Regulation of excitation-contraction coupling at the Drosophila neuromuscular junction.

The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic alterations effect neuromuscular transduction and muscle contractility, which ultimately dictate behavioural output. Here we develop and use a force transducer system to characterize excitation-contraction coupling at Drosophila larval neuromuscular junctions (NMJs), examining how specific neuronal and muscle manipulations disrupt muscle contractility. Muscle contraction force increased with motoneuron stimulation frequency and duration, showing considerable plasticity between 5 and 40 Hz and saturating above 50 Hz. Endogenous recordings of fictive contractions revealed average motoneuron burst frequencies of 20-30 Hz, consistent with the system operating within this plastic range of contractility. Temperature was also a key factor in muscle contractility, as force was enhanced at lower temperatures and dramatically reduced with increasing temperatures. Pharmacological and genetic manipulations of critical components of Ca2+ regulation in both pre- and postsynaptic compartments affected the strength and time course of muscle contractions. A screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation and modulation to alterations in excitation-contraction coupling. KEY POINTS: Larval muscle contraction force increases with stimulation frequency and duration, revealing substantial plasticity between 5 and 40 Hz. Fictive contraction recordings demonstrate endogenous motoneuron burst frequencies consistent with the neuromuscular system operating within the range of greatest plasticity. Genetic and pharmacological manipulations of critical components of pre- and postsynaptic Ca2+ regulation significantly affect the strength and time course of muscle contractions. A screen for modulators of the excitation-contraction machinery identified a FMRFa peptide, TPAEDFMRFa and its associated signalling pathway, that dramatically increases muscle performance. Drosophila serves as an excellent model for dissecting components of the excitation-contraction coupling machinery.

Glial ER and GAP junction mediated Ca2+ waves are crucial to maintain normal brain excitability.

Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca2+ fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca2+ dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca2+ signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca2+ signaling. Here we identify a role for ER store-operated Ca2+ entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. We show that PG cells display diverse Ca2+ activity that varies based on their locale within the brain. Ca2+ signaling in PG cells does not require extracellular Ca2+ and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca2+ release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.

Synaptic Plasticity Induced by Differential Manipulation of Tonic and Phasic Motoneurons in Drosophila.

Structural and functional plasticity induced by neuronal competition is a common feature of developing nervous systems. However, the rules governing how postsynaptic cells differentiate between presynaptic inputs are unclear. In this study, we characterized synaptic interactions following manipulations of tonic Ib or phasic Is glutamatergic motoneurons that coinnervate postsynaptic muscles of male or female Drosophila melanogaster larvae. After identifying drivers for each neuronal subtype, we performed ablation or genetic manipulations to alter neuronal activity and examined the effects on synaptic innervation and function at neuromuscular junctions. Ablation of either Ib or Is resulted in decreased muscle response, with some functional compensation occurring in the Ib input when Is was missing. In contrast, the Is terminal failed to show functional or structural changes following loss of the coinnervating Ib input. Decreasing the activity of the Ib or Is neuron with tetanus toxin light chain resulted in structural changes in muscle innervation. Decreased Ib activity resulted in reduced active zone (AZ) number and decreased postsynaptic subsynaptic reticulum volume, with the emergence of filopodial-like protrusions from synaptic boutons of the Ib input. Decreased Is activity did not induce structural changes at its own synapses, but the coinnervating Ib motoneuron increased the number of synaptic boutons and AZs it formed. These findings indicate that tonic Ib and phasic Is motoneurons respond independently to changes in activity, with either functional or structural alterations in the Ib neuron occurring following ablation or reduced activity of the coinnervating Is input, respectively.SIGNIFICANCE STATEMENT Both invertebrate and vertebrate nervous systems display synaptic plasticity in response to behavioral experiences, indicating that underlying mechanisms emerged early in evolution. How specific neuronal classes innervating the same postsynaptic target display distinct types of plasticity is unclear. Here, we examined whether Drosophila tonic Ib and phasic Is motoneurons display competitive or cooperative interactions during innervation of the same muscle, or compensatory changes when the output of one motoneuron is altered. We established a system to differentially manipulate the motoneurons and examined the effects of cell type-specific changes to one of the inputs. Our findings indicate Ib and Is motoneurons respond differently to activity mismatch or loss of the coinnervating input, with the Ib subclass responding robustly compared with Is motoneurons.

Modulation of neuromuscular synapses and contraction in Drosophila 3rd instar larvae.

