P2X receptors Distribution

P2X receptors are membrane ion channels that open in response to the binding of extracellular ATP. Seven genes in vertebrates encode P2X receptor subunits, which are 40-50% identical in amino acid sequence. Each subunit has two transmembrane domains, separated by an extracellular domain (approximately 280 amino acids) (North, 2002).

These receptors belong to the family of cation channels plays a significant role in the function of the peripheral and central nervous system is gated by Adenosine 5’-triphosphate (ATP).

            The first indication of a transmitter role for Adenosine 5′-triphosphate (ATP) in the nervous system was presented 40 years ago, with the demonstration of ATP release from sensory nerves during antidromic stimulation (Holton, 1959). ATP can function as a fast synaptic transmitter through its actions on ionotropic (P2X) and metabotropic (P2Y) receptors in neuronal tissue. It is now well accepted that ATP is involved in cell to cell communication in the peripheral nervous system (for review, see Dubyak and el-Moatassim, 1993; Burnstock, 1999).

However, ATP-mediated fast neurotransmission has only recently been described in the CNS (Edwards et al., 1992; bardoni et al., 1997; Nieber et al., 1997; Pankratov et al., 1998, 1999; Robertson, 1998) although its role is not yet well understood. Purinergic P2X receptors are ligand-gated ion channels that are activated by extracellular adenosine triphosphate (ATP) and are widely expressed not only in the central and peripheral nervous system but also in tissues throughout the body, playing an important role in the transfer of nociceptive information (Kitahara et al.,2003).

The distribution of the P2X2 purinoceptor subunit protein which permeates cationic ions in response to stimulation by ATP and mediates fast synaptic transmission was examined in the adult rat and guinea-pig cerebellum using two novel antisera generated against separate 18 amino acid sequences located in the predicted extracellular domain of this subunit, the results revealed the existence of P2X2 receptor immunoreactivity in the soma and dendrites, neurons in the granular and molecular layers of purkinje cells and in deep cerebellar nuclei as well (Kanjhan et al., 1996).

The distribution of the P2X₂ receptor subunit of the adenosine 5-triphosphate (ATP)-gated ion channels was examined in the adult rat central nervous system (CNS) by using P2X₂ receptor-specific antisera and riboprobe-based in situ hybridisation. P2X₂ receptor mRNA expression matched the P2X₂ receptor protein localisation. An extensive expression pattern was observed, including: olfactory bulb, cerebral cortex, hippocampus, habenula, thalamic and subthalamic nuclei, caudate putamen, posteromedial amygdalo-hippocampal and amygdalo-cortical nuclei, substantia nigra pars compacta, ventromedial and arcuate hypothalamic nuclei, supraoptic nucleus, tuberomammillary nucleus, mesencephalic trigeminal nucleus, dorsal raphe, locus coeruleus, medial parabrachial nucleus, tegmental areas, pontine nuclei, red nucleus, lateral superior olive, cochlear nuclei, spinal trigeminal nuclei, cranial motor nuclei, ventrolateral medulla, area postrema, nucleus of solitary tract, and cerebellar cortex.

In the spinal cord, P2X₂ receptor expression was highest in the dorsal horn, with significant neuronal labeling in the ventral horn and intermediolateral cell column. The identification of extensive P2X₂ receptor immunoreactivity and mRNA distribution within the CNS demonstrated here provides a basis for the P2X receptor antagonist pharmacology reported in electrophysiological studies.

These data support the role for extracellular ATP acting as a fast neurotransmitter at pre- and postsynaptic sites in processes such as sensory transmission, sensory-motor integration, motor and autonomic control, and in neuronal phenomena such as long-term potentiation (LTP) and depression (LTD). Additionally, labelling of neuroglia and fibre tracts supports a diverse role for extracellular ATP in CNS homeostasis. (Kanjha, 1998)

P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function.(Vulchanova et.al, 1996)

Result

P2X2 receptor subunit immunoreactivity in the NTS

At the light microscope level P2X2 receptor subunit immunoreactivity detected using Cy3 conjugated secondary antibody was seen in the tractus solitarius at all rostro-caudal levels(Fig 1). At more caudal levels bands of P2X2 subunit immunoreactivity were observed to run from the tractus towards the NTS . A dense network of P2X2 receptor subunit immunoreactive fibres and, at higher magnification, presumptive terminals was also seen throughout the NTS and DVN at all rostrocaudal levels(fig.). Dense labelling could be seen in the area postrema and in the subpostremal area of the NTS (Fig).

P2X2 receptor subunit immunoreactivity in the DVN

Fluorescence immunohistochemistry technique enabled us to characterize the P2X2 receptor subunit immunoreactive in the DVN at all different levels of rat medulla oblongata, (predominantly in the medrostral, medial aspects of the nucleus).  The P2X2 receptor immunoreactivity is also seen at the DVN and NTS connection. In addition, numerous P2X2 receptor subunit immunoreactive fibres were observed throughout the DVN at all rostro-caudal levels.  The distribution of P2X2 receptor subunit immunoreactivity was detected using fluorescent Cy3 secondary antibody and streptavidin Alexa in the DVN.

No staining was observed in control sections which were incubated in PBS in place of the primary antibody for 12-24 h at 4Cْ, suggesting the secondary antibody produced no background staining neither was there any labeling in control sections incubated in primary antibody(1: 2000 in PBS) pre-absorbed with its specific peptide indicating that the primary antibody was specific to the antigen and that no other contaminating antibodies were present.

