ArticleHistochemistry and cell biology2012
Double and triple in situ hybridization for coexpression studies: combined fluorescent and chromogenic detection of neuropeptide Y (NPY) and serotonin receptor subtype mRNAs expressed at different abundance levels.
Article in Histochemistry and cell biology, 2012. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 10 papers.
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Who cites it
10 citing papers in PubMed, 12 citations in OpenAlex.
- The dorsal arcopallium of chicks displays the expression of orthologs of mammalian fear related serotonin receptor subfamily genes.Scientific reports · 2020Article
- ModifiedFrontiers in molecular neuroscience · 2020Article
- Sensing Glucose in the Central Melanocortin Circuits of Rainbow Trout: A Morphological Study.Frontiers in endocrinology · 2019Article
- A PCR-Based Method for RNA Probes and Applications in Neuroscience.Frontiers in neuroscience · 2018Article
- The basolateral amygdala γ-aminobutyric acidergic system in health and disease.Journal of neuroscience research · 2016Review
- Neuronal localization of the 5-HT2 receptor family in the amygdaloid complex.Frontiers in pharmacology · 2014Review
- The Histochemistry and Cell Biology compendium: a review of 2012.Histochemistry and cell biology · 2013Review
- Serotonergic innervation of the amygdala: targets, receptors, and implications for stress and anxiety.Histochemistry and cell biology · 2013Review
- Serotonergic innervation and serotonin receptor expression of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei.Brain structure & function · 2013Article
- GABA concentration and GABAergic neuron populations in limbic areas are differentially altered by brain serotonin deficiency in Tph2 knockout mice.Histochemistry and cell biology · 2013Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Multiple fluorescence in situ hybridization is the method of choice for studies aimed at determining simultaneous production of signal transduction molecules and neuromodulators in neurons. In our analyses of the monoamine receptor mRNA expression of peptidergic neurons in the rat telencephalon, double tyramide-signal-amplified fluorescence in situ hybridization delivered satisfactory results for coexpression analysis of neuropeptide Y (NPY) and serotonin receptor 2C (5-HT2C) mRNA, a receptor subtype expressed at high-to-moderate abundance in the regions analyzed. However, expression of 5-HT1A mRNA, which is expressed at comparatively low abundance in many telencephalic areas, could not be unequivocally identified in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in fluorescent receptor mRNA detections. Parallel chromogenic in situ hybridization provided clear labeling for 5-HT1A mRNA and additionally offered the possibility to monitor the chromogen deposition at regular time intervals to determine the optimal signal-to-noise ratio. We first developed a double labeling protocol combining fluorescence and chromogenic in situ hybridization and subsequently expanded this variation to combine double fluorescence and chromogenic in situ hybridization for triple labelings. With this method, we documented expression of 5-HT2C and/or 5-HT1A in subpopulations of telencephalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and fluorescence microscopy, combines advantages of fluorescence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to previously published multiple labeling methods for coexpression analyses in which one mRNA species requires highly sensitive detection.
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