Refinement of Tools for Targeted Gene Expression in Drosophila

Pfeiffer, Barret D.; Ngo, Teri-T.B.; Hibbard, Karen L; Murphy, Christine; Jenett, Arnim; Truman, James W.; Rubin, Gerald M.

doi:10.1534/genetics.110.119917

Cited by 1,060 publications

(1,180 citation statements)

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“…The following stocks were obtained from the Bloomington Drosophila Stock Center (Indiana University): UAS-CD4-tdGFP (Han et al, 2011), UAS-ChR2-H134R (Pulver et al, 2009), UAS GFP.nls, LexAop2-CsChrimson, and GMR50G08-LexA (Pfeiffer et al, 2010), which was used to drive expression in looper interneurons. UAS-dEaat1 RNAi flies (transformant ID 109401) were obtained from the Vienna Drosophila Resource Center (Dietzl et al, 2007).…”

Section: Transgenic Fliesmentioning

confidence: 99%

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment

MacNamee

Liu

Gerhard

et al. 2016

J of Comparative Neurology

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Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron glia interactions. Here, we show the first electrophysiological recordings from Drosophila astrocytes and characterize their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic pre-motor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via Eaat1, and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory post-synaptic currents in motor neurons. The neuronal synapses were always located within a micron of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters. Graphical AbstractCorresponding Author: Sarah E. MacNamee, University of Arizona Dept. of Neuroscience, 1040 E 4 th St GS Rm 611, Tucson AZ 85721, (520) 621 6671, Smac3@email.arizona.edu. Role of AuthorsAll authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: SEM, LAO. Acquisition of data: SEM, KEL, SG, CTT, RDF, AC. Analysis and interpretation of data: SEM, KEL, CTT. Drafting of the manuscript: SEM. Critical revision of the manuscript for important intellectual content: LAO, LPT, AC. Statistical analysis: SEM. Obtained funding: LAO, LPT, AC. Administrative, technical, and material support: none. Study supervision: LAO, LPT. Conflict of Interest StatementThe authors have no conflict of interest to disclose. Data AccessibilityData generated in the lab and the subsequent analyses will be made available upon request to the Oland/Tolbert laboratory by qualified individuals as long as doing so would not compromise intellectual property interests or interfere with publication. Any shared data would include notations, standards, and the like that are necessary for interpretation of the data. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptThe authors show the first recordings from Drosophila astrocytes and find that their electrophysiological properties are strongly analogous to those of vertebrate astrocytes. Using correlative electron microscopy and physiology, they found no glial ensheathment of glutamatergic synap...

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Section: Transgenic Fliesmentioning

confidence: 99%

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment

MacNamee

Liu

Gerhard

et al. 2016

J of Comparative Neurology

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“…1A) (25). We constructed transgenic animals in which the smGFPs were expressed under the control of either GAL4 (26) or LexA (25,27) binary systems.…”

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confidence: 99%

“…1A) (25). We constructed transgenic animals in which the smGFPs were expressed under the control of either GAL4 (26) or LexA (25,27) binary systems. In addition to this transcription control, we engineered a second, orthogonal level of control by incorporating a transcriptional interruption cassette into the smGFP constructs (22,28).…”

mentioning

confidence: 99%

Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system

Nern

Pfeiffer

Rubin

2015

Proc. Natl. Acad. Sci. U.S.A.

496

672

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We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.neuroanatomy | Drosophila | interneuron diversity | light microscopy | recombinase N ervous systems contain numerous and diverse cells displaying complex anatomical relationships. The specification and patterning of these cells must be generated by the execution of a much smaller set of instructions encoded in the genome. How many different genetic algorithms are needed? How precise are their outcomes? What types of rules do they follow? Answering such questions requires knowledge of the anatomy of neuronal processes for many different cell types, for numerous cells of the same type, and in multiple individuals. We describe here the development of a set of methods for collecting such data by light microscopy and their application in the adult visual system of Drosophila.Neuronal morphology is often sufficiently stereotyped to identify cell types. For example, many cell populations in vertebrate and invertebrate visual systems can be reliably distinguished on the basis of cell shape (1-3). In at least some cases, these anatomical cell types have been shown to correlate well with classifications based on genetic marker expression or functional properties. Important anatomical features are not limited to cell shape, but also include the spatial relationships between cells. One critical ...

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“…Thousands of different GAL4 fly lines have been raised, so that virtually any tissue and developmental stage can be targeted. To refine this approach, a number of tricks can be used to achieve even higher spatial or temporal resolution (del Valle Rodriguez et al, 2012;Pfeiffer et al, 2010): (1) The temperature-sensitive GAL4 repressor Gal80 ts can be coexpressed, so that GAL4-induced expression is active only during periods when the experimental animals are shifted to 30°C (McGuire, Mao, & Davis, 2004). (2) The GeneSwitch system uses tissue-specific GAL4 lines which are inactive under normal conditions but can be activated by supplementing the food with the steroid hormone mifepristone (Nicholson et al, 2008).…”

Section: P0105mentioning

confidence: 99%