We developed a dual-adeno-associated-virus expression system that enables strong and sparse labeling of individual neurons with cell-type and projection specificity. We demonstrated its utility for whole-brain reconstruction of midbrain dopamine neurons and striatum-projecting cortical neurons. We further extended the labeling method for rapid reconstruction in cleared thick brain sections and simultaneous dual-color labeling. This labeling system may facilitate the process of generating mesoscale single-neuron projectomes of mammalian brains.
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The data generated in this study are available from the corresponding authors upon request. The AAV vectors of both Cre-FLPo and DreO-vCre versions of our sparse labeling system are available from Addgene (118026–118030).
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We thank J. Liang, F. Yin, N. Li and W. Shi for their assistance with experiments; K. Deisseroth (Stanford University, Stanford, CA, USA) for the pAAV-EF1α-DIO-hChR2(H134R)-mCherry plasmid; and J.H. Snyder for manuscript editing. This work was supported by China MOST (grants 2012YQ03026005, 2013ZX0950910 and 2015BAI08B02 to M.L.; grant 2015CB755602 to H.G.), NNSFC (grant 91432114 to M.L.; grant 91632302 to M.L. and H.G.), the Science Fund for Creative Research Group of China (61721092 to H.G.) and the Beijing Municipal Government (M.L.).
The authors declare no competing interests.
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Integrated supplementary information
Supplementary Figure 1 The dual-AAV sparse labeling system specifically labeled dopamine (DA) neurons in DAT-Cre mice with tunable sparseness.
(a) Injection of sparse labeling viral vectors into the ventral tegmental area (VTA) in a wildtype C57BL/6 mouse brain. (b) The sparse labeling viruses were injected into the VTA of the DAT-Cre mouse. The controller vector was serially diluted. (c) Images of the VTA in DAT-Cre mouse brain injected with a virus mixture containing sparse labeling viruses and AAV-EF1a-DIO-mCherry. The right panel shows the zoom-in view of the boxed region in the left panel. (d) Tunable sparse labeling of DA neurons with GFP was achieved by the dual-AAV sparse labeling system with different titers of the controller vector (VG: viral genomes). The DA neuron population in the VTA was bulk labeled with mCherry. Scale bars, 500 µm (a), 100 µm (c, d).
Supplementary Figure 2 Sparse labeling by the dual-AAV system facilitates the discrimination and reconstruction of single neurons at the whole-brain level.
(a) Sparse labeling viruses were injected into the ventral tegmental area (VTA) in DAT-Cre mice to sparsely label midbrain dopamine (DA) neurons. (b) Images of the VTA in DAT-Cre mouse brain injected with the virus mixture as shown in (a). The right panel shows the 3D rendering of an fMOST image stack overlaid with the reconstruction. The left panel shows the maximum-intensity projection of the boxed stack (480 × 480 × 200 µm3) in the right panel. (c) The serial reconstruction of the local fibers in the boxed region (240 × 240 µm2) in the left panel of (b). (d) Overlay of all 15 completely reconstructed DA neurons registered to a referential brain. The coronal view is shown from the rostral perspective. Scale bars, 100 µm (b), 50 µm (c), and 1,000 µm (d).
Supplementary Figure 3 Example views of the reconstructed individual midbrain dopamine neurons registered in the reference brain.
Neuron IDs are labeled in the top left corner. To show the position of each soma with respect to key brain nuclei, the surface boundaries of the nucleus accumbens (NAc, green), dorsal striatum (DS, yellow), ventral tegmental area (VTA, red), substantia nigra (SN, blue), retrorubral field (RRF, magenta), and central linear nucleus (CLi, cyan) were extracted from the Allen Mouse Common Coordinate Framework version 3 (CCFv3), manually adjusted against PI stained coronal image slices, and are displayed from the dorsal-ventral axial angle (upper right panel). The coronal view of the CCFv3 at specific slices containing the soma are also shown (middle right panel). The red triangles in these panels indicate the location of corresponding soma. MB: midbrain, PH: posterior hypothalamic nucleus, MRN: midbrain reticular nucleus. Scale bar, 1,000 µm.
Supplementary Figure 4 Complete morphological reconstruction of the terminal arborizations allows quantitative analysis and comparison of the target field of individual striatum-projecting dopamine (DA) neurons.
(a) The reconstruction and quantification of the terminal arborizations of the 3 reconstructed DA neurons shown in Fig. 1h. The bottom left panel shows the detailed distribution of the terminal arborizations in the medial shell of the nucleus accumbens (NAcShMed; A: anterior; P: posterior; M: medial; L: lateral). Fiber densities of each arborization were computed over anterior-posterior direction of NAcShMed. The top left panel shows the projection of an fMOST image stack (240 × 240 × 100 µm3) of the boxed region in the bottom left panel. The top right panel demonstrates the serial reconstruction of the arborizations in the boxed region (60 × 60 µm2) in the top left panel. Image stacks of both fMOST GFP signal (upper) or reconstruction overlay (lower), with increasing thickness of 1 µm, 10 µm, 50 µm, and 100 µm are shown. (b) The axon arborizations of 3 neurons shown in (a) have a layered-organization with partial overlapping in NAcShMed. (c, d) The alpha-shapes of DA neurons (c) 1–3 and (d) 5–7. An alpha-shape encompassing the reconstructed points was computed using the MATLAB generic function with the parameter alpha equal to critical alpha, which ensures that the alpha-shape encloses one region. The volume and critical alpha were indicated at the left. (e) The fiber density of DA neurons 5–7 in the dorsal striatum. The surface boundaries of the nucleus accumbens (NAc, green) and dorsal striatum (DS, yellow) were extracted from the Allen Mouse Common Coordinate Framework version 3 (CCFv3), manually adjusted against PI stained coronal image slices, and are displayed from the dorsal-ventral axial angle. Scale bars, 50 µm (a, top left), 10 µm (a, top right), 250 µm (a, bottom left), and 500 µm (e).
