**4.2.1.2 Examination of synaptic formations by immunocytochemical staining and TEM**

To further understand functional activities of neurons on the grid pattern, we observed the microstructure of the neuronal cell body, neuritis extension and processes of the synapse formation by using laser confocal microscope after 7 days in culture. It is found that several neural cell bodies aggregated on the cross point of the grid pattern (Fig.17.b), which neurites extended along the lines clearly by cytoskeletal proteins MAP2 and Synaptophysin double immunocytochemical staining of patterned neurons on PEI(Fig.17.a). Some processes can even gather into a bundle, span the distance between two cross-points and make connection with another neuron. Synaptophysin was a kind of granular protein scattered around the cell bodies and neurites, The neurites from neuron at the cross-point seemed to communicate with those at the lines which express a large number of synaptic vesicle protein(Fig.17.c).

(a) neurites extended along the lines and synaptic vesicle protein around

(b) neural cell bodies at the cross-point and synaptic vesicle protein scattering around them

(c) synapse connection between two neurons at the grid pattern bar=25μm

Fig. 17. Immunofluorescent image of anti-MAP2(red fluorescence) labeled neurons and antisynaptophysin(green fluorescence) labeled synaptic vesicle protein cultured for 7 days on PEI patterns x400

We chose PEI group which can construct more clearly neural network and continued to observe intercellular ultrastructure under transmission electron microscope (Figure18). Two periphery of neurites thicken show high electron density to form a synaptic contact (Red border). Width of synaptic cleft was measured to 30 ~ 50nm. Figure18(a) shows clear synaptic vesicles. Figure18(b) shows the synaptic cleft is relatively narrow and suspected to be electrical synapse structure due to double-membrane structure adjacent closely.
