Changes in expression of P2X purinoceptors in rat cerebellum during postnatal development

doi:10.1016/j.devbrainres.2005.02.015

Developmental Brain Research

Volume 156, Issue 2, 12 May 2005, Pages 147-157

https://doi.org/10.1016/j.devbrainres.2005.02.015 Get rights and content

Abstract

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.

Introduction

Extracellular ATP is now established as an excitatory neurotransmitter or neuromodulator in both the peripheral and central nervous systems that acts via nucleotide receptors [6], [9], [10], [12]. Much evidence indicates that ATP is released as a co-transmitter with other neurotransmitters such as noradrenaline, acetylcholine, nitric oxide, glutamate, γ-amino butyric acid, dopamine and 5-hydroxytryptamine [3], [7], [8], [11]. Purinergic neurotransmission may play an important role in the cerebellum, since high expression levels have been described for different P2X receptors, especially in Purkinje cells, and to a lesser extent, in the granular layer [14], [15], [25], [32]. Seven different members of the P2X family of ligand-gated ion channels have been cloned [13]. Of the seven cloned P2X receptors, P2X₁, P2X₂, P2X₄ and P2X₆ receptors have been reported as being expressed in the cerebellum of adult rat [4], [14], [32], [40]. mRNA expression for P2X₁ and P2X₂ receptors in cerebellum seems to be relatively high in postnatal day 5 rats, although it is much lower in adult animals [25], [26]. P2X₄ and P2X₆ receptors are highly expressed by Purkinje cells in adult rats [14]. Mateo et al. used single-cell fluorescence microscopy and a fura-2 method to study [Ca²⁺]_i signals elicited by extracellular ATP in cultured Purkinje cells from postnatal days 7–8 rat cerebellum [33]. Extracellularly applied ATP evoked fast [Ca²⁺]_i rises revealed by a rapid and transient increase in fura-2 F340/F380 ratio in all Purkinje cells tested. The receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2′, 4′disulphonic acid effectively blocked the responses elicited by ATP [33]. These results demonstrate that the functional ionotropic P2X purinoceptor in the cerebellar Purkinje cells is the P2X₂ receptor as P2X₄ and P2X₆ receptors cannot be effectively blocked by these antagonists in the rat [33], [35]. These data imply that a switch from P2X₂ to P2X₄ and P2X₆ receptors may take place during the development of the cerebellum. In the present study, using single- and double-labeling immunofluorescence, we carried out a detailed analysis of changes in expression of all the P2X receptors during postnatal development in order to clarify the expression patterns of P2X receptors and confirm whether there is a switch from P2X₂ to P2X₄ and P2X₆ receptors on cerebellar Purkinje cells during the postnatal period.

Section snippets

Animals and tissue preparation

Breeding, maintenance and killing of the animals used in this study followed principles of good laboratory animal care and experimentation in compliance with UK Home Office regulations (Schedule One Procedures). Twenty-five Wistar rats were killed by asphyxiation with CO₂ at P3, P7, P14, P21 and P60 (n = 5 for each age group) and perfused through the aorta with 100 ml 0.9% NaCl solution and 250 ml 4% paraformaldehyde in 0.1 mol/L phosphate buffer, pH 7.4. The brains were removed, postfixed

Quantitative analysis

The sections stained by P2X receptor, calbindin D-28k and calretinin antibodies were also used to perform a quantitative analysis. Positively-stained neuronal cell bodies in the cerebellar Purkinje cell layer and granular cell layer were counted per visual field. Ten randomly chosen fields in each section and 5 sections for each rat were analyzed. The percentage of neurons immunoreactive for a P2X receptor that were also immunoreactive for calbindin D-28k or calretinin and the percentage of

Results

A summary of the expression of P2X receptors at postnatal stages P3 to P60 is shown in Table 1 and selected examples of P2X receptor-ir are illustrated in Fig. 1, Fig. 2.

At P3, all P2X receptors immunoreactivities, except for P2X₃, were found in Purkinje cells and deep cerebellar nuclei, although the immunostaining intensity of each receptor was different (Figs. 1A–H). The highest level of expression among the P2X receptors was for the P2X₅ receptor at this stage (Fig. 1D). Staining was

Discussion

This paper is the first to study systematically the changes in expression of P2X receptor in rat cerebellum during postnatal development, using immunocytochemistry. We found that all P2X receptors, with the exception of P2X₃ receptors, were expressed in the cerebellum. This is consistent with the lack of cerebellar P2X₃ receptors described by Kidd et al. [27], although Garcia-Lecea et al. [20] claimed that P2X₃ receptors were expressed in Purkinje cells, cultured from 7-day newborn rats. Among

Acknowledgments

The authors thank Dr. Chrystalla Orphanides for her excellent editorial assistance. Zhenghua Xiang is the recipient of a Wellcome Trust Traveling Fellowship #064931/Z/01/Z.

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Research reportChanges in expression of P2X purinoceptors in rat cerebellum during postnatal development

Abstract

Introduction

Section snippets

Animals and tissue preparation

Quantitative analysis

Results

Discussion

Acknowledgments

Prog. Neurobiol.

FEBS Lett.

Neuroscience

Neuropharmacology

Brain Res. Bull.

Neuropharmacology

Neurosci. Lett.

Neuropharmacology

Neuroscience

Prog. Brain Res.

Gastroenterology

Brain Res.

Differential expression of five N-methyl-d-aspartate receptor subunit mRNAs in the cerebellum of developing and adult rats

J. Comp. Neurol.

Enrichment of unipolar brush cell-like neurons in primary rat cerebellar cultures

Anat. Embryol.

An antagonist-insensitive P2X receptor expressed in epithelia and brain

EMBO J.

Purinergic nerves

Pharmacol. Rev.

Purinergic mechanisms

Ann. N. Y. Acad. Sci.

Purinergic neurotransmission

Semin. Neurosci.

Introduction: P2 receptors

Curr. Top. Med. Chem.

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

J. Neurosci.

Developmental changes in the expression of the chemokine receptor CCR1 in the rat cerebellum

J. Comp. Neurol.

NMDA-receptor channel diversity in the developing cerebellum

Nature

The unipolar brush cells of the rat cerebellar cortex and the cochlear nucleus are calretinin-positive: a study by light and electron microscopic immunocytochemistry

Anat. Embryol.

Research report
Changes in expression of P2X purinoceptors in rat cerebellum during postnatal development

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