Supplementary Figure 5 Example views of the individual reconstructed striatum-projecting intratelencephalic and pyramidal tract neurons registered in the reference brain.
Neuron IDs are labeled in the top left corner. (a) Intratelencephalic. (b) Pyramidal tract. To show the relative position of each soma with respect to key nuclei, the surface boundaries of the nucleus accumbens (NAc, green), dorsal striatum (DS, yellow), anterior cingulate cortex (ACC, yellow), motor cortex (MO, blue), and somatosensory cortex (SS, magenta) were extracted from the Allen Mouse Common Coordinate Framework version 3 (CCFv3), manually adjusted against PI stained coronal image slices, and are displayed from the dorsal-ventral axial angle (upper right panel). Coronal views of the Atlas at specific slices containing the soma are also shown (middle right panel). The red triangle in these panels indicates the corresponding soma location. Scale bar, 1,000 µm.
Supplementary Figure 6 Quantification of fiber densities on striatal projections of individual intratelencephalic neurons over different axes.
The left panel shows the reconstructed axons in the striatum. The right panel shows the quantification of fiber densities. Scale bars, 1 mm.
Supplementary Figure 7 Cell-type-specific sparse labeling of striatal interneurons in PV-Cre and SOM-ires-Cre mice.
(a) Sparse labeling viruses and AAV-EF1a-DIO-mCherry were mixed and injected into the dorsal striatum (DS) in PV-Cre and SOM-ires-Cre mice. (b) Images of the DS in PV-Cre (up) and SOM-ires-Cre (bottom) mouse brain injected with the virus mixture as shown (a). mCherry was expressed only in Cre+ neurons. Scale bars, 100 µm (b upper), 50 µm (b bottom).
(a) PV+ interneurons. (b) SOM+ interneurons. The upper panels show the maximum-intensity projection images of individual neurons. The bottom panels show the corresponding reconstructions. Scale bars, 50 µm.
(a) No significant difference was observed between the total dendritic length of PV+ and SOM+ interneurons (n = 11 for PV+ interneurons; n = 10 for SOM+ interneurons; P value as listed; two-sided unpaired t-test). (b) Striatal PV+ interneurons have a significantly higher number of dendritic branches compared with striatal SOM+ interneurons (n = 11 for PV+ interneurons; n = 10 for SOM+ interneurons; P value as listed; two-sided unpaired t-test). (c) Sholl analysis reveals more compact dendritic fields of striatal PV+ interneurons compared with striatal SOM+ interneurons. The error bars indicate s.e.m.
Supplementary Figure 10 The orthogonality of the DreO-vCre version and the Cre-FLPo version of the dual-AAV sparse labeling system.
(a) The Cre-FLPo version viruses and AAV-CaMKIIa-DreO were mixed and injected into the anterior cingulate cortex (ACC) in DAT-Cre mouse brain. The DreO-vCre version viruses were injected into the ventral tegmental area (VTA) in DAT-Cre mouse brain. (b) No neurons were labeled in either the ACC or the VTA of DAT-Cre mouse brains injected with the virus mixtures as shown in (a). Scale bars, 200 µm.
Supplementary Figures 1–10, Supplementary Tables 2 and 3
Axonal projections of individual reconstructed neurons
Illustration of whole-brain single-neuron reconstruction pipeline. Illustrative video showing the workflow of the single-neuron reconstruction pipeline, related to Fig. 1e. The pipeline includes virus injection, embedding, fMOST imaging, reconstruction and registration. The brain sample shown in this video (0–27s) is NIBS_DA_1 (refer to Supplementary Table 1 for details), in which five neurons were reconstructed in total. Fifteen neurons reconstructed from three brain samples were registered to a referential brain. In the referential brain, the boundaries of the nucleus accumbens (NAc; green), dorsal striatum (DS; yellow), ventral tegmental area (VTA; red), substantia nigra (SN; blue), retrorubral field (RRF; magenta), and central linear nucleus (CLi; cyan) were defined with respect to the Allen Reference Atlas (ARA) and CCFv3.
Highlighting three reconstructed midbrain dopamine neurons projecting to the medial shell of the nucleus accumbens (NAcShMed). Three dopamine neurons reconstructed from brain sample NIBS_DA_1 are highlighted, related to Fig. 1h. The terminal arborizations were unambiguously reconstructed, as is shown in the sliding image stack. The size of the image stack is 750 × 750 × 300 μm3.
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Lin, R., Wang, R., Yuan, J. et al. Cell-type-specific and projection-specific brain-wide reconstruction of single neurons. Nat Methods 15, 1033–1036 (2018). https://doi.org/10.1038/s41592-018-0184-y
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