Christopher J. H. Elliott

Neuromuscular organization and aminergic modulation of contractions in the Drosophila ovary

BackgroundThe processes by which eggs develop in the insect ovary are well characterized. Despite a large number of Drosophila mutants that cannot lay eggs, the way that the egg is moved along the reproductive tract from ovary to uterus is less well understood. We remedy this with an integrative study on the reproductive tract muscles (anatomy, innervation, contractions, aminergic modulation) in female flies.ResultsEach ovary, consisting of 15–20 ovarioles, is surrounded by a contractile meshwork, the peritoneal sheath. Individual ovarioles are contained within a contractile epithelial sheath. Both sheaths contain striated muscle fibres. The oviduct and uterine walls contain a circular striated muscle layer. No longitudinal muscle fibres are seen.Neurons that innervate the peritoneal sheath and lateral oviduct have many varicosities and terminate in swellings just outside the muscles of the peritoneal sheath. They all express tyrosine decarboxylase (required for tyramine and octopamine synthesis) and Drosophila vesicular monoamine transporter (DVMAT). No fibres innervate the ovarioles. The common oviduct and uterus are innervated by two classes of neurons, one with similar morphology to those of the peritoneal sheath and another with repeated branches and axon endings similar to type I neuromuscular junctions.In isolated genital tracts from 3- and 7-day old flies, each ovariole contracts irregularly (12.5 ± 6.4 contractions/minute; mean ± 95% confidence interval). Peritoneal sheath contractions (5.7 ± 1.6 contractions/minute) move over the ovary, from tip to base or vice versa, propagating down the oviduct. Rhythmical spermathecal rotations (1.5 ± 0.29 contractions/minute) also occur. Each genital tract organ exhibits its own endogenous myogenic rhythm.The amplitude of contractions of the peritoneal sheath increase in octopamine (100 nM, 81% P < 0.02) but 1 μM tyramine has no effect. Neither affects the frequency of peritoneal sheath contractions.ConclusionThe muscle fibres of the reproductive tract are circular and have complex bursting myogenic rhythms under octopaminergic neuromodulation. We propose a new model of tissue-specific actions of octopamine, in which strengthening of peritoneal sheath contractions, coupled with relaxation of the oviduct, eases ovulation. This model accounts for reduced ovulation in flies with mutations in the octopaminergic system.

The mechanics of stridulation of the cricketGryllus campestris

SummaryDetails in the stridulatory movement ofGryllus campestris were investigated using an improved high resolution miniature angle measurement system. The following results were obtained: During the closing (sound producing) stroke, the speed of the plectrum always has the same value (within measuring accuracy) at a given position. Plectrum speed is directly proportional to tooth spacing, which is known to vary along the file. The only exception to this rule were occasions when closing velocities of precisely 2 times the standard value were found. In between values were never recorded. While temperature has a large effect on the opening speed and duration, the closing speed has a very smallQ10 (0.07) which is equal to theQ10 of the resonance frequency of the harp. When the harps are removed, the proportionality between tooth spacing and scraper velocity is lost; the velocity is much increased (up to 3-fold) and the variance of the speed is enhanced 5-fold.These results are discussed with respect to 3 hypothetical models explaining the function of the sound generator system. The model describing the cricket sound generator as a clockwork with an escapement system is capable of accommodating all experimental data without any extra assumptions.

Loss and gain of Drosophila TDP-43 impair synaptic efficacy and motor control leading to age-related neurodegeneration by loss-of-function phenotypes.

