# 0.05 and ## 0.01 compared with corresponding nicotine-treated wild-type mice. Discussion Nicotine, like other drugs of abuse, activates the mesolimbic dopaminergic system and increases extracellular dopamine levels in the NAc (Pontieri et al., 1996), a feature related to its rewarding properties. Institutional Animal Care and Use Committee of Kanazawa University or college. Preparation of neuronal cultures. Main neuronal cultures were prepared from hippocampus of 15-d-old embryonic mice (di Lynestrenol Porzio et al., 1980). Hippocampi were dissected from embryonic ICR mice and incubated with Versene (Invitrogen, Carlsbad, CA) at room heat for 12 min. Cells were then mechanically dissociated with a fire-narrowed Pasteur pipette in the culture medium and plated at a density of 1 1.5 Lynestrenol 105 cells/cm2 on a 24-well dish in basal DMEM/Nutrient Combination F-12 (DMEM/F-12) supplemented with Lynestrenol 10% fetal calf serum (FCS) (Dainippon Pharmaceutical, Osaka, Japan), 33 mm glucose, 2 mm glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 5 mm HEPES, and 0.11% sodium bicarbonate. Before use, dishes were sequentially coated with 10 g/ml poly-l-lysine. After 18 h in culture, the culture medium was replaced with basal DMEM/F-12 made up of 5% FCS, 33 mm glucose, 2 mm glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 5 mm HEPES, 0.11% sodium bicarbonate, 25 g/ml transferrin, 250 ng/ml insulin, 0.5 pm -estradiol, 1.5 nm triiodothyronine, 10 nm progesterone, 4 ng/ml sodium seleniate, and 50 m putrescine. Cells were treated with 5 m cytosine arabinoside for 24 h during 2C3 d (DIV). Cultures were kept in serum-free medium, basal DMEM supplemented with 25.5 mm glucose, 0.5 mm glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, 0.11% sodium bicarbonate, 50 g/ml transferrin, 500 ng/ml insulin, 1 pm -estradiol, 3 nm triiodothyronine, 20 nm progesterone, 8 ng/ml sodium PIK3R5 seleniate, and 100 m putrescine after 3 DIV. The culture medium was replaced with a freshly prepared medium of the same composition every 3 d. Cultures were usually managed at 37C in a 5% CO2/95% air-humidified incubator. microdialysis. Animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and a guide cannula (MI-AG-6; Eicom, Kyoto, Japan) was implanted in the NAc (+1.5 mm anteroposterior, +0.8 mm mediolateral from bregma, ?4.0 mm dorsoventral from your skull) according to the mouse brain atlas (Franklin and Paxinos, 1997). On recovery from your medical procedures, a dialysis probe equipped with a microinjection tube (MIA-6-1; 1 mm membrane length; Eicom) was inserted through the guideline cannula and perfused with an artificial CSF (aCSF) (in mm: 147 NaCl, 4 KCl, and 2.3 CaCl2) at a flow rate of 1 1.0 l/min (Nagai et al., 2004). The microdialysis probes were constructed of three stainless steel tubes, two silica tubes (inlet and store) for microdialysis with a 75 m outer diameter, and a microinjection silica tube with a 75 m outer diameter. The microinjection tube was place in parallel with the tubes for microdialysis. The microinjection tube was half the length of the dialysis membrane. These three silica tubes were sealed together with epoxy resin, and each one was secured with stainless steel tubing at the top of the probe. The outflow fractions were collected every 20 min. After the collection of three baseline fractions, plasminogen activator inhibitor-1 (PAI-1) (1C3 ng; Calbiochem, Darmstadt, Germany), human recombinant tPA (30C100 ng; provided by Eisai, Tokyo, Japan), human plasmin (30C100 ng; Chromogenix, Molndal, Sweden), or tyrTRAP7 [(tyr?1)-thrombin receptor-activating peptide 7; YFLLRNP] (3C10 ng; Bachem, Bubendorf, Switzerland) dissolved in 1 l of aCSF answer was injected during a 10 min period through the microinjection tube into the NAc. Ten minutes after the microinjection, a dialysis probe was perfused with 1 mm nicotine made up of aCSF for 20 min. Dopamine levels in the dialysates were analyzed using an HPLC system equipped with an electrochemical detector. For analysis of ACh release, a guide cannula (AG-4; Eicom) was implanted in the hippocampus (?3.3 mm anteroposterior, +3.2 mm mediolateral from bregma, ?2.5 mm dorsoventral from your skull) or striatum (+0.5 mm anteroposterior, +2.0 mm mediolateral from bregma, ?2.8 mm dorsoventral from your skull). On recovery from your medical procedures, a dialysis probe (AI-4-2; 2 mm membrane length; Eicom) was inserted through the guideline cannula and perfused with an aCSF made up of 10 m eserin at a circulation rate of 1 1.0 l/min. Outflow fractions were collected every 15 min. After the collection of three baseline fractions, nicotine-containing aCSF (3 mm) was perfused for 30 min. ACh levels in the dialysates were analyzed using an HPLC system equipped with an electrochemical detector (Tran et al., 2001). zymography. Mice were transcardially perfused with isotonic PBS, pH 7.4, 30 or 60 min after receiving an injection of nicotine (0.5 mg/kg, s.c.). The brain was then removed, immediately frozen in O.C.T. compound (Sakura Finetechnical, Tokyo, Japan), and stored at ?80C. Cryostat sections (14 m) were analyzed for proteinase activity as explained previously (Scott et al., 2001), with modifications. Briefly, 100 l overlays of 1% agarose in PBS made up of 10 g/ml BODIPY TR-X casein (Invitrogen) and 5 mm.