Extended Data Figure 2: TRIO applied to rat primary motor cortex.

a, Schematic of injection sites used for TRIO in rat motor cortex (see Fig. 1c for details of the viruses). Two different C regions were tested: striatum or contralateral motor cortex (cMC). b, c, Coronal section of rat motor cortex stained with DAPI (blue). Starter pyramidal neurons projecting to contralateral motor cortex (b) or striatum (c) (yellow, a subset indicated by arrowheads) can be distinguished from neurons receiving CAV-Cre and AAV-FLEx^loxP-TC (red) or GFP from RVdG (green). Bottom insets, coronal sections showing representative presynaptic GFP⁺ cells in somatosensory cortex (SC) or thalamus (Th). These data indicate that callosal-projecting neurons and striatum-projecting neurons in rat motor cortex both receive direct synaptic input from somatosensory cortex and thalamus (n = 2 animals for cMC C region; n = 3 animals for striatum C region). d, e, Omitting CAV-Cre for TRIO in the rat also resulted in local non-specific infection of RVdG. On average ∼200 cells were observed (n = 4 animals) within 800 µm from the injection site in these control experiments. By comparison, 1,392 GFP⁺ neurons were counted in the same region of a TRIO sample that has the lowest starter cells among the 5 brains analysed. Scale bars, 100 µm. Error bars, s.e.m.