The same intesity and pattern of staining were reported in the distribution of P2X2 receptor subunit immunoreactivity in the NTS and DVN when we used diaminobenzidine (DAB) or fluorescent secondary antibody, and the same result was observed in tissue sections obtained from 12 animals in 12 separate experiments. The specificity of the P2X2R antibody was compared between two different P2X2 R antisera ( neuromics P2X2 R and Alomone). The same pattern of staining was observed with either antisera, although the intensity of the staining was almost higher with neuromics one in all examined areas at the same concentration.

P2X2 receptor immunoreactivity in the mice brain

In this study we have also employee a tissue sections from different phenotypic brain mice (wild type;WT, transgenic;TG, knok in; KI, and knok out; KO) to evaluate the specificity of the P2X2R antisera. At the level of light microscope P2X2 receptor immunoreactivity (P2X2R-ir) detected using Streptavidin Alexa488 was seen in the in the dorsal vagal comples (DVC) (Fig.5.3.1.4) and the hippocampus regoin (Fig.5.3.1.5). The staining was significant in WT and TG, weak to bareley seen in the KI while in the P2X2R- difficient mice (KO) the P2X2R immunoreactivity was absent in both examined areas indicating that the results obtained by this antibody so far is due to nothing except the the real reaction between the P2X2R antibody and its antigen in the examined tissues.

P2X2R immunoreactivity in the rat dorsal vagal complex (DVC) with varoius neurochemical markers

At the light microscope level, P2X2R immunoreactivity detected using either by DAB, Cy3 conjugated secondary antibody or by streptavidin Alexa as a dense band of fibres running in the tractus solitarius and from this nucleus into the NTS. A dense network of P2X2R-ir fibres and, at higher magnification, putative terminals was also seen throughout the NTS and DVN at all rostrocoudal levels and at the area postrema dense labeling could be seen in the subpostremal region of the NTS and also in the AP itself (Fig. 5.3.2a).

In ordre to prove the nature of the P2X2 immunopositive structures, double label immunofluorescence experiments were performed using P2X2 atiserum coupled to antisera raised against several ceelular markers (Fig. 5.3.2). P2X2 immunofluorescence (detected using Cy3, red) was coupled either to immunofluorescence(Alexa, green) for isolectin B4 binding (IB4)(Fig.5.3.2a), synaptic visecle marker (SV2)(Fig.5.3.2b), neurofilament 200 (NF200)(Fig.5.3.2c), calcium gene related peptide(CGRP)(Fig.5.2.2d), v.glut1,v.glut2, (Fig. 5.3.2e) GAD67, GAD65/67(5.3.2f), vanilloid receptor antibody(VR-1)(5.3.2g) and neuronal nitric oxide synthase (nNOS)(5.2.2h)

P2X2R immunoreactivity in the rat sensory ganglia

Fluorescence immunohistochemistry for the P2X2 receptor subunit in nodose ganglia (obtained from 3 animals in 3 separate experiments) detected using a Cy3 conjugated secondary antibody, revealed that the vast majority of cell bodies throughout the nodose ganglion contain P2X2 subunit immunoreactvity (Figure 5.3.). In order to validate the results obtained from P2X2 R subunit double labelling with neurochemical markers in the DVC (Fig. 5.3.3.1) and to interpretate their immunoreactive coexpression in diffirent loci of this complex as a functional implication we tested also the coexpression of P2X2R immunoreactivity some neuropeptides and neurotransmitters in the dorsal root ganglia (DRG) and nodose ganglia (NG)

P2X2-ir was observed in scattered cells of the anterior pituitary, neurons in the hypothalamic arcuate and paraventricular nuclei, and catecholaminergic neurons in the olfactory bulb, the substantia nigra, ventral tegmental area, and locus coeruleus. A plexus of nerve fibers and terminals in the nucleus of the solitary tract contained P2X2-ir. This staining disappeared after nodose ganglionectomy, consistent with a presynaptic function.(Vulchanova et.al, 1993) verifying that the co-localisation was not an artifact of the staining procedure.

Discussion

            The axotomy aspect of the isolation of cultured neurons (Michael & PrIestley, 1999) as well as the very young age of the source of animals (Dunn et.al, 2001) may influence substantially the receptor expression at the time of the assay.

            It is considered that ATP acting at P2x receptors is the transmitter responsible for sympathetically mediated vasoconstriction and excitatory junction potentials.  The only neuron showing P2x2R-ir were primary afferents; immunoreactivity was observed within nerve fibers and terminals in the dorsal vagal complex.  It is also evident that in the there is an evident  vast majority of cell bodies throughout the nodose ganglion  that contains P2X2 subunit immunoreactivity.  , P2X2R immunoreactivity was tested by examining the dorsal root ganglia(DRG and the nodose ganglia (NG) verified that the co-localization was not an artifact of the staining procedure.

Conclusion

            The distribution of P2X receptor subunit in the dorsal vagal complex in different variety of rats are not the same. There are rats that do not have the presence of the P2x receptors  when examined and checked under various kinds of test.  This knowledge about the presence or absence of the   P2X receptor subunit in the dorsal vagal complex can help the future studies under this topic.

Bibliography

Dunn PM, Zhong Y, Burnstock G (2001) P2X receptors in peripheral neurons. Prog Neurobiol 65: 107–134.[CrossRef][ISI][Medline]

Michael GJ, Priestley JV (1999) Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 19: 1844–1854.[Abstract/Free Full Text]

North RA (2003a) P2X3 receptors and peripheral pain mechanisms. J Physiol (Lond) 554: 301–308.[CrossRef][ISI]

North RA (2003b) The P2X3 subunit: a molecular target in pain therapeutics. Curr Opin Invest Drugs 4: 833–840.[Medline]