Over the past four decades, Drosophila melanogaster has become an increasingly important model system for studying the modulation of chemical synapses and muscle contraction by cotransmitters and neurohormones. This review describes how advantages provided by Drosophila have been utilized to investigate synaptic modulation, and it discusses key findings from investigations of cotransmitters and neurohormones that act on body wall muscles of 3rd instar Drosophila larvae. These studies have contributed much to our understanding of how neuromuscular systems are modulated by neuropeptides and biogenic amines, but there are still gaps in relating these peripheral modulatory effects to behavior.

Characterizing the physiological and behavioral roles of proctolin in Drosophila melanogaster.

The neuropeptide proctolin (RYLPT) plays important roles as both a neurohormone and a cotransmitter in arthropod neuromuscular systems. We used third-instar Drosophila larvae as a model system to differentiate synaptic effects of this peptide from its direct effects on muscle contractility and to determine whether proctolin can work in a cell-selective manner on muscle fibers. Proctolin did not appear to alter the amplitude of excitatory junctional potentials but did induce sustained muscle contractions in preparations where the CNS had been removed and no stimuli were applied to the remaining nerves. Proctolin-induced contractions were dose-dependent, were reduced by knocking down expression of the Drosophila proctolin receptor in muscle tissue, and were larger in some muscle cells than others (i.e., larger in fibers 4, 12, and 13 than in 6 and 7). Proctolin also increased the amplitude of nerve-evoked contractions in a dose-dependent manner, and the magnitude of this effect was also larger in some muscle cells than others (again, larger in fibers 4, 12, and 13 than in 6 and 7). Increasing the intraburst impulse frequency and number of impulses per burst increased the magnitude of proctolin's enhancement of nerve-evoked contractions and decreased the threshold and EC50 concentrations for proctolin to enhance nerve-evoked contractions. Reducing proctolin receptor expression decreased the velocity of larval crawling at higher temperatures, and thermal preference in these larvae. Our results suggest that proctolin acts directly on body-wall muscles to elicit slow, sustained contractions and to enhance nerve-evoked contractions, and that proctolin affects muscle fibers in a cell-selective manner.

Cell-selective modulation of the Drosophila neuromuscular system by a neuropeptide.

Neuropeptides can modulate physiological properties of neurons in a cell-specific manner. The present work examines whether a neuropeptide can also modulate muscle tissue in a cell-specific manner using identified muscle cells in third-instar larvae of fruit flies. DPKQDFMRFa, a modulatory peptide in the fruit fly Drosophila melanogaster, has been shown to enhance transmitter release from motor neurons and to elicit contractions by a direct effect on muscle cells. We report that DPKQDFMRFa causes a nifedipine-sensitive drop in input resistance in some muscle cells (6 and 7) but not others (12 and 13). The peptide also increased the amplitude of nerve-evoked contractions and compound excitatory junctional potentials (EJPs) to a greater degree in muscle cells 6 and 7 than 12 and 13. Knocking down FMRFamide receptor (FR) expression separately in nerve and muscle indicate that both presynaptic and postsynaptic FR expression contributed to the enhanced contractions, but EJP enhancement was mainly due to presynaptic expression. Muscle ablation showed that DPKQDFMRFa induced contractions and enhanced nerve-evoked contractions more strongly in muscle cells 6 and 7 than cells 12 and 13. In situ hybridization indicated that FR expression was significantly greater in muscle cells 6 and 7 than 12 and 13. Taken together, these results indicate that DPKQDFMRFa can elicit cell-selective effects on muscle fibers. The ability of neuropeptides to work in a cell-selective manner on neurons and muscle cells may help explain why so many peptides are encoded in invertebrate and vertebrate genomes.

Mode of action of a Drosophila FMRFamide in inducing muscle contraction.

Drosophila melanogaster is a model system for examining the mechanisms of action of neuropeptides. DPKQDFMRFamide was previously shown to induce contractions in Drosophila body wall muscle fibres in a Ca(2+)-dependent manner. The present study examined the possible involvement of a G-protein-coupled receptor and second messengers in mediating this myotropic effect after removal of the central nervous system. DPKQDFMRFamide-induced contractions were reduced by 70% and 90%, respectively, in larvae with reduced expression of the Drosophila Fmrf receptor (FR) either ubiquitously or specifically in muscle tissue, compared with the response in control larvae in which expression was not manipulated. No such effect occurred in larvae with reduced expression of this gene only in neurons. The myogenic effects of DPKQDFMRFamide do not appear to be mediated through either of the two Drosophila myosuppressin receptors (DmsR-1 and DmsR-2). DPKQDFMRFamide-induced contractions were not reduced in Ala1 transgenic flies lacking activity of calcium/calmodulin-dependent protein kinase (CamKII), and were not affected by the CaMKII inhibitor KN-93. Peptide-induced contractions in the mutants of the phospholipase C-ß (PLCß) gene (norpA larvae) and in IP3 receptor mutants were similar to contractions elicited in control larvae. The peptide failed to increase cAMP and cGMP levels in Drosophila body wall muscles. Peptide-induced contractions were not potentiated by 3-isobutyl-1-methylxanthine, a phosphodiesterase inhibitor, and were not antagonized by inhibitors of cAMP-dependent or cGMP-dependent protein kinases. Additionally, exogenous application of arachidonic acid failed to induce myogenic contractions. Thus, DPKQDFMRFamide induces contractions via a G-protein coupled FMRFamide receptor in muscle cells but does not appear to act via cAMP, cGMP, IP3, PLC, CaMKII or arachidonic acid.

