Excitation-contraction coupling and its relation to synaptic dysfunction in Drosophila

The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic deficits alter neuromuscular transduction and muscle contractility that ultimately dictate behavioural output. Here we develop a system for detailed characterization of excitation-contraction coupling at Drosophila larval NMJs and demonstrate how specific synaptic and neuronal manipulations disrupt muscle contractility. Muscle contraction force increases with motoneuron stimulation frequency and duration, showing considerable plasticity between 5-40 Hz, while saturating above 50 Hz. Temperature is negatively correlated with muscle performance and enhanced at lower temperatures. A screen for modulators of muscle contractility led to the identification and characterization of the molecular and cellular pathway by which a specific FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to relate synaptic dysfunction to alterations in excitation-contraction coupling.


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Neuromuscular systems that regulate stereotyped motor behaviours are composed of 52 multiple component parts that interact in a highly coordinated and synchronized fashion to 53 control muscle contraction and displacement (Goulding, 2009;Selverston, 1980). Central pattern 54 generators (CPGs) initiate stereotyped behaviours such as locomotion, feeding, and swimming 55 (Katz and Frost, 1995;Schwarz et al., 2017). CPGs relay rhythmic output through interneurons 56 to motoneurons, controlling the timing and magnitude of muscle contraction (Selverston, 2010). 57 In turn, the peripheral nervous system (PNS) signals the dynamic state of the muscle back to the 58 CPG to modulate circuit output (Singhania and Grueber, 2014). A large body of work on the (contracting to a length less than 50% of resting length) striated multinucleated muscle fiber 79 (Keshishian et al., 1996) attached directly to the cuticle through apodemes (Koh et al., 2000).

Results
: 111 To explore the contractile properties of Drosophila 3 rd instar larval NMJs, muscle 112 contractions were elicited in semi-intact preparations with the CNS removed. A force transducer 113 with 10 µN resolution was modified to attach to the posterior end of a larvae ( Figure 1A). 114 Previous studies in Drosophila examined contractile properties by continually stimulating 115 motoneurons at the same intraburst frequency and duration (Ormerod et al., 2016). An example 116 of a single contraction induced from the setup, highlighting the amplitude, rise tau, and decay tau 117 is shown in Figure 1B. To systematically characterize muscle contraction performance, bodywall neuronal input whose firing pattern is ultimately controlled by the locomotor CPGs within the 123 ventral nerve cord (Song et al, 2007). We recorded fictive locomotor patterns within 124 motoneurons encoded from the CPGs from bodywall muscles via intracellular recordings from 125 semi-intact preparations with the CNS and VNC left intact ( Figure 1D). These patterned outputs 126 displayed widely varying intraburst frequencies from 1 to 150 Hz. Given this variable 127 endogenous activity, we explored a more dynamic approach to eliciting muscle contractions to 128 determine the range of muscle force bodywall muscles are capable of producing under a variety 129 of motoneuron stimulation frequencies. 130 Initially, 25 impulses were delivered to motoneurons and the stimulation frequency was 131 varied from 1 to 150 Hz. For each experiment, 6 replicate contractions were induced at each 132 stimulation frequency, followed by the next stimulation frequency, et cetera ( Figure 1E). 133 Contractions were averaged across the 6 replicate stimuli for a given stimulation frequency and 134 the resulting trace with 95% confidence interval (CI) was determined ( Figure 1F). A force-135 frequency curve of the force generated from this stimulation paradigm was then plotted as a 136 percentage of the magnitude of force generated at the highest stimulation frequency of 150 Hz 137 ( Figure 1I, N=8). 25 stimuli at 1 Hz stimulation induced 3.5 + 1.1% of the maximum force 138 produced at 150 Hz, while 2 Hz stimulation was sufficient to induce 5.2 + 1.1% of the maximum 139 force. Force generation at 2 Hz was not significantly greater than 1 Hz stimulation. In contrast, 25 stimuli at 5 Hz produced a resultant force of 11.0 + 1.1% of the force at 150 Hz, indicating 141 this stimulation frequency is sufficient for temporal summation for contractile force. From 10 to 142 50 Hz stimulation, a nearly linear increase in force was produced at each successive increase in 143 frequency (10, 15, 20, 25, 30, 40, 50 Hz), producing a maximal force at 50 Hz. Increasing the 144 stimulation frequency beyond 50 Hz resulted in a reduction in force production. Therefore, when 145 total stimuli number remains constant, increasing stimulation frequency increases force 146 production until saturation is reached at 50 Hz.

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Although 50 Hz drove maximal muscle contraction, it was unclear whether this saturation 148 of force generation was a consequence of the stimulus frequency or stimulus duration. To 149 explore this relationship further, burst duration was held constant at 600 ms and the frequency of 150 stimulation was varied from 1 to 150 Hz. Figure 1G depicts the raw data from each individual 151 contraction within a single trial, and Figure 1F depicts averaged traces from the replicate stimuli 152 at a given stimulation frequency with a corresponding 95% CI. Under these conditions, 5 Hz 153 stimulation produced a force of 6.0 + 3.1% of that produced during 150 Hz stimulation (Table 1).

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Comparable to the previous experiment, force production increased in a linear fashion as the 155 frequency increased from 10-50 Hz. However, increasing stimulation frequency to 100 and 150 156 Hz increased the force. Thus, both the duration and frequency of stimulation are critical factors 157 in determining the absolute magnitude of force production.

