Journal: Frontiers in Molecular Neuroscience
Article Title: Regeneration of Cochlear Synapses by Systemic Administration of a Bisphosphonate
doi: 10.3389/fnmol.2020.00087
Figure Lengend Snippet: Zoledronate treatment regenerates afferent synapses on IHCs after neuropathic noise exposure in mice. (A) Representative cochlear whole mounts illustrating maximal projections of the IHC area in the 32-kHz region immunostained for CtBP2 (red), GluA2 (green), and Myo7A (blue) revealed the synapses between auditory nerve terminals and IHCs in mice exposed to 97 dB SPL octave band white noise (8–16 kHz) for 2 h followed by subcutaneous injection with zoledronate (“Zole,” top) or saline (bottom) 1, 2, and 3 days after noise exposure, and sacrificed 2 weeks after noise exposure. Scale bar: 10 μm. (B) CtBP2-positive IHC synaptic ribbon counts 2 weeks after noise exposure revealed significantly larger counts at 32 kHz in animals receiving zoledronate than in control animals receiving saline. **** P < 0.0001. Data are presented as means ± SEM. N = 16 ears for Noise + Zoledronate group; N = 14 ears for Noise + Vehicle group. (C) Synaptic counts of CtBP2-positive pre-synaptic ribbons and juxtaposed GluA2-positive post-synaptic densities at 32 kHz confirmed significantly more synapses in animals receiving zoledronate than in control animals receiving saline. ∗∗ P < 0.01. Data are presented as means ± SEM. N = 6 ears for each group.
Article Snippet: Cochleae were extracted and post-fixed for 2 h in 4% PFA and decalcified in 0.12 M EDTA for 72 h. Whole mounts of the organ of Corti were prepared by microdissecting the spiraling cochleae into 6 pieces, blocking them with 5% normal horse serum (NHS) and 0.3% Triton X-100 (TX-100; Integra Chemical, WA, United States) in PBS for 1 h at room temperature, and immunostaining overnight at 37°C with the following primary antibodies diluted in 1% NHS with 0.3% TX-100 rabbit anti-myosin 7A at 1:200 (#25-6790 Proteus Biosciences, CA, United States) to label hair cells, mouse (IgG1) anti-CtBP2 (C-terminal Binding Protein) at 1:200 (#612044, BD Transduction Labs, CA, United States) to label pre-synaptic ribbons, and mouse (IgG2a) anti-GluA2 (Glutamate receptor subunit A2) at 1:2000 (#MAB397, Millipore Sigma, MA, United States) to label post-synaptic receptor patches.
Techniques: Injection
