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Endpoints for Neural Connectivity Including Neurite Outgrowth, Synapse Formation, and Function
Citation:
MUNDY, W. R. Endpoints for Neural Connectivity Including Neurite Outgrowth, Synapse Formation, and Function. Presented at TestSmart DNT2 Meeting, Reston, VA, November 12 - 14, 2008.
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Description:
A strategy for alternative methods for developmental neurotoxicity testing (DNT) focuses on assessment of chemical effects on conserved neurodevelopmental processes. The development of the brain is an integrated series of steps from the commitment of embryonic cells to become neural progenitor cells, to the generation and proliferation of neurons and glia, to the establishment of precise patterns of neural connectivity which underlie a properly functioning nervous system. These events are generally conserved between species and to some extent can be recapitulated in vitro. In order to facilitate the development of rapid and economical model systems for chemical assessment, the formation of functional connections between nerve cells will be discussed in terms of morphologic and biochemical endpoints that are amenable to measurement using higher throughput technologies. These endpoints include the phenotypic maturation of neurons to 1) elaborate neurites which become morphologically distinct axons and dendrites (neurite outgrowth), 2) form presynaptic and postsynaptic terminals (synapse formation), and 3) exhibit neurotransmission between cells (function). Neurite outgrowth begins with the extension of sheet-like lamellipodia that subsequently condense into short processes. As cells mature the processes increase in length and complexity, and for primary neurons, will become polarized by developing a single long axon and several shorter dendrites. The initiation and elongation neurites is controlled by intrinsic, transcriptionally-regulated signaling pathways as well as extrinsic signals such as growth factors, extracelluar matrix proteins, and electrical activity. These signals ultimately regulate the assembly of cytoskeletal elements into neurofilaments and microtubules that determine the shape and length of the neurites. Microscopic assessment of the morphological changes accompanying neurite outgrowth can be quantified at various levels of analyses, ranging from simple scoring of the number of cells exhibiting neurites to the enumeration of the number, length and branching of axons and dendrites. There are numerous examples of chemical effects on neurite outgrowth, and automated imaging systems are available that can accurately quantify these morphologic endpoints. Neurite outgrowth can also be assessed using a variety of biochemical markers (e.g., neurotypic proteins of the cytoskeleton, growth-associated proteins) which correlate with differentiation and neurite growth. Synapse formation occurs when the axonal growth cone contacts a postsynaptic cell, and triggers differentiation into a presynaptic terminal. At the same time, the target neuron creates a specialized zone that will serve as the postsynaptic terminal. These events are regulated by reciprocal signaling between the presynaptic and postsynaptic terminals that initially involves diffusible factors including neurotransmitters, neurotrophins, Wnts, and agrin. Further specialization is promoted by signaling initiated by the contact of cell adhesion molecules including neurexins, neuroligins, and N-cadherins. The result of this signaling is the recruitment to the pre- and postsynaptic terminals of synaptic vesicles, neurotransmitter release apparati, postsynaptic scaffold proteins, and receptors. Chemical effects on synapse formation can be monitored by microscopic analysis of the localization of presynaptic (e.g., synapsin) and postsynaptic (e.g., PSD95) proteins that occurs during development. The accumulation of these synaptic proteins during synapse formation can also be assessed biochemically. Information processing in the nervous system is based on the synaptic transmission of electrical signals from one cell to another. These synaptic connections control communication by conversion of the action potential into chemical signals that traverse the synapse. Synaptic neurotransmission between cells ultimately results in a functionally connected network. The appearance of synaptic neurotransmission can be monitored at the cellular level using both imaging and electrophysiological techniques. Presynaptic neurotransmitter release can be assessed using epiflourescent microscopy to monitor the uptake and release of fluorescent dyes (e.g., FM1-43) in response to depolarization. Electrophysiological methods can be used to assess postsynaptic potentials. In some developing neurons in culture, synapse formation and functional connectivity is accompanied by the formation of spontaneous network activity and synchronized firing (network bursts). Automated systems are being developed to assess the effects of chemicals on the dynamics of network development in neuronal preparation in culture. This abstract does not necessarily reflect USEPA policy.