You are here:
ASSESSMENT OF SYNAPSE FORMATION IN RAT PRIMARY NEURAL CELL CULTURE USING HIGH CONTENT MICROSCOPY.
Citation:
JOSHUA, HARRILL A., T. M. FREUDENRICH, B. ROBINETTE, AND W. R. MUNDY. ASSESSMENT OF SYNAPSE FORMATION IN RAT PRIMARY NEURAL CELL CULTURE USING HIGH CONTENT MICROSCOPY. Presented at Annual Meeting of the Society of Toxicology, Baltimore, MD, March 15 - 19, 2009.
Impact/Purpose:
NONE
Description:
Cell-based assays can model neurodevelopmental processes including neurite growth and synaptogenesis, and may be useful for screening and evaluation of large numbers of chemicals for developmental neurotoxicity. This work describes the use of high content screening (HCS) to detect chemical effects on synaptogenesis in vitro. HCS was performed with a Cellomics ArrayScan VTiTM, an automated epifluorescence microscope and image analysis system using cells in a microtiter plate format. Cerebellar granule cells (CGCs) from PND7 LE rats were seeded at 1.33x105 cells/cm2 on laminin coated 96-well plates and grown for 2, 3, 5, 7, 9 or 14 days in vitro (DIV). Cells were then fixed and fluorescently labeled with antibodies against III-tubulin and syanpsin I to visualize neuronal cells bodies and qualitatively assess the development of pre-synaptic terminals. Synapsin staining was diffuse up to DIV3. Synapsin puncta appeared at DIV5 and were observed up to DIV14. DIV7 CGCs were then used to optimize a protocol for neurite and syanpsin I quantification using an ArrayScan VTiTM. The number of synapsin puncta and total area of synapsin staining associated with neuronal cell bodies and neurites were used as endpoints to measure synaptogenesis. CGCs were then treated with 3 or 10 µM of the PKC inhibitor Bis1, the MEK inhibitor U0126, the Src kinase inhibitor SU6656 or vehicle control (0.1% DMSO) at 2 h post-seeding and evaluated on DIV7 with the ArrayScan protocol (n = 4-6 wells/group). 3 µM SU6656 increased the density of synaptic puncta associated with neurites and cell bodies on DIV7. 3 µM Bis1 and 10 µM U0126 decreased the number and total area of synapsin puncta associated with cell bodies and neurites. 10 µM Bis1 produced large reductions in synapsin staining and decreases in total neurite length (16% of control), indicative of overt cellular toxicity. These data demonstrate that this method can be used as a screening tool to detect chemical effects on synaptogenesis in primary cultures. This abstract does not necessarily reflect USEPA policy.