Getting recordings, glass pipettes (borosilicate, 1.5 mm external diameter; Hilgenberg) was in fact filled with 3 m NaCl and you will placed in the latest thorax, given that a resource, as well as on the brand new travel vision, some acute the new cornea to own recordings. Responses to a repeated light stimulus (1 s) provided by an eco-friendly white-giving off diode (look for Figs. 1B, 3B) was in fact submitted using AxoScope ten.5 and examined playing with Clampfit 10.5 software (Molecular Gizmos). Tracks had been increased playing with good Warner DP311 Air-con/DC amplifier (Warner Instruments) and you can digitized by using the minidigi 1A (Molecular Gadgets). Raw study traces was indeed moved to Igor Specialist 6.thirty-six (Wavemetrics) for contour thinking.
Two-electrode voltage-clamp (TEVC) experiments were performed as described previously (Volders et al., 2012). Third-instar larvae were dissected in HL3 solution (in m m : 110 NaCl, 5 KCl, 10 MgCl2 · 6H2O, 10 NaHCO3, 30 sucrose, 5 trehalose, and 10 HEPES, pH 7.2), and motor neurons from segments A2–A3 were isolated using a suction electrode. Axons were stimulated at least 50% above threshold at 0.2 Hz (TEVC) in the presence of 0.2–0.5 m m CaCl2 respectively. For miniature analysis, recordings were performed in the presence of 1 ? m TTX. Responses were measured in muscle 6 using sharp microelectrodes (±30 M? for current clamp and the voltage sensing electrode for TEVC, 10–15 M? for the current passing electrode for TEVC) filled with a 3:1 solution of 3 m KCl and 3 m KAc. Signals were amplified using the Axoclamp900A amplifier (Molecular Devices), filtered using a 1 kHz Bessel filter, and digitized at 10 kHz using a Digidata 1440A (Molecular Devices). For all recordings, membrane potentials were between ?65 and ?75 mV, with input resistance ?4 M?. For TEVC, membrane potentials were clamped at ?70 mV. (more…)
