Electrophysiological recordings were made with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA), a Powerlab 4/30 A/D converter (ADInstuments, Sydney, Australia), using LabChart software (ADInstruments, Sydney, Australia). L3 larvae were dissected in Ca2+-free HL3.1 saline (70 mM NaCl, 5 mM KCl, 4 mM MgCl2, 10 mM NaHCO3, 5 mM Trehalose, 115 mM sucrose, 5 mM HEPES, pH 7.2) to expose the body wall musculature and the CNSs were removed (Feng et al., 2004 (link)). The saline was then changed to HL3.1 with 1mM CaCl2. Intracellular recordings were made from muscle fiber 6 of abdominal segments 3 and 4 using 10–25 MΩ intracellular electrodes that were pulled using a Sutter model P-97 micropipette puller (Novato, CA) and filled with 3 parts 2 M K3C6H5O7 to 1 part 3 M KCl. The resting potential was held at −65 mV by applying no more than ±1 nA of current. The nerve fiber was stimulated using an A360 stimulus isolator (World Precision Instruments, Sarasota, FL) through a glass suction electrode filled with HL3.1 containing 1mM CaCl2 and broken to have an ~1 micron tip. Ten excitatory junction potentials (EJPs) at 0.04 Hz intervals were recorded and then averaged for each muscle fiber.
A360 stimulus isolator
Manufactured by World Precision Instruments
Sourced in Germany
The A360 Stimulus Isolator is a lab equipment device designed to provide controlled electrical stimulation to biological and electrophysiological samples. It is capable of delivering constant current or constant voltage stimulation through a pair of output electrodes.
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4 protocols using a360 stimulus isolator
1
Electrophysiological Recordings of Larval Neuromuscular Junction
2
Deep Brain Stimulation Protocol for Auditory Processing
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DBS was performed with bipolar, concentric electrodes using monophasic rectangular pulses. The electrical stimulus pulses were created by an A310 acupulser and an A360 stimulus isolator (World Precision Instruments, Berlin, Germany). During DBS, stimuli were given with a frequency of 100 Hz (HFS) and 10 Hz (LFS) with an amplitude of 100 µA and a pulse width of 60 µs. Electrodes are gold-plated with platinum–iridium inner wire (negative contact) and stainless steel outer part (positive contact). The inner and outer electrodes are insulated except for a 75 µm exposed tip (Tan et al., 2010 (link)).
Rats were divided in two groups, one group received implantation of electrodes in the CIC (n = 5) and the other group in the DCBN (n = 5). In the off-stimulation state, designated as the control situation, no electrical stimulation was given. During stimulation-off state, LFS and HFS, ABRs were recorded in separate sessions.
Rats were divided in two groups, one group received implantation of electrodes in the CIC (n = 5) and the other group in the DCBN (n = 5). In the off-stimulation state, designated as the control situation, no electrical stimulation was given. During stimulation-off state, LFS and HFS, ABRs were recorded in separate sessions.
3
Pruritus Induction by Pruritogens
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The pruritogens BAM8–22, β-ALA, ET-1 and HIS were investigated. BAM8–22 was purchased from GenScript (custom synthesis services, Leiden, Netherlands) and all other compounds were supplied from Merck KGaA (Darmstadt, Germany). Concentrations of BAM8–22 (0.4 µg/µl), β-ALA (9 µg/µl), and ET-1 (1 µM) were prepared in sterile ECF (for composition see10 (link)). A 1% histamine di-hydrochloride solution was prepared in de-ionized water (Merck Millipore, Germany) and delivered into the skin by iontophoresis (1 mA for 20 s, equaling 20 mC) (World Precision Instruments, A360 Stimulus Isolator, Friedberg, Germany) to serve as positive pruritus control to BAM8–22, β-ALA, and ET-1, respectively. For iontophoresis, a silver-anode (diameter 5 mm) was equipped with a HIS-soaked cotton swap and as cathode a Kendall® ECG electrode (diameter 24 mm, Covidien Medtronic, Meerbusch, Germany) was attached to the skin surface distally to the anode.
4
Intracellular Recordings of Drosophila Muscle
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Electrophysiological recordings were made with an Axoclamp 2B amplifier (Molecular Devices, Sunnyvale, CA), a Powerlab 4/30 A/D converter (ADInstruments, Sydney, Australia), using LabChart software (ADInstruments, Sydney, Australia). L3s were dissected in Ca2+-free HL3.1 saline to expose the body wall musculature and the CNSs were removed. The saline was then changed to HL3.1 with 1mM Ca2+. Intracellular recordings were made from muscle fiber 6 of abdominal segments 3 and 4 using 10–20 MΩ intracellular electrodes that were pulled using a Sutter model P-97 micropipette puller (Novato, CA) and filled with 3 parts 2 M K3C6H5O7 to 1 part 3 M KCl. The resting potential was held at -65 mV by applying no more than ±1 nA of current. The nerve fiber was stimulated using an A360 stimulus isolator (World Precision Instruments, Sarasota, FL) through a glass suction electrode filled with HL3.1 containing 1mM Ca2+ and broken to have an ~1 micron tip.
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