Improved Inhibitory and Absorption, Distribution, Metabolism, Excretion, and Toxicology (ADMET) Properties of Blebbistatin Derivatives Indicate That Blebbistatin Scaffold Is Ideal for drug Development Targeting Myosin-2.

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 studies were limited to a single myosin-2 isoform. Here we evaluated the potential of blebbistatin scaffold for drug development and found that D-ring substitutions can fine-tune isoform specificity, absorption-distribution-metabolism-excretion, 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, whereas 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.

Developmental expression of human tau in Drosophila melanogaster glial cells induces motor deficits and disrupts maintenance of PNS axonal integrity, without affecting synapse formation.

Tauopathies are a class of neurodegenerative diseases characterized by the abnormal phosphorylation and accumulation of the microtubule-associated protein, tau, in both neuronal and glial cells. Though tau pathology in glial cells is a prominent feature of many of these disorders, the pathological contribution of these lesions to tauopathy pathogenesis remains largely unknown. Moreover, while tau pathology is predominantly found in the central nervous system, a role for tau in the cells of the peripheral nervous system has been described, though not well characterized. To investigate the effects of glial tau expression on the development and maintenance of the peripheral nervous system, we utilized a Drosophila melanogaster model of tauopathy that expresses human wild-type tau in glial cells during development. We found that glial tau expression during development results in larval locomotor deficits and organismal lethality at the pupal stage, without affecting larval neuromuscular junction synapse development or post-synaptic amplitude. There was, however, a significant decrease in the decay time of synaptic potentials upon repeated stimulation of the motoneuron. Behavioral abnormalities were accompanied by glial cell death, disrupted maintenance of glial-axonal integrity, and the abnormal accumulation of the presynaptic protein, Bruchpilot, in peripheral nerve axons. Together, these data demonstrate that human tau expression in Drosophila glial cells does not affect neuromuscular junction synapse formation during development, but is deleterious to the maintenance of glial-axonal interactions in the peripheral nervous system.

A scalable, high resolution strain sensing matrix suitable for tactile transduction.

The integration of tactile information, such as contact area, displacement magnitude, velocity, and acceleration, is paramount to the optimization of robotics in human-centric environments. Cost effective embeddable sensors with scalable receptive field size and strain sensitivity are not readily commercially available and would benefit investigations of in situ tissue mechanics. We describe the design and performance of a scalable sensor matrix that transduces fine parameters of strain and is made of combinable "modules". The sensors transduce static and dynamic strains of both uniaxial and multi-dimensional nature. Modules consist of three silicon wafers placed on top of and three on the bottom of a hexagonal collar, wafers are thus positioned 120° to one another to facilitate force vector extrapolation. Analog signals from each sensor can be easily compared to neighboring sensor output to determine mechanical phenomena such as slip or shear. The smallest of our prototype multiunit matrices consisted of seven hexes in a honeycomb orientation of 4.1mm diameter (containing 42 silicon gauges). Unamplified, unshielded output from this embodiment (3 Vexc button cell) yielded 1 mV from 5 µm displacement. Transduction linearity was high (R>0.99 nearest displacement) and exhibited nominal hysteresis. Modules may be placed upon or embedded into a multitude of materials and the size of individual hexagons may be scaled for favorable stiffness to strain ratio and to scale receptive field. Given the scalability of matrix size and resolution, we believe the sensor matrices could benefit the fields of prosthetics, robotics, and physiologic investigation of tissue mechanics.

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.

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.

Hysteresis in the production of force by larval Dipteran muscle.

We 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 approximately 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.

The resting membrane potential of Drosophila melanogaster larval muscle depends strongly on external calcium concentration.

The resting membrane potential (RMP) of most cells is not greatly influenced by the transmembrane calcium gradient because at rest, the membrane has very low permeability to calcium. We have observed, however, that the resting membrane potential of muscle cells in the larval bodywall of Drosophila melanogaster varies widely as the external calcium concentration is modified. The RMP depolarized as much as 21.8 mV/mM calcium at low concentrations, and on average, about 10 mV/mM across a range typical of neurophysiological investigations. The extent to which muscle RMP varies has important implications for the measurement of synaptic potentials as well. Two parameters of excitatory junctional potential (EJP) voltage were compared across a range of RMPs. EJP amplitude (DeltaV) and peak voltage (maxima) change as a function of RMP; on average, a 10 mV change in RMP elicits a 4-5 mV change in EJP amplitude and peak voltage. The influence of the calcium gradient on resting and synaptic membrane potentials led us to investigate the endogenous ion concentrations of larval hemolymph. In addition to the major monovalent ions and calcium, we report the first voltammetric analysis of magnesium concentration in larval fruit fly hemolymph.

