Extended Data Fig. 2: Monosynaptic connection between rACC and Pn.

a, Schematic illustration of the viral strategy to trace projections from the rACC and motor cortex to the Pn. b, Injection sites in the motor cortex (top), rACC (middle), and the overlay (bottom). Scale bars, 500 μm. c, Projections from the rACC and motor cortex to the Pn. Note that the infected neurons in the motor cortex and rACC do not overlap and that the rACC only targets the rostral part of the Pn. Scale bars, 100 μm. d, Schematic illustration of the viral strategy to identify the brain areas that project to the Pn. SL1, stem-loop 1. e, Representative photomicrographs of labelled Pn projection neurons in several brain areas. M1, primary motor cortex; ACC, anterior cingulate cortex; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; V1, primary visual cortex; CeA, central nucleus of the amygdala; Crus I, crus I of the ansiform lobule; Sp5I, interpolar nucleus. Scale bars, 100 μm. f, Schematic illustration of the strategy to test the synaptic connections between ACC projection neurons and neurons in the Pn. g, Confocal micrograph of two Pn neurons filled with biocytin during recording and labelled using a fluorescent conjugate of streptavidin. Scale bar, 50 μm. h, Average EPSC (black) of a Pn neuron holding at −70 mV evoked by photostimulation of rACC fibres. Adding CNQX (10 μM) in the perfusion solution abolished the EPSCs (red; left). EPSCs in a Pn neuron evoked by photostimulation of rACC fibres (top). TTX (1 μM) abolishes this response (middle), while 4-AP (1 mM) restores the response blocked by TTX (red, bottom).