Cv 7b headstage

Electrical signals were acquired through an Axon instruments CV‐7B headstage, connected to the pipette solution by a chlorided silver wire, and a Multiclamp 700B amplifier. Each primary output was connected to a Humbug to reduce 50Hz background noise. Signals were filtered at 3–6 kHz, digitized and sampled through an analogue‐to‐digital converter, either an Axon CNS 1440A, or National Instruments BNC 2090, at 10 KHz. Software used for acquisition was either WinCP 4.2.1 or Clampex 10.2. Excitatory post‐synaptic field potentials (fEPSPs) were recorded through glass micropipettes of 1‐2 MΩ resistance. For Schaffer collateral stimulation, axonal fibers were stimulated with a bipolar electrode from a Digitimer DS3 constant current stimulator box. The stimulating electrode was placed in stratum radiatum, 100‐200 μm closer to CA3 then the recording electrode. The configuration was allowed to settle for up to 10 minutes and then the stimulus intensity was gradually increased until no further increase in the fEPSP slope was seen. The stimulus power was then adjusted to give 50‐60% of the maximal fEPSP slope – stimulus power did not exceed 70 μA and lasted 100 μs. Paired‐pulse stimuli were given with an inter‐stimulus interval of 50 ms and repeated every 15 s.

EPGs were performed as previously described37 (link)67 (link). We performed EPGs on intact first day adult worms. We created recording chamber by making a ring of vacuum grease on a cover slide, then filled the chamber with Dent’s saline67 (link) containing 10 mM serotonin. 10–15 worms were added to the chamber for each experiment. Worms were then sucked into glass microelectrodes, which were connected by a silver chloride coated silver wire to an Axon Instruments CV-7B headstage. The headstage was connected to Axon Instruments MultiClamp 700B amplifier and DigiData 1440A digitizer. Electrodes were fabricated on a Sutter P-1000 micropipette puller using borosilicate glass with an inner diameter of 0.5 mm. Electrodes were pulled to an inner diameter of approximately 20 μm. We performed optogenetic stimulation25 (link)46 (link)66 (link), using 5 s light pulses separated by 5 s, with the exception of the experiments with worms expressing the pan-neuronal HisCl channel, where we used 200 ms light pulses. All recordings were performed in current clamp mode. E1 spikes were identified by manual observation of the EPG traces using the Axon Instruments pCLAMP 10 software package.

Somatic whole-cell recordings were obtained using patch pipettes pulled from thick-walled borosilicate glass capillaries (1.5 mm outer diameter; Science Products, Germany). All recordings were at 30°C ± 0.5°C maintained with a temperature control unit (Luigs & Neumann, Rattingen). For current-clamp experiments the pipette solution contained (in mM): 130 K–gluconate, 6 KCl, 2 MgCl₂, 4 NaCl, and 10 Hepes, with pH adjusted to 7.25 with KOH. Pipettes had resistances of 5–7 MΩ when filled with this solution supplemented with Fura-2 (Molecular Probes). Recordings were made using a Multiclamp 700B amplifier (Molecular Devices) equipped with CV-7B headstage (Molecular Devices). Data were low–pass–filtered at 10 kHz (−3 dB), single-pole Bessel filter and digitized at 20 kHz using Digidata 1,322 A digitizer driven by PClamp 10 software (Molecular Devices). Care was taken to maintain the access resistance below 10 MΩ.