Hypoxia, Leptin and Hippocampal Neurons Firing Photograph of an hippocampal primary culture on DIV 18, plated over an array of planar multi-electrodes (top). Examples of spontaneous electrical activity recorded from three different channels of the same MEA, on DIV 18 (bottom). Inset: magnification of a burst, the typical pattern of activity in elder cultures. doi:10.1371/journal.pone.0041530.g001 hypoxia, although at a different extent (54% versus 69% reduction, respectively). Leptin, on its own, exerts different effects on normoxic and hypoxic neurons. It significantly decreases the firing frequency in normoxic neurons but, when applied during hypoxia, leptin either counteracts the hypoxia-induced reduction of spontaneous activity or even potentiates the firing frequency of 11 DIV hippocampal neurons. Thus, even if with different effects on younger and elder neurons, overall leptin reverses the inhibitory action of hypoxia on the firing properties of hippocam- pal neurons. On the contrary, application of leptin together with the BK channel blocker paxilline (1 mM), prevents leptin action and restores the hypoxia-induced reduction of spontaneous firing. Materials and Methods Cell Culture on MEAs All experiments were performed in accordance with the guidelines established by the National Council on Animal Care and approved by the local Animal Care Committee of Turin University. Hippocampal neurons were obtained from black-six mouse 18-day embryos. Hippocampus was rapidly dissected under sterile conditions, kept in cold HBSS (4uC) with high glucose, and then digested with papain (0,5 mg/ml) dissolved in HBSS plus DNAse (0,1 mg/ml) [16,17]. Isolated cells were then plated at the final density of 1200 cells/mm2 [18] onto the MEA (previously coated with poly-DL-lysine and laminine) and allowed to adhere on the centre of the chip by using a ring made of Sylgard 184 (Dow Corning), 4.5 mm internal diameter (11 mm external diameter), which was removed after 4 hours. The cells were incubated with 1% penicillin/streptomycin, 1% glutamax, 2.5% fetal bovine serum, 2% B-27 supplemented neurobasal medium in a humidified 5% CO2 atmosphere at 37uC. Each MEA dish was covered with a fluorinated ethylene-propylene membrane (ALA scientific, Westbury, NY, USA) to reduce medium evaporation and maintain sterility, thus allowing repeated recordings from the same chip. Recordings were carried out since DIV 11 until DIV 18. Culture medium was partially (1/3) changed once a week, depending on the age of the culture (young cultures did not need weekly change of medium). Following experiments, MEA dishes were re-used by cleaning overnight in 1% Tergazyme (Sigma- Aldrich, St. Louis, MO), rinsing in distilled water and then sterilizing overnight under UV ray. MEA Recordings Multisite extracellular recordings were performed using the MEA-system, purchased from Multi-Channel Systems (Reutlin- gen, Germany). The 60 electrodes array (TiN/SiN) is composed by a 868 square grid with 200 mm inter-electrode spacing and 30 mm electrode diameter. Data acquisition was controlled through MC_Rack software (Multi-Channel Systems Reutlingen, Germany), setting the threshold for spike detection at 215 mV and sampling at 10 kHz. Experiments were performed in a non- humidified incubator at 37uC and with 5% CO2, without replacing the culture medium. Before starting the experiments, cells were allowed to stabilize in the non-humified incubator for 90 seconds; then recordings of the spontaneous activity was carried out for 6 minutes. For hypoxic treatment, cells were kept for 2 hours into a humified incubator with 5% CO2 and 3% O2 (37uC); after that, spontaneous activity was measured under normoxic condi- tions. The recovery was recorded 2 hours after hypoxia. Murine recombinant leptin (PeproTech, London, UK) was used at a final concentration of 50 nM, being applied 1 hour before the recordings, under hypoxic or normoxic conditions, and main- tained in the MEA dish for the whole recording. Then it was washed out and we waited 2 hours before recording the recovery. Paxilline (Sigma-Aldrich, St. Louis, MO) was dissolved in DMSO (Sigma-Aldrich, St. Louis, MO) and used at the final concentration of 1 mM [19]. Cultures were kept into the normoxic incubator between recording sessions. It is worth noticing that, due to the variability among different MEAs, the effects of any treatment (hypoxia, leptin) have been evaluated with respect to their own control MEAs. Analysis of MEA Activity Bursts analysis was performed using Neuroexplorer software (Nex Technologies, Littleton, MA, USA) after spike sorting operations. A burst consists of a group of spikes with decreasing amplitude [20], thus we set a threshold of at least 3 spikes and a minimum burst duration of 10 ms. We set interval algorithm specifications such as maximum interval to start burst (0.17 sec) and maximum interval to end burst (0.3 sec) recorded in 0.02 s bins. We performed the burst analysis using two different approaches, either by considering the contribution of each recording channel or by calculating for each MEA one mean value over the active recording channels; we obtained comparable results of the spike parameters, suggesting a reliable homogeneity of data. Burst analysis has been performed by monitoring the following parameters: number of spikes, frequency, number of bursts and percentage of spikes in bursts. Cross-correlation probability vs. time diagrams were con- structed by means of Neuroexplorer software (Nex Technologies, Littleton, MA, USA), using 60.5 s and 63.5 s and 5 ms bin size. Data are expressed as means 6 S.E.M and statistical significance was calculated either by using Student’s paired t-test or with a one way ANOVA followed by a Bonferroni post-hoc analysis. Values of p,0.05 were considered significant. Figures were edited by means of Corel Draw Graphics Suite 12 (Corel Corporation, Ottawa, Canada). Cell Viability Assay Cell viability was assessed using the Trypan blue exclusion assay and plating hippocampal neurons on plastic dishes (IBD, Germany) patterned with a grid of 49 square areas of 5006500 mm2. Neuronal cultures were stained with 0.4% Trypan blue (Sigma-Aldrich, St. Louis, MO). Cells that were stained blue were considered dead cells and not included in the cell count, while those with a translucent clear appearance were considered alive and, therefore, included in the cell count. Neurons were counted via a light microscope under620 magnification; ten non- overlapping fields of four different dishes were analyzed, considering the same fields of the dish before and after hypoxia (2 h 3% O2). This assay was performed either on 11 DIV (four dishes) or on 18 DIV neurons (four dishes). PLoS ONE | www.plosone.org 3 July 2012 | Volume 7 | Issue 7 | e41530