High-content imaging analysis for detecting the loss of drebrin clusters along dendrites in cultured hippocampal neurons.

INTRODUCTION: Detection of drug effects on neuronal synapses is important for predicting their adverse effects. We have used drebrin as a marker to detect the synaptic changes in cultured neurons. High concentration of glutamate decreases the amount of drebrin in synapses. To increase the availability of this method for high throughput analysis, we applied the drebrin-based evaluation of synapses to high-content imaging analysis using microplates. METHODS: Three weeks old cultured neurons were fixed and processed for immunocytochemistry to visualize drebrin clusters, dendrites and neuronal cell bodies. After automated image acquisition, total number of drebrin clusters per fields, linear density of drebrin cluster along dendrites, dendrite length and neuron number were automatically measured by a custom-designed protocol. RESULTS: Automated image acquisition and analysis showed that dendrite length and drebrin cluster density along dendrites are measured consistently and reproducibly. In addition, application of 10-100 µM glutamate for 10 min or 0.5-50 µM latrunculin A for 5 min significantly decreased drebrin cluster density without affecting neuron number. These results were consistent with our previous results using manual image acquisition and analysis with regular fluorescence microscope and image analysis software. Furthermore, 0.3 or 1.0 µM staurosporine for 24 h significantly decreased neuron number. DISCUSSION: The present study demonstrates that this high-throughput imaging analysis of drebrin cluster density along dendrites for detecting the effects of substances on synapses is sensitive enough to detect the effects of glutamate receptor activation and latrunculin A treatment, and indicates that this analysis will be useful for safety pharmacology study.

X Irradiation Induces Acute Cognitive Decline via Transient Synaptic Dysfunction.

Cranial X irradiation can severely impair higher brain function, resulting in neurocognitive deficits. Radiation-induced brain injury is characterized by acute, early and late delayed changes, and morbidity is evident more than 6 months after irradiation. While the acute effects of radiation exposure on the brain are known, the underlying mechanisms remain unclear. In this study, we examined the acute effect of X radiation on synaptic function using behavioral analysis and immunohistochemistry. We found that 10 Gy whole-brain irradiation immediately after conditioning (within 30 min) impaired the formation of fear memory, whereas irradiation 24 h prior to conditioning did not. To investigate the mechanisms underlying these behavioral changes, we irradiated one hemisphere of the brain and analyzed synaptic function and adult neurogenesis immunohistochemically. We focused on drebrin, whose loss from dendritic spines is a surrogate marker of synaptopathy. The intensity of drebrin immunoreactivity started to decrease in the irradiated hemisphere 2 h after exposure. The immunostaining intensity recovered to preirradiation levels by 24 h, indicating that X radiation induced transient synaptic dysfunction. Interestingly, the number of newly generated neurons was not changed at 2 h postirradiation, whereas it was significantly decreased at 8 and 24 h postirradiation. Because irradiation 24 h prior to conditioning had no effect on fear memory, our findings suggest that radiation-induced death of newly-generated neurons does not substantially impact fear memory formation. The radiation-induced synaptic dysfunction likely caused a transient memory deficit during the critical period for fear memory formation (approximately 1-3 h after conditioning), which coincides with a change in drebrin immunostaining in the hippocampus, a structure critical for fear memory formation.