Primary neuronal cultures are an invaluable in vitro tool for examining the fundamental physiological changes that occur in diseases of the central nervous system. In this work, we have used a microfluidic device to grow twin cultures of primary hippocampal neuronal/glia cells which are synaptically connected but environmentally isolated. Immunocytochemical staining, for β-III-Tubulin and synaptophysin, indicated that the two neuronal populations were physically connected and that synapses were present. By dispensing predefined volumes of fluids into the device inlets, one culture was chemically stimulated and the consequent increase in neuronal activity in the opposing culture was monitored using calcium imaging. To optimise the experimental procedures, we validated a numerical model that estimates the concentration distribution of substances under dynamic fluidic conditions, proposing that no cross contamination of chemical stimuli occurred during the experiments. Calcium imaging and local chemical stimulation were used to confirm synaptic connectivity between the cultures. Chemical stimulation of one population, using KCl or glutamate, resulted in a significant increase of calcium events in both neurons and astrocytes of the connected population. The integration of the system and techniques described here presents a novel methodology for probing the functional synaptic connectivity between mixed primary hippocampal co-cultures, creating an in vitro testing platform for the high-throughput investigation of synaptic activity modulation either by novel compounds or in in vitro disease models.