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Mossy fiber sprouting (MFS) is a pathological phenomenon that is commonly observed in temporal lobe epilepsy (TLE). However, the molecular mechanisms underlying MFS remain unclear. It has been demonstrated that the tau protein is important in the progression of MFS by the regulation of microtubule dynamics and axonal transport, with all of these functions of tau modulated by its site-specific phosphorylation. Glycogen synthase kinase-3β (GSK-3β) is an active kinase that regulates the phosphorylation of tau protein. Therefore, it was hypothesized that GSK-3β contributes to MFS by phosphorylating tau protein. The aim of the present study was to determine the expression and activity of GSK-3β at different regions in the rat hippocampus during the pentylenetetrazole (PTZ)-kindling process in order to demonstrate the possible correlation with MFS, and to investigate the involvement of GSK-3β in epileptogenesis. Male Sprague-Dawley rats (n=180) were randomly divided into the control and PTZ-treated groups. The chronic epileptic model was established by intraperitoneal injection of PTZ and the hippocampus was observed for the presence of MFS using Timm staining. GSK-3β mRNA, protein and activity were analyzed in various regions of the hippocampus using in situ hybridization, immunohistochemistry and immunoprecipitation followed by a kinase assay and liquid scintillation counting, respectively. MFS was observed prior to kindling and an increased distribution of Timm granules were observed in the CA3 region of the PTZ-treated rats; however, this was not demonstrated in the supragranular layer of the dentate gyrus. The expression of GSK-3β mRNA and protein, as well as the GSK-3β activity, increased significantly from 3 days to 4 weeks in the PTZ group, and this was correlated with the progression of MFS in the CA3 area. In addition, it was demonstrated that MFS did not result from TLE. GSK-3β may therefore be involved in the progression of MFS and is important in epileptogenesis. An understanding of the molecular mechanisms underlying MFS may lead to the identification of a novel therapeutic target to limit epileptogenesis.

Objective: To observe the expression of cyclin-dependent kinase 5 (Cdk5), p35, tau protein and the activity of Cdk5 in rat hippocampus during pentylenetetrazole (PTZ) kindling process and their correlation with mossy fiber sprouting (MFS) so as to investigate the role of Cdk5/p35 in epileptogenesis., Methods: A total of 240 healthy male SD rats were divided randomly into normal controls and pentylenetetrazole (PTZ) treatment groups. The epileptic models were established by injection of PTZ intraperitoneally. At Day 3, Weeks 1, 2, 4 & 6 after a daily injection of PTZ, Timm staining was scored in the CA3 region and dentate gyrus. At the same time, the mRNA and protein of Cdk5 and p35, total tau protein and its phosphorylation at ser202 and Cdk5 activity were analyzed in the hilus and stratum granulosum of dentate gyrus and the CA1, CA3 regions of hippocampus. The methods of in situ hybridization, immunohistochemistry, Western blot and immuno-precipitation and liquid scintillation counter were employed respectively., Results: Prominent MFS was observed in area CA3 rather than the inner molecular layer in PTZ-treated rats. And the degree of MFS progressed with the development of behavioral kindled seizures. The expressions of Cdk5/p35 mRNA and protein, tau protein and its phosphorylation at Ser202 significantly increased from Day 3 to Week 4 in the PTZ treatment group. It was in accordance with the progression of MFS in area CA3., Conclusion: Cdk5/p35 and its substrate tau protein may be involved in MFS. Understanding the molecular mechanisms of MFS may lead to therapeutic interventions for limiting epileptogenesis.

Background: The most well-documented synaptic reorganization associated with temporal lobe epilepsy is mossy fiber sprouting (MFS), which is believed to play a critical role in epileptogenesis. However, the molecular mechanisms underlying this phenomenon remain unclear. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase which is found to be crucial in axon growth and synaptic plasticity. We hypothesized that Cdk5 contributed to MFS via phosphorylating its substrate tau protein, which was known to facilitate microtubule stabilization and axonal elongation., Methods: 240 male SD rats were randomly divided into the control group and PTZ group. The epileptic models were established by intraperitoneal PTZ injection, while the control rats were injected with an equal dose of saline. At different time points, Cdk5/p35 mRNA and protein, total tau protein and its phosphorylation at Ser202 (p-tau) and Cdk5 activity were analyzed in different regions of hippocampus by in situ hybridization, immunohistochemistry, Western blot, immuno-precipitation and liquid scintillation counter. Hippocampus was also evaluated for MFS with Timm stain., Results: Prominent MFS was observed in area CA3 rather than the inner molecular layer in PTZ treated rats and the degree of MFS progressed with the development of behavioral kindled seizures. The expression of Cdk5/p35 mRNA and protein, tau protein and its phosphorylation at Ser202 significantly increased from 3 days to 4 weeks in the PTZ group, which was in accordance with the progression of MFS in area CA3., Conclusions: Cdk5/p35 and its substrate tau protein may be involved in MFS. Understanding the molecular mechanisms underlying MFS may lead to therapeutic interventions that limit epileptogenesis.

Objective: To observe the expression of cyclin-dependent kinase 5 (Cdk5) and p35 in rat hippocampus during pentetrazole kindling process and their relation with mossy fiber sprouting (MFS), and to investigate the role of Cdk5/p35 in epileptogenesis., Methods: Altogether 120 healthy male SD rats were randomly divided into a control group and a pentylenetetrazole (PTZ) treated group. The epileptic models were established by the injection of PTZ intraperitoneally while the control rats were injected with an equal dose of saline. At the 3rd day, 1st week, 2nd week, 4th week, and 6th week after daily injection, Timm staining was performed in area CA3 and dentate gyrus, and the mRNA and protein of Cdk5 and p35 were analyzed in the hilus and stratum granulosum of dentate gyrus and area CA1 and CA3 of hippocampus, by in situ hybridization and immunohistochemistry, respectively., Results: The expression levels of Cdk5 and p35 mRNA were significantly higher in the PTZ treated subgroups of the 3rd day, 1st week, 2nd week, and 4th week than those in the controls. Thereafter, the expression decreased to the level of controls. The expression level of Cdk5 and p35 protein increased from the 3rd day to 2nd week, and then gradually decreased to the level of the controls. Timm scores for PTZ groups were 1 to approximately 4 before kindling and 4~5 after kindling in area CA3., Conclusion: Change of Cdk5/p35 expression in the hippocampus may play a role in epileptogenesis by influencing the process of mossy fiber sprouting.

Objective: To determine the changes of mossy fiber sprouting in hippocampus of pre-kindling and post-kindling rats of chronic epilepsy induced by pentylenetetrazole (PTZ)., Methods: Sixty rats were randomly divided into a control group and a PTZ group (PTZ 30 mg/kg, intraperitoneal injection, once daily). The changes of mossy fiber sprouting in hippocampus of pre-kindling and post-kindling rats were examined by Timm staining., Results: Before the occurrence of convulsion confirmed by behavior and EEG, the mossy fiber sprouting was found in the PTZ group. The grade of the mossy fiber sprouting increased with the gradual establishment of kindling effect., Conclusion: Mossy fiber sprouting may play an important role in the onset and development of epilepsy.