Zhenghua Xiang

Localization of ATP-gated P2X receptor immunoreactivity in rat sensory and sympathetic ganglia

The distribution of P2X1, P2X2, P2X3, P2X4, P2X5 and P2X6 receptors, a family of ATP-gated cation channels, in rat trigeminal, dorsal root, nodose, superior cervical, and coeliac ganglia was studied immunohistochemically. It was found that polyclonal antibodies for the six P2X receptor subtypes could label over 90% of neurones in these ganglia to different intensities. There was also considerable variation in intensity of immunoreactivity in individual neurones within each ganglion. In dorsal root and trigeminal ganglia expression of the P2X3 receptor was much higher than for the other five subtypes. P2X3 immunoreactivity was much more intense in the small-diameter neurones than in the large-diameter neurones. In nodose ganglion, both P2X2 and P2X3 antibodies labelled the neurones intensely. In sympathetic superior cervical and coeliac ganglia, immunoreactivity to five P2X receptor subtypes were detected (exception, P2X5), with P2X2, P2X4 and P2X6 showing higher intensity. Low level expression of P2X3 receptor in sympathetic ganglia indicates that this receptor subtype is not limited to the sensory ganglia where it was highly expressed. The results have demonstrated that both sensory and sympathetic ganglia express a variety of P2X receptor subtypes and that different subtypes are expressed to different levels and by different subpopulations of neurones.

Localization of ATP-gated P2X2 and P2X3 receptor immunoreactive nerves in rat taste buds.

P2X receptors have been suggested to play a role in the transduction of sensory signals such as pain and sound. In the present study, polyclonal antibodies against P2X1 to P2X6 receptors were used to localize P2X receptors in circumvallate and fungiform papillae of rats. Nerve fibres innervating the taste buds stained intensely with P2X3 receptor antibodies. P2X3 receptor-positive nerves were observed in the intra- and subgemmal regions. The nerve fibres were also stained with P2X2 receptor antibodies, but the intensity was much lower. The distribution of P2X2 receptor immunoreactivity overlaps with that of P2X3. These results suggest that ATP might be a neurotransmitter in taste reception cells in the taste buds, where it transducts the taste signals to the afferent taste nerves by activating P2X receptors at the synapses. This is the first experiment indicating such a role for ATP, although supplementary functional studies are required.

Expression of P2X receptors on rat microglial cells during early development.

We have used single‐ and double‐labeling immunfluorescence and reverse transcription‐polymerase chain reaction (RT‐PCR) methods to examine expression of P2X receptor subtypes on microglial cells of brain in late embryonic and postnatal rat, in the N9 microglial cell line and primary cultured microglial cells. P2X1, P2X4, and P2X7 receptors were shown on microglial cells from late embryonic day 16. Almost all the microglial cells that were positive for the marker ED1, expressed P2X1 and P2X4 receptors, whereas only about 30% of the cells with ED1‐immunoreactivity were found to express the P2X7 receptor. Positive cells were localized mainly in the white matter and around ventricles. From postnatal day 7, many microglial cells with P2X4 receptor‐immunoreactivity were seen around the blood vessels. At postnatal day 30, microglial cells with P2X1 receptor‐immunoreactivity disappeared and the cells with P2X4 receptor‐immunoreactivity were mainly localized around blood vessels and lining the subarachnoid space. From postnatal day 30, the microglial cells with P2X7 receptor‐immunoreactivity were found to be distributed widely in the forebrain. Cells with P2X7 receptor‐immunoreactivity from P30 were not labeled by ED1, but some were labeled by isolectin B4. The expression of P2X1, P2X4, and P2X7 receptor mRNA and protein on primary cultures of rat microglial cells and on the N9 microglial cell line was demonstrated with immunocytochemistry and RT‐PCR. This is the first report that the P2X1 receptor is expressed on microglial cells, at least in early development, before postnatal day 30.

P2X2 and P2X3 purinoceptors in the rat enteric nervous system

Adenosine 5′-triphosphate receptors are known to be involved in fast excitatory postsynaptic currents in myenteric neurons of the digestive tract. In the present study, the distribution of P2X2 and P2X3 receptor mRNA was examined by in situ hybridisation while P2X2 and P2X3 receptor protein was localised by immunohistochemical methods. In addition, P2X2 and P2X3 receptors were colocalised with calbindin and calretinin in the myenteric and submucosal plexus. P2X2- and P2X3-immunoreactive neurons were found in the myenteric and submucosal plexuses throughout the entire length of the rat digestive tract from the stomach to the colon. Approximately 60%, 70% and 50% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 56% and 45% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X2 receptor. Approximately 10%, 2% and 15% of the ganglion cells in the myenteric plexus of the gastric corpus, ileum and distal colon, and 62% and 40% in the submucosal plexus of the ileum and distal colon, respectively, showed positive immunoreactivity to the P2X3 receptor. Double-labelling studies showed that about 10–25% of the neurons with P2X2 immunoreactivity in myenteric plexus and 30–50% in the submucosal plexus were found to express calbindin or calretinin. About 80% of the neurons with P2X3 receptor immunoreactivity in the myenteric plexus and about 40% in the submucosal plexus expressed calretinin. Approximately 30–75% of the neurons with P2X3 receptor immunoreactivity in the submucosal plexus expressed calbindin, while none of them were found to express calbindin in the myenteric plexus.

