Pv800 pneumatic picopump

Mitral cells were labeled using retrograde tract tracing from their axons in the telencephalon as described previously (Fuller et al. 2006 (link)). Fish were over-anesthetized with 0.03% MS222 and perfused transcardially with PBS. The brains were dissected, and approximately 0.05μl−0.1μl of Texas Red dextran (10,000 MW, 5mg/ml in PBS, ThermoFisher Scientific) was injected into the medial and lateral olfactory tracts using a PV800 Pneumatic Picopump (World Precision Instruments). To allow active transport of the tracer molecule throughout the length of the neurons brains were cultured using a method modified from Tomizawa et al. (2001) (link) and Fuller et al. (2006) (link). The brains were placed on a 3μm polycarbonate filter in a sterilized six-well culture dish (Costar) containing artificial fish cerebrospinal fluid (100mM NaCl, 2.46mM KCl, 1mM MgCl₂H₂O, 0.44mM NaH₂PO₄H₂O, 1.13mM CaCl₂H₂O, 5mM NaHCO₃) and incubated at 28.5°C with 3–5% CO₂ for approximately 4 hours. Following fixation in 4% paraformaldehyde for 24 hours at 4°C, the tissue was rinsed in PBS and mounted between two coverslips in a polyvinyl alcohol (PVA)-based aqueous hardening mounting medium (13% polyvinyl alcohol, 1.7% 1,4-diazobicyclo[2,2,2]octane, 33% glycerol in 0.2M Tris-HCl) and viewed using confocal microscopy.