being extensively washed in TBS 1X with 0.1% Tween 20, the membrane was incubated for 2 h at room temperature with the proper secondary HRP-conjugated antibodies anti-mouse (1:2000) or anti-rabbit (1:3000; both purchased from Abcam). Finally, the membrane was developed by using enhanced chemiluminescence substrate (ECL from Invitrogen). Bands were revealed through a Versadoc (Bio-Rad Laboratories) and the corresponded digital images were analyzed with Quantity One Software (Bio-Rad Laboratories). Rats and Surgical Procedures Adult male Sprague-Dawley rats (300–350 g) were obtained from Harlan Italy. They were housed in a pathogen-free barrier facility under a 12-h light/dark cycle, with ad libitum access to food and water. Rats (n = 40) were anesthetized i.p. with pentobarbital (60 mg/kg). They were then placed in a stereotaxic frame and inoculated with human Ab (1–42) (Tocris Cookson) into the CA1 area (AP 23.3, ML 21.2, DV 22.6) The peptide was dissolved in artificial cerebrospinal fluid to the concentration of 10 mg/ml, and 3 ml was injected using a microdialysis pump, keeping the flow to the constant value of 0.5 ml/min. Control rats (n = 8) were treated according to the same procedure, and they were inoculated with an equivalent volume of artificial cerebrospinal fluid. In Vivo Treatments After full recovery from stereotaxic surgery, depending upon the experimental protocol design, rats were i.p. administered for 15 days with: CBD 10 mg/kg, MK886 10 mg/kg, and GW9662 1 mg/kg. All coumpounds were dissolved in PEG/Tween80/ saline (Sigma Aldrich), 5:5:90. Control rats were i.p. given an equivalent volume of the proper vehicle. All drug solutions were freshly prepared on the day of the experiment. The drug concentrations were chosen according to their IC50, and on the basis of the results of our preliminary experiments. Rats to be subjected to immunoblotting experiments underwent deep anaes- thesia and sacrificed by decapitation. Their brains were extracted and ipsilateral hippocampi to injection site were dissected and frozen in liquid N2 before the tissue lysis for immunoblot analysis and/or ELISA assay. Rats to be subjected to morphological analysis were sacrificed through transcardiac perfusion with paraformaldehyde 4%. The extracted brains were post-fixed overnight with the same fixative, protected with 30% sucrose, and frozen using 2-methylbutane. Immunohistochemistry Immunohistochemistry analyses were performed on hippocam- pal coronal sections adjacent to the site of the injection, obtained References 1. Prins ND, Visser PJ, Scheltens P (2010) Can novel therapeutics halt the amyloid cascade? Alzheimers Res Ther 2: 5. 2. Iuvone T, Esposito G, De Filippis D, Scuderi C, Steardo L (2009) Cannabidiol: a promising drug for neurodegenerative disorders? CNS Neurosci Ther 15: 65–75. 3. Scuderi C, Filippis DD, Iuvone T, Blasio A, Steardo A, et al. (2009) Cannabidiol in medicine: a review of its therapeutic potential in CNS disorders. Phytother Res 23: 597–602. 4. Iuvone T, Esposito G, Esposito R, Santamaria R, Di Rosa M, et al. (2004) Neuroprotective effect of cannabidiol, a non-psychoactive component from Cannabis sativa, on b-amyloid-induced toxicity in PC12 cells. J Neurochem 89: 134–141. 5. Esposito G, Scuderi C, Savani C, Steardo L, Jr., De Filippis D, et al. (2007) Cannabidiol in vivo blunts b-amyloid induced neuroinflammation by suppress- ing IL-1b and iNOS expression. Br J Pharmacol 151: 1272–1279. 