Electron microscopic localisation of P2X₄ receptor subunit immunoreactivity to pre- and post-synaptic neuronal elements and glial processes in the dorsal vagal complex of the rat

Abstract

P2X receptors are ligand gated ion channels activated by extracellular ATP. There are seven P2X subunits, P2X_1–7, and all are expressed in the CNS. The P2X₄ receptor subunit (P2X₄R) is likely to be important in the CNS as it has been reported to be expressed throughout the brain and spinal cord. However, P2X₄Rs have been identified as restricted to neurones, only in glia or expressed in both neurones and glia with no discernible relationship to CNS region or epitope target of antibodies used for staining. In addition, although there are particularly high levels of mRNA encoding P2X₄R in the brainstem, previous immunohistochemical studies have revealed only indistinct staining. We therefore examined the distribution of P2X₄R in the dorsal vagal complex (DVC) of the brainstem using immunohistochemistry in sections obtained from adult Wistar rats transcardially perfused with aldehyde fixatives. When this revealed staining identifiable only as small puncta at the light microscope level, we examined the area with electron microscopy. This ultrastructural study revealed that P2X₄R immunoreactivity (IR) was present in neurones at both pre- and post-synaptic sites as well as in glial cell processes and somata. This P2X₄R-IR was localised adjacent to plasma membranes, as well as internally in membrane bound structures resembling endosomes. Immunoreactivity in endosomes was more prominent following antigen retrieval protocols. Localisation of P2X₄R-IR in astrocytes, identified by the presence of glial fibrillary acidic protein (GFAP), was confirmed using immunofluorescence. The presence of P2X₄Rs in the dorsal vagal complex is consistent with expression studies, but some reasons for a lack of correlation with pharmacological studies are discussed. The P2X₄R is therefore expressed by neurones and glia in the dorsal vagal complex and may play a role in mediating extracellular signalling by ATP in this region.

Introduction

The possibility that ATP acts as an extracellular signalling molecule in the CNS was suggested when it was found to be released upon stimulation of sensory (auricular) nerves [20] and it was subsequently proposed to act as a neurotransmitter [9]. It is now known that ATP can act as a fast neurotransmitter at excitatory purinergic synapses (e.g. Refs. [40], [55], [56]) where it activates ligand gated cationic channels, the P2X receptors [24], [34], [38]. The P2X receptor family consists of seven cloned subtypes (P2X_1–7), some of which have been localised to neurones in the CNS at both pre- and post-synaptic sites [1], [11], [28], [29], [30], [52] where they mediate both postsynaptic responses [3], [13], [22], [33], [36] and presynaptic release of neurotransmitters [24], [25], [39], [44]. There has been a great deal of effort dedicated to determining the P2X subunits that contribute to the trimeric receptors underlying presumed P2X mediated responses in native tissue (e.g. Ref. [34]). However, in many cases, the pharmacological profile of native responses does not match those of P2X receptors in expression systems (e.g. Refs. [31], [34], [44]). Since some of this mismatch may be accounted for by lack of knowledge of which P2X subunits are present in native tissue, many studies have examined the distribution of P2X receptor subunits. However, conflicting results can be encountered in such studies (see below), which in some cases may be attributable to methodology. We have therefore focussed on one subunit, P2X₄, since mRNA for this subunit has a widespread distribution throughout the CNS [6], [8], [11], [46], yet localisation of protein varies (see below). Furthermore, this P2X subunit is currently the subject of much interest since it has recently been shown to be required in the spinal cord for the development of tactile allodynia [48].

Widespread distribution of mRNA encoding P2X₄ subunits throughout the brain [6], [8], [11], [46] is consistent with an immunohistochemical study which localised P2X₄ receptor subunit (P2X₄R) protein to neurones throughout the CNS, visualised with electron microscopy in pre- and post-synaptic neuronal profiles in various areas including the dorsal horn of the spinal cord [28]. In contrast, a recent study in the dorsal horn of the spinal cord using confocal microscopy indicated that P2X₄Rs were absent from neurones identified by the presence of the neuronal nuclei marker NeuN, but present only in cells positive for the microglial marker OX42 [48]. In addition, in the gerbil hippocampus, P2X₄R-IR was co-localised with isolectin B4 (IB4) positive microglia but not with oligodendrocytes or astrocytes [10]. However, P2X₄R-IR has also been detected in juvenile rat hippocampal astroglia [27]. Therefore, contradictory evidence localises P2X₄Rs only to microglia, to neurones or to both types of cells.

One CNS region that contains high levels of mRNA encoding P2X₄R is the medulla oblongata [6], [8], [11], [46]. However, this intensity is not reflected in protein levels since immunohistochemistry with an antibody directed against the c-terminus of the P2X₄R revealed that only a few brainstem nuclei showed significant cellular staining and that in these, labelled cells often poorly stood out against intense surrounding neuropil staining [28]. In addition, using an antibody directed against an extracellular epitope of the P2X₄R a closer examination of one part of the medulla oblongata, the dorsal vagal complex (DVC), comprising the nucleus tractus solitarius (NTS) and the dorsal vagal nucleus (DVN), indicated that P2X₄R-IR neurones were markedly less numerous than other P2X subtypes [54].

The DVC is an important site for investigation in purine research since several lines of evidence indicate that P2X receptors are important in mediating signalling in the NTS. The first evidence to suggest a role for ATP in the NTS was obtained when application of ATP onto dissociated rat NTS neurones was found to activate cation channels that were highly Ca²⁺ permeable [49]. An ATP gated current has also been reported in DVN neurones [31]. Further, microinjection of ATP or its analogues (e.g. αβ-meATP) into the NTS of anaesthetised rats elicits significant dose-related reductions in blood pressure and heart rate, including a fast component proposed to be mediated by P2X receptors [4], [14], [42], [43]. These P2X receptors may play a role in normal cellular signalling as microinjections of the broad spectrum antagonist suramin into the NTS reduced baroreceptor reflex induced changes in heart rate and blood pressure [43]. Similarly, P2X receptors in the NTS are involved in mediating selective components of the peripheral chemoreceptor reflex since microinjection of suramin or pyridoxal-5-6 azophenyle-2-4 disulphonic acid (PPADS) into the NTS depressed the reflex bradycardia, but not tachypnoea, evoked by peripheral chemoreceptor activation. In addition, the bradycardia resulting from pharyngeo-oesophageal stimulation was not affected by P2X antagonists [37]. Finally, the importance of P2X receptors in the DVC is further highlighted by a recent study which shows that activation of P2X receptors can cause action potential independent release of glutamate from nerve terminals in the NTS [44].

Unfortunately the lack of definitive pharmacological tools (agonists and antagonists) makes it impossible to determine which P2X receptor subunit(s) or receptor(s) is involved in purinergic signaling in the NTS. Moreover, the nature of microinjection studies makes it impossible to determine the precise location of the P2X receptors activated. The availability of subtype specific antibodies to the seven P2X receptor subunits along with the neuroanatomical studies has enabled their distribution to be determined by immunohistochemistry (e.g. Refs. [1], [29], [51], [52]). Considering the diversity of opinion regarding the neuronal or glial location of P2X₄Rs in the CNS, and the fact that the level of P2X₄R mRNA is high in the medulla but protein staining appears indistinct, we examined the localisation of P2X₄Rs in the DVC by utilising the high resolution of electron microscopy. We have found that P2X₄R-IR is present in neurones both in presynaptic terminals and at postsynaptic densities. In addition, glial cells and their processes also contain P2X₄R-IR.