Increment of Synapsin I Protein in the Hippocampus of Kindled Rats

Abstract

Introduction: Synapsin is a vesiele‐associated protein considered to be involved in synaptogenesis and neurotransmitter release. Previously, we havc reported that synapsin I mRNA is significantly increased in the bilalcral dentate gyrus after amygdala‐kindled scizures. In this study, we investigated amygdala kindling‐induced changes i n synapsin I protein using immunohistochemistry. Quantitativc densitomctry (Mathern ct al., Ncurosciene, 1997) was performed to evaluatc changes i n synapsin I protein i n the hippocampus.Method: Fifteen inale Sprague‐Dawley rats weighing 300–350 g wcre uscd. A tripolar elcctrode was implanted stcreotaxically in the hasolateral amygdala under pcntobarbital anesthesia. After a recovery period, the rats were subjected to daily kindling stimuli (a 2‐second train of 50 HL, I‐ins rectangular waves at a currcnt intensity of 75–250 PA). Animals with implanted electrodes without atimulation were used as controls (n = 7). They werc then pcrfused 8 hours aftcr the 24–32nd (average 293th) stage 4 or 5 (Racine's classification) generalized seiztirc under anesthesia. The brains were removed and placed in 4% paraloriiialdehyde for 2 days and then transferred to 15% sucrose dissolved in 0.1 M phosphate buffer (PB) for I week. They werc then quickly frozen with dry ice powder, and coronal scctions 20 +in thick wcrc cut with a cryostat. Immunohistochemistry was performcd with polyclonal synapsin I antibody (Calhiochem). The optical density of hippocampus staining was mcasured by a computer‐bascd image analyhtem as an average of the gray value, compensated by the original control sample between white (0) and black (255). Sections were imaged using a chat‐ged‐couplcd devicc camera attached to a microscope and captured and digitized using a Macintosh computer. Illuminance waa unformly maintained and checkcd after cvery I0 measurements using the original control samplc. The image was analysed using imaging software (NIH Image). Measurements were taken at the right and left hippocampus in >2 coronal sections for each animal. Results and Discussion: The kindled slice exhibited bilatcrd increases i n synapsin I protein of approximately 20%–30% in the bilateral str. lucidurn of CA3, and by approximately 20%–40% in the bilateral dentate gyrus. In the hippocampus, neuronal plastic changes including sprouting have becn shown to develop as generalized kindled scizures are repeated. Therefore, the increased synapsin I protein in these brain regions may reilect the enhanced synaptic activity and the prcsynaptic plastic changes in the specific neural network of the hippocampus of kindled rats.