Localization of ATP-gated P2X2 receptor immunoreactivity in the rat hypothalamus.

Previous pharmacological studies have indicated that ATP receptors may be involved in the regulation of physiological functions in hypothalamus. In the present study, the distribution of P2X2 receptor in the rat hypothalamus was studied with immunohistochemistry. It was shown that P2X2 immunoreactivity-positive neurons and nerve fibres were localized in many hypothalamic nuclei. Intense labelling of both neuronal cell bodies and nerve fibres was observed in the paraventricular nucleus, arcuate nucleus, retrochiasmatic area, periventricular nucleus, and the ventral part of tuber cinereum area. In supraoptic, circular, and ventral tuberomammillary nuclei the neuronal cell bodies were strongly positive, but few nerve fibres were positive. Axons with strong P2X2 immunoreactivity were found in the organum vasculosum of the lamina terminalis and median eminence. Some scattered positive neurons and nerve fibres were found in many hypothalamic nuclei including preoptic nucleus. The results of the present study demonstrated the existence of P2X receptors in hypothalamus, as a basis for detailed studies of the roles of P2X receptors in the regulation of hypothalamic functions.

P2X receptor immunoreactivity in the rat cochlea, vestibular ganglion and cochlear nucleus

P2X receptors have been reported to be involved in neurotransmission in both central and peripheral nerves. In the present study, polyclonal antibodies against P2X1, P2X2, P2X3, P2X4, P2X5, and P2X6 were used to study the distribution of P2X receptors in rat cochlea and vestibulocochlear nerve pathways. It was found that in the vestibular ganglion all six types of antibodies stained the neurons to different intensities. The strongest signal was obtained with the P2X2 antibodies, followed by P2X3 antibodies. The other four antibodies produced weak signals, of approximately the same intensity. In the spiral ganglion, the six types of antibodies also stained almost all neurons. However, the rank order of intensity was different from that in the vestibular ganglion: the strongest signal was still obtained with P2X2 antibodies, followed by P2X4, P2X1, and P2X3 antibodies. The immunolabelling was much weaker with P2X5, and P2X6 antibodies compared with the other four types of antibodies. In the cochlea, besides the spiral ganglion neurons, other tissues such as stria vascularis, the organ of Corti and the tectorial membrane were labelled intensively with P2X2 antibodies only. High density P2X2 immunoreactivity was also observed in the vestibulocochlear nerve fibres. In the cochlear nucleus, neurons and nerve fibres were stained with the P2X2 antibodies, as were the neurons in the trapezoid body. These results suggested that P2 receptors, especially the P2X2 receptors, may play important roles in the signal transduction involved in the perception of sound and balance.

Pharmacological and molecular characterization of P2X receptors in rat pelvic ganglion neurons

1 The presence and characteristics of P2X receptors on neurons of the rat major pelvic ganglia (MPG) have been studied using whole cell voltage‐clamp, in situ hybridization and immunohistochemistry. 2 Rapid application of ATP (100 μM) to isolated rat MPG neurons induced moderately large inward currents (0.33–5.3 nA) in 39% of cells (108/277). The response to ATP occurred very rapidly, with an increase in membrane conductance, and desensitized slowly. 3 The concentration‐response curve for ATP yielded an EC50 of 58.9 μM. The agonist profile was ATP2MeSATP=ATPγS>BzATP, while α,β‐MeATP, β,γ‐MeATP, UTP and ADP were all inactive at concentrations up to 100 μM. 4 The response to ATP was antagonized by suramin (pA2=5.6), reactive blue‐2 (IC50=0.7 μM) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS). 5 Lowering the pH from 7.4 to 6.8 produced a marked potentiation (to 339% of control) of the responses to ATP (30 μM), while raising the pH to 8.0 attenuated the responses (to 20% of control). The EC50s for ATP were 28.8, 58.9 and 264 μM at pH 6.8, 7.4 and 8.0, respectively. 6 Co‐application of ATP with Zn2+ produced a marked enhancement of the responses to ATP, with an EC50 of 9.55 μM. In the presence of Zn2+ (30 μM), the EC50 for ATP was decreased to 4.57 μM. 7 In situ hybridization revealed that the P2X receptor transcripts levels in rat MPG neurons are P2X2>P2X4>P2X1, P2X3, P2X5 and P2X6. The immunohistochemical staining revealed a small number of neurons with strong P2X2 immunoreactivity. 8 In conclusion, our results indicate that there are P2X receptors present on MPG neurons. The pharmacological characteristics of these receptors, the in situ hybridization and immunohistochemical evidence are consistent with them being of the P2X2 subtype, or heteromultimers, with P2X2 being the dominant component.

