neurogenesis and plasticity. These results suggest that Bdnf, Homer-1 and Grin1 play important roles in synaptic remodeling and homeostasis in the cochlear nucleus following severe noise-induced afferent degeneration.

Keywords: dorsal cochlear nucleus; gene expression; noise-induced hearing loss; synaptic plasticity; ventral cochlear nucleus.

Figure 1:

Fold change in expression of synaptic plasticity genes relative to actin housekeeping gene in DCN compared to VCN. Relative abundance of synaptic plasticity genes in the DCN was similar to the VCN as indicated by blue open circles within the 95% confidence interval for VCN. The thick dashed line shows the linear regression fit to the data and the shaded area represents the 95% confidence interval. Red open circles along with gene symbol identify genes that were more abundant in VCN than DCN (above 95% confidence interval) or less abundant in the VCN than DCN (below the 95% confidence interval).

Figure 2:

Mean (+/− SEM, n=6) auditory brainstem response thresholds measured in the right ear of the sham control group and the right ear of the noise exposed group 28 d post-exposure. Results were analyzed by two-way ANOVA, frequency repeated measure, F(1,30) =1211, p<0.0001, Bonferroni post-hoc, p<0.001). Asterisks (p<0.001) indicate thresholds that were significantly different between the control group and noise-exposed group.

Figure 3:

Volcano plots showing the negative logarithm base 10 of the p-value (left ordinate) or p value (right ordinate) versus log 2 fold change in synaptic plasticity gene expression in the 2 d post-exposure group relative to the sham control group. Data shown for the DCN (A) and VCN (B). To be considered significant, the fold change had to be greater than 0.5 fold (i.e., 50% increase, red circles) or less than 0.5 (i.e., 50% decrease, blue circles) as indicated by the red or blue dashed vertical lines respectively. In the VCN, six genes, identified with their gene symbols, met the criteria for a significant increase whereas none of the genes in the DCN met the criteria for significant increase or decrease at 2 d post-exposure. See Appendix Table A.2 and Table A.3 for details.

Figure 4:

Volcano plots showing the negative logarithm base 10 of the p-value (left ordinate) or p value (right ordinate) versus log 2 fold change in synaptic plasticity gene expression in the 28 d post-exposure group relative to the sham control group. Data shown for the DCN (A) and VCN (B). To be considered significant, the fold change had to be greater than 0.5 fold (i.e., 50% increase, red circles) or less than 0.5 (i.e., 50% decrease, blue circles) as indicated by the red or blue dashed vertical lines respectively. In the DCN, two genes, identified with their gene symbols met the criteria for a significant increase while in the VCN, 66 genes met the criteria for a significant decrease at 28 d post-exposure. See Appendix Table A.4 and Table A.5 for details.

Figure 5.

Representative photomicrographs of sections from the posterior ventral cochlear nucleus (PVCN) showing (A) positive and (B) negative assay controls for the in situ hybridization kit. Note reddish/brown spots (arrows) of in situ mRNA probe in panel A and complete absence of reddish/brown dots in panel B. Sections counterstained with Gills hematoxylin. Scale bar shown in each panel.

Figure 6:

Representative photomicrographs of sections from the ventral portion of cochlear nucleus close to auditory nerve root entry showing reddish/brown puncta of in situ Bdnf mRNA probe (arrows). Sections counterstained with Gills hematoxylin. (A) Many reddish/brown puncta present around nuclei of sham control whereas few puncta were present on section from 28d post-exposure group (B) consistent with the noise-induced decrease in Bdnf gene expression (see Appendix Table A.4). Scale bar shown in each panel.

Figure 7:

Representative photomicrographs of sections from the middle layer of dorsal cochlear nucleus (DCN) showing reddish/brown puncta (arrows) of in situ Grin1 mRNA probe. Sections counterstained with Gills hematoxylin. (A) Many reddish/brown puncta present around the nuclei of sham control and (B) 28d post-exposure group consistent with an absence of significant change in Grin1 gene expression (see Appendix Table A.4). Scale bar shown in each panel.

Figure 8:

Representative photomicrographs of sections from the posterior ventral cochlear nucleus (PVCN) close to auditory nerve root entry showing reddish/brown puncta (arrows) of in situ Homer1 mRNA probe. Sections counterstained with Gills hematoxylin. (A) Many reddish/brown puncta of Homer1A mRNA present around nuclei of sham controls whereas at 28 d post-exposure, fewer Homer1A-labeled puncta were present in the VCN, consistent with Homer1 gene expression (see Appendix Table A.5). Scale bar shown in each panel.

Figure 9:

Representative photomicrographs of sections from the middle layer of the dorsal cochlear nucleus (DCN) showing reddish/brown puncta of in situ Homer1 mRNA probe. Sections counterstained with Gills hematoxylin. (A) Many reddish/brown Homer1 puncta present around nuclei of neurons in the DCN of sham controls and (B) nuclei of DCN neurons in the 28 d post-exposure group consistent with an absence of significant change in Homer 1 gene expression. Scale bar shown in each panel.