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An intact insect embryo as assay for developmental neurotoxicity testing

Project Summary:

Developmental neurotoxicity (DNT) of industrial chemicals poses a serious threat to the health of children. Current in vivo test methods for assessing DNT require the use of high numbers of laboratory animals. Alternative in vitro testing methods monitor mainly readily quantifiable toxicological endpoints, such as cell viability, proliferation, and neurochemical differentiation. The formation of a functional brain requires, however, the precisely timed navigation of axons within the complex neuronal tissue environment. We address this complexity by monitoring defects in axonal navigation of pioneer axons of intact locust embryos after exposure to chemicals. Neural pathways in the leg of embryonic locust are established by a pair of afferent pioneer neurons. These two neurons extend axons along a well-defined pathway through the limb bud to the central nervous system. Correct axonal navigation depends on stripes and gradients of semaphorin cell recognition molecules, that were originally discovered by American and Canadian researchers in the locust embryo. A member of semaphorin family of cell recognition molecules also helps to guide the neurites of pyramidal neurons in mammalian cortex.

Locust embryos are kept in serum-free culture in the presence of test chemicals, followed by immunolabeling of leg bud pioneer neurons. Defects in axonal outgrowth and navigation of pioneer axons are quantified in dose-response curves and compared to the general viability of the embryo, as measured by a biochemical assay.

We show that selected chemical compounds interfering with calcium signaling and the cytoskeletal organization can be classified as DNT positive. We calibrate our test system against a range of positive compounds with known DNT potential and negative compounds, which are toxic, but have no specific DNT potential. Comparing the reduction of viability and neurite length, our test results closely match findings obtained from cell culture studies using mammalian neurons. For example, the mitochondrial respiratory chain inhibitor rotenone inhibits both pioneer neuron growth and correct pathfinding in the same concentration range as found in human neurons. The Rho kinase blocker Y27632 can partially rescue outgrowth inhibition, thus excluding a pure metabolic effect of rotenone. This identifies rotenone as a specific developmental neurotoxicant. We plan to monitor birth and death of the transient pioneers, but also of other sensory neurons at defined developmental stages. This will incorporate toxicological endpoints for neurogenesis and apoptosis in our insect embryo assay. Since mechanisms of axonal guidance, such as growth cone navigation along molecular semaphorin gradients are conserved between locust and mammalian nervous systems, we propose that the insect assay will be useful to identify a DNT potential of chemicals in humans.

 

Funding:

German Ministry of Education and Research (031L0062A)

 

Project Lead:

Prof. Dr. Gerd Bicker (gerd.bicker@tiho-hannover.de; 0511-856-7765)

PD Dr. Michael Stern (michael.stern@tiho-hannover.de; 0511-856-7767)

Institute of Physiology and Cell Biology, University of Veterinary Medicine Hannover, Germany

 

 

 

 

 

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