Muscle-fiber specific genetic manipulation of Drosophila sallimus severely impacts neuromuscular development, morphology, and physiology.

The ability of skeletal muscles to contract is derived from the unique genes and proteins expressed within muscles, most notably myofilaments and elastic proteins. Here we investigated the role of the sallimus (sls) gene, which encodes a structural homologue of titin, in regulating development, structure, and function of Drosophila melanogaster. Knockdown of sls using RNA interference (RNAi) in all body-wall muscle fibers resulted in embryonic lethality. A screen for muscle-specific drivers revealed a Gal4 line that expresses in a single larval body wall muscle in each abdominal hemisegment. Disrupting sls expression in single muscle fibers did not impact egg or larval viability nor gross larval morphology but did significantly alter the morphology of individual muscle fibers. Ultrastructural analysis of individual muscles revealed significant changes in organization. Surprisingly, muscle-cell specific disruption of sls also severely impacted neuromuscular junction (NMJ) formation. The extent of motor-neuron (MN) innervation along disrupted muscles was significantly reduced along with the number of glutamatergic boutons, in MN-Is and MN-Ib. Electrophysiological recordings revealed a 40% reduction in excitatory junctional potentials correlating with the extent of motor neuron loss. Analysis of active zone (AZ) composition revealed changes in presynaptic scaffolding protein (brp) abundance, but no changes in postsynaptic glutamate receptors. Ultrastructural changes in muscle and NMJ development at these single muscle fibers were sufficient to lead to observable changes in neuromuscular transduction and ultimately, locomotory behavior. Collectively, the data demonstrate that sls mediates critical aspects of muscle and NMJ development and function, illuminating greater roles for sls/titin.

Developmental and physiological impacts of pathogenic human huntingtin protein in the nervous system.

Huntington's Disease (HD) is a neurodegenerative disorder, part of the nine identified inherited polyglutamine (polyQ) diseases. Most commonly, HD pathophysiology manifests in middle-aged adults with symptoms including progressive loss of motor control, cognitive decline, and psychiatric disturbances. Associated with the pathophysiology of HD is the formation of insoluble fragments of the huntingtin protein (htt) that tend to aggregate in the nucleus and cytoplasm of neurons. To track both the intracellular progression of the aggregation phenotype as well as the physiological deficits associated with mutant htt, two constructs of human HTT were expressed with varying polyQ lengths, non-pathogenic-htt (Q15, NP-htt) and pathogenic-htt (Q138, P-htt), with an N-terminal RFP tag for in vivo visualization. P-htt aggregates accumulate in the ventral nerve cord cell bodies as early as 24 hours post hatching and significant aggregates form in the segmental nerve branches at 48 hours post hatching. Organelle trafficking up-and downstream of aggregates formed in motor neurons showed severe deficits in trafficking dynamics. To explore putative downstream deficits of htt aggregation, ultrastructural changes of presynaptic motor neurons and muscles were assessed, but no significant effects were observed. However, the force and kinetics of muscle contractions were severely affected in P-htt animals, reminiscent of human chorea. Reduced muscle force production translated to altered locomotory behavior. A novel HD aggregation model was established to track htt aggregation throughout adulthood in the wing, showing similar aggregation patterns with larvae. Expressing P-htt in the adult nervous system resulted in significantly reduced lifespan, which could be partially rescued by feeding flies the mTOR inhibitor rapamycin. These findings advance our understanding of htt aggregate progression as well the downstream physiological impacts on the nervous system and peripheral tissues.

Action of octopamine and tyramine on muscles of Drosophila melanogaster larvae.

