We recently demonstrated the P2X₇ receptor subunit is present in presynaptic terminals in the CNS which have the morphology and localisation of excitatory terminals (Deuchars et al. 2001; Sperlçgh et al. 2002). Here we first aimed to confirm this localisation using a new antibody to the P2X₇ receptor. Secondly we examined if the receptor subunit is present in glutamatergic excitatory terminals identified using antisera to the vesicular glutamate transporters VGluT1 and VGluT2.

Male Wistar rats (150-200 g) were humanely killed by anaesthesia with sodium pentobarbital (Sagatal, 60 mg kg^-1 I.P.) and transcardial perfusion with 500 ml of fixative (4 % paraformaldehyde/0-0.1 % glutaraldehyde). To confirm presynaptic localisation of the P2X₇ receptor, 50 mm sections of the medulla and forebrain were prepared for light and electron microscopy (Deuchars et al. 2001), using an antibody raised against amino acids 60-323 of the P2X₇ receptor (Kim et al. 2001). To detect glutamatergic terminals, sections were incubated for 12-24 h with rabbit anti-P2X₇ (Alomone Labs, 1:1000, aa 576-595) and guinea-pig anti-VGluT1 or anti-VGluT2 (1:3000; Varoqui et al. 2002) and primary antibodies detected with donkey anti-rabbit Cy3 (1:1000, Jackson Immunochemicals) or biotinylated anti-guinea-pig (1:200, Jackson) followed by Streptavidin ALEXA⁴⁸⁸.

At light microscope level both primary antibodies for the P2X₇ receptor subunit revealed prominent staining of glial cells, well known to express P2X₇ receptors. In addition, both resulted in fine punctate staining of the neuropil which, at the electron microscopic level, was revealed to be due to immunoproduct within presynaptic terminals, confirming our previous findings with the anti-rabbit antisera. In the medulla oblongata the P2X₇ receptor was co-localised with VGluT2 but not VGluT1. In a surprising contrast, P2X₇ was co-localised with VGluT1 but not VGluT2 in the hippocampus.

These data are consistent with the P2X₇ receptor having a role in modulating glutamate release from presynaptic terminals in the CNS. However, it is clear that not all glutamate-releasing terminals bear the P2X₇ receptor. Furthermore, the expression of the P2X₇ receptor may be correlated with that of either VGluT1 or VGluT2 depending on the CNS region examined.

This work was supported by The Wellcome Trust.

All procedures accord with current UK legislation.