The application of TG or 2-APB before WIN abolished the calcium rise

The application of TG or 2-APB before WIN abolished the calcium rise. DPBS before incubation in 2 ml of DPBS containing 2.0 to 3.0 M Fura-2 AM and 0.066% Pluronic F-127 (Invitrogen). After incubating for 60 to 75 min at 37C in darkness, cultures were washed twice in DPBS to remove extracellular dye and kept Cyclothiazide at room temperature in the dark for more than 30 min before use in the experiments. All measurements were performed in DPBS or, where specified, in Ca2+-free DPBS. Drugs were added in a volume of 200 l to cells in 3 ml of DPBS to make the final volume less than 4 ml in the Petri dishes. The dishes with Cyclothiazide dye-loaded cells were mounted on the stage of Nikon TS-100 fluorescence inverted microscope with a Cohu model 4915 charge-coupled device (CCD) camera (Nikon, Melville, NY). Fluorescent images were captured alternately at the excitation wavelengths of 340 and 380 nm with an emission wavelength of 520 nm, which were analyzed with InCyt Im2 version 4.62 imaging software (Intracellular Imaging). A standard curve was used to derive experimental [Ca2+]i values. The standard curve was generated by using various concentrations of Ca2+ (Calcium Calibration Buffer Kit) in the presence of indicator dye Fura-2 free acid (Invitrogen). During each experiment, background fluorescence was estimated for a region without cells, and this value was automatically subtracted from the measured emission of each channel. The F340/F380 ratios of cell emissions were compared with the standard curve stored in the computer, and both the ratio and [Ca2+]i were displayed on screen. Preliminary measurement of [Ca2+]i was taken on various cells in the field before any drug application. Only cells with basal [Ca2+]i in the range of 90 to 120 nM were chosen for the experiments described here. Experimental Paradigm. Cyclothiazide All pharmacological agents were dissolved in DPBS and applied by brief microperfusion from micropipettes placed near the cells of interest. The concentration and duration ( 2 s) of application were adjusted under control conditions for each experiment to produce Ca2+ signals with peak amplitude (150C350 nM) that could be easily quantified. Ca2+ levels in the presence of TG and cannabinoids were typically measured 5 Cyclothiazide to 20 min after the initial drug exposure. NMDA was added 10 min after the responses returned to baseline. For a majority of the experiments, the bath saline (e.g., DPBS) used during control recordings contained DMSO concentration equivalent to that used in the presence of thapsigargin or cannabinoid agents. Separate vehicle control experiments showed that DMSO ( 0.15%) did not affect the measurements under study. In general, Ca2+ levels at rest or in response to challenges were measured simultaneously for 10 to 30 cells within a microscopic field, with three to five microscopic fields measured per condition. One microscopic field was measured in each Petri dish. Each cell was tested under only one condition. Resting Ca2+ levels were subtracted from amplitude measurements for individual cells to yield peak Ca2+ values. Data Analysis. A between-cell comparison was used to determine the effects of the tested compounds on Ca2+ levels or cytotoxicity. For each group of studies, data from at least five individual Petri dishes were pooled for summary analysis. Each drug was tested on at least two different days, with concurrent interleaved controls. Averages are reported as the mean S.E.M., and the number of cells and/or cultures studied is given. Raw data were analyzed with appropriate parametric tests: paired or unpaired test or analysis of variance (performed with SPSS software; SPSS Cyclothiazide Bmpr2 Inc., Chicago, IL). When analysis of variance was used, post hoc analysis for group differences was performed by using Scheffe’s test or Dunn’s test for unequal sample sizes. Statistical significance was determined at a significance level of 0.05. Results Cannabinoid 0.05). The protective effects of WIN were reversed by CB1 receptor antagonist SR141716A (500 nM) ( 0.05) but not by CB2 receptor antagonist SR144528 (500 nM) ( 0.05), suggesting a specific CB1 receptor-mediated effect. Figure 2 shows representative microphotographs of DRG neuron cultures in control (Fig. 2A), treated with NMDA (100 M) (Fig. 2B), treated with the combination of NMDA (100 M) and WIN (500 nM) (Fig. 2C), or treated with WIN (500 nM) alone. After 24 h of drug treatment, DRG neurons exposed to NMDA exhibited signs of cytotoxicity that was prevented by.