Selective Chemogenetic Blockade of Cerebello-Rubral Pathway by Double-Virus Infection Method Using Lentivirus and...

Double-virus infection method of lentivirus and adeno-associated virus was used to reveal the contribution of cerebellum in rehabilitative training after intracerebral hemorrhage (ICH) in rats. Chemogenetic blockade of cerebello-rubral pathway was achieved by the injection of a lentiviral vector pseudotyped with fusion glycoprotein type E and carrying Cre recombinase into the red nucleus and an adeno-associated viral vector encoding hM4D(Gi)-mCherry in the double-floxed, inverted orientation into the cerebellar lateral nucleus. This silencing system induced by clozapine-N-oxide significantly reduced the success rate of forelimb reaching test that is improved by forced-limb use after ICH. As a good application example of loss-of-function technique, the protocol of our experiment by double-virus infection method will be explained in detail.