Assay for neurite outgrowth quantification

Proper neuronal migration and establishment of circuitry are key processes for nervous system functioning. During development, neurons extend numerous processes that differentiate into den-drites and axons. These processes, also termed neurites, are critical for communication between neurons. The characterization of neurite formation , maturation, and collapse/resorp-tion is an area of intense interest because these cellular processes are essential for the interconnection of neuronal cell bodies. Neurites are particularly interesting in relation to neuropathological disorders , neuronal injury/regeneration, and neuropharmacologic research and screening. Nerve transection was once believed to be irreversible, but it has recently become apparent that the inability of damaged nerve fibers to regenerate is an active process under the control of molecules able to inhibit and repulse growing neurites (1–4). Therefore, major efforts in central nervous system drug discovery research are focused on the identification of compounds that affect the growth of neurites. However, the study of neurites is greatly hampered by the difficulty of isolating and quantifying these minute organelles. Currently available methods for measuring neurite outgrowth consist of the manual examination of individual cells under a microscope or the measurement of total fluo-rescence from a labeled neuronal cell population using a fluorescence plate reader (5,6). The caveats of these methods are that the former is labor-intensive and subjective and the latter cannot discriminate between fluorescently labeled neuronal cell bodies and neurites. Consequently , the lack of a means to isolate and purify sufficient neurite material and the lack of a uniform and highly reproducible method of neurite quantifica-tion have impeded an understanding of the role of these organelles in development , injury, and disease states. Using an extension of the traditional Boyden Chamber technology (7), we developed an assay that is quick and efficient for the quantification of neurite outgrowth in a 24-well format. This method is based on the use of a trans-well cell culture insert (chamber), containing a permeable, polycarbonate membrane with 3-µm pores at its base. The inserts are designed to fit into a receiver vessel, which in use contains a solution intended to contact the bottom of the membrane. These permeable membranes should allow for the discrimination between neurites and cell bodies, since neurites have cross sections of less than 0.8 µm while cell bodies average approximately 20 µm. Therefore, by inducing neurites to traverse these membrane pores, a purified population of neurites would appear on the underside of the membrane, distal …