for 3 minutes. To monitor the kinetics and timing of the perfusion, we included a fluorescent dye (Alexa Fluor 568) together with the glutamate. One hour after the glutamate application, neurons were fixed and immunostained with a pCREB antibody. Analysis of pCREB signal revealed a significant increase in the cell body following spaced application of glutamate. A single, massed application of glutamate for 3 minutes or a spaced application of a vehicle solution did not produce such an increase. These results suggest that in vitro localized spaced stimulation activated new gene expression, paralleling long-term memory formation in whole organisms. These results further demonstrate the benefits of the μLP chamber for studying the cellular mechanisms of synaptic plasticity using more physiologically relevant parameters (i.e., improved spatial and temporal resolution) than traditional culture methods.
