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. 2021 Nov 5;7(45):eabj2225. doi: 10.1126/sciadv.abj2225

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Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution License 4.0 (CC BY).

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Fig. 3. — (A) Summary of MAPseq procedure. (B) Percentage of barcodes detected in the mPFC also detected in brain sites listed on y axis. Data were collected from n = 2 mice. (C) Heatmap representation of mPFC barcodes (% total number) detected in brain regions shown on y axis and in regions shown on x axis. (D) Histogram of connectome of mPFC_➔PVT, mPFC_➔VTA, and mPFC_➔LHb neurons. Shown is the distribution of barcodes (%) for mPFC cells that project to the PVT, VTA, and LHb. (E) Summary of injection procedure to label cortical-habenular neurons that project to VTA and fluorescence image of mPFC from mice injected with CTb retrograde tracers in the LHb and VTA. Shown are CTb-594 (red; mPFC_➔LHb neurons)–labeled and CTb-488 (green; mPFC_➔VTA neurons)–labeled cells. White arrows identify dual-labeled cells (yellow; mPFC_➔LHb^VTA neurons) in the mPFC. A total of n = 5 mice were imaged. (F) Summary of injection procedure to label cortical-habenular neurons that project to the LC and associated fluorescence image of mPFC from injected mice (red; mPFC_➔LHb neurons) and GFP (green; mPFC_➔VTA neurons). White arrows identify dual-labeled cells (yellow; mPFC_➔LHb^LC neurons) in the mPFC. A total of n = 5 mice were imaged. (G) Injection procedure (the tracing the relationship between input and output method, also known as the TRIO method) to label cortico-habenular neurons that project to VTA. Shown are mCherry⁺ cells in the mPFC, which are cortico-habenular neurons that synapse into LHb neurons that project to VTA. A total of n = 4 mice were imaged. (H) Injection procedure to label cortico-habenular neurons that project to the LC. mCherry⁺ cells were detected in the mPFC (right), which are cortico-habenular neurons that synapse into LHb neurons that project to the LC. A total of n = 3 mice were imaged. (I) Graphical representation of the cortico-habenular connectome. A population of mPFC neurons (shown in blue) projects concurrently to the LHb and then to the same monoaminergic brain centers to which LHb neurons also project (shown in red).