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, 28 (6), 709-719

Three-Dimensional Analysis of Mouse Habenula Subnuclei Reveals Reduced Volume and Gene Expression in the Lipopolysaccharide-mediated Depression Model

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Three-Dimensional Analysis of Mouse Habenula Subnuclei Reveals Reduced Volume and Gene Expression in the Lipopolysaccharide-mediated Depression Model

Esther Yang et al. Exp Neurobiol.

Abstract

The habenula (Hb) is small but important brain structure, anatomically and functionally links the forebrain with the midbrain to modulate various neuropsychiatric functions associated with drug addiction and emotion-associated dysfunctions. Several reports suggested that the dysfunction of Hb-related functions affected the Hb structurally and functionally. However, the technical limitation has awaited the solid conclusion of whether Hb change due to depression is likely to occur in certain subnuclei of the Hb. To probe this possibility, we developed 3-dimensional reconstruction methods for the high-resolution volumetric analysis of Hb and the mRNA levels at the given volume in normal or lipopolysaccharide (LPS)-mediated mouse model of depression. Notably, we discovered that the volume reduction was prominent in medial Hb but not in lateral Hb after LPS treatments. On the other hand, the RNA expression levels of known Hb regional markers such as Tac1 (dorsal part of medial Hb), ChAT (ventral part of medial Hb), and Tacr1 (medial and lateral Hb) were all decreased in all Hb subnuclei in LPS-injected mice. Accordingly, accurate volumetry with marker labeling was not feasible. Collectively, these established 3D analyses of mouse Hb successfully and precisely determine the volume-based changes of small brain structure, which should be applicable in a wider range of mouse models or pathological specimens.

Keywords: 3-dimensional structure; Depression; Gene expression; Habenula.

Figures

Fig. 1
Fig. 1
Data acquisition and 3D reconstruction of mouse habenula. (A) Brain was removed from the head and cut into a 2-mm thick coronal section (line 5 and 7) including the habenula (Hb) using coronal mouse brain matrix at 1-mm intervals. Dissected brain was processed with 3 hours of ACT (before and after ACT shown) and nuclear staining (SYTO16) was performed. (B) The assessment of volume and shape in the Hb: Brain samples were processed following the steps depicted in serial image acquisition and 3D segmentation to obtain 3D volumetric models of the Hb. (C) Dorsal view of representative volumetric reconstructions of the Hb (purple, MHb; yellow, LHb) with rostral to the top (caudal).
Fig. 2
Fig. 2
Volume comparison of left and right habenula. Left and right volume differences for MHb (A) and LHb (B) were not statistically significant for both PBS- and LPS-injected mice. Dots represent values of individual mice and vertical lines indicate SEM (PBS, n=5, LPS, n=5 mice).
Fig. 3
Fig. 3
Quality assessment of LPS-induced mouse model at 10 days post-LPS peritoneal injection. (A) Timeline of the experimental design. Eight-week-old mice were injected with LPS or PBS; then behavior tests were done at day 10 post injection. The animals were sacrificed immediately after behavior test for volumetric analysis. (B) Volumetric data does not correlate with body weight, brain weight or habenula size. (C) Duration of immobility was measured during the last 4 min of the 6-min test session. Dots represent values of individual mice and vertical lines indicate SEM (PBS, n=8, LPS, n=7 mice; **p<0.01).
Fig. 4
Fig. 4
Volume measurements of habenula. Following 3D reconstruction, volumes of whole (A), medial (B, MHb), and lateral habenula (C, LHb) were measured in adult mice injected with PBS and LPS. MHb (right and left) volumes are significantly larger in control mice than in LPS-treated mice (D). Volumes of LHb are not significantly different between control and LPS-treated mice (E). Dots represent values of individual mice and vertical lines indicate SEM (PBS, n=5, LPS, n=5 mice; **p<0.01).
Fig. 5
Fig. 5
3D reconstruction and shape analysis of MHb. (A) Schematic definition of length (“L”) from rostral to caudal end, width (“W”), height (“H”) after surface cut at the center. The measured lengths in the left MHb (B) and right MHb (C) were reduced in the LPS model compared with control mice. (D) MHb and LHb subdivisions were rostral, intermediate, and caudal as shown in separate colors: purple, rostral; yellow, intermediate; red, caudal. (E and F) There are no volume differences seen for the rostral and middle parts between the control and LPS groups. The volumes of the caudal region in LPS-treated mice are smaller than those of the control mice. Dots represent values of individual mice and vertical lines indicate SEM (PBS, n=5, LPS, n=5 mice; *p<0.05).
Fig. 6
Fig. 6
Distribution and mRNA expression patterns of Tac1, ChAT, and Tacr1. (A) ISH images were acquired by serial sections (14 μm thick). The total number of sections were 88~90 and 75~80 in PBS and LPS groups, respectively. The images of the serial sections were stacked using the Image J program. Stacked images were reconstructed using the Amira program. (B) Each gene is expressed in a region-specific pattern; Tac and ChAT were enriched in dorsal and ventral portions of MHb, respectively, and Tacr1 was expressed in MHb and LHb. (C) For quantitative analysis, mRNA dot counting was performed on 10× magnified photographs.
Fig. 7
Fig. 7
3-D RNAscope analysis of Tac1, ChAT, and Tacr1 mRNA expression from in situ hybridization assays. Quantification of each mRNA dot for (A) Tac1 and ChAT, (B) Tacr1. (C~F) Quantification of regional mRNA dot changes for each gene in rostral, intermediate, and caudal regions. Dots represent values of individual mice and vertical lines indicate SEM (PBS, n=3, LPS, n=3 mice; *p<0.05, **p<0.01, ***p<0.001).

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