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

Injection of biocytin provides an effective method for labeling axonal proj ections. Several difficulties arise when this technique is employed in feta l or early postnatal animals in vivo, including limited access to injection sites and extended post-injection survival periods. To circumvent these pr oblems, we adapted the technique of extracellular biocytin injection for us e in explanted brain hemispheres of developing mice. Briefly, entire brain hemispheres from perinatal mice (E16-P9) were removed and placed in oxygena ted aCSF in a brain slice recording chamber. Following visually guided inje ction of biocytin (2%) into the prelimbic cortex, the brains were then incu bated in oxygenated artificial cerebrospinal fluid (aCSF) for varying perio ds of time and then immersion-fixed in 4% paraformaldehyde and 0.5% glutara ldehyde. The next day, the brains were sectioned and processed for biocytin histochemistry using the avidin-biotin-complex method. We examined the met hod of injection, electrode type, time of injection, and post-injection inc ubation period. We found that in E16-P9 animals iontophoresis of biocytin u sing 8- to 12-megaohm patch clamp electrodes for a duration of 10 min provi des optimal axonal labeling. Post-injection incubation times of four or mor e hours are sufficient for labeling fine caliber collaterals as well as axo n bundles that reach distances over 3 mm. In vitro injection of biocytin in to explanted brain hemispheres provides a quick and easy method for tract t racing in developing brains. (C) 2000 Elsevier Science B.V. All rights rese rved.