Elsevier

Neuroscience

Volume 115, Issue 2, 2 December 2002, Pages 425-434
Neuroscience

Cerebellar lesion up-regulates P2X1 and P2X2 purinergic receptors in precerebellar nuclei

https://doi.org/10.1016/S0306-4522(02)00397-4Get rights and content

Abstract

ATP released in the extracellular space by neuronal injury can influence neighboring neurons via activation of purinergic receptors. In vitro data suggest the involvement of ATP and purinergic receptors as trophic agents in different biological events such as neuritogenesis and cell survival. Recently, in vivo studies have demonstrated modifications in the glial expression of ionotropic purinergic receptors after CNS lesions. In the present study, we investigated the effects of CNS lesion on the neuronal expression of P2X1 and P2X2 receptor subunits by immunohistochemistry and western blotting techniques. In the precerebellar structures of normal animals the expression of P2X1 and P2X2 was lower than previously reported. P2X1 immunostaining was confined only to fibers, while P2X2 immunostaining demonstrated a neuronal expression. After unilateral cerebellar lesion (hemicerebellectomy) axotomized precerebellar neurons underwent marked cell loss; however, some precerebellar neurons did not degenerate. Seven to 35 days after hemicerebellectomy, a transient, time-dependent, marked increase in the number of immunopositive P2X1 and P2X2 neurons was observed in the precerebellar nuclei of the experimental side. An even distribution of immunopositive neurons was present in almost all precerebellar nuclei examined, except for the inferior olive. In this latter structure, differences in the distribution of immunopositive neurons were evident among the subnuclei. Up-regulation of immunoreactivity over relatively long time periods, distribution selectivity and absence of degenerating morphological features in immunopositive neurons suggest that purinergic receptors may have a role in mediating the survival of neuronal responses to axotomy.

The present findings are the first report in the CNS of P2X1 and P2X2 receptor subunit involvement in neuronal reaction to axotomy. They provide in vivo evidence of a correlation between purinergic receptor subunit up-regulation and survival of injured neurons.

Section snippets

Animals and surgical procedures

Experiments were performed using 21 adult Wistar rats (body weight 200–250 g; Harlan, Italy); 18 animals were used for cerebellar lesions (three for immunohistochemistry at each time point: days 7, 14, 21, 28 and 35; three for western blot on day 14) and three as controls for immunohistochemistry. Experiments were performed according to the guidelines of the Italian law for care and use of experimental animals, approved by the Italian Ministry of Health. All efforts were made to minimize both

P2X1 and P2X2 receptor expressions in normal animals

In the precerebellar nuclei of control animals, we observed a widespread network of lightly labeled fibers with no neuronal staining in P2X1-immunoreacted material. In P2X2-immunoreacted material, we observed lightly stained neurons and some sparse positive fibers. In particular, although we did not observe P2X2-immunopositive cells in the ECu, we observed sparse P2X2-immunopositive neurons in the SpV and in the IO. In the LRn, the immunostaining pattern was characterized by a widespread and

Discussion

Our data show a transient and time-dependent up-regulation of P2X1 and P2X2 expression in the precerebellar nuclei of the experimental side after axotomy. Side differences in the number of immunoreactive neurons in the precerebellar nuclei became evident on day 7 and persisted until 35 days after the lesion. In the IO the number of P2X1-immunopositive neurons peaked at 28 days, whereas the number of P2X2-immunopositive neurons peaked at 14 days post-lesion (Fig. 5). In general in IO and Pn,

Acknowledgements

We are grateful to Prof. G. Bernardi for his continuous support and encouragement. This work was supported by Italian Ministry of Health Grant RA0085M-2000 to M.M. and by a grant from MURST Cofin 2001 on ‘Purinoceptors and Neuroprotection’ to C.V.

References (37)

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    Citation Excerpt :

    Resting microglia, characterised by a complex network of processes, migrate to the site of damage after brain trauma, where they are transformed into the activated amoeboid form; ATP has been shown to replicate this transformation (Xiang et al., 2006). Cerebellar lesions produce up-regulation of P2X1 and P2X2 receptors in precerebellar nuclei (Florenzano et al., 2002). Stab wound injury in the nucleus accumbens led to increase in expression of subtypes of P2X and P2Y receptors (Franke et al., 2006a).

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