No shal staining was evident on either the PD cell body or its neurites
No shal staining was evident on either the PD cell body or its neurites. channels are controlled by long-term actions of modulatory inputs, we removed these inputs from the pyloric network and, after 6 d was cut (before (was cut, and from the same preparation on day 6 in organ culture (Standard electrophysiological techniques were used in all experiments. Nerves were recorded extracellularly using custom built amplifiers and stainless steel electrodes isolated from the bath with petroleum jelly. Intracellular recordings were made using glass microelectrodes filled with a mixture of 2 m potassium acetate (KAc) and 2 10?2m KCl (9C40 M) and an Axoclamp 2A or 2B amplifier (Axon Instruments, Foster City, CA). The PD neuron was identified by a 1:1 correspondence of action potentials that were recorded intracellularly in the soma with those recorded extracellularly from the pyloric dilator motor nerve and by its characteristic phasing and synaptic input during the pyloric motor pattern. Data other TRi-1 than voltage-clamp recordings were recorded TRi-1 on a PC with a data acquisition system (1401 CED; Cambridge Electronic Design, Cambridge, UK) and analyzed using Spike 2 (CED) software. PD neurons, isolated from chemical synaptic input by the presence of CdCl2, picrotoxin (PTX), and tetrodotoxin (TTX) in the bathing saline (see below), were impaled with two electrodes (9C11 M, filled with 2.5 m KCl) for voltage recording and current recording or injection, and voltage-clamped using an Axoclamp-2B amplifier. A Digidata 1200 interface and pClamp 6 software (Axon Instruments) were used to generate the voltage protocols and to acquire data. Data were sampled at 100 sec intervals and filtered at 1.5 kHz with an eight-pole Bessel filter. Linear leakage and capacitative currents were digitally TRi-1 subtracted using a P/6 protocol (Armstrong and Bezanilla, 1974). (assuming curve was fitted to a third-order (= 3) and first-order (= 1) Boltzmann equation of the form: is usually a slope factor. For the third-order Boltzmann fit, = 1), based on the model of Hodgkin and Huxley (1952). Detection of shal type transient K+ channels was performed using indirect immunofluorescence. We used a rabbit anti-shal antibody that was generated against the carboxy portion of the lobster shal peptide (Baro et al., 2000). For shal detection, stomatogastric ganglia were fixed in 4% paraformaldehyde in 0.1m phosphate buffer, pH 7.4, for 1C2 hr at 4C and then rinsed at least five times in PBS with 0.3% Triton X-100 (PBST) over 2 hr. Then, the tissue was incubated for 48 hr in the primary antibody diluted (final concentration, 0.5 g/ml) in PBST with 5% normal goat serum (NGS). After 2 hr of PBST rinsing, the tissue was incubated for an additional 24 hr in goat Rabbit polyclonal to AGPS anti-rabbit fluorescein (Sigma, St. Louis, MO)-conjugated, Texas Red (Vector Laboratories, Burlingame, CA)-conjugated, or Cy5 (Jackson ImmunoResearch)-conjugated Igs (1:200 in PBST with 10% NGS). Finally, the tissue was thoroughly rinsed in PBS (2 hr), dehydrated (in a TRi-1 30, 50, 70, 90, 95, and 100% ethanol series, 10 min each), cleared in pure methylsalicylate (Sigma) and mounted with permount (Fisher Scientific, Houston, TX) on microscope slides. Controls included omission of primary antibody, incubation in primary antibody that had been preabsorbed with the shal fusion protein (10 g/ml, overnight at 4C), and incubation in primary antibody followed by.