Hypoxia, Leptin and Hippocampal Neurons Firing Figure 9. Pre-treatment with leptin partially retrieves the depressive action of hypoxia. A. Outline of the experimental protocol used to study the effect of leptin pre-treatment before exposure to hypoxic injury (top). Below: representative recordings in control condition and after hypoxic treatment in presence of leptin (11 and 18 DIV). The reduction of the firing frequency is still present but not so marked as with hypoxia. B. Main parameters characterizing electrical spontaneous activity of the hippocampal culture at 11 DIV and 18 DIV (n = 9 and n = 6 MEAs, respectively) in control conditions (white bars) and after pre-treatment with leptin before hypoxia (light grey bars). Regarding 18 DIV neurons, firing frequency decreased from to 1.560.2 Hz to 1.060.2 Hz (n = 6, p,0.01) after hypoxia. The number of spikes was reduced from 131615 to 84615 (p,0.01). On the contrary, the number of bursts and the percentage of spikes in bursts were not significantly reduced by the hypoxic treatment. In 11 DIV cultures, firing frequency and number of spikes were both increased after hypoxia: the former from 0.4060.09 Hz to 0.9060.13 Hz (n = 9; p,0.05), the latter from 3868 to 84611 (n = 9, p,0.05). The number of bursts was reduced from 2.660.9 to 1.560.5 (p,0.01). The percentage of spikes in bursts was not significantly altered by hypoxia. Data are given as mean 6 S.E.M. (* p,0.05, **p,0.01 and ***p,0.001; using Student’s paired t-test). doi:10.1371/journal.pone.0041530.g009 Hypoxia Decreases the Firing Frequency and Synchronizes the Events Our data on hippocampal neuronal activity demonstrate that chronic oxygen reduction significantly decreased both firing frequency and burst activity at any age (11 and 18 DIV), with more pronounced in elder cultures. Even though patch-clamp studies would be required for investigating the molecular basis of this process, our findings are in good agreement with the observation that, in response to oxygen deprivation, rat CA1 pyramidal neurons exhibit an hyperpolarization (hypoxic hyper- polarization), associated with a reduction of cell input resistance [37]. This hyperpolarization is mediated by the activation of both ATP-sensitive and Ca2+-dependent K+ channels, due to the rise of Ca2+ released from intracellular stores [37]. These findings could thus explain the reduced firing frequency and burst activity recorded in our experiments after the hypoxic period. On the contrary, acute exposure to oxygen-glucose deprivated medium (OGD) produces opposite effects, by transiently increasing the firing frequency [12], up-regulating L- and N–type Ca2+ channels in CA1 hippocampal neurons [38], or activating a TRPM7 channel, permeable to Ca2+ and Mg2+ ions [26]. It is worth noticing that this initial up-regulation of the firing frequency, which occurs following acute instead of chronic hypoxia, might downstream activate Ca2+-dependent K+ channels, thus reducing the firing frequency on a longer time scale, as we observed in hippocampal neurons after 2 h hypoxia. Moreover, we show that hypoxia increases the synchronization of neuronal spontaneous activity either at 11 or 18 DIV. The alteration of the functional network connectivity after hypoxia was previously reported by Figure 10. Leptin counteracts the effect of hypoxia on hippocampal neurons. A. Summarizing pie charts showing the percentage of electrodes potentiated (striped area), inhibited (black area) and unaltered (white area) by hypoxia, hypoxia+leptin and hypoxia+leptin+paxilline, either at 11 or 18 DIV. B. Top. Percentage of firing frequency reduction with respect to controls under the indicated conditions: hypoxia (black bars), leptin + hypoxia (light grey bars), leptin+hypoxia+paxilline (white bars). Leptin lowered the firing frequency reduction, either at 11 or 18 DIV (p,0.05 and p,0.01 respectively), while the addition of paxilline removed this effect. doi:10.1371/journal.pone.0041530.g010 PLoS ONE | www.plosone.org 14 July 2012 | Volume 7 | Issue 7 | e41530