Table 5.

Key neurodevelopmental processes modeled in the DNT in vitro battery [160].

Process	Description
Proliferation	Division of neural stem cells and neural progenitor cells resulting in an increase in cell number. Changes in proliferation can result in an incorrect cell number (increase or decrease) and altered brain growth. It is measured directly by assessing the number of cells undergoing DNA replication or inferred by measuring an increase in cell number over time.
Apoptosis	Programmed death of cells resulting in a decrease in cell number. Changes in apoptosis can result in increases or decreases in neuron cell numbers and altered brain growth. It is measured by assessment of cell nucleus morphology or detection of biochemical markers specific to the apoptosis pathway.
Migration	Movement of neural progenitor, glial, or neuronal cells from their point of origin to a final location. Changes in migration can result in cells in the wrong location, resulting in abnormal brain structure and function. It is measured by assessing the number of cells moving into a defined area or the distance moved by individual cells.
Neuronal Differentiation	Process in which a neural progenitor cell changes to a specific type of neuron. Changes in differentiation can result in altered cell numbers for specific populations of neurons, resulting in altered brain structure and function. It is measured by assessment of the number of cells expressing markers specific for neurons and neuronal subtypes.
Neurite Growth	Outgrowth of morphological processes relatively early in neuronal differentiation. Neurites eventually develop into dendrites or axons. Changes in neurite growth can alter the number and length of axons and dendrites, changing brain structure and connectivity between neurons. It is measured by counting the number of cells elaborating processes or morphological assessment of neurite length.
Neuronal Maturation & Synaptogenesis	Maturation of neurites into the specialized processes of dendrites and axons which then form synapses responsible for communication between neurons. Changes in neuronal maturation and synaptogenesis alter neuronal connectivity, changing network formation and brain function. They are measured by morphological assessment of axon and dendrite length and counting of the number of synapses.
Glial Differentiation & Maturation	Process in which a neural progenitor cell changes to a specific type of glia (radial glia that support formation of cortical architecture, astrocytes that support neuronal function, and oligodendrocytes that myelinate axons). Changes in differentiation can result in altered numbers of glia and reduced myelination, changing brain structure and function. Differentiation and maturation are measured by assessment of the number of cells expressing markers specific for glial subtypes.
Network Formation	Process in which neurons and glia grow and make functional contacts with each other, exemplified by spontaneous generation and propagation of electrical action potentials within a network. Changes in network formation and function can result in reduced neural connectivity and altered brain function. It is measured by electrophysiological assessment of coordinated electrical activity of neurons and glia grown on microelectrode arrays.
Viability/Cytotoxicity	Test methods for each neurodevelopmental process should also include at least one concurrent measure of cell viability (or its converse, cytotoxicity) as a baseline for comparison of potential non-specific effects of chemical exposure. Cell viability can be assessed by counting cells with normal morphology (based on cell body and nucleus size), delineation of live/dead cells based on uptake or exclusion of vital dyes, or biochemical assessment of active cell metabolism. Cytotoxicity is typically assessed by measuring parameters associated with loss of cell membrane integrity, including leakage of intracellular proteins and enzymes, or exposure of DNA.