Evidence for P2X3 receptors in the developing rat brain

doi:10.1016/S0306-4522(98)00294-2

Neuroscience

Volume 87, Issue 3, 4 August 1998, Pages 533-539

https://doi.org/10.1016/S0306-4522(98)00294-2 Get rights and content

Abstract

P2X receptor-mediated responses to the ATP analogue, α,β-methylene ATP, in rat brain1, 6, 10, 11, 12, 14, 15, 17, 23, 24cannot be accounted for by the receptor proteins known to be present.9, 16, 26Such experiments are often performed on cells from neonates1, 10, 12, 17, 24and, since differential developmental regulation of P2X₁ and P2X₂ receptor messenger RNAs has already been demonstrated,[18]this is likely to be the case for other P2X receptors. This study was designed to address the possible existence of α,β-methylene ATP-sensitive P2X₃ receptors in rat brains of various ages using a P2X₃ receptor-selective antibody. P2X₃ receptor protein was found in discrete regions of the embryonic (E16) and neonatal rat brain (P7 and P14) but was not detectable in adult animals.

This is the first demonstration of the presence of these receptors in brains from various ages of rat and the differential expression of these receptors in neonates may account for some reported electrophysiological responses to α,β-methylene ATP.

Section snippets

Conclusion

The results presented here show that fast-desensitizing, α,βMeATP-sensitive P2X receptors, namely P2X₃ subunits, do exist in the rat brain but apparently only in embryonic or neonatal animals and further support the role of ATP as a fast neurotransmitter in the CNS.[4]Clearly the regulation of the expression of P2X receptors in development and disease has potentially important physiological consequences and warrants further investigation.

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Cited by (47)