Teaching insect retinal physiology with newly designed, inexpensive micromanipulators.

In this article, we detail how to produce two inexpensive micromanipulators that offer high precision (approximately 25 microm) along a single axis of movement. The more expensive of the designs provides improved versatility along multiple axes. Both manipulators offer substantial savings over commercially available micromanipulators with comparable capabilities. Plans and instructions are given such that a novice can produce the manipulators with simple tools. The manipulators are designed to serve undergraduate teaching exercises in physiology. An electroretinogram exercise is suggested in adult house flies (Musca) or flesh flies (Neobellieria). Measuring the intensity-response function and temporal characteristics of visual transduction are discussed. A brief introduction to the field of visual transduction and the physiology of the laboratory exercises is provided as well.

Variability of motoneuron activation and the modulation of force production in a postural reflex of the hermit crab abdomen.

The tri-phasic reflex in hermit crab (Pagurus pollicarus) abdomen is triggered by local mechanoreceptors and is essential for postural control. The reflex consists of three stereotypical phases: a brief, high-frequency burst, a transient cessation of firing, and a late-discharge that is much lower in frequency than the initial burst. To better understand the reflex generation of force, variability of motoneuron discharge in each of five parameters of reflex activation was assessed. An intracellular current injection routine was used to correlate each of these parameters with force production. Phase 3 motoneuron firing frequency showed the greatest correlation with force production. Phase 3 spike rate increased as a function of phase 2 duration, but the relationship between phase 2 duration and force produced by the reflex was weak. Junction potential amplitude decreased as phase 2 duration increased, and we hypothesize that this trend counteracts the increased phase 3 frequency, explaining the weak relationship of phase 2 duration and force production. Surprisingly, when phase 3 frequency was held constant and phase 2 was increased in duration, the concurrent decrease in junction potential amplitude did not reduce force production.

Tools for physiology labs: an inexpensive means of temperature control.

We describe a simple means of modulating preparation temperature, which may be useful in undergraduate physiology laboratories. The device was developed in an effort to make teaching exercises that involve temperature modulation accessible at low cost. Although we were interested in using the device specifically with the larval fruit fly preparation, it is applicable to many preparations and temperature sensitive phenomena. Feedback driven thermoregulators offer superior precision in experiments requiring temperature control, but can be prohibitively expensive, require power supplies and circuitry, and often generate large switching transients (artifacts) during physiological recording. Moreover, many interesting exercises involving temperature control can be carried out with a slightly reduced level of temperature precision.

Demonstrating the temperature sensitivity of synaptic transmission in a Drosophila mutant.

We describe exercises that illustrate the temperature sensitivity of synaptic transmission. The temperature dependence of synaptic transmission is demonstrated by cooling the larval Drosophila melanogaster preparation and recording excitatory junction potentials. Vesicle recycling is explored by utilizing a mutation of the shibire gene. This shibire mutant shows a robust reduction in synaptic vesicle recycling when temperature exceeds a known threshold (∼29° C). Students gain proficiency with the Drosophila larval neuromuscular junction preparation while investigating principles of vesicle release, vesicle recycling, synaptic facilitation and synaptic depression. We show that the viability of the larval preparation is prolonged in vitro with moderate cooling, which is particularly important when introducing the preparation as a novel exercise.

The action of spike frequency adaptation in the postural motoneurons of hermit crab abdomen during the first phase of reflex activation.

Cuticular strain associated with support of the shell of the hermit crab, Pagurus pollicarus, by its abdomen activates mechanoreceptors that evoke a stereotyped reflex in postural motoneurons. This reflex consists of three phases: a brief high-frequency burst of motoneuron spikes, a pause, and a much longer duration but lower frequency period of spiking. These phases are correlated with a rapid increase in muscle force followed by a slight decline to a level of tone that is greater than that at rest but less than maximal. The present experiments address the mechanisms underlying the transition from the first to second phase of the reflex and their role in force generation. Although centrally generated inhibitory post-synaptic potentials (IPSPS) are present during the pause period of the reflex, intracellular current injection of motoneurons reveals a spike frequency adaptation that rapidly and substantially reduces motoneuron firing frequency and is unchanged in saline that reduces synaptic transmission. The adaptation is voltage sensitive and persists for several hundred milliseconds upon repolarization. Hyperpolarization partially restores the initial response of the motoneuron to depolarizing current. Spike frequency adaptation and synaptic inhibition are important mechanisms in the generation of force that maintains abdominal stiffness at a constant, submaximal level.