Role of calcium in the regulation of mechanical power in insect flight

Abstract

Most flying insect species use "asynchronous" indirect flight muscles (A-IFMs) that are specialized to generate high mechanical power at fast contraction frequencies. Unlike individual contractions of "synchronous" muscles, those of A-IFMs are not activated and deactivated in concert with neurogenically controlled cycling of myoplasmic [Ca(2+)] but rather are driven myogenically by oscillatory changes in length. The motor neurons of the A-IFMs, which fire at a rate much slower than contraction frequency, are thought to play the limited role of maintaining myoplasmic [Ca(2+)] above the critical threshold that maintains the muscle in a stretch-activatable state. Despite this asynchronous form of excitation-contraction coupling, animals can actively regulate power output as required for different flight behaviors, although the neurobiological and biophysical basis of this regulation is unknown. While presenting tethered flying fruit flies, Drosophila melanogaster, with visual stimuli, we recorded membrane potential spikes in identified A-IFM fibers. We show that mechanical power output rises and falls in concert with the firing frequency of all A-IFM fibers and cannot be explained by differential recruitment of separately innervated motor units. To explore the hypothesis that myoplasmic [Ca(2+)] might similarly rise and fall in concert with firing frequency, we genetically engineered Drosophila to express the FRET-based Ca(2+) indicator cameleon selectively within A-IFMs. The results show that Ca(2+) levels increase in proportion to muscle firing rate, both during spontaneous flight and when muscle spikes are elicited electrically. Collectively, these experiments on intact animals support an active role for [Ca(2+)] in regulating power output of stretch-activated A-IFM.

Fig. 1.

Mechanical power covaries with A-IFM firing rate. (a and b) Muscle fibers were recorded during flight in tethered flies. Changes in flight behavior were elicited visually by using a visual grating pattern that drifted alternately up and down. Stroke frequency and amplitude were measured with an optical sensor. (c) Flies increased and decreased stroke amplitude and stroke frequency to effect changes in mechanical power in response to the upward and downward motion of the visual stimulus. Power modulations correlated with changes in muscle firing rates. (d) In flight segments selected for rapid changes in A-IFM firing rate, the time course of mechanical power rises much more rapidly than it falls (mean ± SEM, dark shading). Red lines indicate first-order exponential fits through the data. The up stimulus is indicated by light gray background shading (n = 30). The down stimulus has no background shading (n = 22). (e) Steady-state power plotted versus steady-state A-IFM rate. Sixty-two flight sequences were averaged over a 4-s window (Fig. 3b shows seconds 2–6 after the onset of the up stimulus). Data were binned by mean A-IFM firing rate; error bars represent SEM.

Fig. 2.

Intracellular calcium correlates with muscle activity. (a) Cameleon 3.1 expressed specifically in A-IFM (both DLMs and DVMs) but not synchronous muscle (e.g., tergotrochanteral muscle, TTM). (b) During flight, increased FRET from YFP to CFP indicated that calcium levels varied with A-IFM spike rate. (c) Current injected in the brain evoked simultaneous activity in A-IFMs by means of the giant fiber (gf) system. mn, motorneuron; psi, peripherally synapsing interneuron; e, electrical synapse; c, chemical synapse. (d) In the absence of flight, calcium responses to single pulses (arrows) and trains of electrical stimulation at 4, 2, 1.5, 3, 2.5, 1, 5, 20, 12.5, 15, 10, 8, and 25 Hz. Bar height is only qualitatively proportional to stimulus frequency. (e) Steady-state calcium levels in response to brain stimulation (mean of stimulus pulses 2–10 in each train) plotted versus stimulus frequency.