Modulation of the neuronal network activity by P2X receptors and their involvement in neurological disorders
2015, Pharmacological Research
Citation Excerpt :
P2X receptors are widely expressed through the organism, in SNC the 7 subunits are expressed, although with differential levels in distinct area; for example, in some neurons of rat DRG only P2X2 and 3 are expressed [35], while in glia mainly P2X7 is present. However, it is important to note that the level of expression of P2XR depends of the specie, maturational and physiological state, among other consideration; for example, in rat is reported that P2X3 is present in brain from P7 to P14, but not in adult brain [36]; therefore this subject has been already reviewed elsewhere (see [37]). All the P2X subunits are expressed in neurons, but their expression is not homogeneous between the distinct brain regions [38], which implies a great variability in their responses to ATP.
ATP is a key energetic molecule, fundamental to cell function, which also has an important role in the extracellular milieu as a signaling molecule, acting as a chemoattractant for immune cells and as a neuro- and gliotransmitter. The ionotropic P2X receptors are members of an ATP-gated ion channels family. These ionotropic receptors are widely expressed through the body, with 7 subunits described in mammals, which are arranged in a trimeric configuration with a central pore permeable mainly to Ca²⁺ and Na⁺. All 7 subunits are expressed in different brain areas, being present in neurons and glia. ATP, through these ionotropic receptors, can act as a neuromodulator, facilitating the Ca²⁺-dependent release of neurotransmitters, inducing the cross-inhibition between P2XR and GABA receptors, and exercising by this way a modulation of synaptic plasticity. Growing evidence shows that P2XR play an important role in neuronal disorders and neurodegenerative diseases, like Parkinson's and Alzheimer's disease; this role involves changes on P2XR expression levels, activation of key pathways like GSK3β, APP processing, oxidative stress and inflammatory response. This review is focused on the neuromodulatory function of P2XR on pathophysiological conditions of the brain; the recent evidence could open a window to a new therapeutic target.
P2 receptor expression in the dopaminergic system of the rat brain during development
2007, Neuroscience
Citation Excerpt :
More recently, P2X3 subunit-IR was found on rat midbrain single synaptic terminals (Diaz-Hernandez et al., 2001), and emulsion radioautographs revealed a low but significant level of transcription on forebrain regions (Seguela et al., 1996). P2X3 receptors are highly expressed in embryonic animals (E9.5–18.5), then they decline in P7 rats, and in P14 animals there is little detectable P2X3 receptor expression (Kidd et al., 1998; Cheung and Burnstock, 2002). We observed a low P2X3-IR in the organotypic slice cultures and in P3–5 rats.
Extracellular ATP facilitates the release of dopamine via P2 receptor activation in parts of the mesolimbic system. To characterize P2X/Y receptor subtypes in the developing dopaminergic system, their expression in organotypic slice co-cultures including the ventral tegmental area/substantia nigra (VTA/SN) complex and the prefrontal cortex (PFC) was studied in comparison to the receptor expression in 3–5 day-old and adult rats.
Reverse transcriptase–polymerase chain reaction (RT-PCR) with specific primers for the P2X_1,2,3,4,6,7 and P2Y₁ receptors in the tissue extracts of organotypic co-cultures revealed the presence of the P2X and P2Y receptor mRNAs investigated. Multiple immunofluorescence labeling of the P2X/Y receptor protein indicated differences in the regional expression in the organotypic co-cultures after 10 days of cultivation (VTA/SN, P2X_1,2,3,4,6,7, P2Y_1,6,12; PFC, P2X_1,3,4,6,7, P2Y_1,2,4,6,12). At postnatal days 3–5, an immunofluorescence mostly comparable to that of adult rats was observed (VTA/SN and PFC: P2X_1,2,3,4,6,7, P2Y_1,2,4,6,12). There was one important exception: the P2X₇ receptor immunocytochemistry was not found in adult tissue, suggesting a potential role of this receptor in the development.
Only few P2 receptors (e.g. P2X₁, P2Y₁) were expressed at fibers interconnecting the dopaminergic VTA/SN with the PFC in the organotypic co-cultures. The treatment of the cultures with the ATP analogues 2-methylthio-ATP and α,β-methylene-ATP induced an increase in axonal outgrowth and fiber density, which could be inhibited by pre-treatment with the P2X/Y receptor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid. The co-localization of the dopamine-(D1) receptor with the P2X₁ receptor in organotypic slice cultures was evident. In the PFC of the co-cultures, and that of young but not adult rats, a number of tyrosine hydroxylase (TH)–positive cells also possessed P2Y₁-immunoreactivity (IR). Additionally, a strong P2Y₁-IR was observed on astrocytes.
The present results show a time-, region- and cell type–dependent in vitro and in vivo expression pattern of different P2 receptor subtypes in the dopaminergic system indicating the involvement of ATP and its receptors in neuronal development and growth.
Involvement of P2 receptors in the growth and survival of neurons in the CNS
2006, Pharmacology and Therapeutics
Extracellular adenosine 5′-triphosphate (ATP) has been recognized as a ubiquitous, unstable signalling molecule, acting as a fast neurotransmitter and modulator of transmitter release and neuronal excitability. Recent findings have demonstrated that ATP is a growth factor participating in differentiation, cell proliferation, and survival, as well as a toxic agent that mediates cellular degeneration and death. Potential sources of extracellular purines in the nervous system include neurons, glia, endothelium, and blood. A complex family of ectoenzymes rapidly hydrolyzes or interconverts extracellular nucleotides, thereby either terminating their signalling action or producing an active metabolite of altered purinoceptor selectivity. Most effects are mediated through the 2 main subclasses of specific cell surface receptors, P2X and P2Y. Members of these P2X/Y receptor families are widely expressed in the central nervous system (CNS) and are involved in glia–glia and glia–neuron communications, whereby they play important physiological and pathophysiological roles in a variety of biological processes. After different kinds of “acute“ CNS injury (e.g., ischemia, hypoxia, mechanical stress, axotomy), extracellular ATP can reach high concentrations, up to the millimolar range, flowing out from cells into the extracellular space, exocytotically, via transmembrane transport, or as a result of cell damage. In this review, P2 receptor activation as a cause or a consequence of neuronal cell activation or death and/or glial activation is described. The involvement of P2 receptors is also described under different “chronic” pathological conditions, such as pain, epilepsia, toxic influence of ethanol or amphetamine, retinal diseases, Alzheimer's disease (AD), and possibly, Parkinson's disease. The relationship between changes in P2 receptor expression and the specific response of different cell types to injury is extremely complex and can be related to detrimental and/or beneficial effects. The present review therefore considers ATP acting via P2 receptors as a potent regulator of normal physiological and pathological processes in the brain, with a focus on pathophysiological implications of P2 receptor functions.
