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The output NIFTI files of the co-registered ×4 brain scans were uploaded into the Arivis Vision 4D software (V.3.3.0). Individual neurons were identified using the Blob Finder feature in the software and their xyz coordinates were extracted. A Python script was used to visualize the 3D scatterplots for each neuronal group, where the quality of registration was checked once again. For the coronal view of the 3D distribution, the 3D scatterplots of three mice (per group) were plotted and a coronal snapshot was created from rostral locations within the ARC comparable to that of the two-dimensional (2D) coronal distribution. The coordinates of the 2D coronal distributions were also extracted using the Arivis software from the Halo output files (‘Imaging and quantification of RNA ISH’) that had been previously aligned manually. The final distribution graph was plotted using the Prism software.

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