D3308

Procedures used to label the CT terminal field with fluorescent tracers were similar to that described previously in mouse (Sun et al., 2015 (link), 2017 (link); Skyberg et al., 2017 (link)). Briefly, 24 h after PBN injections, animals were anesthetized as described above, placed into a non-traumatic head holder (Erickson, 1966 (link)) and maintained at 36°C with a water-circulating heating pad. The animal was placed into the supine position and an incision was made along the ventral midline of the neck. Musculature was retracted to expose the right tympanic bulla. The bulla was opened and the CT was cut peripheral to the geniculate ganglion. Crystals of 3-kDa tetramethyrhodamine dextran amine (TMR; Thermofisher Scientific; D3308, RRID: AB_2315472) were placed on the central stump of the CT, and the opening of the bulla was filled with Kwik-Sil (World Precision Instruments) to prevent crystals from diffusing from the site of the intended label. The incision was then sutured and the animal was revived using Antisedan (atipamezole hydrochloride, i.m.; Pfizer Animal Health).

Whole brains were dissected in Hanks’ balanced salt solution from P14.5 Neurog2^CreER(G2A); Cdhr1^tTA; TRE^{tdTomato-sypGFP} triple heterozygotes that had been given TM at E15.5. A small crystal of tetramethylrhodamine or fluorescein-conjugated fixable dextran (Thermo Fisher catalog #D3308, D3306) was prepared following the described methods (Glover, 1995 ; Sato et al., 1998 (link)) and inserted into the anterior most position of the OT that was accumulated with tdTomato-expressing axons under a fluorescence dissection microscopy (MZFLII, Leica). Until the dextran was transported through the axons, the brains were roll-bottle cultured for 3–4 h at 37°C in Neurobasal medium (Thermo Fisher) containing B27 supplements (Thermo Fisher) in an atmosphere of 95% O₂/5% CO₂ using a whole embryo culture apparatus (10-310, Ikemoto Rica Kogyo). The brains were then fixed with 4% PFA/PBS and subjected for histochemical analyses as described above. The dextran injections were performed on the both sides of 12 brains, and a confined labeling of the targeted cell cluster in the most anterolateral OT was successful in nine brains (at least one side). In all these successful cases, a small bundle of dextran-labeled axons coursing through the polymorph layer of the OT was observed as shown in the Figure 12C,D.

The chorda tympani and greater superficial petrosal nerves (gustatory branches of cranial nVII) were exposed in anesthetized mice and cut immediately distal to the tympanic bulla. Crystals of fluorescent dextran were placed on the cut proximal end as previously described^{21 (link)}. Dyes selected were lysine fixable 3 kDa dextrans, conjugated to fluorescein or tetramethylrhodamine (ThermoFisher D3306, D3308). After 5–7 h to allow dextran to accumulate in the neuronal soma, mice were fixed by perfusion and ganglia were harvested and processed for immunohistochemical analyses as described above.

Permeability assays were performed as described previously [15 (link)] in infected or healthy animals (Fig. 4a). TMR 3kD dextran (200 µl of 2 mM stock in PBS, D3308, Thermo Fisher, Germany) was injected intraperitoneally, followed by anesthesia 5 min later. After a circulation time of 20 min for the tracer, the animals were prepared for transcardial perfusion and ~ 300 µl blood was collected from the chest cavity just after atrial puncture, followed by perfusion for 3 min with PBS. Hemibrain (free of the olfactory lobes, cerebellum and hindbrain) and a single kidney were collected and immediately frozen on dry ice and stored at − 80 °C. For fluorescence measurement, samples were thawed on ice, weighed and homogenized in PBS (200 µl for hemi-cerebrum and 300 µl for kidney), followed by centrifugation for 20 min, 4 °C at 15,000×g. Supernatants (40 µl) as well as equal volumes of serum (1/5 dilution in PBS) were loaded into a 384-well black plate and fluorescence was measured at the corresponding excitation (550 nm)/emission (580 nm) in a plate reader (Tecan, Switzerland). Sham animals (without tracer injection) were used to subtract autofluorescence values. Permeability index (ml/g) was calculated as the ratio of (tissue RFUs/g tissue weight) to (serum RFUs/ml serum).

Anesthetic cream was applied to the tail 20 min prior to i.v injections. Tracers were injected via the tail vein at 4 μl/g body weight. Doses and circulation times are summarized in figure legends for each experiment. The following tracers were used: Goat anti Rabbit IgG-A488 (A11008; Thermo Fisher Scientific), Goat anti Human IgG-A647 (109-605-003; Jackson Immuno), BSA-A488 (A13100; Thermo Fisher Scientific), BSA-A647 (A34785; Thermo Fisher Scientific), 3 kD dextran-TMR (D3308; Thermo Fisher Scientific), 70 kD dextran-TMR (D1818; Thermo Fisher Scientific), 500 kD dextran-FITC (D7136; Thermo Fisher Scientific), and 2,000 kD dextran-FITC (D7137; Thermo Fisher Scientific). Mice were subsequently sacrificed and transcardially perfused with PBS only and immediately dissected (flow cytometry analysis) or PBS followed by 4% formaldehyde (IHC analysis) followed by 24 h after fixation in 4% formaldehyde.

Retrograde-labeling was performed as described previously [61 (link)] with slight modifications as follows. E11.5 mouse embryos were dissected and cultured in mouse Ringer’s solution oxygenated with carbogen (95% O₂ plus 5% CO₂) for 30 minutes to heal any cut edges that may incorporate the dye. Then a slit was made from the surface ectoderm overlying somites through DRG at the level of the thoracolumbar region and filled with lysine-fixable 3000 MW tetramethylrhodamine dextran (D3308, Molecular Probes/Invitrogen). The embryos were cultured for another 6 to 10 hours at room temperature to permit retrograde transport of the label and then fixed for immunofluorescence.