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.

proliferation, neurochemical differentiation, migration of neural precursor cells, and electrical activity in randomly formed neural networks (Frank et al., 2018). Most in vitro systems do not recapitulate many of the complex cell-cell and cell-matrix interactions or morphogen gradients in the intact organism that are necessary for normal brain formation and may be subject to significant influence by toxicants (Lein et al., 2005). To address possible adverse effects of chemicals on these complex mechanisms, non-mammalian models such as zebrafish embryos can be successfully employed (Dach et al., 2019). Alternatively, the applicability of invertebrate models such as Caenorhabditis (Avila et al., 2012), planarians (Hagstrom et al., 2019), or Drosophila (Rand, 2010) is recognized. In spite of the phylogenetical distance between vertebrates and invertebrates, mechanisms of neural development appear to be highly conserved (Sánchez-Soriano et al., 2007). Comparative DNT studies between a zebrafish and a

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 welldefined 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. The developing fetal and juvenile human brain is more sensitive to exposure to industrial chemicals than the adult. Investigations of developmental neurotoxicity (DNT) deal with any adverse effects on the structure or function of the nervous system during gestational-or lactation-periods 1. DNT positive chemicals pose a serious threat to the health of our children, because maldevelopment of the brain will eventually result in behavioral abnormalities, such as attention deficit, hyperactivity, and autism spectrum disorder 2,3. Since there is growing concern that the increase of those neurologic defects may, at least in part, be caused by a cocktail of DNT positive compounds used in our daily life, there is now an urgent demand for risk assessment of industrial chemicals. Until 2017, only 13 different substances have been identified as developmental neurotoxicants in humans by epidemiological approaches. In contrast, tens of thousands industrial compounds have not been studied so far and remain uncertain 2,4. To evaluate these potentially toxic compounds large numbers of vertebrates over long testing periods are needed. Due to limitations of traditional animal testing by guidelines of the OECD, alternative assays have to be developed. Alternative in vitro testing methods focus on key aspects in brain development that can be readily quantified in cell culture assays. They include, for example, measurements of cell viability, cell proliferation, neurite extension, development of neurochemical phenotype, and electrical activity in randomly formed neural network as toxicological endpoints 5-8. However, there are also non-mammalian organ...

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.

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.