Electrophysiological Recording of The Central Nervous System Activity of Third-Instar Drosophila Melanogaster (original) (raw)
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Journal of Visualized Experiments
The majority of the currently available insecticides target the nervous system and genetic mutations of invertebrate neural proteins oftentimes yield deleterious consequences, yet the current methods for recording nervous system activity of an individual animal is costly and laborious. This suction electrode preparation of the third-instar larval central nervous system of Drosophila melanogaster, is a tractable system for testing the physiological effects of neuroactive agents, determining the physiological role of various neural pathways to CNS activity, as well as the influence of genetic mutations to neural function. This ex vivo preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of insect neuronal activity. A wide variety of chemical modulators, including peptides, can then be applied directly to the nervous system in solution with the physiological saline to measure the influence on the CNS activity. Further, genetic technologies, such as the GAL4/UAS system, can be applied independently or in tandem with pharmacological agents to determine the role of specific ion channels, transporters, or receptors to arthropod CNS function. In this context, the assays described herein are of significant interest to insecticide toxicologists, insect physiologists, and developmental biologists for which D. melanogaster is an established model organism. The goal of this protocol is to describe an electrophysiological method to enable the measurement of electrogenesis of the central nervous system in the model insect, Drosophila melanogaster, which is useful for testing a diversity of scientific hypotheses.
Neuropeptides in interneurons of the insect brain
Cell and Tissue Research, 2006
A large number of neuropeptides has been identified in the brain of insects. At least 35 neuropeptide precursor genes have been characterized in Drosophila melanogaster, some of which encode multiple peptides. Additional neuropeptides have been found in other insect species. With a few notable exceptions, most of the neuropeptides have been demonstrated in brain interneurons of various types. The products of each neuropeptide precursor seem to be co-expressed, and each precursor displays a unique neuronal distribution pattern. Commonly, each type of neuropeptide is localized to a relatively small number of neurons. We describe the distribution of neuropeptides in brain interneurons of a few well-studied insect species. Emphasis has been placed upon interneurons innervating specific brain areas, such as the optic lobes, accessory medulla, antennal lobes, central body, and mushroom bodies. The functional roles of some neuropeptides and their receptors have been investigated in D. melanogaster by molecular genetics techniques. In addition, behavioral and electrophysiological assays have addressed neuropeptide functions in the cockroach Leucophaea maderae. Thus, the involvement of brain neuropeptides in circadian clock function, olfactory processing, various aspects of feeding behavior, and learning and memory are highlighted in this review. Studies so far indicate that neuropeptides can play a multitude of functional roles in the brain and that even single neuropeptides are likely to be multifunctional. Keywords Insect brain. Neuropeptide. G-protein-coupled receptor. Drosophila melanogaster. Schistocerca gregaria. Leucophaea maderae (Insecta) The original research in the authors' laboratories was supported by DFG grants HO 950/14 and 950/16 (U.H.) and Swedish Research Council grant VR 621-2004-3715 (D.R.N).
Invertebrate Neuroscience, 2012
The goal of this research was to induce neuronlike properties in Sf21 cells, an insect ovarian cell line, which could lead to a new high-throughput insecticide screening method and a way to mass produce insect neuronal material for basic research. This study applied differentiation agents to produce viable neuron-like cells. In the presence of the molting hormone 20-hydroxyecdysone (20-HE), or insulin, in the growth medium, a maximum of ca. 30 % of Sf21 cells expressed an apparent neuronal morphology of unipolar, bipolar, or multipolar axon-like processes within 2-3 days. Maximal differentiation occurred after 2 days in the presence of 50 lM 20-HE or 3 days in 10 lM insulin. Both 20-HE and insulin displayed time-and concentration-dependent differentiation with biphasic curves, suggesting that two binding sites or processes were contributing to the observed effects. In addition, combinations of 20-HE and insulin produced apparent synergistic effects on differentiation. Caffeine, a central nervous system stimulant, inhibited induction of elongated processes by 20-HE and/or insulin, with an IC 50 of 9 nM for 20-HE, and the inhibition was incomplete, resulting in about one-quarter of the differentiated cells remaining, even at high concentrations (up to 1 mM). The ability to induce a neural phenotype simplifies the studies of insect cells, compared to either the use of primary nervous tissue or genetic engineering techniques. The presence of ion channels or receptors in the differentiated cells remains to be determined.
2016
The molecular transformation of an external stimulus into changes in sensory neuron activity is incompletely described. Although a number of molecules have been identified that can respond to stimuli, evidence that these molecules can transduce stimulation into useful neural activity is lacking. Here we demonstrate that pickpocket1 (ppk1), a Drosophila homolog of mammalian Degenerin/epithelial sodium channels, encodes an acid-sensing sodium channel that conducts a transient depolarizing current in multidendritic sensory neurons of Drosophila melanogaster. Stimulation of Ppk1 is sufficient to bring these sensory neurons to threshold, eliciting a burst of action potentials. The transient nature of the neural activity produced by Ppk1 activation is the result of Ppk1 channel gating properties. This model is supported by the observation of enhanced bursting activity in neurons expressing a gain of function ppk1 mutant harboring the degenerin mutation. These findings demonstrate that Ppk...