Cytoplasmic accumulation and nuclear clearance of TDP-43 characterize familial and sporadic forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, suggesting that either loss or gain of TDP-43 function, or both, cause disease formation. Here we have systematically compared loss- and gain-of-function of Drosophila TDP-43, TAR DNA Binding Protein Homolog (TBPH), in synaptic function and morphology, motor control, and age-related neuronal survival. Both loss and gain of TBPH severely affect development and result in premature lethality. TBPH dysfunction caused impaired synaptic transmission at the larval neuromuscular junction (NMJ) and in the adult. Tissue-specific knockdown together with electrophysiological recordings at the larval NMJ also revealed that alterations of TBPH function predominantly affect pre-synaptic efficacy, suggesting that impaired pre-synaptic transmission is one of the earliest events in TDP-43-related pathogenesis. Prolonged loss and gain of TBPH in adults resulted in synaptic defects and age-related, progressive degeneration of neurons involved in motor control. Toxic gain of TBPH did not downregulate or mislocalize its own expression, indicating that a dominant-negative effect leads to progressive neurodegeneration also seen with mutational inactivation of TBPH. Together these data suggest that dysfunction of Drosophila TDP-43 triggers a cascade of events leading to loss-of-function phenotypes whereby impaired synaptic transmission results in defective motor behavior and progressive deconstruction of neuronal connections, ultimately causing age-related neurodegeneration.

Neural Network Analysis in the Snail Brain

How can we hope to use the techniques of cellular neurobiology, which have been successfully used to analyze the properties of single neurons or small networks, to eventually understand a structure such as the snail brain which contains about 25,000 neurons? Our approach has been to carry out detailed analyses of specific neural networks underlying several types of function in the brain of the pond snail, Lymnaea, while collecting less specific information on more global aspects of brain organization and the larger scale of interactions within it. The concentration of the CNS into a compact brain and the distribution of cells of the same type over several ganglia (see, for instance, the neurosecretory cells of Fig. 1A) makes such a whole brain analysis almost inevitable even for simple mapping studies, but the global aspect of organization within the snail brain is also emphasized by our analysis of two interneurons that have follower cells in at least five of the ganglia of the CNS (Fig. 1C,D). Even neural systems underlying specific behavioral acts can be widely distributed and motoneurons responsible for whole body withdrawal responses occur in all nine ganglia of the central ganglionic ring (Fig. 1B).

Distance and force production during jumping in wild type and mutant Drosophila melanogaster

SUMMARY In many insects renowned for their jumping ability, elastic storage is used so that high forces can be developed prior to jumping. We have combined physiological, behavioural and genetic approaches to test whether elastic energy storage makes a major contribution to jumping in Drosophila. We describe a sensitive strain gauge setup, which measures the forces produced by tethered flies through their mesothoracic legs. The peak force produced by the main jumping muscle of female flies from a wild-type (Canton-S) strain is 101±4.4 μN [and this is indistinguishable from a second wild-type (Texas) strain]. The force takes 8.2 ms to reach its peak. The peak force is not affected significantly by altering the leg angle (femur–tibia joint angle) in the range of 75–120°, but the peak force declines as the leg is extended further. Measurements of jumping ability (distance jumped) showed that female Drosophila (with their wings removed) of two wild-type strains, Canton-S and Texas, produced jumps of 28.6±0.7 and 30.2±1.0 mm (mean ± s.e.m.). For a female wild-type Drosophila, a jump of 30 mm corresponds to a kinetic energy of 200 nJ on take-off (allowing 20% of the energy to overcome air resistance). We develop equations of motion for a linear force–time model of take-off and calculate that the time to take-off is 5.0 ms and the peak force should be 274 μN (137 μN leg–1). We predicted, from the role of octopamine in enhancing muscle tension in several locust muscles, that if stored elastic energy plays no part in force development, then genetic manipulation of the octopaminergic system would directly affect force production and jumping in Drosophila. Using two mutants deficient in the octopaminergic system, TbhnM18 (M18) and TyrRhono (hono), we found significantly reduced jumping distances (20.7±0.7 and 20.7±0.4 mm, respectively) and force production (52% and 55%, respectively) compared with wild type. From the reduced distance and force production in M18, a mutant deficient in octopamine synthesis, and in hono, a tyramine/octopamine receptor mutant, we conclude that in Drosophila, as in locusts, octopamine modulates escape jumping. We conclude that the fly does not need to store large quantities of elastic energy in order to make its jump because (1) the measured and calculated forces agree to within 40% and (2) the reduction in distances jumped by the mutants correlates well with their reduction in measured peak force.

