Hypoxia, Leptin and Hippocampal Neurons Firing Figure 8. Leptin reduces firing activity in hippocampal cultures at any age. A. Top. Experimental protocol used to study the effect of leptin (10 minutes incubation) on hippocampal primary cultures. Below: recordings from two representative MEAs in control conditions and after application of leptin (10 min), at 11 or 18 DIV. The insets show a magnification of a single burst: it is clearly visible the reduction of bursts duration after leptin’s treatment at 18 DIV. B. Mean frequency, spikes number, bursts number and bursts duration, in control conditions (white bars) and after 10 minutes application of leptin (grey bars). Data averaged from n = 7 and n = 10 MEAs, respectively, for 11 DIV and 18 DIV cultures. Data are given as mean 6 S.E.M. (* p,0.05 and **p,0.01; using Student’s paired t-test). C. Mean firing frequency measured in control conditions (white bars) and after addition of leptin 50 nM and paxilline 1 mM to the culture medium (light grey bars). At both ages, paxilline together with leptin significantly potentiated the mean frequency with respect to controls in approximately half of the electrodes (n = 8 MEAs for 11 DIV and n = 7 for MEAs 18 DIV; p,0.01 using Student’s paired t-test). In the remaining, paxilline plus leptin either had no effect (18 DIV) or strongly limited the reduction (see text). doi:10.1371/journal.pone.0041530.g008 Our findings on the reduced firing frequency and burst duration by leptin are in good agreement with recent data, showing that in rat hippocampal cultures and slices leptin has a partial inhibitory action which is ascribed to a PI3-kinase-dependent activation of BK channels [30,2]. Activation of BK channels is also in line with the reduction of bursts duration that we measured after leptin treatment. Block of BK channels in fact induces prolonged bursts in hippocampal neurons [31]. Taken together, our data support a role of BK channels as one of the molecular targets of leptins action. Leptin Reverses the Hypoxic Inhibition through the Activation of BK Channels In order to assess whether leptin (50 nM) could limit the hypoxic-induced effects, we applied the adipokine 1 h before exposing the hippocampal culture to 3% of O2 [8] and maintained the same leptin concentration during the hypoxic treatment (Fig. 9A). Under these conditions, on 18 DIV (n = 6 MEAs), hypoxia still inhibited the firing frequency compared to controls (Fig. 10A), as shown in Figure 9A. Interestingly, this reduction occurred with a drastically lower extent than the one observed without leptin (31% versus 69%, p,0.05, Fig. 10B). Furthermore, pre-treatment with leptin preserved the bursts activity in elder cultures, thus completely reversing the effects of hypoxia on the number of bursts and the number of spikes in the burst. The effects of leptin on DIV 11 hypoxic neurons (n = 9 MEAs) were significantly different with respect to elder cultures. Firing frequency was potentiated in 76% of cases (Fig. 10A), with more than a two-fold increase versus controls (from 0.4060.09 Hz to 0.9060.13 Hz; p,0.05; Fig. 9B). In the remaining we found that, similarly to the elder group, hypoxia still caused a reduction of the firing frequency (from 0.4460.12 to 0.2960.09 Hz), even if significantly lower than the one in the absence of leptin (3367% versus 5469%, p,0.05, Fig. 10B). Representative traces of this dual pattern of activity are shown in Figure 9A, where the firing frequency decreases in the upper trace while increases in the remaining. Taken together these findings suggest that the overall action of leptin is to reverse the inhibitory effects of hypoxia on spontaneous firing, even though with more pronounced effects on younger versus elder cultures. Although in the presence of leptin a slowdown of firing persisted during hypoxia in elder cultures, the percentage of reduction was significantly attenuated as compared to the leptin-free hypoxic group. Interestingly, when paxilline was applied together with leptin, the inhibitory effects of hypoxia were completely restored, as the firing frequency decreased by 61610% (18 DIV) or by 68613% (11 DIV) with respect to controls. This pattern of activity, characterized by a strong reduction of spontaneous activity was present in the majority of electrodes (85% at 18 DIV and 87% in younger cultures, Fig. 10A). The counteracting effects of leptin versus hypoxia and the role of BK channels in the leptin-mediated pathway are summarized respectively in Fig. 11 A and Fig. 11 B. The presence of leptin during hypoxia restores the firing frequency observed under control conditions, at both ages. When comparing the firing frequency of controls (white areas) versus leptin+hypoxia (grey areas), channel activity remains unaltered, as clearly visible in Figure 11 A. On the contrary, when paxilline is added together with leptin on hypoxic neurons (Fig. 11 B), firing frequency is severely impaired: spontaneous activity of hippocampal neurons is reduced to a comparable extent occurring during hypoxia alone (Fig. 5). Overall these data suggest that: i) leptin opposes to the hypoxia- induced inhibition, and ii) leptin-mediated pathway involves the activation of paxilline-sensitive BK channels. Discussion Using the MEAs, we have provided evidence that chronic hypoxia affects neuronal excitability of mouse hippocampal primary cultures, mainly reducing the firing rate of DIV 11 and DIV 18 neurons and increasing their synchronized firing. Moreover, we investigated from a functional point of view the effects of leptin, demonstrating that leptin pre-treatment success- fully opposes the depressive effects induced by hypoxic injury on hippocampal excitability. These findings may be associated to the functional impairment of central neuronal populations occurring at the first symptoms of neurodegenerative disorders, and thus address to the potential neuroprotective role of leptin. Network Burst Activity Under Normoxia Our data clearly show that hippocampal network activity changes along with maturation, switching from single spikes to synchronized bursts. This pattern of activity, also present in many brain areas such as cortex [32] and midbrain dopaminergic neurons [33], has been matter of debate for a long time and seems to derive from the balance between neuronal excitation and synaptic inhibition, rather than being the consequence of strong depolarization [20]. During a burst, the amplitude of APs is typically reduced, due to a balance between excitation and inhibition [34], while burst duration is regulated by vesicle pools availability [35]. We pointed out that: i) network synchronization is tightly correlated to the development of the network, as indicated by the net increment of the cross-correlation index between the recording electrodes at DIV 18, probably ascribed to the increased number of connections between neurons in culture [14], and ii) bursts are the main feature of spontaneous activity in mature hippocampal cultures, in agreement with the fact that bursts are triggered when neuronal spike activity is quicker, during mature stages of neuronal networks development [34]. These changes in the firing patterns of hippocampal networks are relevant for a variety of physiological mechanisms, like synaptic plasticity and circuits development [36]. Furthermore, bursts synchronization in neuronal networks can be promoted by learning, as shown by Li et al. [25] taking advantage of the MEA technology. PLoS ONE | www.plosone.org 12 July 2012 | Volume 7 | Issue 7 | e41530