Muscle twitch speeds were recorded in situ (Figure 4). For all preparations, birds were restrained on a soft foam pad that was securely attached to the surgical bench. Birds were then anesthetized with isoflurane (2–4% in O 2 ). To prepare the PEC and SH, we first cut a small (1 cm) incision in the skin. We then exposed the moist surface of the muscle by gently pushing apart the skin. Once the muscle was in clear view, we implanted it with the stripped ends (1–2 mm) of two insulated silver wire electrodes (diameter: 0.14 mm). These electrodes were then connected to a nearby stimulator (Model 2100, A-M Systems, WA, USA). We next fastened a stainless steel hook (0.1 mm diameter) to the muscle directly adjacent to the implanted electrodes. This hook was connected through a monofilament line to a force transducer (Model FT03, Grass Technology, RI, USA), which was tightly clamped to a heavy stand (≈6 kg). As soon as this preparation was completed, we placed a drop of normal avian saline (0.9%) over the exposed muscles to prevent tissues desiccation during the recording session. We adjusted the slack in the line to the force transducer by slightly moving the heavy stand; this allowed us to maintain tension in the line between the muscle and force transducer, and thus optimize the sensitivity of muscle twitch recordings without overloading the force transducer signal. When the recordings were completed, the electrodes and monofilament line from the force transducer were removed from the muscle, and the incision was closed using Vetbond tissue adhesive. All surgical preparations occurred at room temperature, which was similar to the outside ambient temperature (≈30°C).

Figure 4 Download asset Open asset Schematic representation of the experimental set up used to record muscle twitch in situ. Birds were deeply anesthetized with isoflurane; their muscles were exposed, attached to the force transducer, and implanted with silver electrode wires. Data were collected on a nearby laptop computer. Note that the elements in this figure are not drawn to scale. https://doi.org/10.7554/eLife.13544.009

We prepared the SC in a similar manner to the PEC and SH; however, we made a few modifications to this surgery, given that the SC lies deep to the PEC and is more difficult to access (Figure 1). First, we cut a small (1.5 cm) incision in the skin above the furcula. We then gently moved aside the underlying fat-pad and exposed the inter-clavicular air-sac. To gain access to the SC, we carefully moved the membrane of this air-sac to the side, taking great caution not to puncture it. From this angle, we could then see the SC positioned above the keel and below the PEC. We quickly implanted the SC muscle with the stripped ends of the silver electrodes, and allowed the inter-clavicular air-sac to fall back into position. Finally, we gently moved the fat pad back over the furcula to prevent desiccation during the recordings and placed a small drop of normal avian saline over this tissue. Given the deep (and difficult) position of the SC, it was not possible to fasten the hook from the force transducer directly to the muscle for twitch speed recordings. Instead, we attached the hook to the bird’s elbow, and recorded the action of the stimulated SC as movement of the wing. When recordings were completed, we lightly pulled the electrodes from the muscle, removed the force transducer line from the elbow, and closed the dissection with Vetbond glue.

Both the stimulator and force transducer were connected to a laptop through an A-D converter (Model NI USB-6212, National Instruments, TX, USA). The signal from the force transducer was first amplified (5K–10K) and low-pass filtered (3000 Hz) using an AC/DC strain gage amplifier (Model P122, Grass Technologies). We collected all recordings in AviSoft-RECORDER (v.4.2.22) and measured the data in Praat software (v.5.4.21, P. Boersma and D. Weenink). This was accomplished by measuring the percentage at which muscles relax after each stimulatory pulse within a given stimulation train. Complete muscle relaxation (100% relaxation) occurred when the extra tension detected by the force transducer in response to stimulation was fully relieved (i.e., the signal returned to its baseline level). Partial muscle relaxation occurred when the extra tension placed on the force transducer in response to stimulation was less than fully relieved (i.e., summation occurred). Inasmuch, the values of partial relaxation ranged between 99% and 0% (full fusion) and were calculated by dividing the actual amount of relaxation by the amount of relaxation that would otherwise be necessary for full recovery.

For each stimulation train, we averaged the percent recovery values for the first eight stimulations. This corresponds to similar numbers of wing-oscillations that golden-collared and red-capped manakins use in their forelimb displays (Bostwick and Prum, 2003; Fuxjager et al., 2013). Notably, in our calculations, we used baseline as the measure of a fully relaxed (un-stimulated) muscle, even though summation changes peak force production over the course of the stimulation train. As such, saturation levels may not always reflect 0% recovery.