significantly contributing to a proper neuronal connectivity in neuronal networks [38]. According to these observations, results from the current investigation demonstrate that CBD-mediated activation of PPARc is associated with a significant neurogenic activity in the granule cell layer of the hippocampal DG. Results indicate that CBD markedly counteracts the massive reduction of neurogenesis in the DG caused by Ab exposure towards control animals and this effect is consequential, as expected, to a selective PPARc involvement. It has been recently asserted that CBD could promote adult hippocampal neurogenesis by activating CB1 receptors [39]. Such an assumption was supported by the observation that the CBD neurogenic effect was lost in CB1 knock-out mice, suggesting that the pro-neurogenic action of this phytocannabinoid was clearly dependent on the interaction at the CB1 receptor, which shows a wide expression over the entire DG, including the neuronal precursor cells. However, the observation that D9-tetrahydrocan- nabinol, a preferential agonist at CB1 receptor site, failed in the same investigation to affect hippocampal neurogenesis, tempers the notion that CB1 receptor activation and CBD neurogenetic properties are linked in a straightforward way. Since CBD exhibits negligible affinity to CB1 receptors and the majority of actions appears to be cannabinoid receptor independent, it is possible to assume that the CBD effect on neurogenesis could only involve CB1 receptor sites indirectly. In conclusion, results of the present research demonstrate that CBD may exert protective functions through a PPARc dependent activation, which leads to a reduction in reactive gliosis and consequently in neurodegeneration. Moreover, in the current experimental conditions this phytocannabinoid appears to stimu- late neurogenesis since it increases DCX immunopositive cell proliferation rate in rat DG. Innovative therapeutic approaches which could significantly improve AD course require new molecules that will be able to have an impact on different pathological pathways, which converge at the progressive neurological decline. CBD has shown a capability to profoundly reduce reactive astrogliosis and to guarantee both direct and indirect neuronal protection in Ab induced neuroinflamma- tion/neurodegeration. So far, the lack of understanding of the precise molecular mechanism involved in CBD pharmacological actions, has had limited interest and has puzzled investigators. Currently, findings of the present study throw some light on the issue, and frame CBD as a new PPARc activator. Materials and Methods Ethics Statement All experiments were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and those of the Italian Ministry of Health (D.L. 116/92), and they were approved by the Institutional Animal Care and Use Committees (Centro Servizi Veterinari-University of Naples Federico II; PROT. 012F). Astrocyte culture Rat primary astroglial cultures were obtained from newborn Sprague-Dawley rats (two days old) according to Vairano et al. [40]. Brains were mechanically processed to obtain single cells that were seeded in 75 cm2 flasks at a density of 36106 cells/flask with 15 ml of culture medium (DMEM, 10% inactivated FBS, 100 IU/ ml penicillin, 100 mg/ml streptomycin; all from Sigma Aldrich) and incubated at 37uC in a humidified atmosphere containing 5% CO2. Astrocytes were mechanically separated from microglia and CBD Promotes Neuronal Survival and Neurogenesis oligodendrocytes, and plated onto 10 cm ø Petri dishes at a density of 16106 cells/dish or onto eight chambers polystyrene culture slides at a density of 36104 cells/chamber, depending upon the experimental procedure. Purity of cell cultures was tested by immunofluorescence with monoclonal anti-glial fibrillary acidic protein (GFAP) and only cultures with more than 95% GFAP- positive cells were employed for the experiments. Treatments in isolated primary astrocytes Confluent primary astrocyte cultures were treated with 1 mg/ml Ab (1–42) (Tocris Bioscience) in the presence or absence of the following substances: CBD (1029–1027 M), MK886 (3 mM), the selective PPARa antagonist, and GW9662 (9 nM), the selective PPARc antagonist (all purchased from Tocris Bioscience). The concentrations of the substances were chosen according to the results of a series of pilot experiments aimed at identifying the lowest effective concentration (data not shown). No significant variation versus control was observed when CBD, MK886, or GW9662 were given alone (data not shown). Nitric oxide (NO) measurement Production of nitric oxide (NO) was determined by measuring the amount of nitrite (NO22) accumulated in supernatants of primary rat astroglial cells 24 h after treatments and in the inoculated ipsilateral hippocampi homogenates deriving from in vivo experiments. A spectrophotometer assay based on the Griess reaction was used [41]. The absorbance of controls and unknown samples was measured at 540 nm. The NO22 concentration was thus determined using a standard curve of sodium nitrite (NaNO2) and referred to 16106 cells for the in vitro experiments or expressed as NO22 referred to mg of homogenized hippocampal protein content for the in vivo experiments. ELISA Quantitative determination of Tumor Necrosis Factor alpha (TNFa), interleukin 1b (IL1b), and S100B was carried out performing Enzyme Linked-Immuno-Sorbent Assay (ELISA) assays in vitro and in ex vivo deriving samples from homogenated hippocampi ipsilateral to the inoculation site; (TNFa and IL b kits purchased from Invitrogen; S100B kit from BioVendor). In in vitro experiments, 24 h after treatments, cell culture medium was collected and ELISA assay was performed according to kit instruction. Similarly, samples collected by hippocampal homog- enates deriving from the in vivo experiments, also undergo ELISA. Western Blot analysis Twenty-four hours after treatment, harvested astrocytes (16106 cells) were washed twice with ice-cold PBS, and collected by centrifugation at 180 g for 10 min at 4uC. Pellets were resuspended in 50 ml of ice-cold hypotonic lysis buffer (Tris/HCl pH 7.5 50 mM; NaCl 150 mM; EDTA 1 mM; Triton X-100 1%) supplemented with the proper protease inhibitor cocktail (Roche) and incubated on ice for additional 15 min. Ipsi- lateral hippocampi to injection site, deriving from in vivo experiments, were dissected from frozen excised rat brains and lysed with the same buffer. Cells or hippocampi were then mechanically lysed, and the total protein extracts were obtained by centrifugation at 13,000 g for 15 min at 4uC. Samples were subjected to SDS- polyacrylamide gel electrophoresis, and proteins were transferred onto nitrocellulose membrane and incubated with one of the following antibodies: anti-GFAP 1:50000; anti-iNOS 1:200; anti- p50 1:400, anti p65 1:1000, anti-calbindin 1:1000, anti-S100B 1:1000, anti b-actin 1:1000 (all purchased from Abcam). After PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28668