Purinergic Signalling (2012) 8 (Suppl 1):S61–S79 was used as a positive control, and indeed, P2X7 expression was demonstrated by Western blot analysis with both the intracellular and extracellular antibodies. In addition, immunocytochemistry with anti-P2X7 receptor (extracellu- lar)-FITC demonstrated P2X7 membrane expression in living J744 cells [72]. P2X7 in the CNS Although P2X7 is clearly expressed in brain tissue, the question whether its expression is restricted to non-neuronal cells is a matter of ongoing debate (see below). In ependymal cells along the lateral ventricle of the mouse brain, P2X7 immunoreactivity was observed in brain slices, which was absent in KO mice. Using gold labeling and electron micrographs, it was shown that P2X7 is expressed in the plasma membrane of an ependymal cell specifically localized on the surface of the cilia and microvilli of these barrier cells [79]. Western blot analysis of mouse microglial cells (as well as in macrophages) showed that P2X7 is highly expressed in control, but not in KO mice preparations. In addition, using immunoprecipitation methods, it was revealed that homotrimers of P2X7 were able to co-immunoprecipitate with P2X4, suggesting that an interaction occurs between rather than within receptor complexes. P2X7 receptors were predominantly at the cell surface, whereas P2X4 receptors were predominantly intracellular [64]. Several splice variants of P2X7 were discovered and characterized to a certain extent, but in most cases, their differential functions, physiological roles, and localization are still a matter of research. However, the availability of two antibodies directed against different domains of the channel may serve as a tool for detecting the differential localization S75 of these splice isoforms. Western blot analysis of lysates from astrocytes in cultures originating from either the cortex or the hippocampus revealed the possible presence of different isoforms of the P2X7 receptor in astrocytes cultured from distinct brain regions. Anti-P2X7 receptor antibody detected the presence of a protein band of the predicted molecular weight of 66 kDa in both hippocampal and cortical astrocytes. The presence of a band corresponding to the full-length receptor in both astrocyte cultures was confirmed by the anti-P2X7 receptor (extracellular) antibody. This antibody recognized in the hippocampal, but not in cortical cultures, a major band of about 27 kDa, likely corresponding to a cytoplasmic tail-deleted variant of the receptor, given it was not detected by the anti-P2X7 receptor antibody [85]. In cortex–striatum–subventricular zone organotypic cul- tures, anti-P2X7 receptor antibody in immunocytochemistry studies was used to show that P2X7 expression is upregu- lated and that its staining is co-localized with microglial marker OX42 following 40 min of oxygen and glucose deprivation (OGD) [86]. This upregulation contributes to microglia activation and the following neuronal damage. A similar upregulation was evaluated in the mouse N9 micro- glial cell line, as seen in Western blot, following OGD [86]. By performing immunohistochemistry on spinal cord preparations and Western blot analysis on spinal cord microglial cell lysates of rats receiving morphine, P2X7 immunoreactivity was co-localized with the microglial marker OX42, but not the astrocytic marker GFAP, in the spinal cord. The protein level of spinal P2X7R was upregulated after chronic exposure to morphine and contributed to the generation of tolerance (Fig. 9) [87]. Whether the P2X7 receptor is expressed in neurons remains controversial. One of the main reasons for this ongoing debate arises from the fact that P2X7 immunore- Fig. 9 Double immunostaining of P2X7R and cell-specific markers in morphine-tolerated rats. a Schematic diagram of the spinal cord. Black open square (a) marks the corresponding scope of confocal images (b, d) on the spinal cord section. White open squares (b, d) mark the corresponding scope of amplified images (c, e) on the confocal images. b, c Double immunostaining of P2X7R and the microglial marker OX42. d, e Double immunostaining of P2X7R and the astrocytic marker GFAP. Scale bars, 40 μm (b, d) and 10 μm (c, e). Adapted from reference 87 with permission of Society for Neuroscience