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

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 volumebased changes of small brain structure, which should be applicable in a wider range of mouse models or pathological specimens.

Three‐dimensional multi‐gene expression maps reveal cell fate changes associated with laterality reversal of zebrafish habenula

“…To echo our study needs and the facts that 3D gene expression atlases are used progressively more to uncover new features of gene expression patterns (Akiyama-Oda & Oda, 2016;Keranen et al, 2006;Luengo Hendriks et al, 2006;Yang et al, 2019), a method that allows simultaneously quantifying and visualizing multiple gene expressions in developing HA has been developed in this study. Comparing to the sample processing procedures that detected multiple transcripts in whole-mount zebrafish embryos utilizing techniques involving the use of specific fluorophore-labeled RNA hairpins or specifically designed short probes containing specific pre-amplifier-interacting tails for each gene of interests (Choi et al, 2010;Gross-Thebing et al, 2014), our probe detecting procedures could easily be adopted by most laboratories utilizing standard probe generation and detection protocol.…”

Three Dimensional Multi-gene Expression Maps Reveal Cell Fate Changes Associated with Laterality Reversal of Zebrafish Habenula

“…To echo our study needs and the facts that 3D gene expression atlases are used progressively more to uncover new features of gene expression patterns (Akiyama-Oda and Oda, 2016;Keranen et al, 2006;Luengo Hendriks et al, 2006;Yang et al, 2019), a method that allows simultaneously quantifying and visualizing multiple gene expressions in developing HA has been developed in this study. Comparing to the sample processing procedures that detected multiple transcripts in whole-mount zebrafish embryos utilizing techniques involving the use of specific fluorophore-labeled RNA hairpins or specifically designed short probes containing specific pre-amplifier-interacting tails for each gene of interests (Choi et al, 2010;Gross-Thebing et al, 2014), our probe detecting procedures could easily be adopted by most laboratories utilizing standard probe generation and detection protocol.…”

Down-regulation of habenular calcium-dependent secretion activator 2 induces despair-like behavior

“…The slides were post-fixed in 4% paraformaldehyde (PFA) and dehydrated in ascending concentration of ethanol, then treated with protease. For RNA detection, RNAscope fluorescent Multiplex detection reagents (ACDBio, Newark, CA, US) were used as in previous studies and following the manufacture’s recommendations 54 . Detailed methods and probes used for RNAscope are described in the supplementary information.…”