In vitro biocytin injection into perinatal mouse brain: a method for tract tracing in developing tissue

Injection of biocytin provides an effective method for labeling axonal projections. Several difficulties arise when this technique is employed in fetal or early postnatal animals in vivo, including limited access to injection sites and extended post-injection survival periods. To circumvent these problems, we adapted the technique of extracellular biocytin injection for use in explanted brain hemispheres of developing mice. Briefly, entire brain hemispheres from perinatal mice (E16–P9) were removed and placed in oxygenated aCSF in a brain slice recording chamber. Following visually guided injection of biocytin (2%) into the prelimbic cortex, the brains were then incubated in oxygenated artificial cerebrospinal fluid (aCSF) for varying periods of time and then immersion-fixed in 4 % paraformaldehyde and 0.5 % glutaraldehyde. The next day, the brains were sectioned and processed for biocytin histochemistry using the avidin–biotin-complex method. We examined the method of injection, electrode type, time of injection, and post-injection incubation period. We found that in E16–P9 animals iontophoresis of biocytin using 8- to 12-megaohm patch clamp electrodes for a duration of 10 min provides optimal axonal labeling. Post-injection incubation times of four or more hours are sufficient for labeling fine caliber collaterals as well as axon bundles that reach distances over 3 mm. In vitro injection of biocytin into explanted brain hemispheres provides a quick and easy method for tract tracing in developin