Dataset Information

Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

ABSTRACT: The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila melanogaster. The purpose of this study was to identify which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakB(N+16) isoform only, at its output synapses in the thorax. The shakB2 mutation disrupts these GF outputs and also abolishes JON-GF synaptic transmission. However, the identity of the innexin that forms the presynaptic hemichannels in the JONs remains unknown. We used electrophysiology, immunocytochemistry and dye injection, along with presynaptically-driven RNA interference, to investigate this question. The amplitude of the compound action potential recorded in response to sound from the base of the antenna (sound-evoked potential, or SEP) was reduced by RNAi of the innexins Ogre, Inx3, Inx6 and, to a lesser extent Inx2, suggesting that they could be required in JONs for proper development, excitability, or synchronization of action potentials. The strength of the JON-GF connection itself was reduced to background levels only by RNAi of shakB, not of the other seven innexins. ShakB knockdown prevented Neurobiotin coupling between GF and JONs and removed the plaques of ShakB protein immunoreactivity that are present at the region of contact. Specific shakB RNAi lines that are predicted to target the ShakB(L) or ShakB(N) isoforms alone did not reduce the synaptic strength, implying that it is ShakB(N+16) that is required in the presynaptic neurons. Overexpression of ShakB(N+16) in JONs caused the formation of ectopic dye coupling, whereas ShakB(N) prevented it altogether, supporting this conclusion and also suggesting that gap junction proteins may have an instructive role in synaptic target choice.

SUBMITTER: Pezier AP

PROVIDER: S-EPMC4833477 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

Pézier Adeline P AP Jezzini Sami H SH Bacon Jonathan P JP Blagburn Jonathan M JM

PloS one 20160404 4

The Johnston's Organ neurons (JONs) form chemical and electrical synapses onto the giant fiber neuron (GF), as part of the neuronal circuit that mediates the GF escape response in Drosophila melanogaster. The purpose of this study was to identify which of the 8 Drosophila innexins (invertebrate gap junction proteins) mediates the electrical connection at this synapse. The GF is known to express Shaking B (ShakB), specifically the ShakB(N+16) isoform only, at its output synapses in the thorax. Th ...[more]

PMID: 27043822

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Project description:The synapse between auditory Johnston's Organ neurons (JONs) and the giant fiber (GF) of Drosophila is structurally mixed, being composed of cholinergic chemical synapses and Neurobiotin- (NB) permeable gap junctions, which consist of the innexin Shaking-B (ShakB). Previous observations showed that misexpression of one ShakB isoform, ShakB(N+16), in a subset of JONs that do not normally form gap junctions results in their de novo dye coupling to the GF. Misexpression of the transcription factor Engrailed (En) in these neurons also has this effect, and in addition causes the formation of new chemical synapses. These results, along with earlier studies suggesting that gap junctions are required for the development of some chemical synapses, led to the hypothesis that ShakB would, like En, have an instructive effect on the distribution of mixed chemical/electrical contacts. To test this, we first confirmed quantitatively that ShakB(N+16) misexpression increased the dye-coupling of JONs with the GF, indicating the formation of ectopic gap junctions. Conversely, expression of the 'incorrect' isoform, ShakB(N), abolished dye coupling. Immunocytochemistry of the ShakB protein showed that ShakB(N+16) increased gap junctional plaques in JON axons but ShakB(N) did not. To test our hypothesis, fluorescently-labeled presynaptic active zone protein (Brp) was expressed in JONs and the changes in its distribution on the GF dendrites was assayed with confocal microscopy in animals with misexpression of ShakB(N+16), ShakB(N) or, as a positive control, En. Using different methods of image analysis, we confirmed our previous result that En misexpression increased the chemical synapses with the GF and the amount of GF medial dendrite branching. However, contrary to our hypothesis, misexpression of ShakB did not increase these parameters. Immunostaining showed no association between presynaptic active zones and the new ShakB plaques, further evidence against the hypothesis. We conclude that both subsets of JON form chemical synapses onto the GF dendrites but only one population forms gap junctions, comprised of ShakB(N+16). Misexpression of this isoform in all JONs does not instruct the formation of new mixed chemical/electrical synapses, but results in the insertion of new gap junctions, presumably at the sites of existing chemical synaptic contacts with the GF.

