Abstracts

RATIONALE: The serine 845 (ser-845) of the AMPA receptor subunit GluR1 is phosphorylated by protein kinase A (PKA). The phosphorylation state regulates AMPA receptor function. To examine the possible role of PKA-dependent phosphorylation of GluR1 in kindling, we investigated whether activation of PKA increases phosphorylation of GluR1 at postsynaptic densities (PSDs) and whether there is a relationship between GluR1 phosphorylation and tetanus-induced afterdischarges. We have previously shown that pretreatment with forskolin (FSK), an adenylate cyclase activator, for 30 min occludes kindling development.
METHODS: Hippocampal slices were obtained from Long Evans rats (postnatal day 15-22). Repetitive tetanic stimuli (100 Hz, 2 sec, twice maximal intensity, 10 min apart) were delivered to CA3 stratum radiatum to induce afterdischarges. FSK was administered by bath application. Correlation of FSK-induced PSD GluR1 phosphorylation and responses to tetanic stimulation was examined. Parallel experiments evaluated phosphorylation state and responses to tetanic stimulation after 30 min of FSK. To evaluate how long lasting the FSK effects were, phosphorylation state and responses to tetanic stimulation were evaluated in slices treated with FSK for 30 min, followed by 4 hr washout. Slices were collected for western blot analysis. Synaptosomes obtained from sucrose gradient centrifugation were treated with Triton X-100 to extract PSDs. Samples were blotted and probed with antibodies against GluR1 and phospho-ser-845 GluR1. An I-125 labeled secondary antibody was used to reveal signal. The intensity of signal was analyzed by densitometry.
RESULTS: FSK (10-50 [mu]M) for 30 min increased ser-845 phosphorylation in PSDs to 531% of the control value (p [lt] 0.0002, n = 7). Numbers of spikes induced by the first tetanus were significantly higher in the FSK group (control = 11.8[plusminus]3.3, FSK = 80.2[plusminus]17.1, p [lt] 0.05). In addition, kindling development was occluded by FSK. Prolonged wash in control media resulted in a return of phosphorylation of ser-845 to less than 110% of the control value (p [gt] 0.05 vs control) and the increase in initial evoked spike numbers caused by FSK was significantly reduced (33.2[plusminus]6.5, p [lt] 0.05 vs FSK). However, the number of spikes induced by a first tetanus was still greater than control (p [lt] 0.05) and kindling development was observed in only half slices (n = 6).
CONCLUSIONS: 1. Activation of PKA increases ser-845 phosphorylation of GluR1 at PSDs.
2. A 30 min exposure to FSK causes a transient phosphorylation of ser-845 at PSDs; there is a return to baseline after 4 hr.
3. Phosphorylation of ser-845 may have a modulating role in kindling development.
Support: NS31718(FEJ).