February 15, 2015 (Vol. 35, No. 4)

A Kinetic Co-Culture Assay for Investigating Neurite Development and Toxicity Responses

Regulated changes in axon and dendrite (neurite) morphology over days and weeks are required for the developmental organization and continual remodeling of the nervous system that supports adaptation, function, and maturation. The ability to measure alterations in neurite morphology can provide information regarding changes leading to disease states or effects following exposure to toxic agents, thus providing a rationale for developing technologies to observe and quantify these events.

Studying changes in neurite morphology can be performed in vivo, however these approaches are costly and afford limited throughput. Here we describe a medium throughput in vitro assay providing kinetic measurements of neurite dynamics in a co-culture model of neurons and astrocytes.

An ideal in vitro method to track neurite dynamics would allow continuous, automated measurement of structural parameters, including length and number of branch points, in a nonperturbing manner. This can be achieved in neuronal monocultures using phase images obtained with Essen Bioscience’s IncuCyte ZOOM™ live-cell imaging platform followed by analysis with IncuCyte NeuroTrack™ software. However, a limitation of a monoculture approach is that it does not detect the effects of glial cells on neurite development and function.

In this tutorial, we describe a method for visualizing and quantitating neurite outgrowth in co-culture with astrocytes, utilizing a novel fluorescent label for neuronal processes and the IncuCyte ZOOM live-cell imaging system. The fluorescent reagent, NeuroLight Red™, specifically labels both primary neurons and human iPSC derived neurons when grown in co-culture with astrocytes, allowing for quantitation using the IncuCyte NeuroTrack™ software. This kinetic, 96-well assay method provides dynamic information on neurite outgrowth that is not available from monoculture or end-point experiments and enables pharmacological studies of neuroactive agents.

Labeling Primary Neurons

A co-culture model containing rat E18 primary forebrain neurons grown in co-culture with rat cortical astrocytes (NeuroPrime™ Cell Kit) was used to examine neurite dynamics while capturing important cell-cell interactions. Neurons can be visualized in the phase image shown at the top right panel in Figure 1, although reliable quantification of neurite length was problematic due to obscuring textures presented by astrocytes.

NeuroLight Red is a lentiviral-based reagent that has been designed to efficiently transduce multiple types of neurons with low toxicity. The reagent uses a synapsin promoter to drive neuronal expression of the red fluorescent protein mKate2 and minimize crossover to non-neuronal cells.

NeuroLight Red enables clear visualization and quantification of neurites from primary neurons (compare same fields shown in upper left panel and upper right panel in Figure 1). The fluorescent images of neurons provided sufficient signal amplitude over background to enable the masking of neurites (bottom left, yellow) and cell body clusters (bottom right, blue) using NeuroTrack software and user-defined processing definitions.

Masking these structures allows for the quantitation of several metrics, including neurite length, neurite branching, number of cell body clusters, and cell body cluster area.
In addition, values of neurite length or branching can be normalized to cell body cluster number or area. In addition to rat primary forebrain neurons, NeuroLight Red can label other primary neuronal subtypes (hippocampal, cortical, and dorsal root ganglion) as well as iPSC-dervied neurons (data not shown). 

Figure 1. Fluorescence (upper left) and phase (upper right) images of a rat forebrain neuron and rat cortical astrocyte co-culture. Imaging occurred seven days after plating and infecting with NeuroLight Red. NeuroTrack™ software was used to mask neurites (bottom left, yellow) and cell body clusters (bottom right, blue) using fluorescent imaging. All images show same field.

Kinetic Assay for Neurite Dynamics

Labeling neurons with a flueorescent reagent such as NeuroLight Red provides a means to develop a medium-throughput assay to study neurite dynamics in a co-culture setting. Figure 2 shows data from a 96-well assay measuring neurite length over twelve days using NeuroLight Red labeled rat forebrain neurons with rat cortical astrocytes (NeuroPrime™ Cell Kit). Cells were imaged in an Incucyte ZOOM and neurite length was measured using NeuroTrack software.

An advantage of this approach is that kinetic changes in neurites are characterized over extended times with minimal disturbance as the cells remain in the incubator while measurements are obtained. The top panel in Figure 2 shows a whole-plate view of a 96-well experiment illustrating consistent well-to-well performance.

The pharmacologic sensitivity of this assay was evaluated with agents known to affect neurite length and neuron survival. Historically, excessive glutamate release is associated with neurodegenerative diseases and neuronal injury. Here, glutamate addition causes time and concentration-dependent reductions in neurite length (Figure 2, middle panel; glutamate added 9 days after plating neurons). This glutamate toxicity occurs in the presence of prolonged, chronic exposure (hours to days), where the presence of astrocytes may alter neuronal sensitivity to glutamate treatment.

Paclitaxel is a successful chemotherapeutic, but it can produce peripheral neuropathies and other adverse effects on neuronal function (FDA package insert on TAXOL™). Assessments of these neurotoxic effects are typically done using in vivo models, which provide limited evaluative throughput. CNS assay systems, such as the NeuroPrime Cell Kit, may provide a useful model for studying the off-target neurotoxicity of paclitaxel (Figure 2, bottom panel) and related agents.

Addition of paclitaxel after three days in vitro caused a concentration- and time-dependent decline in neurite length with an estimated EC50 value of 11 nM at 12 days in vitro. Blood levels in clinical settings can exceed this concentration (FDA/CDER TAXOL label).

These data, taken together, suggest that the NeuroPrime Cell Kit in conjunction with the IncuCyte ZOOM live-cell imaging system offers a robust method to continuously measure long term kinetic changes in neurite morphology. The measurements are obtained while the cells remain in a physiologic setting (the cell culture incubator), resulting in minimal disturbance to the underlying biology while providing a quantitative, pharmacologic assay for neurite dynamics.  

Figure 2. Performance of NeuroPrime Cell Kit. Whole plate view of 96-well assay showing changes in neurite length over 12 days for each well. Glutamate added at day 9 (middle panel) and paclitaxel added at day 3 (lower panel) reduced neurite length. Note that each plot point indicates a mean ± SEM (n = 4).

Owen B. McManus ([email protected]) is director, ion channel drug discovery and screening, and Dyke McEwen is principal scientist at Essen BioScience.

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