Immunostaining study of the presynaptic vesicle membrane protein synaptophysin (green), and the postsynaptic marker PSD-95 (red) in control and AgNP-treated neurons. To examine how AgNPs affect synaptic structural and functional components, cells were first cultured for 10 days and allowed to develop synaptic structures. Cells were then exposed to AgNPs at 1, 5, and 10 μg/ml for another 2 days. Neurons were then incubated with antibodies against synaptophysin and PSD-95. Neurons kept under control culture conditions (A, also see inserts) showed extensive neuritic processes in which dense puncta labeling of the synaptic vesicle protein synaptophysin (green) was observed. In addition, both cell bodies and dendritic neurites exhibit high intensity of red fluorescent labeling of PSD-95. Co-localized spots (yellow) represent the location of potential synapses. However, AgNP treatment even at low concentration of 1 μg/ml drastically reduced both the green fluorescent synaptophysin and red fluorescent PSD-95 (B). The reduction in fluorescent intensity of synaptic proteins by AgNPs at the 10 μg/ml was accompanied by injured cell morphology and compromised network integrity (C). The data shows that the mean fluorescent intensity of both the synaptophysin (D) and PSD-95 (E) was significant decreased by AgNPs at the concentrations examined. Scale bars, 20 μm (A,B,C) and 5 μm (inserts).