(A and B) Representative images of TH immunolabeled 50 μm brain slices of the LSO region from a control (A) and a sound exposed mouse (B, 2 hr exposure). Sound exposure occurred at 7 weeks of age, and immunolabeling at 8 weeks. Each number identifies a TH+ LOC intrinsic neuron. (C) A representative brain slice of the LSO region from a sound exposed mouse (2 hr exposure) expressing tdTomato (Ai9) driven by ChatiresCre to label cholinergic LOC intrinsic neurons (red). Co-labeling with TH immunostaining (green) demonstrates that TH expression is found in existing cholinergic LOC intrinsic neurons. (D) Box plots demonstrating the number of TH+ LOC intrinsic neurons identified within LSO from either side of the brain, in control versus sound exposed mice for three sets of experiments. For both, 2 hr 110 dB SPL exposure (red) (control: n = 22 LSOs, 11 mice, exposed: n = 22 LSOs, 11 mice) and 5 × 12 hr 90 dB SPL exposure (orange) (control: n = 14 LSOs, 8 mice, exposed: n = 16 LSOs, 8 mice), the exposed groups have significantly larger numbers of TH+ LOC neurons than control groups, when examined one week from the beginning of the exposure (2 hr 110 dB SPL exposure, control: 11 ± 7, exposed: 47 ± 35) (5 × 12 hr exposure, control: 15 ± 11, exposed: 31 ± 46). In contrast, when examined three weeks from the beginning of the 5 × 12 hr 90 dB SPL exposure (yellow) (control: n = 12 LSOs, 6 mice, exposed: n = 12 LSOs, 6 mice), the number of TH+ LOC neurons is not significantly different between exposed and control groups (5 × 12 hr exposure w/long wait, control: 15 ± 11, exposed: 19 ± 9). Linear mixed model with sound exposure as the fixed effect and a random intercept for each mouse to account for the correlation among two measurements from the same mouse, ***p<0.0005; *p<0.05; n.s. not significant p>0.05. Presented data indicate median ±IQR per LSO. (E). Modified schematic drawing demonstrating the hypothesis that after sound exposure, a subset of cholinergic LOC intrinsic neurons will become dopaminergic and cholinergic. (F). An apical cochlear segment of a Th2A-CreER; Ai3 mouse raised in the institutional vivarium. Tamoxifen was administrated between 1–3 weeks. The mouse was sacrificed at the age of 1 month. Comparing mouse line labeling (top, a GFP antibody recognizes the EYFP reporter protein) and TH immunostaining (middle) suggests that some of the LOC efferent terminals that expressed Th at the time of tamoxifen injection did not express Th anymore at 1 month. See also Figure 5—figure supplement 1.
© 2019 Tim Phelps, JHU AAM. Illustration in panel E: Tim Phelps © 2019 JHU AAM (Department of Art as Applied to Medicine, Johns Hopkins University School of Medicine), published with permission. These illustrations are not covered by the CC-BY 4.0 licence and may not be separated from the article.