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