Octopamine (OA) and tyramine (TA) play important roles in homeostatic mechanisms, behavior, and modulation of neuromuscular junctions in arthropods. However, direct actions of these amines on muscle force production that are distinct from effects at the neuromuscular synapse have not been well studied. We utilize the technical benefits of the Drosophila larval preparation to distinguish the effects of OA and TA on the neuromuscular synapse from their effects on contractility of muscle cells. In contrast to the slight and often insignificant effects of TA, the action of OA was profound across all metrics assessed. We demonstrate that exogenous OA application decreases the input resistance of larval muscle fibers, increases the amplitude of excitatory junction potentials (EJPs), augments contraction force and duration, and at higher concentrations (10(-5) and 10(-4) M) affects muscle cells 12 and 13 more than muscle cells 6 and 7. Similarly, OA increases the force of synaptically driven contractions in a cell-specific manner. Moreover, such augmentation of contractile force persisted during direct muscle depolarization concurrent with synaptic block. OA elicited an even more profound effect on basal tonus. Application of 10(-5) M OA increased synaptically driven contractions by ≈ 1.1 mN but gave rise to a 28-mN increase in basal tonus in the absence of synaptic activation. Augmentation of basal tonus exceeded any physiological stimulation paradigm and can potentially be explained by changes in intramuscular protein mechanics. Thus we provide evidence for independent but complementary effects of OA on chemical synapses and muscle contractility.

Thermal games in crayfish depend on establishment of social hierarchies.

An unequal resource distribution is commonly seen in dominance hierarchies, in which the individual with the higher status is more successful in obtaining the resource. One possible resource is preferred temperature. When situations allow, ectotherms regulate their body temperature by behaviourally selecting different environmental conditions, achieving, when possible, a preferred temperature. Using a shuttlebox, the preferred temperature for Procambarus clarkii was determined to be 23.9°C with upper and lower voluntary escape temperatures of 25.9 and 21.8°C, respectively. If this preferred temperature zone (21.8-25.9°C) was valued as a resource, given the choice between a preferred temperature and a non-preferred temperature, crayfish should compete over the preferred temperature, with the dominant individual of dyadic pairs achieving the preferred temperature more often than the subordinate. Using a dual-choice experimental tank, competition over a binary temperature choice between rank-established paired crayfish was determined under both warm and cold challenge conditions (warm vs preferred temperature and cold vs preferred temperature, respectively). In naive pairings, similar levels of competition over the preferred temperature occurred in both warm and cold challenge trials, as predicted by game theory. In established pairings, however, dominant crayfish gained significantly greater access to preferred temperature in both warm and cold challenge conditions. These results demonstrate that crayfish engage in a cost-benefit assessment during their initial agonistic contests over temperature, but as hierarchies mature, these thermal games are decided by the dominant animal gaining primary access to the temperature resource.

Glial Ca2+signaling links endocytosis to K+ buffering around neuronal somas to regulate excitability.

Glial-neuronal signaling at synapses is widely studied, but how glia interact with neuronal somas to regulate their activity is unclear. Drosophila cortex glia are restricted to brain regions devoid of synapses, providing an opportunity to characterize interactions with neuronal somas. Mutations in the cortex glial NCKXzydeco elevate basal Ca2+, predisposing animals to seizure-like behavior. To determine how cortex glial Ca2+ signaling controls neuronal excitability, we performed an in vivo modifier screen of the NCKXzydeco seizure phenotype. We show that elevation of glial Ca2+ causes hyperactivation of calcineurin-dependent endocytosis and accumulation of early endosomes. Knockdown of sandman, a K2P channel, recapitulates NCKXzydeco seizures. Indeed, sandman expression on cortex glial membranes is substantially reduced in NCKXzydeco mutants, indicating enhanced internalization of sandman predisposes animals to seizures. These data provide an unexpected link between glial Ca2+ signaling and the well-known role of glia in K+ buffering as a key mechanism for regulating neuronal excitability.

Astrocyte Ca2+ Influx Negatively Regulates Neuronal Activity.

Maintenance of neural circuit activity requires appropriate regulation of excitatory and inhibitory synaptic transmission. Recently, glia have emerged as key partners in the modulation of neuronal excitability; however, the mechanisms by which glia regulate neuronal signaling are still being elucidated. Here, we describe an analysis of how Ca2+ signals within Drosophila astrocyte-like glia regulate excitability in the nervous system. We find that Drosophila astrocytes exhibit robust Ca2+ oscillatory activity manifested by fast, recurrent microdomain Ca2+ fluctuations within processes that infiltrate the synaptic neuropil. Unlike the enhanced neuronal activity and behavioral seizures that were previously observed during manipulations that trigger Ca2+ influx into Drosophila cortex glia, we find that acute induction of astrocyte Ca2+ influx leads to a rapid onset of behavioral paralysis and a suppression of neuronal activity. We observe that Ca2+ influx triggers rapid endocytosis of the GABA transporter (GAT) from astrocyte plasma membranes, suggesting that increased synaptic GABA levels contribute to the neuronal silencing and paralysis. We identify Rab11 as a novel regulator of GAT trafficking that is required for this form of activity regulation. Suppression of Rab11 function strongly offsets the reduction of neuronal activity caused by acute astrocyte Ca2+ influx, likely by inhibiting GAT endocytosis. Our data provide new insights into astrocyte Ca2+ signaling and indicate that distinct glial subtypes in the Drosophila brain can mediate opposing effects on neuronal excitability.

