Use of high content image analyses to detect chemical-mediated effects on neurite sub-populations in primary rat cortical neurons

Abstract

Traditional developmental neurotoxicity tests performed in vivo are costly, time-consuming and utilize a large number of animals. In order to address these inefficiencies, in vitro models of neuronal development have been used in a first tier screening approach for developmental neurotoxicity hazard identification. One commonly used endpoint for assessing developmental neurotoxicity in vitro is measurement of neurite outgrowth. This biological process is amenable to high-throughput measurement using high content imaging (HCI) based methodologies. To date, a majority of HCI studies of neurite outgrowth have focused on measurements of total neurite outgrowth without examining whether stereotypic neuronal growth patterns are disrupted or whether specific sub-populations of neurites (i.e. axons or dendrites) are selectively affected. The present study describes the development and implementation of two HCI based analysis methods for assessing chemical effects on neuronal maturation. These methods utilize the stereotypical growth pattern of primary rat cortical neurons in culture (i.e. the Staging Method), as well as the differential cytoplasmic distribution of β(III)-tubulin and MAP2 (i.e. the Subtraction Method), to quantify inhibition of neurite initiation, axon outgrowth and secondary neurite (or dendrite) outgrowth in response to chemical exposure. Results demonstrate that these distinct maturational processes are differentially affected by pharmacological compounds (K252a, Na(3)VO(4), Bis-1) known to inhibit neurite outgrowth. Furthermore, a group of known developmental neurotoxicants also differentially affected the growth of axons and secondary neurites in primary cortical culture. This work improves upon previous HCI methods by providing a means in which to rapidly and specifically quantify chemical effects on the growth of axons and dendrites in vitro.