Reanalysis of P2X₇Receptor Expression in Rodent Brain

Abstract

P2X receptors are cationic-selective ion channels gated by extracellular ATP. There are seven subunits (P2X 1-7 ), the first six of which are expressed throughout the peripheral and central nervous systems. P2X 7 receptors are rapidly upregulated and activated as a result of inflammatory stimuli in immune cells, where they act not only as cationic channels but uniquely couple with rapid release of proinflammatory cytokines, cytoskeletal rearrangements, and apoptosis or necrotic cell death. The P2X 7 receptor has been termed the cytolytic non-neuronal P2X receptor because it had not been detected in neurons until recently when it has been immunolocalized to several brain regions, particularly the hippocampus, and has been suggested to be involved in presynaptic modulation of transmitter release. Because its expression in brain neurons may have substantial functional implications, we have performed detailed immunocytochemical, immunoblot, and immunoprecipitation studies on brain and non-neuronal tissue using all currently available antibodies. We first examined rats, but staining patterns were inconsistent among antibodies; we therefore studied mice for which there are two P2X 7 knock-out mice constructs available, one expressing the LacZ transgene. We found that P2X 7 receptor protein is strongly and reliably detected in the submandibular gland and lung of wild-type mice but not in either of the P2X 7 Ϫ/Ϫ mice. However, we failed to find evidence for P2X 7 receptor protein in hippocampal neurons or their input-output projections. Either the P2X 7 protein in the hippocampus is below the limits of detection by the currently available methods or it is not present.

Expression, signaling, and function of P2X7 receptors in bone

“…This discrepancy could be due to the different strategies used to generate the mice or their different genetic backgrounds. In this regard, the P2rx7 −/− mice characterized by Gartland et al were constructed by insertion of a lacZ gene at the beginning of exon 1 of P2rx7 [44], whereas the P2rx7 −/− mice described by Ke et al carried a deletion of the region encoding C-terminal amino acids 506-532 [41]. Despite these differences, P2X7 receptor protein is not detectable in either model [44].…”

“…In this regard, the P2rx7 −/− mice characterized by Gartland et al were constructed by insertion of a lacZ gene at the beginning of exon 1 of P2rx7 [44], whereas the P2rx7 −/− mice described by Ke et al carried a deletion of the region encoding C-terminal amino acids 506-532 [41]. Despite these differences, P2X7 receptor protein is not detectable in either model [44]. It is noteworthy that the decrease in bone density observed by Ke et al was more pronounced in adult male mice, whereas Gartland et al analyzed the bones of a relatively small number of animals, the gender of which was not specified.…”

The P2X7 receptor in retinal ganglion cells: A neuronal model of pressure-induced damage and protection by a shifting purinergic balance

“…Patterns of staining in the hippocampus produced by different antibodies were inconsistent, while staining was similar in P2X 7 -/-and wildtype mice. However, several lines of evidence support the presence of the receptor in retinal ganglion cells.…”

“…While mRNA message for the P2X 7 receptor was detected in rat retinas throughout life, expression was higher in neonatal retinas; expression at postnatal day (PD) 2 was higher than on PD 7, although staining at PD 7 was similar to that found at PD 14 and in adult rats, suggesting expression stabilized around PD 7 [35]. While the loss of cholinergic cells in the ganglion cell layer over PD 1-2 was reduced by blocking P2X 7 receptors, these cells were likely related to displaced amacrine cells not ganglion cells, and block did not change the number of cells identified as retinal ganglion cells over this period [5].…”

“…It should be noted when assessing immunologic studies of the P2X 7 receptor that the specificity of some antibodies raised against the receptor is uncertain, and neuronal staining has been questioned [7]. Patterns of staining in the hippocampus produced by different antibodies were inconsistent, while staining was similar in P2X 7 -/-and wildtype mice.…”