Analytical Chemistry, 2011
Drosophila, the fruit fly, is a common model organism in biology, however quantifying neurotransmitters in Drosophila is challenging because of the small size of the central nervous system (CNS). Here, we develop neurotransmitter quantification by capillary electrophoresis with fast-scan cyclic voltammetry detection, which allows peak identification by both migration time and the cyclic voltammogram, in contrast to traditional amperometric detection which provides no chemical identification. Tissue content of biogenic amine neurotransmitters was determined in a single CNS dissected from a Drosophila larva. Low detection limits, 1 nM for dopamine and serotonin, 2.5 nM for tyramine, and 4 nM for octopamine, were achieved using field-amplified sample stacking by diluting the homogenized tissue with percholoric acid and acetonitrile. Two different strains of wild-type flies, Oregon R and Canton S, have similar dopamine and serotonin levels but different octopamine content. When flies are fed NSD-1015, which inhibits dopamine decarboxylase (Ddc) a synthesis enzyme in the dopamine and serotonin pathways, dopamine significantly decreases by 52%. A genetically altered driver line, Ddc-GAL4, had lower serotonin and dopamine content as did w 118 flies. When the Ddc-GAL4 line was used to produce flies overexpressing the serotonin synthesis enzyme tryptophan hydroxylase (Ddc-GAL4;UAS-Trh), serotonin tissue content was greater than for Ddc-GAL4, but not significantly different than wildtype. These results show that CE-FSCV is useful for monitoring the impact of genetic and pharmacological manipulations on the content of multiple neurotransmitters in a CNS from a Drosophila larva.
Molecular neurobiology: Implications for insecticide action and resistance
Pesticide Science, 1989
Recent advances in molecular neurobiology have provided an unprecedented insight into the structure and function of the three principal target sites for neurotoxic insecticides: acetylcholinesterase, the 4-aminobutyric acid ( G A B A ) receptor-chloride ionophore complex, and the voltage-sensitive sodium channel. This paper reviews some of these advances and their current or potential application to problems in insecticide resistance. I t particularly emphasizes studies of' the molecular biology of voltage-dependent sodium channels in the context of resistance to DDT and pyrethroids resulting from reduced neuronal sensitivity.
The Journal of Neuroscience, 2002
The ability of the excitatory anti-insect-selective scorpion toxin AahIT (Androctonus australis hector) to exclusively bind to and modify the insect voltage-gated sodium channel (NaCh) makes it a unique tool to unravel the structural differences between mammalian and insect channels, a prerequisite in the design of selective pesticides. To localize the insect NaCh domain that binds AahIT, we constructed a chimeric channel composed of rat brain NaCh ␣-subunit (rBIIA) in which domain-2 (D2) was replaced by that of Drosophila Para (paralytic temperaturesensitive). The choice of D2 was dictated by the similarity between AahIT and scorpion -toxins pertaining to both their binding and action and the essential role of D2 in the -toxins binding site on mammalian channels. Expression of the chimera rBIIA-ParaD2 in Xenopus oocytes gave rise to voltage-gated and TTX-sensitive NaChs that, like rBIIA, were sensitive to scorpion ␣-toxins and regulated by the auxiliary subunit  1 but not by the insect TipE. Notably, like Drosophila Para/TipE, but unlike rBIIA/ 1 , the chimera gained sensitivity to AahIT, indicating that the phyletic selectivity of AahIT is conferred by the insect NaCh D2. Furthermore, the chimera acquired additional insect channel properties; its activation was shifted to more positive potentials, and the effect of ␣-toxins was potentiated. Our results highlight the key role of D2 in the selective recognition of anti-insect excitatory toxins and in the modulation of NaCh gating. We also provide a methodological approach to the study of ion channels that are difficult to express in model expression systems.
Fly, 2015
General anesthetics achieve behavioral unresponsiveness via a mechanism that is incompletely understood. The study of genetic model systems such as the fruit fly Drosophila melanogaster is crucial to advancing our understanding of how anesthetic drugs render animals unresponsive. Previous studies have shown that wild-type control strains differ significantly in their sensitivity to general anesthetics, which potentially introduces confounding factors for comparing genetic mutations placed on these wild-type backgrounds. Here, we examined a variety of behavioral and electrophysiological endpoints in Drosophila, in both adult and larval animals. We characterized these endpoints in 3 commonly used fly strains: wild-type Canton Special (CS), and 2 commonly used white-eyed strains, isoCJ1 and w 1118 . We found that CS and isoCJ1 show remarkably similar sensitivity to isoflurane across a variety of behavioral and electrophysiological endpoints. In contrast, w 1118 is resistant to isoflurane compared to the other 2 strains at both the adult and larval stages. This resistance is however not reflected at the level of neurotransmitter release at the larval neuromuscular junction (NMJ). This suggests that the w 1118 strain harbors another mutation that produces isoflurane resistance, by acting on an arousal pathway that is most likely preserved between larval and adult brains. This mutation probably also affects sleep, as marked differences between isoCJ1 and w 1118 have also recently been found for behavioral responsiveness and sleep intensity measures.