Extended Data Fig. 7. Retrograde labeling of aNB-projecting neurons in the midbrain.

a, Schematic of experimental design. Retrobeads were injected into the aNB to retrogradely label the aNB-projecting neurons in PB of ChAT^IRES-Cre or Slc17a6^IRES-Cre mice. ChAT⁺ or vGLUT2⁺ PB neurons were labeled by AAVs encoding Cre-inducible EGFP. b, Images showing red Retrobeads injected into the aNB region (AP −0.8 – −1.1 mm) (repeated in 3 mice). c–f, Coronal brain sections showing the distribution of Retrobeads in the midbrain 10 days after injection (3 mice). g, h, Percentages of aNB-projecting neurons in midbrain nuclei at AP −4.6 – −4.9 mm (g) and −5.1 – −5.4 mm (h) (n = 3 mice). Inset: fraction of Retrobeads⁺ cells in the lateral PB (lPB) and the medial PB (mPB) ipsilateral or contralateral to the injection site. i, Colocalization of Retrobeads⁺ somas and NeuN⁺ nuclei in PB. Arrows indicate Retrobeads⁺NeuN⁺ cells. j, Percentages of NeuN⁺ cells containing Retrobeads (3 mice). k, Colocalization of Retrobeads⁺, EGFP-labeled vGLUT2⁺, and ChAT immunoreactive somas in PB of Slc17a6^IRES-Cre mice. Yellow and magenta arrows indicate vGLUT2⁺Retrobeads⁺ and ChAT⁺Retrobeads⁺ cells, respectively (3 mice). l, Percentages of Retrobeads⁺ cells expressing vGLUT2 and/or ChAT in lPB and mPB (n = 3 mice). m, Confocal images of coronal sections from ChAT^IRES-Cre mice. Upper, colocalization of Retrobeads⁺ and calcitonin gene-related peptide immunoreactive (CGRP⁺) somas in the external lPB (elPB) and the external mPB (emPB) (3 mice). Red and white arrows indicate Retrobeads⁺ and CGRP⁺ cells, respectively. Lower, colocalization of Retrobeads⁺, EGFP-labeled ChAT⁺, and CGRP⁺ somas in mPB (2 mice). Yellow, white, and magenta arrows indicate Retrobeads⁺ChAT⁺CGRP⁻, Retrobeads⁺ChAT⁻CGRP⁻, and Retrobeads⁻ChAT⁻CGRP⁺ cells, respectively. n, Percentages of Retrobeads⁺ cells expressing CGRP in mPB and lPB (2 mice). Mean ± SEM.