Project description:While microRNAs (miRNAs) have recently emerged as critical post-transcriptional modulators of gene expression in neuronal development, very little is known regarding the roles of miRNA-mediated regulation in the specification of cell-type specific dendritic complexity. The dendritic arborization (da) sensory neurons of the Drosophila PNS offer an excellent model system for elucidating the molecular mechanisms governing class specific dendrite morphogenesis and for exploring miRNA-mediated control of this process. To facilitate functional analyses of miRNA regulation in da neurons, we have conducted whole-genome miRNA expression profiling as well as mRNA expression profiling of three distinct classes of da neurons, thereby generating a comprehensive molecular gene expression signature within these individual subclasses of da neurons. To further validate the role of these expressed miRNAs in directing dendritic architecture, we conducted a genome-wide UAS-miRNA phenotypic screen using live-image confocal microscopy followed by semi-automated neurometric quantification, to directly assess the effect of over/mis-expression of individual and clustered miRNAs on neurons of varying dendritic complexity. Through this approach, we have identified numerous miRNAs with previously unknown functions in dendritic development. Gain-of-function and loss-of-function analyses revealed an endogenous role miR-2b and miR-13b (members of the K-box family) and miR-12/283/304 in dendritic patterning in da neuron subclasses. Moreover, using an integrative bioinformatic analysis approach involving inverse correlation between miRNA and mRNA expression profiling data in combination with existing target prediction algorithms, we have identified putative target of these miRNAs in regulating da neuron dendritic development. Validation of these predicted miRNA-target relationships via phenotypic analyses as well as QPCR, revealed the regulatory effect of these molecules in restricting dendritic branching in da neurons.

Project description:Mechanosensitive channels serve as essential sensors for cells to interact with their environment. The identity of mechanosensitive channels that underlie somatosensory touch transduction is still a mystery. One promising mechanotransduction candidate is the Transient Receptor Potential Ankyrin 1 (TRPA1) ion channel. To determine the role of TRPA1 in the generation of mechanically-sensitive currents, we used dorsal root ganglion (DRG) neuron cultures from adult mice and applied rapid focal mechanical stimulation (indentation) to the soma membrane. Small neurons (diameter <27 microm) were studied because TRPA1 is functionally present in these neurons which largely give rise to C-fiber afferents in vivo. Small neurons were classified by isolectin B4 binding. Mechanically-activated inward currents were classified into two subtypes: Slowly Adapting and Transient. First, significantly more IB4 negative neurons (84%) responded to mechanical stimulation than IB4 positive neurons (54%). Second, 89% of Slowly Adapting currents were present in IB4 negative neurons whereas only 11% were found in IB4 positive neurons. Third, Slowly Adapting currents were completely absent in IB4 negative neurons from TRPA1-/- mice. Consistent with this, Slowly Adapting currents were abolished in wild type IB4 negative neurons stimulated in the presence of a TRPA1 antagonist, HC-030031. In addition, the amplitude of Transient mechanically-activated currents in IB4 positive neurons from TRPA1-/- mice was reduced by over 60% compared to TRPA1+/+ controls; however, a similar reduction did not occur in wild-type neurons treated with HC-030031. Transfection of TRPA1 in HEK293 cells did not significantly alter the proportion or magnitude of mechanically-activated currents in HEK293 cells, indicating that TRPA1 alone is not sufficient to confer mechanical sensitivity.These parallel genetic and pharmacological data demonstrate that TRPA1 mediates the Slowly Adapting mechanically-activated currents in small-diameter IB4 negative neurons from adult mice. The TRPA1 protein may also contribute to a complex that mediates Transient mechanically-activated currents in small IB4 positive C fiber type neurons.

Dataset Information

Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

Publications

Shaking B Mediates Synaptic Coupling between Auditory Sensory Neurons and the Giant Fiber of Drosophila melanogaster.

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