Monitoring neurite morphology and synapse formation in primary neurons for neurotoxicity assessments and drug screening

Synapse formation during nervous system development and degeneration in the pathogenesis of human neurological diseases are highly regulated processes. Subtle changes in the environment of the complex neuronal network may cause either breakdown or creation of synaptic connections. Drug discovery screening for neurological diseases and compound neurotoxicity evaluation would benefit from robust, automated, quantitative in vitro assays that monitor neuronal function. We hypothesized that (1) toxic insults to the nervous system will cause neuronal synapses to deteriorate in the early phase of neurotoxicity, eventually leading to neurite degeneration and neuronal cell death if the damage is severe; and (2) an in vitro functional assay for synapse formation and neuronal morphology could be used to monitor and identify such neurotoxic events. We thus developed an automated, functional, high-content screening (HCS) imaging assay to track and quantify the dynamic changes in neurites and synapses. This assay facilitates automation and streamlining of a laborious process in drug discovery screening and compound neurotoxicity assessment. The assay also enables quantitative comparisons between compounds that affect neuronal morphology and function, particularly in neuriteand synapse-associated events. Introduction Neurons in central and peripheral nervous systems function to transmit electrical signals from one location to the other to keep the brain and the body functioning properly. One of the critical structures in the neuron to maintain their proper functional network is the synapse, which is the junction between a nerve cell and the cell that receives an impulse from the neuron. The molecular network between these synapses controls not just synaptic signal transmission and synaptic plasticity but also regulates neuronal growth, differentiation, and death. The microstructure of synaptic junctions has been extensively studied to understand the relationship between synaptic activity and neuropathophysiology, as well as the molecular mechanism involved in synaptogenesis and regulation of the synapse. Once synaptic function is disrupted by natural or manmade neurotoxic substances, it could lead to longlasting and often irreversible neuronal damage. Synaptic damage has often been recognized as the first sign of neurodegeneration in many different pathological conditions, including traumatic nerve injury, ischemic stroke, and many neurodegenerative disorders such as motor neuron diseases, Alzheimer’s, Parkinson’s, and Huntington’s diseases. Many synaptic proteins play an important role in the progression of neurodegenerative diseases. For example, amyloid beta precursor protein, presenilin, alpha-synuclein, huntingtin, ataxin-1, frataxin, and prion proteins are all involved in presynaptic or postsynaptic structure of the neuron and play a role in synaptic damage and neurodegeneration. To measure the synaptic changes that occur in synaptogenesis or synaptic damage, we needed to develop a reliable, accurate, and efficient method to measure accurate synaptic loss, neurite changes, and neuronal death. Here we introduce a new way of measuring synaptic function utilizing the power of automated, quantitative, high-content, cell-based imaging and analysis. Results Thermo ScientificTM HCS synaptogenesis reagents combined with the Thermo ScientificTM ArrayScan VTI HCS Reader and Neuronal Profiling BioApplication enables the quantitation of neuronal morphology and synapses in vitro (Figure 1). On-the-fly automated image analysis and quantitation accompanying the automated image acquisition is done by the Neuronal Profiling BioApplication, which is an automated image analysis software module on the ArrayScan VTI HCS Reader. Using this technology and assay method, we could identify synaptic changes over time and measure synaptic and neurite parameters in an automated manner. The assay identified primary neuronal cells by a neuronspecific marker and detected synapses on the spines of neurites with preand postsynaptic markers (Figure 2 and Table 1). The multiplexed targets, including a nuclear marker, were simultaneously detected with four fluorescent colors, and the fluorescent images of the labeled neurons and synapses were acquired by automated imaging (Figure 3). The phenotypic features of neuronal morphology and the synapse were automatically identified and quantified in real time. Such features are potential indicators for neuronal development, differentiation, and neurotoxicity, and we could quantify changes in these features under different conditions and for different drug treatments (Figures 4 and 5). By monitoring changes in these features, we could also quantitatively evaluate compounds involved in developmental neurotoxicity (Figure 6). Table 1. Potential synaptogenesis HCS assay targets can be detected in four different colors. Fluorescence channel Cellular entity targeted Candidates for cellular target (best target screened in bold) Fluorescent dye and color Channel 1 Nucleus DNA DAPI Channel 2 Cell body, neurite mask MAP-2, ß3-tubulin, neurofilament DyLightTM 488 Channel 3 Postsynaptic marker PSD95, drebrin, spinophilin/ neurabin DyLightTM 549 Channel 4 Presynaptic marker Synaptophysin, synapsin1, synaptobrevin, synaptotagmin DyLightTM 649 Automated plate delivery: ArrayScan VTI HCS Reader Automated image acquisition Real-time quantitative image analysis: BioApplications Decisions: Thermo ScientificTM HCS Explorer Analysis and visualization: vHCS Discovery ToolBox Automated data management: Thermo ScientificTM Store Figure 1. Seamless integration of steps in cellular analysis using the Thermo ScientificTM HCS platform.