A Semi-Automated Approach to Quantifying Synapse Formation in Cultured Iron-Deficient Hippocampal Neurons
Abstract A Semi-Automated Approach to Quantifying Synapse Formation in Cultured Iron-Deficient Hippocampal Neurons Background: Iron plays a critical role in meeting the high energy requirements of neuron structural development, which is required for neural circuit formation and function. Iron deficiency (ID) is one of the most prevalent nutrient deficiencies worldwide. ID is common in pregnant women and children, affecting 40-50% of these populations. ID in fetuses and newborns can result in long lasting and potentially permanent neurobehavioral, cognitive, developmental and metabolic issues, even after iron repletion. Objectives: The aim of this study was to develop a high-throughput method of imaging and quantifying synapses to understand how ID and iron repletion affect the developing neuron. We hypothesized that iron repletion will only partially rescue deficits seen in synapse formation due to deficiency. Methods: Primary neurons derived from the hippocampus of embryonic mice were transfected with a DNA plasmid that expresses a green fluorescent protein-tagged intracellular antibody against postsynaptic density protein 95, PSD95 and grown on coverslips. Cultured neurons were made iron-deficient by treatment with 9μM deferoxamine (DFO), an iron chelator, beginning at 3 days in vitro (DIV). The neurons were treated with iron for one week between 14 DIV and 21 DIV and then fixed for microscopy. A fully automated fluorescent microscope (ZEISS Celldiscoverer 7) was used to image entire neurons with GFP-positive signals at high-resolution. Results: We have developed a semi-automated method to process images of PSD95 puncta. This new method includes applying image deconvolution, background subtraction, and a “top-hat” filter to prepare the images for automated synaptic puncta counting. The results of the effect of ID on neuronal complexity have yet to be analyzed as we are currently in the process of statistically quantifying this data. Conclusions: By using this method, imaging and analysis of neuron structure can be done at increased speed and efficiency. This new approach will allow us to rapidly assess the effects of ID on neuronal development by quantifying the effects of iron status on PSD95 density as a measure of synapse formation. Keywords: iron deficiency, synapse density, PSD95, neuron development, microscopy