High-content imaging analysis for detecting the status of synapse using drebrin in cultured neurons

抄録

The status of the synapses is associated with brain function. We have used an actin-binding protein, drebrin in the postsynaptic sites of glutamatergic synapses as a marker to detect the status of synapses. Our previous studies have shown that activation of NMDA-type glutamate receptors and application of amyloid β oligomers decrease the amount of drebrin in synapses. To increase the availability of this method to detect the effects of various drugs on synapses, we applied the drebrin-based evaluation of synapses to high-content image analysis using microplates. We used frozen stocks of hippocampal neurons to minimize the variation among experiments. After 3 weeks in vitro, neurons were fixed and processed for immunocytochemistry to visualize the synaptic status (drebrin), dendrite length (MAP2) and neuronal cell bodies (nuclear staining). After automated image acquisition, total number of drebrin clusters per fields, drebrin cluster density along dendrites, dendritic length and neuronal number were automatically quantified by a custom designed protocol. To evaluate the efficacy of this analysis, we analyzed the effect of glutamate on these parameters. 30-100 μM glutamate significantly reduced the drebrin cluster density without affecting neuronal number and dendritic length. These results suggest that this high-content analysis for synapses using drebrin will be useful for detecting the effect of drugs that may affect brain function. Supported by AMED 17bk0104077h0001.