Whole-cell recordings had been made from recognized gastric-projecting rat dorsal engine nucleus of the vagus (DMV) neurons. Data were analyzed with the Mini Analysis system (Synaptosoft Leonia NJ). Electrical activation Synaptic currents were evoked using tungsten bipolar stimulating electrodes (World Precision Tools Sarasota FL) placed in the centralis or medialis subnuclei of the NTS. Pairs of stimuli (0.05-1.0 msec 10 μA 100 msec apart) were applied every 20 sec to evoke submaximal IPSCs or EPSCs. When IPSCs were analyzed the Krebs’ remedy contained 1 mm kynurenic acid. Recordings were carried out at ?50 mV (IPSCs) or at ?60 mV (EPSCs). Immunohistochemistry Rats IOX1 (= 14) were injected with IOX1 fluorogold (20 μg/1 ml saline i.p. per rat; Fluorochrome Denver CO) to label vagal preganglionic neurons innervating the subdiaphragmatic viscera therefore allowing delineation of the boundaries of the DMV (Fox and Powley 1985 Zheng et al. 1999 Guo et al. 2001 Three days later on the rats were anesthetized the brainstem was extracted as explained above and 200 μm solid slices were allowed to recover for 90 min in oxygenated Krebs’ at 32°C. Four to six brainstem slices from each of the individual rats were separated into the following groups so that each animal provided slices to two different organizations and each group comprised two IOX1 or three slices from four different animals: group 1 control (incubation in Krebs’ remedy for 60 min at 35°C); group 2 control (incubation in Krebs’ remedy for 120 min at 35°C); group 3 activation of cAMP (incubation in 10 μm forskolin for 60 min at 35°C); group 4 stimulation of cAMP (incubation in 10 μm forskolin for 60 min at 35°C and then washout with Krebs’ for 60 min at 35°C); group 5 stimulation of cAMP (incubation in 10 μmforskolin for 60 min at 4°C); group 6 blockade of PKA (incubation in 1 μm = 4); group 2 the presynaptic effects of ME on the IPSCs amplitude were “uncovered” by forskolin (= 4); group 3 the presynaptic effects of ME on the IPSCs amplitude were not uncovered by forskolin (= 7); group 4 the presynaptic effects of ME on the IPSC amplitude were uncovered by forskolin but the slice was fixed in Zamboni’s fixative 1 hr later (= 4). After overnight fixation at 4°C the slice was washed repeatedly in PBS-Triton-X before incubation in streptavidin-Texas Red (1:100) to visualize the neurobiotin-filled neuron. The slice was then processed for double-labeling immunofluorescence for MOR and GAD as described above using donkey anti-rabbit conjugated with Cy2 (1:400;MOR staining) and donkey anti-mouse Cy5 (1:400; GAD staining) as secondary antibodies. The sections were then washed with PBS (three times for 2 hr each) mounted onto histological slides cleared in alcohol and xylene and coverslipped with DPX (Fluka Ronkonkoma NJ). Mounted tissues were allowed to dry overnight at room temperature and then examined using confocal microscopy. Three sequential Z-stack series of images (40 images per stack gathered at 0.3 μm steps) of the brainstem region containing the neurobiotin-Texas Red-labeled cell and profiles labeled for GAD (Cy5) and MOR (Cy2) were collected using Bio-Rad MRC 1000 confocal scanning laser microscope equipped with Kr/Ar-ion laser (Bio-Rad Richmond CA). Note that we used Z-stack only for 3D volume reconstructions but we did not collapse these Z-stacks because merging different optical layer planes would produce artifact colocalization of immunoreactive profiles. The microscope was equipped with filters for the selective visualization of Texas Red Cy2 and Cy5. Mouse monoclonal to CD53.COC53 monoclonal reacts CD53, a 32-42 kDa molecule, which is expressed on thymocytes, T cells, B cells, NK cells, monocytes and granulocytes, but is not present on red blood cells, platelets and non-hematopoietic cells. CD53 cross-linking promotes activation of human B cells and rat macrophages, as well as signal transduction. Images were collected using 60× lens and a zoom of 2. Sequences of collected images were converted into merged 3D volumes using Volocity software (version 2.6.1; Improvision Lexington MA)provided by the Supercomputer Institute for Digital Simulation and Advanced Computation (University of Minnesota Minneapolis MN). For visualization purposes merged image series were pseudocolored as follows: blue neurobiotin-Texas Red-labeled cell; green GAD-labeled profiles; red MOR-labeled profiles. Digital images were manipulated using Adobe Photoshop CS software (Adobe Systems San Jose CA). Drugs and IOX1 solutions Krebs’ included the next (in mm): 126 NaCl 25 NaHCO3 2.5 KCl 1.2 MgCl2 2.4 CaCl2 1.2 NaH2P04 and 11 dextrose taken care of at pH 7.4 by bubbling with 95% O2/5%CO2. Intracellular remedy contained the next (in mm): 128 K-gluconate (or KCl) 10 KCl 0.3 CaCl2 1 MgCl2 10 HEPES 1 EGTA 2 ATP-Na and 0.25 GTP-Na modified to pH 7.35 with KOH. Zamboni’s fixative.