Whole-brain projectome map of Ventral Tegmental Area neuron types

For Serial Sectioning: 6 brains were sectioned coronally at 30 μm thickness using Leica CM1950, serial sections were collected at a sampling interval of 1:5. Sections were imaged using Olympus VS120 slide scanner (Objectives: UPLSAPO 2 10x N.A. = 0.40, WD = 3.1 mm).Data sample descriptionA total of 12 adult mice (aged 8-10 weeks, female) were utilized. For dual-color tissue clearing, 3 experimental groups (n = 1 per group) are included: TH-FlpO;GAD2-Cre, DAT-FlpO;vGlut2-Cre, and GAD2-FlpO;vGlut2-Cre.

Single-color tissue clearing consists of 3 experimental groups (n = 1 per group): TH-Cre, GAD2-Cre, vGlut2-Cre. Serial Sectioning consists of 3 experimental groups (n = 2 per group): TH-Cre, GAD2-Cre, vGlut2-Cre.Data quality / Technical Validation(1)  Registration Accuracy: Raw 3D images were mapped to the Allen Mouse Brain Common Coordinate Framework version 3 (CCFv3) at 10-µm resolution. Registration was performed using non-rigid automated registration algorithms to precisely correct for nonlinear morphological deformations, supplemented by manual refinement to ensure optimal alignment accuracy in critical brain regions.(2)  Multi-tiered Projection Validation: The projection patterns identified in dual-color PEGASOS-cleared brains from FlpO;Cre double-transgenic mice, which necessitated the parallel use of two distinct recombinase-dependent viruses (expressing eGFP and mCherry), were cross-verified against single-color cleared brains.

These brains were generated across three separate Cre-transgenic lines using an identical Cre-dependent virus expressing eGFP. This specific verification ruled out potential labeling biases or confounding variables arising from the comparison of different viral vectors in the dual-recombinase system. Subsequently, cross-modality validation was performed to assess spatial accuracy.

The morphological integrity and fluorescence distribution observed in the 3D PEGASOS-cleared brains were directly cross-referenced and anatomically confirmed by parallel 2D serial coronal sections.Data volume and data formatThe total volume of this dataset is approximately 355 GB. The 3D volumetric images are stored in .ims format. The CCFv3 registration outputs and transformation fields are available in .tiff format.

The 2D serial slice images are provided in .tiff format.Data usage methods and suggestionsResearchers can use Imaris to view the 3D imaging results and the entire rendered surface of the registered CCFv3 atlas. The Slicer tool within the AllSurface module allows users to view the registered data from the coronal, sagittal and horizontal planes. To render and examine an individual brain region, the Measurement function of the Lit AiBot (Request a trial: sales@lit-cn.com) is required.

The operational workflow is as follows: open the .ims data file, load the corresponding annotation .tiff file into Lit AiBot, execute the Output Brain Tree command, and select the desired brain regions from the tree directory. After rendering, researchers can visualize the rendered individual 3D brain regions in Imaris.