Whole brain mapping of orexin receptor mRNA expression visualized by branchedin situhybridization chain reaction

Abstract

Orexins, which are produced within neurons of the lateral hypothalamic area, play a pivotal role in the regulation of various behaviors, including sleep/wakefulness, reward behavior, and energy metabolism, via orexin receptor type 1 (OX1R) and type 2 (OX2R). Despite the advanced understanding of orexinergic regulation of behavior at the circuit level, the precise distribution of orexin receptors in the brain remains unknown. Here, we develop a new branched in situ hybridization chain reaction (bHCR) technique to visualize multiple target mRNAs in a semiquantitative manner, combined with immunohistochemistry, which provided comprehensive distribution of orexin receptor mRNA and neuron subtypes expressing orexin receptors in mouse brains. Only a limited number of cells expressing both Ox1r and Ox2r were observed in specific brain regions, such as the dorsal raphe nucleus and ventromedial hypothalamic nucleus. In many brain regions, Ox1r-expressing cells and Ox2r-expressing cells belong to different cell types, such as glutamatergic and GABAergic neurons. Moreover, our findings demonstrated considerable heterogeneity in Ox1r- or Ox2r-expressing populations of serotonergic, dopaminergic, noradrenergic, cholinergic, and histaminergic neurons. The majority of orexin neurons did not express orexin receptors. This study provides valuable insights into the mechanism underlying the physiological and behavioral regulation mediated by the orexin system, as well as the development of therapeutic agents targeting orexin receptors.

Spatial mRNA expression patterns of orexin receptors in the dorsal hippocampus

“…Our analysis approach of counting the fraction of positive cells for OXR1 and OXR2 mRNA in a larger cohort of mice allows for a semiquantitative comparison of OXR1 vs. OXR2 levels on specific target regions of the brain related to memory functions. Cells expressing both receptor subtypes simultaneously were rarely observed, in agreement with the results of the brain wide analysis of OXR1 and OXR2 mRNA, demonstrating a co-expression of OXR1 and OXR2 only in the paraventricular thalamus, the ventromedial nucleus of the hypothalamus and the dorsal raphe nucleus of the brain stem 25 .…”

“…In the mPFC, we observed a significant higher expression of OXR2 compared to OXR1 mRNA throughout the Cg, PL and IL subregions. While these regions are not analyzed in detail in the recent study using a high-sensitivity in situ hybridization approach, representative images suggest a similar pattern for OXR1 and OXR2 in the mPFC subregions 25 . A moderate expression of OXR1 in the mPFC subareas of mice is confirmed with OXR1-GFP mice 28 .…”

“…In the rat brain, low expression levels are reported for both OXR1 and OXR2 mRNA in the NRe 22 , similar to our data for the mouse brain. Tsuneoka & Funato 25 indicate low expression of OXR2 mRNA in this area in their comprehensive analysis of the mouse brain, but no expression of OXR1 mRNA in the NRe beyond background levels. However, upon inspection of the representative images few cells positive for OXR1 are visible dependent on the plane analyzed, suggesting that under low expression conditions differences may arise dependent on the image analysis method (plane and number of slices analyzed, segmentation method).…”

“…Using both techniques, the number of punctae usually detected within a single cell is overall low (1-5 punctae in most cells), reflecting a low mRNA content within the cells (Fig. 1+2, 25 ). While we did not detect a significant circadian variability in the expression patterns described by the fraction of cells positive for OXR1 and OXR2 mRNA, a circadian modulation may rather be evident in the amount of OXR1 or OXR2 mRNA produced within a cell.…”

“…High sensitivity in situ approaches like RNAScope or the branched in situ hybridization chain reaction (bHCR) technique utilized by Tsuneoka & Funato 25 enable the detection of few mRNA molecules and the number of punctae detected in a cell corresponds to a certain extend to the amount of mRNA within a cell. Using both techniques, the number of punctae usually detected within a single cell is overall low (1-5 punctae in most cells), reflecting a low mRNA content within the cells (Fig.…”