Wt-RABV tracing:
For the present trans-synaptic tracing (Figure 1), nine out of eleven cases injected were used from the previous study, in which we focused on cerebellar connections into sensorimotor cortices (Aoki et al., 2019). Before surgery, animals were anesthetized with ketamine (80 mg/kg, Imalgene, France) and xylazine (10 mg/kg, Bayer, Germany). Appropriate levels of anesthesia were monitored by the absence of whisker movements and foot-pinch withdrawal reflex. Additional doses of the ketamine-xylazine mixture were administered i.p. when necessary. After placing animals into a stereotaxic frame (David Kopf Instruments), a mixture (0.15 or 0.2 L) of the French subtype of CVS-11 rabies virus (Wt-RABV, Aoki et al., 2019; Coulon et al., 2011; Raux et al., 1997; Ruigrok et al., 2008; Suzuki et al., 2012; Ugolini, 2010) and cholera-toxin b subunit (CTb, low salt; List Biological Laboratories, 1% w/v in 0.2 M phosphate buffer (PB) at pH 7.4: the injection solution consisted of four parts Wt-RABV and one part CTb) was injected by a 1 l Hamilton syringe in the following functional areas of the cerebral cortex: primary motor cortex (M1, n = 4), secondary motor cortex (M2, n = 4), and medial prefrontal cortex (mPFC, n = 3, one centered on prelimbic and two centered on cingulate cortex). As shown in our previous study, coordinates of the injection sites were determined by reference to the standard rat brain atlas (Aoki et al., 2019; Paxinos and Watson, 2004). The viral stock was kept at 80C until use. This CVS-11 strain of RABV has been confirmed to be transported trans-synaptically in a retrograde direction as well as in the time-dependent manner (Aoki et al., 2019; Kelly and Strick, 2004; Ruigrok et al., 2008; Suzuki et al., 2012; Ugolini, 2010). No neighboring neurons are infected unless they have synaptic contacts to the already infected cells. Adding CTb to the injection solution enabled accurate determination of the injection site (Aoki et al., 2019; Suzuki et al., 2012). Upon the injection, the needle was left in place for another 5 min to allow the virus to spread. After surgery, animals were monitored for signs of stress or discomfort. …