parkin-induced defects in neurophysiology and locomotion are generated by metabolic dysfunction and not oxidative stress

Parkinsons disease (PD) is characterized by movement disorders, including bradykinesia. Analysis of inherited, juvenile PD, identified several genes linked via a common pathway to mitochondrial dysfunction. In this study, we demonstrate that the larva of the Drosophila parkin mutant faithfully models the locomotory and metabolic defects of PD and is an excellent system for investigating their inter-relationship. parkin larvae displayed a marked bradykinesia that was caused by a reduction in both the frequency of peristalsis and speed of muscle contractions. Rescue experiments confirmed that this phenotype was due to a defect in the nervous system and not in the muscle. Furthermore, recordings of motoneuron activity in parkin larvae revealed reduced bursting and a striking reduction in evoked and miniature excitatory junction potentials, suggesting a neuronal deficit. This was supported by our observations in parkin larvae that the resting potential was depolarized, oxygen consumption and ATP concentration were drastically reduced while lactate was increased. These findings suggest that neuronal mitochondrial respiration is severely compromised and there is a compensatory switch to glycolysis for energy production. parkin mutants also possessed overgrown neuromuscular synapses, indicative of oxidative stress, which could be rescued by overexpression of parkin or scavengers of reactive oxygen species (ROS). Surprisingly, scavengers of ROS did not rescue the resting membrane potential and locomotory phenotypes. We therefore propose that mitochondrial dysfunction in parkin mutants induces Parkinsonian bradykinesia via a neuronal energy deficit and resulting synaptic failure, rather than as a consequence of downstream oxidative stress.

Dopaminergic expression of the Parkinsonian gene LRRK2-G2019S leads to non-autonomous visual neurodegeneration, accelerated by increased neural demands for energy

Parkinsons disease (PD) is associated with loss of dopaminergic signalling, and affects not just movement, but also vision. As both mammalian and fly visual systems contain dopaminergic neurons, we investigated the effect of LRRK2 mutations (the most common cause of inherited PD) on Drosophila electroretinograms (ERGs). We reveal progressive loss of photoreceptor function in flies expressing LRRK2-G2019S in dopaminergic neurons. The photoreceptors showed elevated autophagy, apoptosis and mitochondrial disorganization. Head sections confirmed extensive neurodegeneration throughout the visual system, including regions not directly innervated by dopaminergic neurons. Other PD-related mutations did not affect photoreceptor function, and no loss of vision was seen with kinase-dead transgenics. Manipulations of the level of Drosophila dLRRK suggest G2019S is acting as a gain-of-function, rather than dominant negative mutation. Increasing activity of the visual system, or of just the dopaminergic neurons, accelerated the G2019S-induced deterioration of vision. The fly visual system provides an excellent, tractable model of a non-autonomous deficit reminiscent of that seen in PD, and suggests that increased energy demand may contribute to the mechanism by which LRRK2-G2019S causes neurodegeneration.

UDCA exerts beneficial effect on mitochondrial dysfunction in LRRK2G2019S carriers and in vivo

