Cholinergic calcium responses in cultured antennal lobe neurons of the migratory locust.

Locusts are advantageous organisms to elucidate mechanisms of olfactory coding at the systems level. Sensory input is provided by the olfactory receptor neurons of the antenna, which send their axons into the antennal lobe. So far, cellular properties of neurons isolated from the circuitry of the olfactory system, such as transmitter-induced calcium responses, have not been studied. Biochemical and immunocytochemical investigations have provided evidence for acetylcholine as classical transmitter of olfactory receptor neurons. Here, we characterize cell cultured projection and local interneurons of the antennal lobe by cytosolic calcium imaging to cholinergic stimulation. We bulk loaded the indicator dye Cal-520 AM in dissociated culture and recorded calcium transients after applying cholinergic agonists and antagonists. The majority of projection and local neurons respond with increases in calcium levels to activation of both nicotinic and muscarinic receptors. In local interneurons, we reveal interactions lasting over minutes between intracellular signaling pathways, mediated by muscarinic and nicotinic receptor stimulation. The present investigation is pioneer in showing that Cal-520 AM readily loads Locusta migratoria neurons, making it a valuable tool for future research in locust neurophysiology, neuropharmacology, and neurodevelopment.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

An intact insect embryo for developmental neurotoxicity testing of directed axonal elongation.

Developmental neurotoxicity (DNT) of chemicals poses a serious threat to human health worldwide. Current in vivo test methods for assessing DNT require the use of high numbers of laboratory animals. Most alternative in vitro testing methods monitor rather simple toxicological endpoints, whereas the formation of a functional brain requires precisely timed navigation of axons within a complex tissue environment. We address this complexity by monitoring defects in axonal navigation of pioneer axons of intact locust embryos after exposure to chemicals. Embryos develop in serum-free culture with test chemicals, followed by immunolabeling of pioneer neurons. Defects in axon elongation of pioneer axons are quantified in concentration-response curves and compared to the general viability of the embryo, as measured by a resazurin assay. We show that selected chemical compounds interfering with calcium signaling, the cytoskeletal organization, and the reference developmental neurotoxicant rotenone, can be classified as DNT positive. The pesticide rotenone inhibits pioneer neuron elongation with a lower IC50 than the viability assay. The rho kinase inhibitor Y27632 can partially rescue outgrowth inhibition, supporting the classification of rotenone as a specific DNT positive compound. Since mechanisms of axonal guidance, such as growth cone navigation along molecular semaphorin gradients are conserved between locust and mammalian nervous systems, we will further explore the potential of this invertebrate preparation as an assay for testing the DNT potential of chemicals in humans.