Cortical spreading depression (CSD) is a robust method for the induction of brain preconditioning, i.e. the adaptive cytoprotection that protects against subsequent, potentially lethal insults (Obrenovitch et al. 2002). We are interested in delineating the mechanisms underlying brain preconditioning with a view to identifying novel neuroprotection strategies. Recent studies have shown that reduction in cell-surface expression AMPA receptors protected primary hippocampal neurons from ischaemic stress (Ralph et al. 2001). In addition, up-regulation of α7 nAChRs has been proposed to explain the neuroprotection afforded by chronic nicotine treatment of neuronal cell cultures (Jonnala & Buccafusco, 2001). Here we report recent findings focusing on effects of CSD-induced preconditioning upon the glutamatergic and cholinergic systems.
All animal procedures complied with the UK Animals (Scientific Procedures) Act, 1986. Adult male C57B1/6 mice (25Ð30 g) were anaesthetized with halothane and placed in a stereotaxic frame. Ten full consecutive recurrent CSDs were elicited by epidural application of 1 M KCl to the right occipital cortex, as previously described (Obrenovitch et al. 2002). In sham-operated controls, 1 M KCl was replaced by physiological saline. Animals were re-anaesthetized 24 h later, killed by decapitation, the brain removed, and the whole hemicortices dissected out and rapidly frozen in liquid nitrogen. P2 membranes from individual sham- and KCl-treated right hemicortices were prepared and subjected to quantitative immunoblotting (Obrenovitch et al. 2002). Four mice were used in each treatment group. Polyclonal anti-rodent NR1 (17Ð35), NR2A (1381Ð1394) and NR2B (46Ð60) antibodies (Chazot et al. 2002) were used at a final protein concentration of 1 mg ml-1. Polyclonal anti-GluR1 and GluR2 antibodies were used at a concentration of 1/3000. Monoclonal anti-α7 nAChR subunit (Martin-Ruiz et al. 2000) was used at 1/5000. Loading variations were standardized by reprobing the immunoblots with monoclonal anti-β-actin antibodies (1/1000). Immunoblots were quantified by densitometry using a Gel Doc 2000 system. Optical density values (standardized with β-actin) were compared using Student’s unpaired t test with a significance level of P < 0.05.
In comparison with the sham-treated samples, KCl treatment resulted in a modest parallel reduction in both GluR1 (30 ± 13 %) and GluR2 (28 ± 13 %) expression (means ± S.D., n = 4 of each group, P < 0.03). No significant changes were detected in NR1, NR2A or NR2B immunoreactivities (n = 4 of each group, P > 0.05). In marked contrast, a profound increase in α7 nAChR subunit expression was observed (15 ± 4-fold; mean ± S.D., n = 4 of each group, P < 0.001) following preconditioning. In conclusion, these changes in AMPA and neuronal nicotinic receptor protein expression may underlie in part the adaptive cytoprotection afforded by CSD. The anatomical profile and the physiological consequences of these changes in protein expression are currently under investigation.
Chazot, P.L. et al. (2002). Neuropharmacology 42, 319Ð324.
Jonnala, R.R. & Buccafusco, J.J. (2001). J. Neurosci. Res. 66, 565Ð572.
Martin-Ruiz, C.M. et al. (2000). Neuropharmacology 39, 2830Ð2839.
Obrenovitch, T.P. et al. (2002). Neurosci. Lett. 320, 161Ð163.
Ralph et al. (2002). Mol. Cell. Neurosci. 17, 662Ð670.
This work was supported by the European Commission, contract no. QLG3-CT-2000-00934 and The Wellcome Trust (UK).
All procedures accord with current UK legislation.