Hypoxia, Leptin and Hippocampal Neurons Firing Figure 5. A period of hypoxia reduces spontaneous electrical activity in hippocampal cultures. A. Outline of the experimental protocol used to study the effect of hypoxia on hippocampal primary cultures (top). Below some examples of recordings from two representative MEAs in control conditions, 10 minutes after hypoxia and 2 hours after the treatment, at 11 and 18 DIV. It is clearly visible the reduction of the firing frequency after the exposure to lower oxygen supply (3% of oxygen for 2 hours). B. Main parameters characterizing electrical spontaneous activity of the hippocampal culture at 11 DIV and 18 DIV (n = 15 and n = 16 MEAs, respectively) in control conditions (white bars), after hypoxia (black bars) and 2 hours after the post-hypoxic recording (striped bars). For 18 DIV neurons, firing frequency decreased from to 1.760.3 Hz to 0.560.2 Hz (p,0.01) after hypoxia. Number of bursts and number of spikes were respectively reduced from 10.761.8 to 2.860.7 (p,0.001) and from 155630 to 48617 (p,0.01). In 11 DIV firing frequency decreased from to 1.160.2 Hz to 0.5160.12 Hz (p,0.01) after hypoxia. Number of bursts and number of spikes were similarly reduced, the first one from 8.361.6 to 3.961.0 (p,0.05), the second one from 102615 to 46611 (p,0.01). At both ages, the percentage of spikes in burst did not change after hypoxia. For all parameters, recovery was not significantly different from the hypoxic group. Data are given as mean 6 S.E.M. (* p,0.05, **p,0.01 and ***p,0.001, using one way ANOVA followed by a Bonferroni post-hoc analysis). doi:10.1371/journal.pone.0041530.g005 parameters characterizing the spontaneous firing were significantly different between younger and elder cultured neurons. At 18 DIV, the firing frequency almost doubled (from 0.8060.13 to 1.760.3 Hz, p,0.01) and this effect was accompanied by an increased number of bursts, from 6.161.4 to 9.461.2 (p,0.05). In agreement with the observation that burst activity is the main feature at 18 DIV, the burst duration and the percentage of spikes within the burst significantly increased, whereas the mean interburst interval (IBI) was reduced at 18 DIV (see legend of Fig. 4). Tetrodotoxin (300 nM) completely blocked the spontane- ous activity, both at DIV 11 and DIV 18, confirming the all-or- none nature of the APs driven by TTX-sensitive Nav1 channels (data not shown). Hypoxia Reduced the Firing Activity but Increased the Synchronization Before focusing on the effects of leptin, we studied how oxygen reduction could alter the network activity, by exposing the hippocampal cultures to hypoxia (3% O2) for 2 h. Since hypoxic-ischemic conditions can be achieved with different incubation times in primary neuronal cultures, ranging from 2 min up to 4 h exposure to low oxygen tension, we decided to use a protocol of 2 h [26–29]. Hippocampal activity was monitored for 90 s under three different conditions: initially under normoxia (controls), immediately after 2 h of hypoxia (3% O2) and after recovery from hypoxia (Fig. 5, top). By measuring the activity of hippocampal neurons at 18 DIV (n = 16 MEAs), we found that hypoxia reduced the mean frequency by 69% (from 1.760.3 Hz to 0.560.2 Hz, p,0.01) in the majority of electrodes (79%, Fig. 5), while leaving the remaining unaffected. This reduction was accompanied by a significant decrease of the number of bursts (from 10.761.8 to 2.860.7; p,0.001), while the percentage of spikes in the burst was not significantly altered (5266% versus 3067%, Fig. 5B). Hypoxia therefore limits bursts onset without significantly affecting burst properties. Two hours after the end of the hypoxic treatment, when returning to normoxic conditions, mean values of firing frequency (1.060.3 Hz) and burst activity (5.761.3 bursts), were still comparable to the hypoxic group (Fig. 5; ANOVA with Bonferroni post-hoc, p.0.05), suggesting that recovery was not complete. Though, a complete recovery from hypoxia could be obtained over a longer period (12–24 h, data not shown). Concerning the pattern of electrical activity, we found that hypoxia drastically increased the occurrence of synchronized events even in younger cultures, as shown by the cross- correlogram plots in Figure 6. When the same hypoxic treatment was performed on younger cultures (11 DIV, n = 15 MEAs), where spontaneous firing occurs through the generation of single spikes rather than synchronized burst activity, we again assessed that hypoxia caused significant effects in 62% of channels, where the firing frequency was reduced by 54% (from 1.160.2 to 0.5160.12, p,0.01). Number of spikes and number of bursts underwent a similar reduction: the former decreased from 102615 to 46611 and the latter from 8.361.6 to 3.961.0 after hypoxia. Similarly to the elder group, recovery after hypoxia was not complete within 2 hours. Remaining electrodes (38%) showed no significant changes. Our data indicate that hypoxia indeed reduces the firing frequency of hippocampal neurons, but at higher degree in elder cultures. Such variability of responses can be attributed to the heterogeneity of the culture, where the mixture of pyramidal neurons and interneurons may be differently firing, or else to the developmental changes of cultured neuronal networks occurring in vitro [24]. In order to assess if the altered firing activity induced by hypoxia was due to cell death, we performed the trypan blue exclusion assay (see Material and Methods). This organic dye selectively stains dead/dying cells. Under control conditions, the density of unstained living cells at 11 DIV was approximately 10267 cells/ mm2 and remained unaltered after the hypoxic treatment (9967 cells/mm2, n = 4 dishes, p.0.05 Student’s paired t-test, Fig. 7B). Concerning the effect of hypoxia on cell density on 18 DIV cultures, again, we did not reveal any statistical significant 2 reduction of this parameter (2362 vs 2262 cells/mm , n = 4 dishes, p.0.05 Student’s paired t-test, Fig. 7B). Moreover, looking at the morphology of the culture, cell bodies and dendritic trees were not affected by the treatment (Fig. 7A). These data indicate that cell density and viability were not affected by hypoxia. Leptin Reduced Spontaneous Activity of Normoxic Hippocampal Neurons A preliminary series of experiments was devoted to investigate the activity of the neuronal network after 10 min application of 50 nM leptin, and keeping the adipokine in the culture medium also during the recordings [5,2,30]. These experiments were performed under normoxic conditions. On average, leptin re- duced the firing frequency both in younger and elder cultures, although the effect was more pronounced on 11 DIV neurons. More specifically, on DIV 11 (n = 7 MEAs), leptin markedly reduced the firing frequency by 71% with respect to controls (from 0.5460.11 Hz to 0.1660.04 Hz; p,0.01). A similar reduction has been observed also for the mean burst duration (70%) and the number of bursts, which underwent 79% decrease after the treatment (Fig. 8B). This reduction of the spontaneous activity was present in most of the microelectrodes (72%), whereas, in the remaining, electrical activity was not affected by leptin. When the BK channel blocker paxilline (1 mM) was added, the leptin- mediated reduction was either drastically lowered (from 71% to 21%), or even completely removed in 45% of cases, where the firing frequency was potentiated (from 0.560.2 Hz to 1.160.2, p,0.01), suggesting a BK-channel mediated action of leptin on normoxic neurons [5]. This opposite modulation of the firing PLoS ONE | www.plosone.org 8 July 2012 | Volume 7 | Issue 7 | e41530