3-mercaptopropionic acid-induced seizures decrease NR2B expression in Purkinje cells: cyclopentyladenosine effect

Abstract

Inhibitory mechanism of cerebellum epileptic activity can be involved depending on the intensity and frequency of seizure convulsions. N-methyl-D-aspartate receptors (NMDARs) play key roles in excitatory synaptic transmission and have been implicated in neurological disorders: in cerebellum, they have specific characteristics. NMDARs are heteromeric complexes, and the expression of functional receptors in mammalian cells requires the subunit NR1 (essential) and one NR2 subtype of the four isoforms: NR2A-NR2D. In mature Purkinje cells, the combination of NR1 with NR2B subunits forms functional NMDARs; NR2B subunit may be altered in exocitotoxic events. Cyclopentyladenosine (CPA), an adenosine analogue, administered to rats, for one or more days, increases seizure threshold induced by the convulsant drug 3-mercaptopropionic acid (MP). In this study, we focused on the expression of NR2B in cerebellum after repetitive seizures induced by MP and the effect of adenosine analogue CPA administered alone or previous to MP (CPA + MP). A significant decrease in NR2B in the whole cerebellum was observed after MP and CPA administration with a tendency to recover to normal values in the combined treatment of CPA administered 30 min before MP by Western blot assay. In immunohistochemical studies, NR2B expression was observed and analysed in Purkinje cells. NR2B expression was decreased after MP (55%) and CPA (12%) administration, and CPA injected 30 min before MP led to 28% reduction in Purkinje cells. These results could be related to Purkinje cell damage or alternatively to avoid the excitotoxic effect. Results recorded after CPA + MP treatment seemed involved in decreasing the convulsant MP effect.

Fig. 1

NR2B cerebellar expression. a Western blot for NR2B. Control(C). 3-mercaptopropionic acid (MP), Cyclopentyladenosine (CPA) and CPA 30 min before (CPA + MP). Each lane represents 23 μg of pooled membrane proteins. b The bar graphs illustrate the percent change with respect to control. P < 0.05

Fig. 2

Photograph of NR2B immunostaining in cerebellum. Different layers are indicated: Molecular (M), Purkinje cell (P), and Granular (G). Original magnification ×40

Fig. 3

Photograph of cerebellum in each treatment. C Control, MP convulsant treatment, CPA cyclopentyladenosine, an adenosine analogue; CPA + MP CPA administered 30 min before MP. Original magnification ×100. White arrows indicate Purkinje cells

Fig. 4

Relative optical density of NR2B immunostained Purkinje cells after Control, MP, CPA and CPA + MP treatment. Values are expressed as the mean ± SEM. ** P ≤ 0.001 and * P ≤ 0.01 related to control. Statistical difference was observed among different treatments (P ≤ 0.01)