Objective: To further characterize mitochondrial dysfunction in LRRK2G2019S mutant Parkinson disease (PD) patient tissue (M-LRRK2G2019S), determine whether ursodeoxycholic acid (UDCA) also exerts a beneficial effect on mitochondrial dysfunction in nonmanifesting LRRK2G2019S mutation carriers (NM-LRRK2G2019S), and assess UDCA for its beneficial effect on neuronal dysfunction in vivo. Methods: Intracellular adenosine 5′-triphosphate (ATP) levels, oxygen consumption, and activity of the individual complexes of the mitochondrial respiratory chain as well as mitochondrial morphology were measured in M-LRRK2G2019S, NM-LRRK2G2019S, and controls. UDCA was assessed for its rescue effect on intracellular ATP levels in NM-LRRK2G2019S and in a LRRK2 transgenic fly model with dopaminergic expression of LRRK2G2019S. Results: Crucial parameters of mitochondrial function were similarly reduced in both M-LRRK2G2019S and NM-LRRK2G2019S with a specific decrease in respiratory chain complex IV activity. Mitochondrial dysfunction precedes changes in mitochondrial morphology but is normalized after siRNA-mediated knockdown of LRRK2. UDCA improved mitochondrial function in NM-LRRK2G2019 and rescued the loss of visual function in LRRK2G2019S flies. Conclusion: There is clear preclinical impairment of mitochondrial function in NM-LRRK2G2019S that is distinct from the mitochondrial impairment observed in parkin-related PD. The beneficial effect of UDCA on mitochondrial function in both NM-LRRK2G2019S and M-LRRK2G2019S as well as on the function of dopaminergic neurons expressing LRRK2G2019S suggests that UDCA is a promising drug for future neuroprotective trials.

The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila

Summary Obscurin (also known as Unc-89 in Drosophila) is a large modular protein in the M-line of Drosophila muscles. Drosophila obscurin is similar to the nematode protein UNC-89. Four isoforms are found in the muscles of adult flies: two in the indirect flight muscle (IFM) and two in other muscles. A fifth isoform is found in the larva. The larger IFM isoform has all the domains that were predicted from the gene sequence. Obscurin is in the M-line throughout development of the embryo, larva and pupa. Using P-element mutant flies and RNAi knockdown flies, we have investigated the effect of decreased obscurin expression on the structure of the sarcomere. Embryos, larvae and pupae developed normally. In the pupa, however, the IFM was affected. Although the Z-disc was normal, the H-zone was misaligned. Adults were unable to fly and the structure of the IFM was irregular: M-lines were missing and H-zones misplaced or absent. Isolated thick filaments were asymmetrical, with bare zones that were shifted away from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depends on the symmetry of adjacent thick filaments; shifted bare zones resulted in abnormally long or short thin filaments. We conclude that obscurin in the IFM is necessary for the development of a symmetrical sarcomere in Drosophila IFM.

Abnormal visual gain control in a Parkinson's disease model

Our understanding of Parkinsons disease (PD) has been revolutionized by the discovery of disease-causing genetic mutations. The most common of these is the G2019S mutation in the LRRK2 kinase gene, which leads to increased kinase activity. However, the link between increased kinase activity and PD is unclear. Previously, we showed that dopaminergic expression of the human LRRK2-G2019S transgene in flies led to an activity-dependent loss of vision in older animals and we hypothesized that this may have been preceded by a failure to regulate neuronal activity correctly in younger animals. To test this hypothesis, we used a sensitive measure of visual function based on frequency-tagged steady-state visually evoked potentials. Spectral analysis allowed us to identify signals from multiple levels of the fly visual system and wild-type visual response curves were qualitatively similar to those from human cortex. Dopaminergic expression of hLRRK2-G2019S increased contrast sensitivity throughout the retinal network. To test whether this was due to increased kinase activity, we fed Drosophila with kinase inhibitors targeted at LRRK2. Contrast sensitivity in both day 1 and day 14 flies was normalized by a novel LRRK2 kinase inhibitor ‘BMPPB-32’. Biochemical and cellular assays suggested that BMPPB-32 would be a more specific kinase inhibitor than LRRK2-IN-1. We confirmed this in vivo, finding that dLRRK− null flies show large off-target effects with LRRK2-IN-1 but not BMPPB-32. Our data link the increased Kinase activity of the G2019S-LRRK2 mutation to neuronal dysfunction and demonstrate the power of the Drosophila visual system in assaying the neurological effects of genetic diseases and therapies.