Dissection of the long-range projections of specific neurons at the synaptic level in the whole mouse brain

Abstract

Through synaptic connections, long-range circuits transmit information among neurons and connect different brain regions to form functional motifs and execute specific functions. Tracing the synaptic distribution of specific neurons requires submicron-level resolution information. However, it is a great challenge to map the synaptic terminals completely because these fine structures span multiple regions, even in the whole brain. Here, we develop a pipeline including viral tracing, sample embedding, fluorescent micro-optical sectional tomography, and big data processing. We mapped the whole-brain distribution and architecture of long projections of the parvalbumin neurons in the basal forebrain at the synaptic level. These neurons send massive projections to multiple downstream regions with subregional preference. With three-dimensional reconstruction in the targeted areas, we found that synaptic degeneration was inconsistent with the accumulation of amyloid-β plaques but was preferred in memory-related circuits, such as hippocampal formation and thalamus, but not in most hypothalamic nuclei in 8-month-old mice with five familial Alzheimer's disease mutations. Our pipeline provides a platform for generating a whole-brain atlas of cell-type-specific synaptic terminals in the physiological and pathological brain, which can provide an important resource for the study of the organizational logic of specific neural circuits and the circuitry changes in pathological conditions.

Keywords: Alzheimer's disease; basal forebrain; synapse; synaptophysin; whole brain.

Fig. 1.

Pipeline for the construction of the axonal projections and synaptic terminals of PV⁺ neurons in the MS/VDB. (A) Virus injection and sample treatment process of the embedding method. (B) Whole-brain imaging with the fMOST system at a resolution of 0.2 × 0.2 × 1 μm and data preprocessing. (C) The 3D dataset was reconstructed, analyzed, and registered to the Allen CCFv3 Brain Atlas. (D) Diagram of the virus for axonal projection and synaptic connection. (E) The procedure of virus expression in the MS/VDB. The red signal denotes tdTomato-labeled fibers projected from the MS/VDB brain area. The green signal denotes EGFP-labeled synaptophysin. (F) Fluorescence images of MS/VDB in the PV mouse brain. (Scale bar: 100 μm.) (G and H) Immunohistochemical staining and quantification of PV⁺ neurons in the MS/VDB. (Scale bar: 20 μm.) (I) Horizontal and sagittal views of the whole brain. The red signals represent axon distribution. The green signals represent synaptic distribution. A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral.

Fig. 2.

Visualization of the synaptic terminal distribution in the whole brain with fluorescence micro-optical sectioning tomography. (A) Schematics illustrating the major projection patterns of the PV⁺ neurons in the MS/VDB. (B) The PV⁺ neurons in the BF projected to special positions of the RSP, MM, and HIP. (Scale bar: 200 μm.) (C and D) Three-dimensional reconstruction of the RSP (blue dots), MM (magenta dots), and HIP (red dots). (E and F) HIP divided into CA1 (magenta dots), CA2 (yellow dots), CA3 (blue dots), and the DG (red dots). (G–I) Enlarged images of CA3 (blue dots), CA2 (yellow dots), and the DG (red dots). po, polymorph layer; slm, stratum lacunosum-moleculare.

Fig. 3.

Quantitative analysis of synaptic terminals in the targeted brain regions. (A) Three-dimensional reconstruction of the whole brain. Different colors represented different nuclei. (B) The data block shows the distribution of GFP⁺ dots in different brain regions from (A). Block size: 100 × 100 × 100 μm. (Scale bar: 20 μm.) (C) The proportion of fluorescence intensity in 34 subregions (intensity, mean gray value per punctum [AU.]) (n = 5). Details of the proportions of fluorescence intensity in HPF subregions. Synapse numbers per unit volume in 34 brain subregions (average number synapse of 1 mm³) (n = 5). A, anterior; D, dorsal; L, lateral; M, medial; P, posterior; V, ventral; BLA, basolateral amygdalar nucleus; BMA, basomedial amygdalar nucleus; CS, superior central nucleus raphe; ECT, ectorhinal area; HY, hypothalamus; IPN, interpeduncular nucleus; LPO, lateral preoptic area; MB, midbrain; OLF, olfactory areas; STR, striatum; PAL, pallidum; TH, thalamus; TT, taenia tecta.

Fig. 4.

Synaptic degeneration of long-range projections in 5×FAD mice. (A) Synaptic distribution in different coronal sections. The red dots represent one synaptic terminal. (B) High-resolution images of the CA1, CA2, CA3, DG, and MM of PV-ires-Cre and 5×FAD; PV-ires-Cre mice. (Scale bar: 500 μm; 25 μm.) (C) Synaptic density comparison between CON and AD mice (n = 4, respectively) (multiple t tests followed by Holm-Sidak comparisons method, *P < 0.05, **P < 0.01, ***P < 0.001). (D) No major loss of PV neurons was observed in the MS/VDB of CON and AD mice. (Scale bar: 200 μm.) (E) Quantitative analysis of the number of PV neurons in the MS/VDB (n = 5, respectively). n.s means no significance. (F) Immunohistochemical staining of Aβ plaques in synaptic terminals brain regions (Scale bar: 50 μm.). ACA, anterior cingulate area; ECT, ectorhinal area; HY, hypothalamus; TH, thalamus.