Fig. 1: Optotagging and characterization of L5–POm projections.

a Labeling of L5–POm axons with rgAAV expressing floxed-mCherry ChR2 (magenta) injected in POm of Rbp4-Cre mice. S1bf, primary somatosensory cortex, barrel field; SC, spinal cord; POm, posteromedial nucleus of the thalamus. b Confocal overview image of mCherry axons (left top). Lightsheet imaging reveals projections to SC (left bottom). mCherry⁺ somata are located in L5, NeuN (green, right). Scale bars, 500 µm, 2 mm, and 50 µm. S2 secondary somatosensory cortex, EC external capsule, Cpu caudate putamen, IC internal capsule, Rt reticular nucleus of the thalamus, LD laterodorsal thalamic nucleus, LP lateral posterior thalamic nucleus, VPM ventral posteromedial thalamic nucleus, VPL ventral posterolateral thalamic nucleus, wm white matter. c Bar plot of mCherry⁺ NeuN⁺ neurons (2491 NeuN⁺ neurons in total in S1bf L5, n = 12 confocal z-stacks, N = 4 mice. d Confocal image of a single mCherry⁺ L5 pyramidal neuron with an illustration of a patch-clamp recording. Scale bar, 50 µm. Recordings replicated in n = 8 neurons, N = 5 mice. e Current clamp recording from a burst-firing mCherry⁺ neuron. Scale bars, 100 ms, 20 mV, and 250 pA. AP burst evoked by a 500 ms and 10 ms blue light stimulation (blue shading) in vitro. Scale bars, 20 mV and 100 ms (f), and 20 ms (g). h Confocal image of a POm neuron (biocytin fill, green) receiving large mCherry⁺ boutons (magenta). Scale bar, 30 µm. In vitro voltage recordings replicated in n = 7 POm neurons from N = 6 mice. i Typical depolarizing and hyperpolarizing responses from a POm neuron. Scale bars, 20 mV, 100 ms, and 200 pA. j Optogenetically evoked AP burst to blue light stimulation (3 ms). Scale bar, 20 mV. k mCherry⁺ boutons positive for vesicular glutamate transporter 1 (VGlut1, green). n = 3 slices, N = 1 mouse. Yellow arrowheads indicate giant (>1 µm diameter) corticothalamic boutons. Scale bar, 5 µm. l Top: Two optical pulses (@5 Hz, 3 ms, blue) induced paired-pulse depression of the EPSPs (average of 40 trials). Bottom: Voltage-clamp recording of EPSCs (@20 Hz, 3 ms) reveal postsynaptic depression (trace average of 4 trials). POm neurons held at –76 mV (Liquid junction potential corrected). m Population data of peak EPSC amplitude reduction (10th /1st EPSC) revealed a frequency-dependent depression. One-way ANOVA P = 0.0079. Tukey’s multiple comparisons test 20 vs. 10 Hz, *P = 0.03, 20 vs. 1 Hz, **P = 0.009, 10 vs. 1 Hz P = 0.83. n = 7 POm neurons from N = 6 mice. Data are presented as mean ± SEM. n Mouse brain schematic (adapted from¹⁰²) with experimental approach and example in vivo juxtacellular recordings (V_Juxta) of L5 (top) and POm neurons (bottom), showing LED-evoked (blue bars, @1 Hz, 5 pulses per trial) and spontaneous spiking. o High magnification of a L5 AP burst (200 Hz) temporally aligned with delayed single AP in the POm. Source data are provided as a Source data file. Atlas diagram in (b) adapted from ref. ¹⁰³.