Hypoxia, Leptin and Hippocampal Neurons Firing Figure 7. Exposure to 2 hours of hypoxia does not affect the viability of hippocampal neurons. A. Images of the mouse hippocampal primary culture in control conditions (top) and after 2 hours exposure to 3% O2 (bottom), at 11 (left) and 18 DIV (right). Cell bodies, axons and dendrites morphology were not affected by the reduction of oxygen supply. B. Bar histogram showing the mean number of cells/mm2, counted before and after the hypoxic treatment by the trypan blue exclusion assay. The number of vital cells did not significantly change after hypoxia, either at 11 DIV (left) or 18 DIV (right) (n = 4 dishes, p.0.05, using Student’s paired t-test). doi:10.1371/journal.pone.0041530.g007 frequency, which can be inhibited by leptin or even potentiated by leptin plus paxilline, is shown in Fig. 8B and 8C, respectively. Regarding 18 DIV neurons, the effects of leptin were milder: spontaneous activity was reduced only by 22% (from 1.260.2 Hz to 1.060.2 Hz; p,0.01) and in a minor percentage of cases (55% versus 72%), the remaining being unaffected. Addition of paxilline together with leptin, not only removed this inhibition, but uniquely led to an increased firing frequency in 50% of the channels (from 0.7460.19 Hz to 1.260.3 Hz; p,0.01, Fig. 8C), suggesting that, also in elder cultures, when BK channels are blocked, leptin action is reversed. In the remaining electrodes no significant effects were caused by adding paxilline to leptin. The effects of leptin (50 nM) were also evaluated after 3 hours of application, in order to assess if prolonged exposures could lead to different effects on hippocampal excitability. We found that spontaneous electrical activity was reduced by leptin independent of the duration of the treatment, at both ages. On DIV 11 (n = 6 MEAs), leptin reduced the firing frequency (from 0.9060.09 Hz to 0.4860.13 Hz; p,0.001) in 60% of the electrodes, without significant effects on the remaining. This behaviour persisted also at DIV 18 (n = 8), where 64% of the electrodes showed a reduction of firing frequency, from 1.160.2 Hz to 0.7660.13 Hz, (p,0.001), while the remaining were unaffected. PLoS ONE | www.plosone.org 10 July 2012 | Volume 7 | Issue 7 | e41530