Hypoxia, Leptin and Hippocampal Neurons Firing Figure 4. Firing frequency and bursts number increment with time in culture. Parameters characterizing electrical spontaneous activity of the hippocampal culture at DIV 11 (n = 29 MEAs; grey bars) and at DIV 18 (n = 36 MEAs; black bars) given as mean 6 S.E.M. The difference between the two groups is statistically significant for all parameters (* p,0.05, **p,0.01 and ***p,0.001; using Student’s paired t-test). Spikes and bursts numbers both increased between 11 and 18 DIV. Number of spikes increased from 74611 to 150623 (p,0.01 ), while the number of bursts from 6.161.4 to 9.461.2 (p,0.05). The Maturation of the culture led to a prolongation of bursts duration (from 0.0960.01 s at 11 DIV to 0.2660.06 s at 18 DIV; p,0.02) and an increment of the percentage of spikes in the burst (4665% vs 6065%; p,0.03). Prolonged bursts and higher frequency on 18 DIV were accompanied by decreased mean interburst interval (IBI). Young cultures presented a mean IBI value of 1562 s while, in mature cells (18 DIV), this interval was reduced to 9.761.4 s (p,0.01). doi:10.1371/journal.pone.0041530.g004 Results Synchronization and Firing Frequency of Hippocampal Neurons Increased with Days in Culture Spontaneous firing of hippocampal neurons was monitored for up to three weeks after plating. We tested 72 MEAs in our experiments, and we found that hippocampal neurons became spontaneously active at DIV 8 (days in vitro). From the analysis we discarded only 10% of the arrays, in which the majority of channels (75% or more) remained silent. In addition, we selected DIV 11 as the day for monitoring the spontaneous firing of younger neurons, and we compared their activity with those of 18 DIV neurons, to assess the in vitro functional changes of the network activity during development. Figure 1 shows two representative photographs of the same hippocampal culture at the two stages of development (11 and 18 DIV). Firing properties of hippocampal neurons significantly varied with time: spontaneous activity was mainly characterized by isolated spikes with limited number of bursts at DIV 11 (Fig. 1A). Neurons spontaneously fired trains of single spikes with the typical shape of extracellularly recorded action potentials (APs), i.e., a large and fast inward deflection followed by a small and slow outward deflection lasting few milliseconds (see inset) that correspond to the negative first derivative of intracellularly recorded APs [19,21–23] One week later (18 DIV, Fig. 1 B), the firing changed significantly. There was a visible increase of spiking frequency, as well as a prevalence of synchronized bursts of APs (Fig. 1 B, inset) that lasted tens of milliseconds and were rarely present in younger cultures. This effect is more evident when comparing the raster plots of Fig. 2, where the APs monitored by each active recording channel are translated in a sequence of brief vertical lines versus time. Neuronal activity is distributed more or less randomly over time in younger cultures, whereas becomes more synchronized at DIV 18, when nearly all recording channels display synchronous AP bursts for tens of seconds (rectangle in Fig. 2). To better quantify the occurrence of synchronized activity with increasing age of the culture, we measured the probability of coincidence of the single events between different electrodes [24,25]. As shown in the cross-correlograms of Fig. 3, calculated at different time windows (60.5 s and 63.5 s with 5 ms bin size), the maximal correlation of neuronal activities at t = 0 s is increased by more than 300% at DIV 18 (from 0.0660.01 to 0.2660.03; p,0.001; see inset in Fig. 3). This indicates that synchronization of APs activity in mouse hippocampal network is greatly enhanced during neuronal maturation in culture. In this regard, represen- tative recordings from the same MEA are shown in Fig. 3C. Firing Properties of Hippocampal Neurons Under Normoxic Conditions Because of the different spontaneous firing activity between young and elder cultures, separated analysis at DIV 11 and DIV 18 networks were carried out. Control data were taken from recordings under normoxic conditions. As shown in Fig. 4, all the PLoS ONE | www.plosone.org 6 July 2012 | Volume 7 | Issue 7 | e41530