To visualize the projections from VCN to MNTB, axons from VCN were labeled in two ways: with lipophilic dyes in fixed tissue or with dextran dyes in fresh tissue slices made in an acute in vitro preparation. In fixed tissue, we used the lipophilic dye NeuroVue Red (PTI Research, Inc., Exton, PA), as previously described (Hsieh and Cramer, 2006 (link); Hsieh et al., 2007 (link)). After perfusion with 4% PFA, the cerebellum was carefully dissected away so that VCN was clearly visible. With fine forceps, a small piece of NeuroVue Red filter paper (100–200 μm2) was placed in VCN. To allow dye transport, brainstems were incubated in 4% PFA at 37° C for 10–14 days. Coronal vibratome sections were then cut at 100 μm, mounted onto slides, and coverslipped with Glycergel mounting medium (Dako).
In fresh tissue, we used dextran dyes in acute brainstem slices maintained in vitro. First, mice were euthanized with an overdose of isoflurane and perfused transcardially with artificial cerebrospinal fluid (ACSF; 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 20 mM NaHCO3, 3 mM HEPES, and 10 mM glucose saturated with 95% O2/5% CO2). Brainstems were quickly removed and thick brainstem slices (0.5–1.0 mm) were cut and maintained in oxygenated ACSF. Dextran amine dyes (Rhodamine or Alexa 488, 3,000 MW; Invitrogen) were used in a 6.25% solution containing 0.4% Triton X-100 in PBS. Brainstems were injected either unilaterally with a single dye or bilaterally with two different dyes. Dye solutions were delivered to VCN using pressure injection through a pulled micropipette attached to a Picospritzer. Current was subsequently applied at the dye injection site. Dye was allowed to transport for 1–3 hours. Tissue was fixed overnight in 4% PFA in PBS at 4°C, rinsed, then sectioned on a vibratome at 100 μm, mounted on slides and coverslipped with Glycergel mounting medium.
In some cases we delivered small deposits of rhodamine dextran amine (RDA) or horseradish peroxidase (HRP) in order to label small numbers of identifiable individual axons in P6–28 ephrin-B2lacZ/+ mice. Using a pulled glass pipette coated with concentrated RDA or HRP, three to four deposits were made in the ventral acoustic stria, just ventromedial to the VCN. Dye was allowed to transport for 2 hours and brainstems were fixed in 4% PFA at 4°C overnight. Brainstems were sectioned coronally at 150–200 μm on a vibratome, reacted for DAB (for HRP deposits), mounted on slides, and coverslipped.
In fresh tissue, we used dextran dyes in acute brainstem slices maintained in vitro. First, mice were euthanized with an overdose of isoflurane and perfused transcardially with artificial cerebrospinal fluid (ACSF; 130 mM NaCl, 3 mM KCl, 1.2 mM KH2PO4, 2.4 mM CaCl2, 1.3 mM MgSO4, 20 mM NaHCO3, 3 mM HEPES, and 10 mM glucose saturated with 95% O2/5% CO2). Brainstems were quickly removed and thick brainstem slices (0.5–1.0 mm) were cut and maintained in oxygenated ACSF. Dextran amine dyes (Rhodamine or Alexa 488, 3,000 MW; Invitrogen) were used in a 6.25% solution containing 0.4% Triton X-100 in PBS. Brainstems were injected either unilaterally with a single dye or bilaterally with two different dyes. Dye solutions were delivered to VCN using pressure injection through a pulled micropipette attached to a Picospritzer. Current was subsequently applied at the dye injection site. Dye was allowed to transport for 1–3 hours. Tissue was fixed overnight in 4% PFA in PBS at 4°C, rinsed, then sectioned on a vibratome at 100 μm, mounted on slides and coverslipped with Glycergel mounting medium.
In some cases we delivered small deposits of rhodamine dextran amine (RDA) or horseradish peroxidase (HRP) in order to label small numbers of identifiable individual axons in P6–28 ephrin-B2lacZ/+ mice. Using a pulled glass pipette coated with concentrated RDA or HRP, three to four deposits were made in the ventral acoustic stria, just ventromedial to the VCN. Dye was allowed to transport for 2 hours and brainstems were fixed in 4% PFA at 4°C overnight. Brainstems were sectioned coronally at 150–200 μm on a vibratome, reacted for DAB (for HRP deposits), mounted on slides, and coverslipped.
