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

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.

Serotonergic innervation and serotonin receptor expression of NPY-producing neurons in the rat lateral and basolateral amygdaloid nuclei

“…Serial coronal sections (10 μm) were cut on a cryostat set at −25 °C and thaw-mounted on slides. Subsequently, sections were fixed in freshly prepared 4 % FA in 0.01 M PBS, transferred to 100 % ethanol and stored at 4 °C as described previously (Bonn et al 2012). If not mentioned otherwise, the following steps were carried out at room temperature.…”

“…Generation of cRNA probes specific for rat NPY (GenBank accession: NM_012614), rat 5-HT1A (GenBank accession: NM_012585.1) and rat 5-HT2C mRNA (GenBank accession: NM_012765.3) was performed as described by Bonn et al (2012). A cDNA fragment of mouse 5-HT3 mRNA (GenBank accession: NM_013561.2; 92 % homology to the corresponding rat sequence) was cloned into pGEM-T vector (Promega, Mannheim, Germany) and further processed for generation of antisense and sense cRNA probes as previously described (Bonn et al 2012).…”

“…A cDNA fragment of mouse 5-HT3 mRNA (GenBank accession: NM_013561.2; 92 % homology to the corresponding rat sequence) was cloned into pGEM-T vector (Promega, Mannheim, Germany) and further processed for generation of antisense and sense cRNA probes as previously described (Bonn et al 2012). …”

“…For triple labelings, NPY cRNA was reacted using biotin-RNA labeling mix, 5-HT2C cRNA with fluorescein-RNA labeling mix and 5-HT1A cRNA with DIG-RNA labeling mix. Rehydration in a graded series of ethanol, acetylation and pre-hybridization were done according to Bonn et al (2012). Sections were covered with hybridization solution containing one, two or three differently labeled cRNA probes of interest.…”

“…Sections were covered with hybridization solution containing one, two or three differently labeled cRNA probes of interest. Hybridization to nucleic acid targets was carried out for 16–20 h at 57–58 °C followed by post-hybridization washes as described by Bonn et al (2012). Subsequently, tissue was treated with blocking buffer according to the manufacturer’s instruction (Perkin Elmer, Rodgau, Germany).…”