|Budget Amount *help
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥1,600,000 (Direct Cost: ¥1,600,000)
Purpose : The aim of present study is to clarify the presynaptic function following epileptic seizures by estimating the change of mints in the brain.
Methods : Adult male Sprague Dawley rate weighing 200-250 g were used. All animals were pretreatment with injections of atropine methylbromide 5 mg/kg intraperitoneally. Epileptic seizures were produced by intraperitoneal injection of pilocarpine hydrochloride 320-350 mg/kg. Seizure activity was monitored behaviorally, and after 3-4 hours of convulsive status epileptics, seizures were terminated with diazepam 10 mg/kg. Control rats were received saline only. Only animals that displayed continuous, convulsive seizure activity after pilocarpine treatment were used in the following experiments. Animals were perfusion-fixed with 4 % paraformaldehyde solution 2, 4, 6 hr, 1, 2, 3, 4, 7, 14 day 6 weeks after epileptic seizures (n=2-3 in each group). Serial coronal sections (20 μm in thickness) were obtained from the fixed brains with a vibratome
and then subjected to immunocytochemistry for mint1, munc18, syp2. Immunostaining was performed with rabbit polyclonal antibody against rat mint1, munc18, syp2 as a primary antibody, followed by the ABC method. After reaction with respective primary antibodies, immunodetection was performed using the Vectastain ABC Elite Kit followed by reaction product visualization using 0.05 % diaminobenzidine.
Results : In control rats (n=3) hippocampus, the immunoreactivity of mint1 was stained in the afferent fiber from the adjacent entorhinal cortex and mossy fiber from granule cell layer, in the molecular layer of the dentate gyres. The immunoreactivity of munc18 was uniformly stained in the hippocampus. Syp2 revealed in the mossy fiber and dentate gyros, In mossy fiber, the immunoreactivity of mint1 increased from 2 to 7 days after epileptic insult. On the other hand, there was no significant different of the immunoreactivity for munc18 and syp2 between control and epileptic mice in this area. In the dentate gyres, the immunoreactivity of mint1, munc18 and syp2 were greatly strained in the molecular layer. At the day 14, they marked distributed in the inner layer of molecular cell layer. These immunoreactivity continued until 6 weeks after initial epileptic status.
Comments : The present study demonstrated that transient presynaptic dysfunction in the excitatory neurons to CA1 pyramidal neurons be prolonged since mint1, but not munc18 nor syp2 in the mossy fiber marked distributed until 7 days. These suggested that mint1 played the important role of neurotransmission from the entorhinal cortex to hippocampus following epileptic seizures. Less