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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 large numbers of laboratory animals. Most alternative testing methods monitor readily quantifiable toxicological endpoints in cell culture, 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 or 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 viability. 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, including a prediction model, for testing the DNT potential of chemicals in humans. [ABSTRACT FROM AUTHOR]

Animal circadian clocks consist of central and peripheral pacemakers, which are coordinated to produce daily rhythms in physiology and behaviour. Despite its importance for optimal performance and health, the mechanism of clock coordination is poorly understood. Here we dissect the pathway through which the circadian clock of Drosophila imposes daily rhythmicity to the pattern of adult emergence. Rhythmicity depends on the coupling between the brain clock and a peripheral clock in the prothoracic gland (PG), which produces the steroid hormone, ecdysone. Time information from the central clock is transmitted via the neuropeptide, sNPF, to non-clock neurons that produce the neuropeptide, PTTH. These secretory neurons then forward time information to the PG clock. We also show that the central clock exerts a dominant role on the peripheral clock. This use of two coupled clocks could serve as a paradigm to understand how daily steroid hormone rhythms are generated in animals. [ABSTRACT FROM AUTHOR]