0.01; KolmogorovCSmirnov test). poorly comprehended (North, 2002; Khakh et al., 2003). Here we show that this major mechanism of the cNTS network excitation by ATP is usually by way of triggering Ca2+-dependent exocytosis of glutamate, but not that of GABA, by Ca2+ access through presynaptic P2X receptors. Surprisingly, this glutamate release produced larger postsynaptic depolarizing potentials than those occurring spontaneously, Relebactam leading to generation of action potentials in postsynaptic cells, in a similar manner to but through unique mechanisms from your recently reported case with presynaptic nicotinic receptors in the hippocampus (Sharma and Vijayaraghavan, 2003). Materials and Methods (1988) and the regulation of the local animal committee. Briefly, the lower brainstem was dissected out under deep ketamine anesthesia (100C150 mg/kg, i.p.), and two to three transverse slices of 400 m thickness made up Relebactam of the cNTS were slice in the ice-cold trimming artificial CSF (ACSF) composed of (in mm): 125 NaCl, 3 KCl, 0.1 CaCl2, 5 MgCl2, 1.25 NaH2PO4, 12.5 d-glucose, 0.4 l-ascorbic acid, and 25 NaHCO3 25 (pH 7.4; bubbled with 95% O2/5% CO2; osmolarity, 330 mOsm/kg). The slices were first incubated in a holding chamber with standard ACSF, of which the concentrations of CaCl2 and MgCl2 were 2 and 1.3 mm, respectively, at 37C for 30C45 min. The slices were then kept at room heat (25C) in the same chamber MGC20461 for 0.5C10 hr until the recording. A slice was transferred to a recording chamber (0.4 ml volume) and submerged in and continuously superfused at a Relebactam rate of 1C2 ml/min with the standard ACSF, which additionally contained 100 m picrotoxin and 1 m 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) to block GABAA/C and adenosine A1 receptors, respectively. Strychnine (1 m) was also added to the ACSF to block glycine receptor currents in some experiments. Tetrodotoxin citrate (TTX; 1 m) was also added to the ACSF after verification of the monosynaptically activated postsynaptic currents around the solitary tract activation (observe below), except for the experiments in which stimulation-evoked responses were analyzed. In these cases, the concentration of MgCl2 was 3 mm to avoid re-excitatory processing of the cNTS network (Fortin and Champagnat, 1993). The ACSF made up of no added Ca2+ contained 0 mm CaCl2 and 3 mm MgCl2. EGTA was not included. We applied ATP or ,-methylene ATP (mATP) (100 or 300 m) to 333 healthy-looking cNTS neurons recorded in 159 slices from 85 rats. Of these neurons, 220 cells (66.1%) responded to ATP (100 or 300 m) or mATP (100 m), or both, with an increase in spontaneous synaptic inputs under voltage-clamp or current-clamp recording conditions. = 93) and whole-cell capacitance were compensated. The input resistance (= 92). In a part of the study in which IPSCs were recorded, the internal answer contained (in mm): 140 CsCl, 1 CaCl2, 2 ATP Mg, 0.3 GTP Na, 10 EGTA, 10 HEPES hemisodium (pH 7.2 as adjusted with CsOH; osmolarity, 310 mOsm/kg). The tip resistance of the electrode was 3C8M. The membrane potential was held at C70 mV during the recordings. The membrane current was recorded with an Axopatch Relebactam 200B amplifier (Axon Devices) or CEZ-2400 (Nihon-Kohden, Tokyo), low-pass filtered at 2 kHz, and sampled at 4 or 10 kHz (for the analysis of event kinetics) with a PowerLab interface (AD Devices) together with the holding potential, the activation timing pulse, and the timing signal from your electromagnetic valve controller utilized for the drug application (VC-6, Warner Devices; observe below). A tip of a bipolar concentric electrode was placed on the solitary tract (TS). In all neurons recorded, the TS was stimulated every 5 sec at a submaximum intensity (0.01C3 mA; 100 sec) to verify monosynaptically evoked EPSCs before adding TTX or CdCl2 to the ACSF. The neurons showing evoked EPSCs with a latency that was not within the range for the monosynaptic input from your TS afferents (Kato and Shigetomi, 2001) or those without TS stimulation-activated evoked EPSCs were discarded at this moment. The effect of TTX or Cd2+ was confirmed by the complete absence of evoked response in all neurons to which these blockers were applied. All recordings were made at room heat (20C25C). = 0.05) for the KolmogorovCSmirnov statistics. Note that each curve is out of this limit, indicating that the amplitude distribution is usually significantly different before and after mATP. = 10) of mEPSCs appearing before and during mATP application. Traces.