Fig. 2. To adjust for the difference in timing between a stimulus applied to the muscle and muscle activation recorded during tethered flight, we recorded extracellular potentials (A) and force (B) from the dl₁ muscles during isometric twitches. Stimuli were delivered through extracellular electrodes spanning all subunits of the dl₁ muscles and were 0.2 ms in duration. Extracellular potentials were recorded from dl_1c. (A) Three extracellular muscle potentials in response to stimuli of increasing amplitude. The stimulus artifact (s) varied in amplitude with the amplitude of the applied stimulus. Subthreshold stimuli (a) produced only a stimulus artifact whereas suprathreshold stimuli (b,c) resulted in a clearly identifiable extracellular spike of constant amplitude. We defined the delay between the onset of the stimulus and the evoked potential (t_ep) as the time between the onset of the stimulus artifact and the peak of the evoked potential. The large voltage deflections of the stimulus artifact in b and c exceeded the range of the data acquisition system and are cut off at ±10 V. (B) Muscle force recorded following the three stimuli shown in A. Note that the time scale differs from that in A. Subunits of the dl₁ muscles responded in an all-or-none manner to stimuli. Subthreshold stimuli (a), defined by the absence of an evoked extracellular spike, generally did not result in twitch forces. Supramaximal stimuli (b,c) consistently produced a maximal twitch of nearly constant amplitude. In some preparations, subthreshold stimuli evoked submaximal twitches with discreet amplitudes. In these cases, the submaximal twitches probably resulted from selective recruitment of subunits other than dl_1c, the subunit in which we had implanted the recording electrodes. Stimulus amplitude in our measurements of mechanical power output was adjusted to the level that produced the maximal twitch force from the whole muscle. We defined the phase of activation in our work loop measurements as the projected time of the evoked potential normalized to the cycle period (see text for details).