Abstract: Objective To establish a primary culture model of neurons of newborn rat and investigate whether the known Netrin-1 receptors, DCC and UNC5H1, are co-receptors for Netrin-4. Method We improved Koh method to cultivate the primary neurons of newborn rat. Evaluation of neurons was made by immunocytochemical techniques. Ligand-receptor binding assays and in situ staining with the alkaline phosphatase (AP4) tagged Netrin-4 fusion protein were used. Reverse transcription polymerase chain reaction (RT-PCR) method was also used to observe the expression level of Netrin-4, DCC, UNC5H1 mRNA in brain during development. Result Cultured neurons were stick to the wall of the cultivation plate when they were growing. The neurons were satiety and with long axon. Immunocytochmical staining showed 90% neurofilament positive staining cell. In situ staining of AP4-Netrin-4 protein bound to COS7 cells transiently transfected with eukaryotic expression coding DCC and UNC5H1 showed that the Netrin-4 could bind to the transfected cells. Conclusion Primary culture of olfactory and cortical neurons of newborn rat by the method is a reliable and high stable way to obtain neurons. Primary culture of olfactory and cortical neurons of newborn rat by the method is a reliable and high stable way to obtain neurons.DCC and UNC5H1 are coreceptors for Netrin-4