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To more fully explore the relationship between stimulus frequency and stimulus duration, 159 we generated a series of force-frequency curves by keeping the burst duration constant through 160 an entire experiment over 200, 250, 300, 500, 600, 750, 900, 1000, 2000, or 5000 ms while 161 increasing stimulation frequency from 1 to 150 Hz. Each of the independent force-frequency 162 curves for a given burst duration generated a sigmoidal curve similar to what was observed at 163 600 ms ( Figure 2A and Table 1). The overall patterns from the stimulation force recordings 164 revealed that increasing stimulus frequency or duration results in a progressive and gradual 165 increase in muscle force production. In all experiments, a single action potential was sufficient to 166 induce a contraction that was between 2.6 and 3.3% of the 150 Hz contraction force (note that 1 167 Hz stimulation at 1000, 2000, and 5000 ms corresponded to 2, 4, and 10 stimuli respectively).

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Stimulus durations of 200, 250, and 300 ms resulted in increased force production with increases 169 in stimulus frequency that did not saturate until 150 Hz (Table 1). Stimulus durations of 600, 750, 900, and 1000 ms resulted in sigmoidal increases in force production with increases in 171 stimulus frequency that saturated at 100 Hz. Increasing the stimulus duration to 2000 and 5000 172 ms resulted in a saturation of force at 40 and 30 Hz, respectively. Given force saturates at or 173 above 100 Hz for all stimulus durations, there should be no differences between these conditions 174 once force reaches maximal. Thus, it is not surprising that the greatest differences between 175 stimulus durations are seen from 10 to 50 Hz, where considerable plasticity in muscle 176 performance is still possible (Fig 2A). Plotting the percent differences between the various 177 stimulation durations as a function of stimulus frequencies generated a bell-shaped curve with 178 peak differences observed at 25 Hz. The greatest effect of stimulus duration was observed at 25 179 Hz, where an increase from 200 to 5000 ms resulted in a 70.3% increase in muscle force. Figure   180 2B depicts an overlay of 25 Hz stimulation from 200, 300, 600, 900, and 2000 ms. A critical 181 feature of the dataset is reflected in the stimulation frequency required to generate half-maximal 182 force (50%) for each stimulus duration, where a linear decrease in the stimulus frequency 183 required to reach 50% is observed (Table 1). Plotting the 50% max value as a function of 184 stimulus duration generated a strong negative correlation (R 2 =0.9) with a slope of -0.016, 185 indicating that every 100 ms increase in duration shifts this value to the left by 1.6 Hz. Despite 186 the dramatic effects that stimulation duration has on force production below 50 Hz, the saturated 187 or maximum raw force generated from a 200 ms duration stimulus was not significant for any of 188 the longer stimulus durations ( Figure 2C, D). These findings indicate maximal force from 189 Drosophila bodywall muscles can be generated by a 200 ms stimulus delivered at 100 Hz.

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Throughout the experiments there was variability in the total raw force generated from 191 one preparation to the next (Fig 2C), differing in magnitude by up to 50%. Given this variability, 192 one potential contributor to the differences in force generation was larval size. To examine this 193 variable, the length and width of 100 larvae was measured and the total force generated by each  conditions. Figure 5B shows the effect of external Ca 2+     Consequently, we repeated this experiment using 25 Hz to determine if temperature might have a 264 more profound effect, particularly at lower temperature. Significant differences were observed at 265 15, 16, 19, and 20°C, indicating more substantial effects of temperature at lower stimulation 266 frequencies ( Figure 6C). Rearing animals at different temperatures, particularly ectothermic 267 animals, has been shown to adapt various aspect of an animal's physiology, shifting their 268 physiological efficiency closer to rearing temperatures (Bennett, 1985). Thus, we reared animals  TPAEDFMRFa's capacity to enhance contraction force, suggesting it acts through multiple 385 receptors and in both synaptic compartments to effect muscle output.

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Our data indicate neither the size of 3 rd instar larvae nor the muscle subgroup examined 387 had any significant effect on the maximal force production or biophysical properties of 388 excitation-contraction coupling across the whole animal (Figures 3, 4). Although muscle have shown that muscle length does not result in greater force production, but rather impacts 391 contraction velocity (Forman et al., 1972). For some fibers, e.g. muscle fiber 6, a clear increase in the resting muscle width is observed, compared to its direct neighbor, muscle fiber 7. It is 393 interesting that a greater number of longitudinal muscles are likely present along the ventral axis 394 of the larvae (Fig. 4,   and 39°C (Fig. 7). Action potential propagation in axons is known to be blocked at higher 437 temperatures due to rapid gating dynamics of Na + channels, reducing positive charge influx (τ-rise) and decay time (τ-decay) (Miller and Rinzel, 1981). Impulse propagation failure is 442 thought to occur at regions of nonuniform morphology causing increases in capacitance, typical 443 at axonal branch points and transitions zones from myelination to demyelination (Hille, 1985).   averaged for each animal. 600 ms duration: intraburst duration at each frequency was 600 ms in duration, 869 6 replicate contractions were elicited at each stimulation frequency and averaged for each animal, N=20). 870   generated for control Canton S, Cpx null mutants (cpx SH1 ), Gbb null mutants (Gbb 1 /Gbb 2 ), Syt1 null 918 mutants (Syt1 AD4 /Syt1 N13 ) and Syt4 null mutants (Syt4 BA1 ). B) The magnitude of force generated from a 919 single stimulus from each genotype is shown. One-way ANOVA, **p<0.01, ***p<0.001. 920   Table: 952 Supplemental Table 1: EC 50 calculations for TPAEDFMRFa's effectiveness for increasing the force of  953 contractions. For each frequency the maximum force increase (typically from 10 -5 M) observed following 954 TPAEDFMRFa was also calculated.