P2X Receptors Are Expressed On Neurons Containing Luteinizin Hormone-releasing Hormone In The Mouse Hypothalamus

There is substantial molecular, cellular and system-level evidence that extracellular ATP, via activation of purinoceptors, plays important roles during physiological and pathological processes. P2X receptors are ATP-gated ion channels which mediate rapid (within 10 ms) and selective permeability to cations (Na+, K+ and Ca2+). Seven distinct P2X subtypes have been cloned from mammalian species, which assemble into ATP-activated ion channels either as homomers or heteromers. Purinergic P2X receptors are expressed throughout the hypothalamus. The luteinizing hormone-releasing hormone (LHRH)-containing neurons are distributed diffusely throughout the hypothalamus and project to the median eminence where they release LHRH from their axon terminals into the hypophysiotropic portal system. But the expression of P2X receptors on LHRH-containing neurons has not yet been studied.Expression of P2X1, P2X2, P2X3, P2X4, P2X5 and P2X6 receptors, members of a family of ATP-gated cation channels, on neurons containing luteinizing hormone-releasing hormone (LHRH) in themouse hypothalamus was studied with double-labeling fluorescence immunohistochemistry. The P2X7 receptor isn't investigated in this study because it is functionally unique among P2X receptors in being able to act as a non-selective pore.LHRH-immunoreactive (-ir) neurons were mainly found in the area around the organum vasculosum of the lamina terminalis (OVLT). And LHRH-ir fibers were mainly found in the OVLT and the median eminence (ME). It was observed that P2X2, P2X4, P2X5 and P2X6 receptor antibodies labeled different subpopulations of neurons with different intensities in the area around the OVLT, no signal was observed with P2X1 and P2X3 receptor antibodies in this area. In the ME and OVLT, where LHRH-ir fibers were found abundantly, only P2X6 receptor antibody labeled fibers, which resembled LHRH-ir fibers.When using double-labeling fluorescence immunohistochemistry, it was observed that all the LHRH-ir neurons were also positive for P2X2, P2X4, P2X5 and P2X6 receptors respectively, although the immunostaining intensity was different. The order of immunostaining intensity with different antibodies was P2X6>P2X4>P2X2>P2X5. All the LHRH-ir fibers were also strongly labeled by P2X6 antibody. Some LHRH-ir fibers, especially these nearby the perikarya, were labeled by P2X2 antibody, but LHRH-ir fibers in the ME and OVLT were not labeled by P2X2 receptor antibody. Almost no LHRH-ir fibers were labeled by P2X4 or P2X5 receptor antibodies. We used double-labeling fluorescence immunohistochemistry to study the colocalization of P2X receptor protein with LHRH-ir neurons of the mouse forebrain. This study provides the first evidence that P2X2, P2X4, P2X5 and P2X6 receptors are expressed onLHRH-containing neurons and hence has provided a substantial neuroanatomical basis for possible functional interactions between the purinergic and the LHRH system in the mouse forebrain.In short, this study demonstrated that different combinations of P2X receptor subunits were expressed on the perikarya and axon terminals of LHRH-producing neurons. It was shown for the first time that P2X2, P2X4, P2X5 and P2X6 receptor subunits were expressed on the perikarya of LHRH-producing neurons and P2X2 and P2X6 on their axon terminals. These results suggest that activation of P2X receptors by ATP via different homomeric or heteromeric P2X receptors at both presynaptic and postsynaptic sites could be involved in the regulation of LHRH secretion at the forebrain level.