Abstracts

RATIONALE: NMDA receptor (NMDAR) function can be modulated by phosphorylation of the NR1 serine 897 site. Forskolin (FSK), an adenylate cyclase activator, increases NR1 phosphorylation through activation of the adenyl cyclase-protein kinase A (PKA) pathway, and we have reported that FSK enhances epileptiform activity in hippocampal slices. The goal of this study was to investigate whether the seizure enhancing effects of FSK are mediated by NMDA receptors. We examined whether FSK increases phosphorylation level of NMDA receptor NR1 subunit in postsynaptic density (PSD) fraction and whether the effect of FSK on tetanus induced afterdischarge is affected by the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV).
METHODS: Hippocampal slices were prepared from 15-22 days old rats. Electrographic seizures were induced by a tetanus (2 s, 100 Hz) delivered by an electrode placed in area CA3. Field potentials were recorded in CA3 pyramidal cell body layer. FSK was administered alone or with APV by bath application 30 min prior to stimulation. Numbers of spikes and the duration of afterdischarges were measured to evaluate the effect of APV. To determine the phosphorylation level of NMDA receptor NR1 subunit, slices were taken 30 min after drug application. Triton X-100 insoluble membrane samples (PSD fraction) were prepared for western blot analysis. Blots were first probed with an antibody against phospho-serine 897 (a PKA site) of NR1 and then reprobed with an antibody against NR1 C terminal. NR1 phosphorylation was calculated using the density ratio of these two signal bands.
RESULTS: Perfusion of hippocampal slices with FSK (50 [mu]M, 30 min) caused an increase in serine 897 phosphorylation of NR1 to 402% of the control level (FSK = 2.37[plusminus]0.39, control = 0.589[plusminus]0.09, p = 0.004, n = 5) in Triton X-100 insoluble membrane samples. The total amount of NR1 in the samples was not altered (102% of the control level). In comparison to the control group (n = 14), FSK treated slices (n = 8) had significantly higher spike numbers (FSK = 223[plusminus]42, control = 53[plusminus]11, p [lt] 0.001) and longer afterdischarge duration (FSK = 96 s[plusminus]25, control = 22 s[plusminus]2, p [lt] 0.001). When slices (n = 14) were treated with APV (100 [mu]M) along with FSK, APV prevented the increase in spike numbers (57[plusminus]6, p [lt] 0.001 versus FSK, p = 0.86 versus control) and afterdischarge duration (20[plusminus]2, p [lt] 0.001 versus FSK, p = 0.915 versus control).
CONCLUSIONS: FSK increases serine 897 phosphorylation of NR1 in the PSD fraction. The NMDA receptor antagonist APV attenuates FSK-enhanced ictal activity. The effects of FSK on ictal activity and NR1 phosphorylation occur within minutes. The results suggest that the phosphorylation state of the NMDAR NR1 serine 897 site may regulate seizure activity. Post-translational modification of NMDARs may be an important early event in epileptogenesis.
[Supported by: NIH grant RO1 NS 31718-10 (FEJ)]