Abstract
A cellular response consequent to the interaction with NMDA receptor is useful for predicting toxicological action of chemicals. Drebrin is an actin-binding protein that governs the dendritic spine formation of CNS neurons and is responsible for the morphological plasticity of dendritic spines. The drebrin exodus from dendritic spines is available as a marker of the activation of NMDA receptors in neurons. The subcellular localization of drebrin is determined by glutamate receptor activity, and when drebrin is lost in dendritic spines, the learning and memory mechanism does not function properly. In the present study, we have observed drebrin and MAP2 distribution in neurons with a confocal quantitative image cytometer, CQ1 (Yokogawa, Japan), and developed new algorithms to quantify the glutamate receptor binding agonist-induced changes in the distribution pattern of their immunoreactivity. Cultured hippocampal neurons (21 DIV) were fixed after glutamate treatments and processed for immunocytochemistry to visualize drebrin, MAP2 and cell nucleus. After automated image acquisitions, neuron number, dendrite length, and drebrin clusters were automatically quantified with originally developed algorithms. In particular, the intensity distribution analysis of drebrin clusters is highly sensitive. Identification of drebrin clusters and analysis of their distribution are promising to detect drebrin exodus induced by a low concentration of glutamate receptor binding agonists.
