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.

SUMMARYWe describe neuromuscular hysteresis -the dependence of muscle force on recent motoneuron activity -in the body wall muscles of larval Sarcophaga bullata and Drosophila melanogaster. In semi-intact preparations, isometric force produced by a train of nerve impulses at a constant rate was significantly less than that produced by the same train of stimuli with a brief (200ms) high-frequency burst of impulses interspersed. Elevated force did not decay back to predicted values after the burst but instead remained high throughout the duration of the stimulus train. The increased force was not due to a change in excitatory junction potentials (EJPs); EJP voltage and time course before and after the high-frequency burst were not statistically different. Single muscle and semi-intact preparations exhibited hysteresis similarly, suggesting that connective tissues of the origin or insertion are not crucial to the mechanism of hysteresis. Hysteresis was greatest at low motoneuron rates -yielding a ~100% increase over predicted values based on constant-rate stimulation alone -and decreased as impulse rate increased. We modulated motoneuron frequency rhythmically across rates and cycle periods similar to those observed during kinematic analysis of larval crawling. Positive force hysteresis was also evident within these more physiological activation parameters.

Ormerod KG, Krans JL, Mercier AJ. Cell-selective modulation of the Drosophila neuromuscular system by a neuropeptide. J Neurophysiol 113: 1631-1643, 2015. First published December 17, 2014 doi:10.1152/jn.00625.2014.-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 nifedipinesensitive 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 cellselective 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.

Blebbistatin, para-nitroblebbistatin (NBleb) and para-aminoblebbistatin (AmBleb) are highly useful tool compounds as they selectively inhibit the ATPase activity of myosin-2 family proteins. Despite the medical importance of the myosin-2 family as drug targets, chemical optimization has not yet provided a promising lead for drug development because previous structure-activity-relationship (SAR) studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that Dring substitutions can fine-tune isoform specificity, ADME and toxicological properties. We defined the inhibitory properties of NBleb and AmBleb on seven different myosin-2 isoforms, which revealed an unexpected potential for isoform specific inhibition. We also found that NBleb metabolizes six times slower than blebbistatin and AmBleb in rats, while AmBleb metabolizes two times slower than blebbistatin and NBleb in human, and that AmBleb accumulates in muscle tissues. Moreover, mutagenicity was also greatly reduced in case of AmBleb. These results demonstrate that small substitutions have beneficial functional and pharmacological consequences, which highlight the potential of the blebbistatin scaffold for drug development targeting myosin-2 family proteins and delineate a route for defining the chemical properties of further derivatives to be developed. Significance statement: Small substitutions on the blebbistatin scaffold have beneficial functional and pharmacological consequences highlighting their potential in drug development targeting myosin-2 family proteins.

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