Ced micro 1401 mkii

Slices were initially visualized under epifluorescence illumination with a high-sensitivity digital frame transfer camera (Cooke SensiCam) mounted on an Olympus BX50-WI epifluorescence microscope with a × 40 long working distance water-immersion lens. Once a GFP+ interneuron was identified, visualization was switched to infrared–differential interference contrast microscopy for the actual patching of the neuron. Micropipettes for whole-cell recording were constructed from 1.2 mm outer diameter borosilicate pipettes on a Narishige PP-83 vertical puller. The standard internal solution for whole-cell current-clamp recording was as follows (in mM): 130 K-gluconate, 10 KCl, 2 MgCl₂, 10 HEPES, 4 Na₂ATP, 0.4 Na₂GTP, pH 7.3. These pipettes had a DC impedance of 3–5 MΩ. Membrane currents and potentials were recorded using an Axoclamp 700B amplifier (Molecular Devices). Recordings were digitized at 20–40 kHz with a CED Micro 1401 Mk II and a PC running Signal, version 5 (Cambridge Electronic Design). Optogenetic stimulation in vitro consisted of 2–5 ms duration blue light pulses delivered using a high-power (750 mW) LED. Sweeps were run at 20 s intervals.

Three-day-old larvae were immobilized with Parafilm on a moistened matchstick. A chloride-coated silver wire reference electrode was inserted into the posterior end of the larva. Borosilicate glass capillary microelectrodes with a tip diameter of less than 1 µm were filled with Drosophila larval ringer solution adjusted to pH 7.1 with HCl or NaOH [13 (link)]. The tip of the microelectrode was inserted into the cuticle at the base of the dorsal organ. Electrical activity was acquired using a Neurolog system (Digitimer). The differential activity from the reference and the recording electrodes was amplified and filtered (filter unit NL125 and 126) and directed to a CED micro 1401 mk II (Cambridge Electronic Design) analogue to digital converter at a sampling rate of 10 kHz. The digital signal was recorded and analysed with CED Spike2 software (v. 7.06). Spike sorting was performed off-line using Spike2. Each recording contained the activity of only one functional OSN. The activity of the functional OSN was extracted on the basis of the amplitude and waveform of the spikes and its responsiveness to odours; where the activity of other OSNs was recorded, it showed unmodulated spontaneous activity irrespective of stimulation. A principal component analysis verified that spikes belonged to the functional OSN [2 (link)].