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[PMC free article] [PubMed] [Google Scholar] 44

[PMC free article] [PubMed] [Google Scholar] 44. induction by patterned activation, whereas K+-induced manifestation was unaffected. Related results were acquired for depolarization-induced manifestation of the immediate early genes andPregnant dams (Sprague Dawley rat; Zivic-Miller, Zelienople, PA) were rapidly killed by exposure to carbon dioxide. The uterine horns were removed and placed into PBS comprising 10% glucose, and the embryos were excised. To assign gestational age groups, the day after mating was designated embryonic day time (E) Parathyroid Hormone 1-34, Human 0.5. E16.5 petrosal ganglia (PG) were digested in Dispase (Roche Molecular Biochemicals, Mannheim, Germany) for 30 min at 37C, followed by trituration through siliconized, fire-polished Pasteur pipettes. Cells were plated onto glass coverslips coated with Growth Element Reduced Matrigel Matrix (diluted 1:10; Becton Dickinson, Bedford, MA) at a denseness of one ganglion per well. Dissociate cultures were cultivated in Neurobasal medium supplemented with B-27 serum-free product, 1% penicillinCstreptomycinCneomycin antibiotic combination, and 0.5 mml-glutamine (Life Technologies, Gaithersburg, MD). All cultures were supplemented with recombinant human being brain-derived neurotrophic element (a gift from Regeneron Pharmaceuticals Inc., Tarrytown, NY) at a concentration of 10 ng/ml. Neurons were cultured for a total of 4 d before activation for 6 or 24 hr with either 40 mm KCl or patterned electrical impulses (observe below). For experiments examining TH protein expression, the medium was replaced immediately after activation, and the cultures were grown for an additional 12 hr in control conditions to permit new protein synthesis. For experiments examining TH mRNA or Nur-related factor (Nurr) expression, cultures were fixed immediately after activation. PG cultures were electrically stimulated in 24-well plates fitted with a pair of platinum electrodes connected in parallel to a stimulator (MultiStim System; Digitimer, Hertfordshire, UK). Cultures were stimulated with 0.2 msec pulses of alternating polarity delivered at 5 Hz continuously or at 25 Hz in 2-sec-long bursts delivered once every 10 sec. These two activation paradigms produce an equivalent quantity of pulses over the 6 hr activation period. The drugs used are as follows. -Conotoxin GVIA (Sigma, St. Louis, MO) and tetrodotoxin (Sigma) were used at final concentrations of 1 1 and Parathyroid Hormone 1-34, Human 1.5 m, respectively. Nimodipine (Sigma), dissolved in methanol, was used at a final concentration of 2 m. PD98059 (Calbiochem, La Jolla, CA) and H-89 (Calbiochem), dissolved in dimethylsulfoxide (DMSO), were used at final concentrations of 50 and 3 m, respectively. Membrane-permeable forms of the protein kinase A (PKA) inhibitor PKI14C22amide-myristoylated (Calbiochem), the protein kinase C (PKC) inhibitor PKC-I19C27-myristoylated (Calbiochem), and the calcium/calmodulin kinase (CaMK) II inhibitor autocamtide-2 inhibitory peptide-myristoylated (Calbiochem) were used at a final concentration of 50 m. These concentrations of kinase inhibitors have been shown previously to be Parathyroid Hormone 1-34, Human efficacious and specific (Eichholtz et al., 1993; Alessi et al., 1995; Ishida et al., 1995;Harris et al., 1997). Actinomycin D (Sigma) was used at a final concentration of 0.5 g/ml.d,l-2-Amino-5-phosphonovaleric acid (APV) (Sigma) was used at a concentration of 50 m. 6-Cyano-7-nitroquinoxaline-2,3-(1H,4H)-dione (CNQX) (Sigma) was dissolved in DMSO and used at a final concentration of 10 m. In each experiment, the final DMSO concentration by no means exceeded 0.02%. Pertussis toxin (Calbiochem) was used at a final concentration of 1 1 g/ml, and cultures were preincubated with the toxin for 20 hr before the onset of activation. All cultures were fixed with 4% paraformaldehyde in 0.1 m sodium phosphate buffer (PFA), pH 7.4, for 30 min. The following antibodies were utilized for double-immunostaining: polyclonal anti-TH (Pel-Freez Biologicals, Rabbit Polyclonal to BAX Rogers, AR), polyclonal anti-Nurr1/Nur77 (Santa Cruz Biotechnology, Santa Cruz, CA), polyclonal anti-phosphorylated cAMP response element-binding protein (pCREB) (Upstate Biotechnology, Lake Placid, NY), monoclonal anti-neurofilament (NF) protein (NF160,68; Sigma), goat anti-rabbit IgG-FITC (Roche Molecular Biochemicals), and goat anti-mouse IgG rhodamine (Cappel, Durham, NC). THCNF immunostaining was performed as explained previously (Brosenitsch et al., 1998). The protocol for Nurr and pCREB immunostaining was the same as for THCNF, except that cells were incubated in 20% goat serum in PBS made up of 0.5% Triton X-100 (PBS-Tx) before incubation in the primary antibodies, which was performed at 4C in anti-Nurr1/Nur77 (1:4000) or anti-pCREB (1:2000) and anti-NF (1:100).