Carbachol

To study the action of cholinergic drugs on degranulation of dural mast cells, hemiskulls of Wistar rats of both sexes were used. One control hemiskull was filled for 20 min with the basic saline solution (BSS), containing (in millimolars) 152 NaCl, 5 KCl, 10 HEPES, 10 glucose, 2.6 CaCl₂, 2.1 MgCl₂ (pH adjusted to 7.4 with NaOH), whereas others were filled with BSS containing 50 µM carbachol or 100 µM nicotine. Then, the hemiskulls were left in 4% PFA solution for at least 4 h. After that meninges were dissected carefully and placed on a microscope slide, where they were stained with 0.1% toluidine blue (26 (link)). Images of meninges were acquired with Olympus AX70 microscope (20× objective). Homogeneously stained and well-shaped mast cells were classified as non-degranulated, whereas pale poorly stained mast cells as well as mast cells with distorted borders were classified as degranulated (22 (link), 26 (link)). To study an effect of carbachol and nicotine on mast cells degranulation, 10 randomly chosen, same in different animals, perivascular areas of meninges enriched by mast cells were analyzed (27 (link)). Then, total number and number of degranulated mast cells were counted in a blind manner.

Neurons were lysed in 1% SDS lysis buffer, sonicated twice at 3.5 power for 5 seconds and the protein quantified using the bicinchoninic acid assay (BCA). Equal amounts of protein were loaded into freshly cast bis-trisacrylamide gels (10%) and proteins were separated by gel electrophoresis then transferred to PVDF membranes. Membranes were blocked in TBST (5% milk) for 1 hour, then probed overnight at 4°C with primary antibodies for synaptophysin (1:500), or PSD-95 (Neuromab, 1:500). Membranes were incubated for 1 hour with HRP-conjugated secondary antibodies (1:1000) and developed. Band densitometry was determined using Image J software, and results normalized to total protein as determined by Coomassie blue protein stain.
For measurement of astrocyte TSP1 levels, astrocytes were plated on PDL (40 μg/mL) pre-coated 100 mm plates at a density of 2.5 × 10⁶ per plate and cultured for 4 days, serum-deprived for 24 hours, then treated for 24 hours with carbachol (1 mM) prepared in serum free medium. Medium was collected and cells were lysed in 1% SDS lysis buffer from treated and untreated plates immediately after the 24h treatment (time 0), and 6 and 24 hours after treatment washout. Intracellular protein was quantified from lysate samples using the BCA method and equal amounts were loaded into 3–8% Tris-Acetate gels and separated by gel electrophoresis. After transfer, PDVF membranes were blocked in TBST (3% BSA) for 1 hour and probed overnight in goat anti-mouse TSP1 (1:1000), then incubated for 1 hour with HRP-conjugated secondary antibodies (1:1000) and developed. TSP1 was detected by chemiluminescence and normalized to β-actin levels using densitometric analysis. For measurements of TSP1 in the astrocytic medium, 7 mL of media from each time point was concentrated to 200 μl using Pierce concentrators (MWCO 9kDa) centrifuged at 4000 × g for 25 minutes at 25 C. After the addition of protease inhibitors, reducing reagents, and sample buffer, samples were denatured by heating (70°C for 10 minutes) and equal volumes (30 μl) of concentrated sample were loaded into 3–8% Tris-Acetate pre-cast gels. After transfer, PDVF membranes were blocked in TBST (3% BSA) for 1–3 hours and incubated overnight in goat anti-mouse TSP1 (1:500). Band densitometry was determined using Image J software and normalized to corresponding cell lysate protein concentrations.

Primary cortical astrocytes were trypsinized from established cultures and sub-cultured in 24 well plates (250,000 cells/mL) for synaptogenesis and electrophysiology experiments, or on the underside of 0.4 μm mesh 6-well plate inserts for protein expression experiments. Wells and inserts were coated with 40 μg/mL PDL. Forty-eight hours prior to co-culture with neurons, astrocytes were serum deprived (DMEM + 0.1% bovine serum albumin and P/S) for 24 hours, and then treated with or without carbachol (0.01, 0.10, 1 mM) for an additional 24 hours. Astrocytes were washed with PBS and incubated in serum-free medium for three hours prior to co-culture with neurons. In some experiments, 30 minutes prior to the addition of carbachol, astrocytes were treated with 10 μM of the acetylcholine receptor antagonists, mecamylamine, gallamine, or 4-DAMP. After treatment washout and medium conditioning, primary hippocampal neurons (12–13 DIC), grown on glass coverslips were inverted over the pre-treated astrocyte monolayer for immunocytochemistry or electrophysiology experiments. Neurons were never in direct contact with astrocytes, nor were they exposed to astrocyte treatments. For Western blot experiments, astrocytes plated on the underside of porous inserts and their medium were transferred to 6-well plates containing primary hippocampal neurons.
To block TSP1 signaling in neurons, after astrocyte carbachol pre-treatment and washout, neurons and astrocytes were pre-treated for half hour with gabapentin (15 or 30 μM) prior to co-culturing; gabapentin remained throughout the co-culture incubation. For all co-culture experiments, astrocytes and neurons were co-incubated for 24 hours.