Of this test most (84%, 96 of 115) from the ascending neurons were cholinergic, whereas most (63%, 40 of 64) from the descending neurons expressed NADPH-diaphorase activity

Of this test most (84%, 96 of 115) from the ascending neurons were cholinergic, whereas most (63%, 40 of 64) from the descending neurons expressed NADPH-diaphorase activity. whereas the 5-HT1P-responsive cells got mainly descending projections and had been nitrergic (67%). Chemical P-positive neurons had been cholinergic; a lot of the cells (75%) exhibited 5-HT3mediated replies and got ascending projections. Muscle tissue strip recordings backed the functional need for the differential area of 5-HT receptor subtypes. Hence, contractile responses of gastric round muscle Ginkgolide B strips were improved with a 5-HT3 and reduced with a 5-HT1P agonist dose-dependently. Outcomes indicated that excitatory ascending enteric pathways comprising cholinergic, chemical Pergic neurons were activated by 5-HT3 receptors, whereas 5-HT1P receptors were involved in activation of inhibitory descending pathways using nitrergic neurons. This suggested that different effects of 5-HT on gastric functions are related to specific activation of receptors located on different subsets of enteric neurons. Experiments were performed on the isolated gastric corpus of the guinea pig The experiments were done under visual control using an Olympus (Tokyo, Japan) IMT 2 inverted microscope equipped with Hoffmann modulation optics. Intracellular recordings were performed with glass microelectrodes filled with 0.5 m KCl containing 1% neurobiotin. The electrodes had resistance between 150 and 200 M. Signals were amplified (Intra 767; WPI, New Ginkgolide B Haven, CT), displayed on an oscilloscope (DSO 420; Gould Instruments, Valley View, OH) and a chart recorder (Gould TA 11), and stored using a digital audio tape recorder (DTR-1202; Biological Science Instruments, Claix, France). No filters were applied to the stored data. Data were analyzed and displayed off-line using a Macintosh computer and a MacLab system (MacLab 4 s/e with Chart 3.5.1 software; AD Instruments, Castle Hill, Australia). Impaled myenteric neurons were first classified by their response to intracellular stimulation (rectangular pulses, 0.1C0.3 nA; duration, 300 msec) according to the method of Schemann and Wood (1989). Neurons that responded with more than three action potentials corresponded to gastric I cells; neurons with only one to three action potentials corresponded to gastric II cells. Neurons in which no Ginkgolide B action potential could be evoked were termed gastric III and were only included if they received synaptic input. The neurons were then filled with neurobiotin using current pulses Flt4 of 0.3 nA, 0.3 Hz, pulse width 300 msec for 3 min. Serotonin (5-hydroxytryptamine creatinine sulfate complex; SigmaCAldrich, Deisenhofen, Germany) was applied to the cells by pressure ejection (50C900 msec) from a spritz pipette directed toward the impaled ganglion cell. Stock solutions from 5-HT (10 mmin 0.9% NaCl) and fast green (Sigma, St. Louis, MO) (10 mmin Ginkgolide B 0.9% NaCl) were diluted in Krebs solution to obtain final concentrations of 1 1 mm for serotonin and 0.5 mmfor fast green in the spritz pipette. Fast green was used for visual control of drug ejection. Pressure ejection with fast green solution only had no effect on the neurons. To detect changes in membrane resistance or excitability induced by 5-HT, short hyperpolarizing or subthreshold depolarizing current pulses were used. At the end of the electrophysiological experiments the tissue was either immediately fixed with 2% paraformaldehyde and 0.2% picric acid in 0.1 m phosphate buffer overnight at 4C or treated with colchicine in an organotypic culture before the fixation. Colchicine treatment was performed to increase the levels of substance P in the myenteric neurons. The tissue was washed with sterile Krebs solution, pinned into a sterile petri dish, and incubated in culture medium (DMEM/F-12 with 10% heat-inactivated fetal calf serum, 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2.6 g/ml amphotericin B, and 50 g/ml gentamicin at pH 7.4; all chemicals by Sigma) containing 60 mcolchicine and 1 m nifedipine. The petri dish was kept in a humidified incubator at 37C in an atmosphere Ginkgolide B of 5% CO2 and air. It was placed on a rocking tray shaking at a frequency of about 0.5 Hz. After 16 hr the tissue was fixed for 4 hr at room temperature. For the immunohistochemistry, fixed tissue was washed three times in 0.1 m phosphate buffer for 10 min and preincubated for 1 hr in 0.1 m PBS containing 4% goat serum and 0.5% Triton X-100. After preincubation, the tissue was exposed to a mixture of primary antisera diluted in PBS containing serum and Triton X-100 for 18 hr at room temperature. The following primary antisera were used: anti-choline acetyltransferase (ChAT) raised in rabbits (P3YEB, 1:2000) (Schemann et al., 1995) and rat.

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