Design of a multiple slice interface chamber and application for resolving the temporal pattern of CREB phosphorylation in hippocampal long-term potentiation.

We describe an improved method for the investigation of time-dependent intracellular events giving rise to long-lasting changes in synaptic efficacy. A new interface chamber for the simultaneous superfusion of approximately 30 rat hippocampal slices was designed. The slice chamber contains an upper and a lower medium reservoir connected by a grooved incubation platform which is mounted at an angle of 7 degrees on a thermoregulation unit. Surface slices placed in the chamber are perfused with oxygenated medium at a rate of 1 ml/min and are maintained synaptically viable for at least 6 h. At different time points after induction of long-term potentiation by stimulation of the Schaffer collateral pathway, slices were either fixed in Zamboni's fixative or the CA1 region was excised and lysed in boiling SDS-sample buffer. Fixed 400 microm hippocampal slices were cut into 30 microm sections and immunocytochemically stained with an anti-serine 133 phosphorylated cAMP-responsive element binding protein (pCREB) antibody. Binding of primary antibody was detected with the avidin-biotinylated peroxidase complex method and enhanced using peroxidase-catalyzed deposition of biotinylated tyramine. Staining was visualized with streptavidin-cyanine 3.18 and observed under a confocal laser scanning microscope. CA1 lysates were electrophoresed and subjected to Western blot analysis. Both pCREB immunocytochemical staining and Western blotting showed that CREB is rapidly and transiently phosphorylated after induction of long-term potentiation. pCREB levels peaked within 30 min and declined back to control after 60 min. Immunocytochemistry also showed that pCREB was localized to the nuclei of CA1 pyramidal cells near the tetanization site.