Intraocular Tracer Injection:
Bats were anesthetized with a pharmacological mixture of tramadol hydrochloride (5 mg/kg i.m.), ketamine (5 mg/kg i.m.), diazepam (0.5 mg/kg i.m.) and xylazine (0.5 mg/kg i.m). While maintained at the appropriate level of anesthesia, the animals received, via needle attached to a micropump, 15 µl injections of unconjugated CTb (List Biological Laboratories, Campbell, CA, United States) in one eye.
The monocular injections were administered into the vitreous humor of the left eye and contained 1% CTb mixed with 5% dimethyl sulfoxide. Five days post-injection, the bats were anesthetized with the same anesthetic protocol as the surgical procedure and subjected to the following procedures.
Perfusions and Immunohistochemistry:
… Frozen brains were cut into 30-m coronal sections. Slices with SCN were collected from 0.45 mm to 1.08 mm post-bregma, approximately at the same level of the anterior commissure in the rostral section. On the other hand, slices containing IGL were collected from 3.84 mm to 5.04 mm post-bregma, approximately at same level of the external medullary lamina in the rostral level. All sections were collected in PB, and a one-in-six series was processed for CTb. Floating sections were incubated with goat anti-CTb antiserum (List Biological Laboratories, Campbell, CA, United States) in PB containing 0.4% Triton X-100 and 5% normal donkey serum for 1824 h. The sections were incubated with a secondary antiserum (biotinylated donkey anti-goat, Jackson Immunoresearch Laboratories, West Grove, PA, United States) in the same medium as above for 90 min.
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