Opioid receptors are G protein-coupled receptors (GPCRs) that modulate brain function at all levels of neural integration, including autonomic, sensory, emotional and cognitive processing. Mu (MOR) and delta (DOR) opioid receptors functionally interact in vivo, but whether interactions occur at circuitry, cellular or molecular levels remains unsolved. To challenge the hypothesis of MOR/DOR heteromerization in the brain, we generated redMOR/greenDOR double knock-in mice and report dual receptor mapping throughout the nervous system. Data are organized as an interactive database offering an opioid receptor atlas with concomitant MOR/DOR visualization at subcellular resolution, accessible online. We also provide co-immunoprecipitation-based evidence for receptor heteromerization in these mice. In the forebrain, MOR and DOR are mainly detected in separate neurons, suggesting system-level interactions in high-order processing. In contrast, neuronal co-localization is detected in subcortical networks essential for survival involved in eating and sexual behaviors or perception and response to aversive stimuli. In addition, potential MOR/DOR intracellular interactions within the nociceptive pathway offer novel therapeutic perspectives.Electronic supplementary materialThe online version of this article (doi:10.1007/s00429-014-0717-9) contains supplementary material, which is available to authorized users.

Spermatogenesis constitutes a remarkable program of cell differentiation, which involves dramatic changes in cell morphology, biochemistry and gene expression 1,2 . But the study of male germ cells is complicated by the exceptional organization of the seminiferous epithelium and by the lack of established cell lines that are able to recapitulate any of the multiple differentiation steps of the spermatogenesis program in vitro. Cell types with differences in their sedimentation properties or cell surface markers can be isolated from the whole tissue by various methods, but these methods do not allow accurate identification of all the differentiation stages. As a consequence of strict paracrine regulation by Sertoli cells, spermatogenesis proceeds in synchronized waves along the seminiferous tubules, and every given cross-section of the tubule contains only certain cell types in a specific combination (Fig. 1). The light absorption pattern of a seminiferous tubule, as seen under a dissection microscope, correlates with defined stages of the spermatogenic wave, which makes it possible to isolate specific stages on the basis of their transillumination properties 3-6 . The accuracy of the isolation of specific stages can be improved by combining it with phase-contrast microscopy of live cell preparations 7-10 . The staging of the spermatogenic cycle is best characterized in rat and mouse 11,12 . Here, we describe a method designed to identify, isolate and characterize mouse male germ cells at specific steps of differentiation by transillumination-assisted microdissection. The distinctive morphological features of germ cells, as detected by phase-contrast microscopy, are detailed. Although the method described here generates small quantities of cells and therefore does not allow for further isolation of the different cell types comprising each differentiation stage, the applications of this method are powerful and diverse. It enables the monitoring of spermatogenic differentiation events, and in combination with biochemical analyses, it allows the characterization of the molecular mechanisms governing these processes. This method can be used for the identification of the effects of cytotoxic and environmental factors on male reproductive function, as well as for the rapid and comprehensive diagnosis and characterization of sperm cell defects attributable to infertility. In combination with gene targeting models, it enables the characterization of the critical factors involved in the cell cycle, chromatin dynamics, spermatid differentiation, stem cell biology and fertility.