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A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age.

ABSTRACT: There have been relatively few studies of how central synapses age in adult Drosophila melanogaster. In this study we investigate the aging of the synaptic inputs to the Giant Fiber (GF) from auditory Johnston's Organ neurons (JONs). In previously published experiments an indirect assay of this synaptic connection was used; here we describe a new, more direct assay, which allows reliable detection of the GF action potential in the neck connective, and long term recording of its responses to sound. Genetic poisoning using diphtheria toxin expressed in the GF with R68A06-GAL4 was used to confirm that this signal indeed arose from the GF and not from other descending neurons. As before, the sound-evoked action potentials (SEPs) in the antennal nerve were recorded via an electrode inserted at the base of the antenna. It was noted that an action potential in the GF elicited an antennal twitch, which in turn evoked a mechanosensory response from the JONs in the absence of sound. We then used these extracellular recording techniques in males and female of different ages to quantify the response of the JONs to a brief sound impulse, and also to measure the strength of the connection between the JONs and the GF. At no age was there any significant difference between males and females, for any of the parameters measured. The sensitivity of the JONs to a sound impulse approximately doubled between 1 d and 10 d after eclosion, which corresponds to the period when most mating is taking place. Subsequently JON sensitivity decreased with age, being approximately half as sensitive at 20 d and one-third as sensitive at 50 d, as compared to 10 d. However, the strength of the connection between the auditory input and the GF itself remained unchanged with age, although it did show some variability that could mask any small changes.

SUBMITTER: Blagburn JM

PROVIDER: S-EPMC6946141 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age.

Blagburn Jonathan M JM

PloS one 20200107 1

There have been relatively few studies of how central synapses age in adult Drosophila melanogaster. In this study we investigate the aging of the synaptic inputs to the Giant Fiber (GF) from auditory Johnston's Organ neurons (JONs). In previously published experiments an indirect assay of this synaptic connection was used; here we describe a new, more direct assay, which allows reliable detection of the GF action potential in the neck connective, and long term recording of its responses to soun ...[more]

PMID: 31910219

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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:Viscoelasticity is a fundamental property of virtually all biological materials, and proteinaceous, fibrous materials that constitute the extracellular matrix (ECM) are no exception. Viscoelasticity may be particularly important in the ECM since cells can apply mechanical stress resulting from cell contractility over very long periods of time. However, measurements of ECM fiber response to long-term constant force loading are scarce, despite the increasing recognition that mechanical strain regulates the biological function of some ECM fibers. We developed a dual micropipette system that applies constant force to single fibers for up to 8?h. We utilized this system to study the time dependent response of fibronectin (Fn) fibers to constant force, as Fn fibers exhibit tremendous extensibility before mechanical failure as well as strain dependent alterations in biological properties. These data demonstrate the Fn fibers continue to stretch under constant force loading for at least 8?h and that this long-term creep results in plastic deformation of Fn fibers, in contrast to elastic deformation of Fn fibers under short-term, but fast loading rate extension. These data demonstrate that physiologically-relevant loading may impart mechanical features to Fn fibers by switching them into an extended state that may have altered biological functions. STATEMENT OF SIGNIFICANCE: Measurements of extracellular matrix (ECM) fiber response to constant force loading are scarce, so we developed a novel technique for applying constant force to single ECM fibers. We used this technique to measure constant force creep of fibronectin fibers since these fibers have been shown to be mechanotransducers whose functions can be altered by mechanical strain. We found that fibronectin fibers creep under constant force loading for the duration of the experiment and that this creep behavior resembles a power law. Furthermore, we found that constant force creep results in plastic deformation of the fibers, which suggests that the mechanobiological switching of fibronectin can only occur once after long-term loading.

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A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age.

Publications

A new method of recording from the giant fiber of Drosophila melanogaster shows that the strength of its auditory inputs remains constant with age.

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