Drosophila development, physiology, behavior, and lifespan are influenced by altered dietary composition.

Diet profoundly influences the behavior of animals across many phyla. Despite this, most laboratories using model organisms, such as Drosophila, use multiple, different, commercial or custom-made media for rearing their animals. In addition to measuring growth, fecundity and longevity, we used several behavioral and physiological assays to determine if and how altering food media influence wild-type (Canton S) Drosophila melanogaster, at larval, pupal, and adult stages. Comparing 2 commonly used commercial food media we observed several key developmental and morphological differences. Third-instar larvae and pupae developmental timing, body weight and size, and even lifespan significantly differed between the 2 diets, and some of these differences persisted into adulthood. Diet was also found to produce significantly different thermal preference, locomotory capacity for geotaxis, feeding rates, and lower muscle response to hormonal stimulation. There were no differences, however, in adult thermal preferences, in the number or viability of eggs laid, or in olfactory learning and memory between the diets. We characterized the composition of the 2 diets and found particularly significant differences in cholesterol and (phospho)lipids between them. Notably, diacylglycerol (DAG) concentrations vary substantially between the 2 diets, and may contribute to key phenotypic differences, including lifespan. Overall, the data confirm that 2 different diets can profoundly influence the behavior, physiology, morphology and development of wild-type Drosophila, with greater behavioral and physiologic differences occurring during the larval stages.

Functional characterization on invertebrate and vertebrate tissues of tachykinin peptides from octopus venoms.

It has been previously shown that octopus venoms contain novel tachykinin peptides that despite being isolated from an invertebrate, contain the motifs characteristic of vertebrate tachykinin peptides rather than being more like conventional invertebrate tachykinin peptides. Therefore, in this study we examined the effect of three variants of octopus venom tachykinin peptides on invertebrate and vertebrate tissues. While there were differential potencies between the three peptides, their relative effects were uniquely consistent between invertebrate and vertebrae tissue assays. The most potent form (OCT-TK-III) was not only the most anionically charged but also was the most structurally stable. These results not only reveal that the interaction of tachykinin peptides is more complex than previous structure-function theories envisioned, but also reinforce the fundamental premise that animal venoms are rich resources of novel bioactive molecules, which are useful investigational ligands and some of which may be useful as lead compounds for drug design and development.

Cholesterol-independent effects of methyl-ß-cyclodextrin on chemical synapses.

The cholesterol chelating agent, methyl-ß-cyclodextrin (MßCD), alters synaptic function in many systems. At crayfish neuromuscular junctions, MßCD is reported to reduce excitatory junctional potentials (EJPs) by impairing impulse propagation to synaptic terminals, and to have no postsynaptic effects. We examined the degree to which physiological effects of MßCD correlate with its ability to reduce cholesterol, and used thermal acclimatization as an alternative method to modify cholesterol levels. MßCD impaired impulse propagation and decreased EJP amplitude by 40% (P<0.05) in preparations from crayfish acclimatized to 14 °C but not from those acclimatized to 21 °C. The reduction in EJP amplitude in the cold-acclimatized group was associated with a 49% reduction in quantal content (P<0.05). MßCD had no effect on input resistance in muscle fibers but decreased sensitivity to the neurotransmitter L-glutamate in both warm- and cold-acclimatized groups. This effect was less pronounced and reversible in the warm-acclimatized group (90% reduction in cold, P<0.05; 50% reduction in warm, P<0.05). MßCD reduced cholesterol in isolated nerve and muscle from cold- and warm-acclimatized groups by comparable amounts (nerve: 29% cold, 25% warm; muscle: 20% cold, 18% warm; P<0.05). This effect was reversed by cholesterol loading, but only in the warm-acclimatized group. Thus, effects of MßCD on glutamate-sensitivity correlated with its ability to reduce cholesterol, but effects on impulse propagation and resulting EJP amplitude did not. Our results indicate that MßCD can affect both presynaptic and postsynaptic properties, and that some effects of MßCD are unrelated to cholesterol chelation.