6. Esposito G, De Filippis D, Carnuccio R, Izzo AA, Iuvone T (2006) The marijuana component cannabidiol inhibits b-amyloid-induced tau protein hyperphosphorylation through Wnt/b-catenin pathway rescue in PC12 cells. J Mol Med 84: 253–258. CBD Promotes Neuronal Survival and Neurogenesis from both control and treated rats. Free-floating sections were treated with PBS containing 15 mM NaN3, 10% albumin, and 0.25% Triton X-100, and then they were incubated at 4uC in a damp chamber by continuous shaking with mouse anti-GFAP antibody (1:400, Sigma Aldrich). After two overnight incubation, sections were treated with the biotinylated secondary antibody (1:200; Vector Laboratories) and with the preformed avidin- biotinylated peroxidase complex (VECTASTAIN ABC kit; Vector Laboratories), and the reaction was revealed by 3,3-diaminoben- zidine tetrahydrochloride (Sigma Aldrich). Sections were mounted upon gelatin-coated slides before coverslipping with a non- aqueous medium. Immmunofluorescence In the same experimental conditions above described, immu- nofluorescence analyses were performed on hippocampal coronal sections adjacent to dentate gyrus (DG). Briefly, slices derived from both control and treated rats were blocked in 10% albumin bovine serum 0.1% Triton-PBS solution for 90 min and subsequently incubated for 1 h with a 10% albumin bovine serum 0.1% Triton- PBS solution of anti-doublecortin (DCX) antibody (dil. 1:250 v/v). DCX antibody was purchased from Cell Signaling. Sections were incubated for 1 h in the dark with the proper secondary antibody: Tetramethyl Rhodamine Isothiocyanate (TRITC) conjugated anti-rabbit 1:100 (both from Abcam). Pictures were taken using a camera (Nikon DIGITAL SIGHT DS-U1) connected with a microscope (Nikon ECLIPSE 80i by Nikon Instruments Europe) provided of the proper fluorescent filters. Slides were thus analyzed with a microscope (Nikon ECLIPSE 80i by Nikon Instruments Europe), and images were captured at 10X magnification by a high-resolution digital camera (Nikon DIGI- TAL SIGHT DS-U1). Statistical Analysis Results were expressed as mean 6 S.E.M. of experiments. Statistical analysis was performed using parametric one-way analysis of variance, and multiple comparisons were performed by Bonferroni’s test. Values of p,0.05 were considered significant. Author Contributions Conceived and designed the experiments: GE CS. Performed the experiments: MV VL GIT DDF MC TI. Analyzed the data: MRC LS. Contributed reagents/materials/analysis tools: GE CS MRC LS. Wrote the paper: GE CS LS. 7. Phiel CJ, Wilson CA, Lee VM, Klein PS (2003) GSK-3alpha regulates production of Alzheimer’s disease amyloid-b peptides. Nature 423: 435–439. 8. Hampson AJ, Grimaldi M, Axelrod J, Wink D (1998) Cannabidiol and (-)Delta9- tetrahydrocannabinol are neuroprotective antioxidants. Proc Natl Acad Sci U S A 95: 8268–8273. 9. Castillo A, Tolo´n MR, Ferna´ndez-Ruiz J, Romero J, Martinez-Orgado J (2010) The neuroprotective effect of cannabidiol in an in vitro model of newborn hypoxic-ischemic brain damage in mice is mediated by CB(2) and adenosine receptors. Neurobiol Dis 37: 434–40. 10. O’Sullivan SE 2007 Cannabinoids go nuclear: evidence for activation of peroxisome proliferator-activated receptors. Br J Pharmacol 152: 576–582. 11. Kersten S 2008 Peroxisome proliferator activated receptors and lipoprotein metabolism. PPAR Res 2008: 132960. 12. Bensinger SJ, Tontonoz P (2008) Integration of metabolism and inflammation by lipid-activated nuclear receptors. Nature 454: 470–477. 13. Bookout AL, Jeong Y, Downes M, Yu RT, Evans RM, et al. (2006) Anatomical profiling of nuclear receptor expression reveals a hierarchical transcriptional network. Cell 126: 789–799. PLoS ONE | www.plosone.org 7 December 2011 | Volume 6 | Issue 12 | e28668