P2 receptor-stimulation influences axonal outgrowth in the developing hippocampus in vitro
2006, Neuroscience
Extracellular ATP might act as a trophic factor on growing axons during development of the CNS via P2 receptors. In the present study the postnatal presence of selected P2 receptor subtypes was analyzed and their putative trophic capacity in entorhino-hippocampal slice co-cultures of mouse brain was tested. The effect of the P2 receptor ligands 2-methylthioadenosine-5′-triphosphate (P2X/Y receptor agonist) and pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid (P2X/Y receptor antagonist) on axonal growth and fiber density of biocytin-labeled hippocampal projections was compared both with untreated cultures and with cultures treated with artificial cerebrospinal fluid. After 10 days in vitro, double immunofluorescence labeling revealed the expression of P2X₁, P2X₂, P2X₄ as well as P2Y₁ and P2Y₂ receptors in the examined regions of entorhinal fiber termination. Further, quantitative analysis of identified biocytin-traced entorhinal fibers showed a significant increase in fiber density in the dentate gyrus after incubation of the slices with the P2 receptor agonist 2-methylthioadenosine-5′-triphosphate. This neurite outgrowth promoting effect was completely abolished by the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid. Our in vitro data indicate that ATP via its P2X and P2Y receptors can shape hippocampal connectivity during development.
Changes in expression of P2X purinoceptors in rat cerebellum during postnatal development
2005, Developmental Brain Research
Changes in expression of P2X receptors (P2X_1–7) during postnatal development of the rat cerebellum are described. At P3, immunoreactivity (ir) to all the P2X receptors, except for P2X₃ receptors, was found in Purkinje cells and deep cerebellar nuclei, P2X₅-ir being most prominent. Granular and microglial cells were labeled for P2X₅ (weakly) and P2X₄ receptors, respectively. At P7, expression of all the P2X receptors (with the exception of P2X₃) was up-regulated, P2X₅ and P2X₆ receptors being most prominent. Scattered P2X receptor-ir in unipolar brush cells in the granular cell layer and P2X₁- and P2X₇-ir of microglial cells was also present. At P14, the dendritic trees of Purkinje cells were intensely labeled by P2X_1–7 receptor antibodies, except for P2X₃, while P2X₁, P2X₄ and P2X₇ receptor immunostaining in microglial cells and P2X₅ receptor immunostaining in granular cells was up-regulated. At P21, expression of all P2X receptors (except P2X₃) was down-regulated in the Purkinje cells and deep cerebellar nuclei; P2X₁, P2X₄ and P2X₇ receptors-ir was present in microglial cells. In contrast, expression of P2X₅-ir in granular cells was up-regulated. At P60, expression levels of all the P2X receptors (except P2X₃) were similar with those at P21. In double-labeling experiments, almost all the P2X-ir Purkinje cells were immunoreactive for calbindin-D28k, while 60–80% of P2X-ir cells in the granular cell layer were immunoreactive for calretinin. The possible short- and long-term functional significance of the changes in expression of P2X receptors during postnatal development is discussed.
Molecular physiology of P2 receptors in the central nervous system
2004, European Journal of Pharmacology
Neurons of the central nervous system (CNS) are endowed with ATP-sensitive receptors belonging to the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) types. Whereas a number of P2X receptors mediate fast synaptic responses to the transmitter ATP, P2Y receptors mediate either slow changes of the membrane potential in response to non-synaptically released ATP or the interaction with receptors for other transmitters. To date seven P2X and seven P2Y receptors of human origin have been molecularly identified and functionally characterized. P2X subunits may occur as homooligomers or as heterooligomeric assemblies of more than one subunit. P2X₇ subunits do not form heterooligomeric assemblies and are uniqe in mediating apoptosis and necrosis of glial cells and possibly also of neurons. The P2X₂, P2X₄, P2X₄/P2X₆ and P2Y₁ receptors appear to be the predominant neuronal types. The localisation of these receptors may be at the somato-dendritic region (postsynaptic) or at the nerve terminals (presynaptic). Postsynaptic P2 receptors appear to be mostly excitatory, while presynaptic P2 receptors may be either excitatory (P2X) or inhibitory (P2Y). Since in the CNS the stimulation of a single neuron may activate multiple networks, a concomitant stimulation of facilitatory and inhibitory circuits as a result of ATP release is also possible. Finally, the enzymatic degradation of ATP may lead to the local generation of adenosine which can modulate via A₁ or A_2A receptor-activation the ATP effect.

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Letter to NeuroscienceEvidence for P2X3 receptors in the developing rat brain

Abstract

Section snippets

Conclusion

Neurosci. Lett.

Fedn Eur. biochem. Socs Lett.

Neuroscience

Neuropharmacology

Eur. J. Pharmac.

Molec. Brain Res.

Neuroscience

Neurosci. Lett.

Eur. J. Pharmac.

Neuropharmacology

New structural motif for ligand-gated ion channels defined by an ionotropic ATP receptor

Nature

Purinergic neurotransmission

Semin. Neurosci.

A P2X purinoceptor expressed by a subset of sensory neurons

Nature

Cloning of P2X5 and P2X6 receptors and the distribution and properties of an extended family of ATP-gated ion channels

J. Neurosci.

Letter to Neuroscience
Evidence for P2X₃ receptors in the developing rat brain

Cloning of P2X₅ and P2X₆ receptors and the distribution and properties of an extended family of ATP-gated ion channels