Histochem Cell Biol (2012) 137:11–24 DOI 10.1007/s00418-011-0882-3 O R I G I N A L P A P E R 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 Maria Bonn • Angelika Schmitt • Esther Asan Accepted: 25 October 2011 / Published online: 11 November 2011 The Author(s) 2011. This article is published with open access at Springerlink.com 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 sat- isfactory 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 abun- dance in many telencephalic areas, could not be unequiv- ocally identiﬁed in NPY mRNA-reactive neurons due to high background and poor signal-to-noise ratio in ﬂuores- cent 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 ﬂuores- cence and chromogenic in situ hybridization and subse- quently expanded this variation to combine double ﬂuorescence and chromogenic in situ hybridization for tri- ple labelings. With this method, we documented expression M. Bonn E. Asan (&) Institute of Anatomy and Cell Biology, University of Wuerzburg, Koellikerstr. 6, 97070 Wuerzburg, Germany e-mail: esther.asan@mail.uni-wuerzburg.de A. Schmitt Department of Psychiatry, Psychosomatics and Psychotherapy, University of Wuerzburg, Fuechsleinstr. 15, 97080 Wuerzburg, Germany of 5-HT2C and/or 5-HT1A in subpopulations of telence- phalic NPY-producing neurons. The method developed in the present study appears suitable for conventional light and ﬂuorescence microscopy, combines advantages of ﬂuores- cence and chromogenic in situ hybridization protocols and thus provides a reliable non-radioactive alternative to pre- viously published multiple labeling methods for coexpres- sion analyses in which one mRNA species requires highly sensitive detection. Keywords Triple in situ hybridization NPY 5-HT1A 5-HT2C Coexpression Introduction Functional properties of neurons in the context of speciﬁc neuronal circuitries are largely determined by their pro- duction of particular neuromediators on the one hand, and of speciﬁc receptors for neurotransmitters from afferent systems on the other hand. Within the entire brain there are numerous subsets of neurons showing differential produc- tion of neuropeptides, which have been implicated in var- ious functions particularly in limbic telencephalic areas (Asan 1998; Muller et al. 2003; Karson et al. 2009; Criado et al. 2011). Morphological studies have indicated that monoaminergic afferents contact subsets of peptidergic neurons in limbic and other cortical and subcortical areas of the rat brain (Asan 1997, 1998; Eliava et al. 2003; Yilmazer-Hanke et al. 2004; Asan et al. 2005). Determi- nation of monoamine receptor expression of target neurons is a necessary prerequisite for further studies assessing the functional impact of this innervation. Multiple immunola- beling or combined immunolabeling/in situ hybridization (ISH), which are methods of choice for these analyses since 123