Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP

Katayama, Yukitoshi; Saito, A; Ogoshi, Maho; Tsuneoka, Yousuke; Mukuda, Takao; Azuma, Morio; Kusakabe, Makoto; Takei, Yoshio; Tsukada, Takehiro

doi:10.1007/s00441-022-03596-y

“…ISH procedures were similar to those previously described with some modifications (29, 67) The free-floating sections were washed with PBS containing 0.2% Triton (PBST) and immersed in methanol for 10 min, followed by PBST washing for 5 min twice. After washing, the sections were prehybridized for 5 min at 37 °C in a hybridization buffer containing 10% dextran sulfate, 0.5× SSC, 0.1% Tween 20, 50 μg/ml heparin, and 1× Denhardt’s solution.…”

Section: Methodsmentioning

confidence: 99%

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

Tsuneoka,

Funato

2023

Preprint

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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.

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“…However, additional hybridization steps are required for every round of ISH. We designed 12 sets of short hairpin DNAs, including those in a previous report (Tsuneoka and Funato, 2020;Katayama et al, 2022). Therefore, four rounds of ISH using three colors in each round should be applicable without additional probe hybridization.…”

Section: Discussionmentioning

confidence: 99%

“…ISH procedures were similar to those previously described with some modifications (Tsuneoka and Funato, 2020;Katayama et al, 2022). The sections were washed with PBS containing 0.2% Triton (PBST) and immersed in methanol for 10 min, followed by PBST washing for 5 min twice.…”

Section: Probe Hybridizationmentioning

confidence: 99%

Fluorescence quenching by high-power LEDs for highly sensitive fluorescence in situ hybridization

Tsuneoka

¹

,

Atsumi

²

,

Makanae

³

et al. 2022

Front. Mol. Neurosci.

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Recent technical advances have made fluorescent in situ hybridization (ISH) a pivotal method to analyze neural tissue. In a highly sensitive ISH, it is important to reduce tissue autofluorescence. We developed a photobleaching device using a light-emitting diode (LED) illuminator to quench autofluorescence in neural tissue. This device was equipped with 12 high-power LEDs (30 W per single LED) and an evaporative cooling system, and these features achieved highly efficient bleaching of autofluorescence and minimized tissue damage. Even after 60 min of photobleaching with evaporative cooling, the temperature gain of the tissue slide was suppressed almost completely. The autofluorescence of lipofuscin-like granules completely disappeared after 60 min of photobleaching, as did other background autofluorescence observed in the mouse cortex and hippocampus. In combination with the recently developed fluorescent ISH method using the hybridization chain reaction (HCR), high signal/noise ratio imaging was achieved without reduction of ISH sensitivity to visualize rare mRNA at single copy resolution by quenching autofluorescence. Photobleaching by the LED illuminator was also effective in quenching the fluorescent staining of ISH-HCR. We performed multiround ISH by repeating the cycle of HCR staining, confocal imaging, and photobleaching. In addition to the two-round ISH, fluorescent immunohistochemistry or fluorescent Nissl staining was conducted on the same tissue. This LED illuminator provides a quick and simple way to reduce autofluorescence and quench fluorescent dyes for multiround ISH with minimum tissue degradation.

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“…ISH procedures were similar to those previously described with some modifications ( Tsuneoka and Funato, 2020 ; Katayama et al, 2022 ). The free-floating sections were washed with PBS containing 0.2% Triton (PBST) and immersed in methanol for 10 min, followed by PBST washing for 5 min twice.…”

Section: Methodsmentioning

confidence: 99%

Whole Brain Mapping of Orexin Receptor mRNA Expression Visualized by Branched In Situ Hybridization Chain Reaction

Tsuneoka,

Funato

2024

eNeuro

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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 branchedin situhybridization 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 bothOx1randOx2rwere observed in specific brain regions, such as the dorsal raphe nucleus and ventromedial hypothalamic nucleus. In many brain regions,Ox1r-expressing cells andOx2r-expressing cells belong to different cell types, such as glutamatergic and GABAergic neurons. Moreover, our findings demonstrated considerable heterogeneity inOx1r- orOx2r-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.Significance statementThe neuropeptide orexin regulates sleep and other behaviors through its receptors, OX1R and OX2R, which are targets for the development of therapeutic agents for sleep and related disorders. However, the cellular distribution of orexin receptors in the brain is only partially known. We applied a newly developed branchedin situhybridization chain reaction (bHCR) technique and conducted a whole-brain mapping of orexin receptor mRNA expression in the brain with neuron subtype markers. Few cells expressed both OX1R and OX2R, and OX1R and OX2R were expressed in the different neuronal subtypes in many brain regions. This study fills an important gap in understanding and modulating the orexin system.

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Gene duplication of C-type natriuretic peptide-4 (CNP4) in teleost lineage elicits subfunctionalization of ancestral CNP

Cited by 7 publications

References 44 publications

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

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

Fluorescence quenching by high-power LEDs for highly sensitive fluorescence in situ hybridization

Whole Brain Mapping of Orexin Receptor mRNA Expression Visualized by Branched In Situ Hybridization Chain Reaction

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