Expression Of P2X₅ Receptors In The Mouse CNS

There is strong molecular,cellular and systems-level evidence that extracellular ATP, via purinoceptors,plays important roles during physiological and pathophysiological processes.P2X receptors are ATP-gated purinoceptors and composed of seven known subunits(P2X^1-7) in mammals.P2X₅ receptor can be found in all vertebrate species (human,mouse,rat,chick,bullfrog and zebrafish),mainly distributing on proliferating cells in skin,gut,bladder,thymus and spinal cord where they are involved in inhibiting cell proliferation and increasing differentiation.It has been accepted that P2X₂,P2X₄ or P2X₄/ P2X₆ subtypes on neurons perform their roles in nervous system,however,controversy exists about the expression and functions of P2X₅ receptor in the central nervous system (CNS).We planed to investigate the expression of P2X₅ receptor in CNS to lay foundation for future functional researches.The present study investigates the P2X₅ receptor expression pattern in the mouse CNS using immunohistochemistry and in situ hybridization histochemistry.To assess the specificity of the polyclonal P2X₅ receptor antibody used here, experiments of peptide block control and Western blotting were performed.This staining for P2X₅ receptor was blocked in control sections exposed to P2X₅ receptor antisera preabsorbed with its specific peptide.An immunoreactive band was detected at about 51 kDa that corresponded to molecular weight of P2X₅ receptor,whereas preadsorption of the antiserum with the peptide antigen resulted in the absence of the band.As for the specificity of the hybridization signal,two antisense digoxigenin-labeled cRNA probes transcripted from rP2X₅ cDNA 1-586 and rP2X₅ cDNA 361-785 were used and we obtained the same distribution patterns of P2X₅ mRNA hybridization signal.Sections, which were incubated in sense digoxigenin-labeled cRNA probe,showed no hybridization signal.We found that P2X₅ receptor were widely distributed in mouse CNS,and the distribution patterns and levels of P2X₅ receptor immunolabeling of native protein had significant correlation with its mRNA counterpart.The main olfactory bulb,accessory olfactory bulb,and primary olfactory cortex showed P2X₅ receptor immunostaining at high levels.The immunostaining was observed mainly in the dendrites and cell bodies,confirming a direct involvement of postsynaptic ATP-gated channels in fast excitatory purinergic transmission in the central olfactory system.Heavy P2X₅ receptor immunostaining was observed in the motor,somatosensory, piriform and entorhinal cortex.Dense immunolabeling in layersâ… ,â…¢andâ…¤,especially in layerâ… (molecular layer),suggests a role for P2X₅ receptors in interconnection of local cortical areas.In the hippocampus,the synaptic region where CA3 pyramidal cell axon(Schaeffer) collaterals form synapses onto distal dendrites of CA1 pyramidal cells had high levels of P2X₅ receptor immunostaining,suggesting that the receptor may contribute to hippocampal excitatory neurotransmission or modulation in Schaeffer collaterals,mossy fibers and the perforant pathway.This study showed that the P2X₅ receptor was expressed extensively in most subnuclei of the thalamus,particularly heavy immunostaining was observed in the anteroventral,anteromediai,anterodorsal and ventral-lateral thalamic nuclei.In different regions of the hypothalamus,P2X₅ receptor immunostaining neurons and fibers were distributed widely,but variably.This result was similar with our previous data from rat.In the hypothalamus,ATP was shown to be involved in regulation of body temperature and hormone secretion and P2X₅ receptor may be involved in those modulation functions.In the midbrain neurons of substantia nigra showed heavy P2X₅ receptor immunostaining.About 80%and 60%of neurons in the compact and reticular part of this nucleus,respectively,were positive for the P2X₅ receptor.P2X₅ receptor immunostaining was widely distributed in the mouse hindbrain and appeared darker than in other parts of the brain.Heavy P2X₅ receptor immunostaining was detected in the pontine nuclei,mesencephalic trigeminal nucleus,motor trigeminal nucleus, ambiguous nucleus,inferior olive,hypoglossal nucleus,dorsal motor vagus nucleus,area postrema,dorsal and ventral cochlear nuclei and vestibular nuclei.In the solitary tract nucleus,ATP,as a cotransmitter with norepinephrine and neuropeptide Y,maybe activate P2X₅ receptor to control the blood pressure.The study showed heavy immunostaining for P2X₅ receptor in the Purkinje cell and granular cell,especially in the cell body and dendritic tree of cerebellar Purkinje cell. Localization of P2X₅ receptor protein on dendrites is compatible with extracellular ATP acting as a fast excitatory neurotransmitter.P2X₅ receptor immunostaining was observed in neurons and neuropil of the spinal gray matter at the cervical,thoracic,lumbar and sacral levels.The most prominent staining was seen in the dorsal horn.Immunolabeling was evident in many motoneurons in the ventral horn of the spinal cord.About 75%of neurons in the ventral horn of the spinal cord were positive for P2X₅ receptors.This is the first extensive study undertaken to investigate the combined distribution of P2X₅ receptor protein and mRNA in the mouse CNS using immunohistochemistry and in situ hybridization histochemistry.The distribution of P2X₅ receptor shown here considerably extends previous studies which reported P2X₅ receptor exists in limited populations of neurons.The result suggests that P2X₅ receptor may be one of purinoceptor subtype on neurons activated by extracellular ATP to perform kinds of functions.