Fig. 3. Long-term stimulation of neuronal cultures. Rat hippocampal neurons were isolated from newborn pups and plated at high density on silicon wafers. After cultures were established (7 DIV) the chips were placed into parallel stimulation devices, the control chip receiving no stimulation whereas the second chip received periodic high frequency stimulation in a circular region at the center of the wafer. Following stimulation for 1–3 days, chips were removed from the device and fixed, and then processed for immunostaining with anti-bassoon antibody. Bassoon is a presynaptic active zone protein, allowing the visualization of synapses. Images are acquired at low magnification and joined together to span the entire chip. (A) Unstimulated network, (B) central region of stimulated chip. Several mathematical methods were used to analyze the synaptic distribution; the first steps in analysis include deconvolution, thresholding and watershed analysis of the synaptic distribution images. (C) Sample synaptic density profiles represented by density distribution, illustrating altered patterns that result from long-term stimulation. Pseudo-color scale represents regions of low to high synaptic density.