As described earlier, one benefit of the three-inlet μLP chamber design is the ability to control perfusions with improved temporal resolution. In order to demonstrate this function, we sought to investigate how different patterns of neurotransmitter stimulation alter transcriptional responses in postsynaptic neurons. Previous work in Drosophila and Aplysia has shown that spaced, but not massed, presentation of conditioning stimuli leads to long-term memory formation, mediated by phosphorylation of CREB and new gene expression (Carew et al., 1972; Yin et al., 1994). Here we examined the ability of spaced versus massed applications of glutamate to increase pCREB in neuronal cell bodies (Figure 7). Using the perfusion channel in the μLP chamber we delivered 3 one minute pulses of glutamate to dendrites with 5 minute rest periods between each pulse (spaced condition). In the massed application condition glutamate was applied continuously 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
