22 controlled without any risk of development of unspeciﬁc reaction products, and the development can be terminated as soon as the optimal signal-to-noise ratio is achieved. Therefore, the working protocol is straightforward, com- paratively simple and reproducible, and can be easily adapted to simultaneous detection of other mRNAs of interest. Additionally, the high and reproducible detection sensitivity in double labelings enables semi-quantitative evaluation of coexpression incidence. Thus, in our experi- ments, semi-quantitative evaluation of the relative number of NPY neurons coexpressing 5-HT1A or 5-HT2C mRNA among all NPY neurons in the different brain areas appears possible. For triple labelings, the protocol takes only about 1 h longer than HS-TSA-FISH/CISH double labelings and no additional IHC procedure or radioactive isotopes are required. Another positive aspect of the protocol is the fact that tissue can be inspected by conventional light and ﬂuorescence microscopy, and thus by eye. Besides allowing for intermittent monitoring of the procedure after all steps, visual examination ensures brief UV and visible light exposition times, thus reducing bleaching and unspeciﬁc chromogen deposition. Moreover, problems of unmixing of autoﬂuorescence and of different ﬂuorescence excitation spectra occurring when multiple ﬂuorophores are applied and analyzed using confocal laser systems (e.g., quantum dots and organic ﬂuorophores; Chan et al. 2005), are avoi- ded. However, before double and triple ISH experiments are performed, some important caveats have to be observed to provide optimal results using this method. Figure 6 shows a concept for planning optimal experimental design. Thus, coexpression studies should be preceded and accompanied by comparative single HS-TSA-FISH and CISH. These experiments serve to establish receptor mRNA expression levels and detectability using different probes and detection protocols as described for the probes used in the present experiments and are a necessary prerequisite for choosing appropriate ISH protocols for multiple labeling. If applica- ble, double FISH is preferable to FISH/CISH combinations since it presents even less pitfalls. For instance, limited stability of the NBT/BCIP under UV light in combination with a ﬂuorescence mounting medium can complicate analysis and documentation. Additionally, semi-quantita- tive analyses of multiple ISH results can only be carried out if the detection sensitivity of mRNA species to be analyzed is comparable in single and multiple labelings. Thus, since in our triple labelings NPY mRNA detection obviously did not detect all NPY-producing neurons, only qualitative analyses should be carried out on these preparations. Also, since each cRNA probe requires a different range of hybridization temperature, multiple labelings yielding identical sensitivity for each probe compared to corre- sponding single labelings might sometimes not be achiev- able. Then, only qualitative evaluation should be done. 123 Histochem Cell Biol (2012) 137:11–24 However, if these caveats are considered, the described method provides a signiﬁcant technical improvement for investigations not only into the receptor complement of identiﬁed neuronal subtypes, but also into coexpression of proteins expressed at different abundance levels in other tissues. Functional implications Sensory and limbic cortical and subcortical regions of the rat brain are innervated by serotonergic afferents arising from the dorsal and median raphe nuclei, which inﬂuence projection cells and interneurons via synaptic contacts and in a paracrine mode (e.g., Beaudet and Descarries 1976; Papadopoulos et al. 1987; Jacobs and Azmitia 1992; Vertes et al. 1999; Michelsen et al. 2007; Muller et al. 2007). Serotonergic signal transduction, particularly via the 5-HT1A and 5-HT2C receptor subtypes, has been shown to be relevant for processing of sensory and emotionally rel- evant stimuli, and imbalances in these systems are thought to be involved in the etiopathology of affective disorders such as depression and anxiety (Lowry et al. 2005; Ogren et al. 2008; Iwamoto et al. 2009). Via activation of the two receptor subtypes, serotonin exerts differential inﬂuence on the target neuron. 5-HT1A activation has been reported to produce cell hyperpolarization due to the opening of G protein-gated inwardly rectifying potassium channels (Luscher and Slesinger 2010). In contrast, 5-HT2C acti- vation facilitates cell depolarization by inhibition of spe- ciﬁc potassium channels promoting potassium outﬂow (Weber et al. 2008). Previous studies have documented expression of 5-HT1A and 5-HT2C in pyramidal cells and GABAergic interneurons in limbic and other cortical areas of the rat brain (Aznar et al. 2003; Amargo´s-Bosch et al. 2004; Puig et al. 2010). Coexpression of both receptor subtypes has to date only been indicated for cortical pyramidal cells (Yuen et al. 2008). The present ﬁndings documenting the expression of 5-HT1A and 5-HT2C mRNA in subpopulations of cortical and subcortical NPY- producing neurons provide the ﬁrst morphological evidence for a functional link between serotonergic and NPY sys- tems. Additionally, our ﬁrst triple ISH experiments supply proof that some NPY mRNA-producing cortical interneu- rons coexpress both 5-HTR. Since NPY neurons in cortical areas represent a small subpopulation of GABAergic interneurons, the results complement previous studies and show that, like pyramidal cells, interneurons may express multiple 5-HTR subtypes. The effects of serotonin on NPY neurons producing one or both 5-HTR may depend on extracellular serotonin concentration and sensitization/ desensitization of these receptors. It also appears possible that NPY neurons bearing different 5-HTR might be innervated by different subsets of serotonergic afferents