Nitrated α-Synuclein Induces the Loss of Dopaminergic Neurons in the Substantia Nigra of Rats

Background The pathology of Parkinsons disease (PD) is characterized by the degeneration of the nigrostriatal dopaminergic pathway, as well as the formation of intraneuronal inclusions known as Lewy bodies and Lewy neurites in the substantia nigra. Accumulations of nitrated α-synuclein are demonstrated in the signature inclusions of Parkinsons disease. However, whether the nitration of α-synuclein is relevant to the pathogenesis of PD is unknown. Methodology/Principal Findings In this study, effect of nitrated α-synuclein to dopaminergic (DA) neurons was determined by delivering nitrated recombinant TAT-α-synuclein intracellular. We provide evidence to show that the nitrated α-synuclein was toxic to cultured dopaminergic SHSY-5Y neurons and primary mesencephalic DA neurons to a much greater degree than unnitrated α-synuclein. Moreover, we show that administration of nitrated α-synuclein to the substantia nigra pars compacta of rats caused severe reductions in the number of DA neurons therein, and led to the down-regulation of D2R in the striatum in vivo. Furthermore, when administered to the substantia nigra of rats, nitrated α-synuclein caused PD-like motor dysfunctions, such as reduced locomotion and motor asymmetry, however unmodified α-synuclein had significantly less severe behavioral effects. Conclusions/Significance Our results provide evidence that α-synuclein, principally in its nitrated form, induce DA neuron death and may be a major factor in the etiology of PD.

Expression of P2X 6 receptors in the enteric nervous system of the rat gastrointestinal tract

Expression of P2X4 and P2X6 receptor subunits in the gastrointestinal tract of the rat was studied with double-labeling fluorescence immunohistochemistry. The results showed that P2X6 receptors were expressed widely in the submucosal and myenteric plexuses. In the myenteric plexus, P2X6 receptors were expressed mainly in large size neurons which resembled Dogiel type II neurons. These P2X6 receptor-immunoreactive (ir) neurons also expressed calbindin 28K, calretinin and neuronal nuclei (NeuN), proteins that are markers of intrinsic sensory neurons. In the submucosal plexus, all the calbindin 28K, calretinin and NeuN-ir cells were immunoreactive for P2X6 receptors. P2X6 receptors do not form homomultimers, but rather heteromultimers with either P2X2 or P2X4 receptors. P2X4 receptors were not expressed in neurons, but were expressed in macrophages of the rat gastrointestinal tract. These data indicate that P2X6 receptors are mainly expressed on intrinsic sensory neurons and that ATP, via P2X6 receptors probably in heteromeric combination with P2X2 receptors, may be involved in regulating the physiological functions of these neurons.

Functional up-regulation of P2X3 receptors in the chronically compressed dorsal root ganglion

Abstract P2X receptors on dorsal root ganglion (DRG) neurons have been strongly implicated in pathological nociception after peripheral nerve injuries or inflammation. However, nothing is known of a role for purinergic receptors in neuropathic pain produced by a chronic compression of DRG (CCD) – an injury that may accompany an intraforaminal stenosis, a laterally herniated disc or other disorders of the spine leading to radicular pain. In a rat model of DRG compression, hyperexcitable neurons retain functioning axonal connections with their peripheral targets. It is unknown whether such hyperexcitability might enhance chemically mediated nociceptive stimulation of the skin. In this study, CCD facilitated the nocifensive behavior and mechanical hyperalgesia‐induced by the P2X3 agonist, α,β‐methylene ATP (α,β‐meATP). An injection of α,β‐meATP into the hind paw of CCD rats resulted in a significantly greater decrease in the mean threshold to von Frey stimuli and a greater duration of paw lifts than in sham‐operated control rats. CCD also increased the levels of P2X3 receptor protein and the number of P2X3 immunoreactive, small diameter DRG neurons in the compressed ganglion. P2X3 receptors were co‐labeled with the isolectin IB4, consistent with a role in nociception. In addition, a α,β‐meATP induced significantly larger fast‐inactivating currents in CCD‐ than in sham‐operated acutely dissociated DRG neurons. These currents were accompanied by the generation of action potentials – but only in the CCD neurons. U0126, a specific inhibitor of the MEK1/2, greatly down‐regulated the enhanced current. Taken together, these observations suggest that enhanced purinergic responses after CCD are mediated by P2X3 receptors.