Sparse Labeling of MNTB Calyces

In four-week-old mice, calyceal innervation levels were determined by sparsely labeling calyces of Held using dye electroporation injections as previously described^{9 (link),14 (link),15} . Mice were transcardially perfused with artificial cerebrospinal fluid (aCSF; 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 20 mM NaHCO3, 3 mM HEPES, 10 mM glucose, 2 mM CaCl2, 1.3 mM MgSO4 infused with 95% O2 and 5% CO2). Brains were quickly dissected and placed in a chamber with oxygenated aCSF. Brains remained in oxygenated aCSF for about 30 min and were then transferred to an aCSF-containing petri dish. A pulled glass micropipette was filled with rhodamine dextran amine (RDA; MW 3000, Invitrogen; in solution of 6.35% RDA with 0.4% Triton-X100 in PBS). RDA was electrophoresed at the rate of 5 pulses/second at 55 V for 50 ms using an Electro Square Porator (ECM830; BTX); multiple pulses were delivered at the midline to target the ventral acoustic stria (VAS). These pulses resulted in sparse dye-labeling of GBC axons and their calyceal terminals in MNTB on both sides. Brains were placed back into the aCSF chamber for approximately 2 h under continuous oxygenation to allow for dye transport. The tissue was then transferred to 4% PFA solution overnight followed by incubation in 30% sucrose solution in 0.1 M PBS. Brainstems were dissected and coronally cryosectioned at 18 µm in a series of 5 alternating slides. Tissue sections containing RDA-labeled calyces were immunolabeled for vesicular glutamate transporter 1/2 (VGLUT1/2) and counterstained with fluorescent Nissl.