Sound localization ability and glycinergic innervation of the superior olivary complex persist after genetic deletion of the medial nucleus of the trapezoid body

The medial nucleus of the trapezoid body (MNTB) in the superior olivary complex (SOC) is an inhibitory hub considered critical for binaural sound localization. We show that genetic ablation of MNTB neurons in mice only subtly affects this ability by prolonging the minimum time required to detect shifts in sound location. Furthermore, glycinergic innervation of the SOC is maintained without an MNTB, consistent with the existence of parallel inhibitory inputs. These findings redefine the role of MNTB in sound localization and suggest that the inhibitory network is more complex than previously thought

MCV sT expression is lethal in mice.

<p>(A) Design of the <i>ROSA26-CAG-LNL-MCVsTco</i>, a ROSA26 knock-in construct encoding codon-optimized MCV sT with flanking loxP-STOP-Neo-loxP (LNL) sequence, recombined with the ROSA26 genomic locus. <i>ROSA26-CAG-LNL-MCVsTco</i> crossed with <i>Ubc</i>^{<i>CreERT2</i>} or <i>Atoh1</i>^{<i>CreERT2</i>} mice allows for TMX-induced Cre recombinase excision of the loxP-STOP-Neo-loxP sequence that results in MCV sT expression under the CAG promoter. (B) MCV sT expression is lethal in <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i> transgenic mice. Kaplan-Meier curve of high dose (0.2 mg/g) and low dose (0.02 mg/g) TMX injected mice. High dose TMX injection caused rapid weight loss and mice reached the euthanasia criterion (20% loss of body weight) within 5 days of the injection (n = 4, solid orange). Survival of the control <i>Ubc</i>^{<i>CreERT2</i>} mice (n = 7, dotted orange) was not affected by TMX injection. <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i> mice injected with lower dose TMX exhibited significantly prolonged survival compared to high dose TMX (n = 18, solid red) and control mice were unaffected (n = 10, dotted red) (C) Multi-tissue MCV sT protein expression in <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i> mice that died immediately after high dose TMX injection (< 5 days). Lower dose TMX injection induces less MCV sT protein expression in a mouse that died at day 7 after the injection as detected by immunoblotting using an antibody raised against MCV sT, CM8E6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142329#pone.0142329.ref022" target="_blank">22</a>]. MCV sT vector or empty vector transfected HEK293 cells were used as a positive and a negative control, respectively. Equal amounts of sT-transfected HEK293 cell lysates were loaded for normalization across different blots. Hsp70/Hsc70 was detected as a protein quality control. (D) MCV sT protein expression was maintained in tissues of <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i> mice that survived over 70 days after low dose TMX injection. Immunoblots were performed as in (C). Tissue lysates from <i>ROSA</i>^<i>sT</i> were used as a negative control.</p

MCV sT induces hyperproliferaton in MEF cells.

<p>(A) Induction of MCV sT in multiple mouse embryonic fibroblast (MEF) cells from littermate by 4-OHTMX. MEFs extracted from <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i> and <i>ROSA</i>^<i>sT</i> embryos were cultured in the presence or absence of 500 nM 4-OHTMX for 7 days. MCV sT and c-Myc protein expression was detected by immunoblot. Hsp/Hsc70 protein expression was detected as a loading control. (B) MCV sT expression accelerates cell proliferation in MEF cells. Proliferation of MEF cells treated with or without 4-OHTMX was evaluated by Wst1 assays.</p

MCV sT transgenic mice develop tumors in a p53 null setting.

<p>(A) p53 ablation does not rescue mice from MCV sT-induced lethality. Kaplan-Meier curve of low dose (0.02 mg/g) TMX injected <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>; p53</i>^{<i>flox/flox</i>} mice (Solid blue line, n = 26) and <i>Ubc</i>^{<i>CreERT2</i>}<i>; p53</i>^{<i>flox/flox</i>} mice (dotted blue line, n = 10). Pink lines indicate the survival of <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>(solid line)</i> and <i>Ubc</i>^{<i>CreERT2</i>} (dotted line) mice with wild type p53 as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142329#pone.0142329.g001" target="_blank">Fig 1B</a> for comparison with p53 knockout background (blue lines). (B) MCV sT expression produces tumors <i>in vivo</i> in <i>p53</i> null setting. 80% (4/5) of mice that survived over 60 days after TMX injection develop grossly visible tumor in the spleen and liver. Representative spleen and liver tissues with tumor nodules from p53.7F are shown with corresponding normal tissues from a C57BL/6 control mouse. (C) Immunoblotting of MCV sT protein expression in liver and spleen tissues from mouse p53.7F) with macroscopic tumors. Spleen, muscle and ear tissues consistently maintained sT expression over 60 days after TMX injection. sT expression was detectable in liver tissues by immunoblotting only from liver with visible nodules (mouse p53.7F). MCV sT protein was detected using CM8E6 antibody, and Hsp/Hsc70 expression was used as a loading control. (D) The top panel shows anaplastic neoplasia in the spleen and liver. A range of proliferative changes was observed in the kidney, where distal tubular epithelia are most severely affected, but glomeruli (black arrowhead), proximal tubular epithelia (*), and interstitial tissues are relatively spared of proliferative changes. The middle panel shows a representative immunohistochemical staining of sT protein expression in liver tumor, spleen tumor and kidney tissues from <i>Ubc</i>^{<i>CreERT2</i>}<i>; ROSA</i>^<i>sT</i><i>; p53</i>^{<i>flox/flox</i>} mice. Tissue samples were immunostained with MCV sT (CM5E1) antibody. (E) The top panel shows a sT-induced liver tumor with immunoreactivity to α-smooth muscle actin (ASMA) and bottom panel shows K14 positivity in scattered tumor cells.</p