Increases in NMDAR1 mRNA Levels are Involved in the Generation of Local Afterdischarges in Amygdaloid‐Kindled Rats
Authors: Kikuchi Shuichi , Iwasa Hiroto , Miyagishima Hiro , Hasegawa Shuji 
Published: August 2009

Purpose: Changes in neural transmission via N‐methyl‐D‐aspartate (NMDA) receptors (NMDARs) play a crucial role in seizure generation and epileptogenesis. Our previous study suggested that NMDAR1 contributes to the acquisition and maintenance of epileptogenesis in the kindled brain. However, the factors involved in the changes of NMDAR1 expression are still unclear. To clarify the relation between the induction of afterdischarges at the primary stimulation site and NMDAR expression in other brain regions, we investigated the levels of gene transcripts (NMDAR1 and NMDAR2A‐2D) during several seizure stages in amygdaloid‐kindled rats by using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). Methods: Twenty male Sprague‐Dawley rats were used. The kindling stimulus was a 50‐Hz biphasic square wave at an intensity abovethe threshold for afterdischarges (fixed at 350 FA). The conditions for sham stimulation were the same as those for kindling, except that theintensity was fixed at 50 μA. The rats in the partially kindled group (K‐P; n = 4) received three stimulations and were analyzed 24 h after the last partial seizure (class 0–2, according to Racine's classification). The rats in the sham‐stimulated group (K‐S; n = 4) were analyzed 24 h after three stimulations without any local afterdischarges or behavioral changes. The rats in the fully kindled group showed 10 consecutive generalized seizures and were analyzed 24 h (K‐I; n = 4) and 4 weeks (K‐11; n = 4) after the last seizure. The rats in the control group (n = 4) were given only a sham operation. Total RNAs were extracted and purified from the cerebral cortex, hippocampus, and cerebellar cortex. The cDNAs were synthesized by using murine leukemia virus reverse transcriptase. PCR was performed with a [32P] deoxycytidine triphosphate (dCTP) and subunit‐specific primers that were reported previously (J Biol Chem 1994;269: 16989–92). P‐Actin was amplified in the same way as a reference. The PCR products were electrophoresed in polyacrylamide gels. The radioactivity of the radiopositive bands was measured with an image analyzer (Fujix Bas 2000). Statistical analysis was done with a Student's unpaired r test. Results: NMDARl mRNA increased significantly in the cerebral cortex bilaterally in the K‐P group (stimulated side, 41%, and unstimulated side, 33%; p < 0.05) in comparison with the control group. whereas no change was observed in the K‐S group. An increase of NMDARl mRNA also was observed bilaterally in the cerebral cortex in rats of both the K‐I (90 and 77%; p < 0.05) and K‐I1 (54 and 49%: p < 0.05) groups. No changes in the NMDARI mRNA levels were detected in other brain regions in any of the groups. NMDAR2C mRNA decreased in the cerebellar cortex of rats in the K‐I group. But in the K‐I1 group, it returned to the same level as that in the controls. The levels of other subunit mRNAs remained unaltered in all brain regions in all of the groups. Conclusions: Because the increase of NMDAR 1 gene transcripts was marked in the K‐P group, but not in the K‐S group, it is suggested that the induction of afterdischarges at the primary stimulation site accelerates the transcription of the NMDARI gene in distant regions transsynaptically. On the other hand, because the decrease in the level of NMDAR2C mRNA in the cerebellum was marked only during the early phase after generalized seizures, the decrease may be related to the seizure activity. As the increase of NMDARI mRNA appeared to be unaffected by any further electrical stimulation over a long period after achievement of full kindling, it is also suggested that NMDAR I plays an important role in the maintenance as well as the acquisition of epileptogenesis in the kindled brain.