leptin levels as good indicators of susceptibility to prevent AD in elderly population: higher plasma concentrations of leptin correlated with a significantly lower risk of dementia and AD [43]. Alzheimer disease could be defined as a brain expression of metabolic disorder, involving the disregulation of lipid metabolism and thus positively correlating with obesity [44]. Moreover, it is proved that leptin ameliorates the pathology of CRND8 trans- genic mice, a model of AD, by reducing the levels of tau phosphorylation and b-amyloid in vitro [45,46]. Thus, there is an increasing interest in exploring the possible use of leptin in therapeutic treatments to prevent this neurodegenerative disorder. A role of leptin as neuroprotective agent of hypoxic-ischemic brain injury is highlighted in recent works, mostly focusing on cell survival and intracellular pathways [47]. Leptin protects hippo- campal neurons against cell death, induced by excitotoxic and oxidative insults, by activating a PI3-kinase-mediated pro-survival signalling pathway, which involves the activation and phosphor- ylation of Akt [28,48]. These issues, concerning the role of leptin against hypoxic injury, have been investigated here for the first time by analyzing the functional properties of hippocampal networks. Our main finding is that leptin differently acts on hippocampal neurons during development. Leptin approximately halves the hypoxia- induced inhibition of the firing frequency on elder neurons (DIV 18). Leptin is supposed to exert its effect through large- conductance BK channels, since the addition of paxilline causes the hypoxic inhibition to be completely restored. In this respect, patch-clamp experiments will be useful to assess whether leptin effectively hyperpolarizes the membrane resting potential against a prolonged depolarization evoked during hypoxia [2,5,30]. A prominent role of BK channels in neuroprotection is supported indeed by a number of works on transient cerebral ischemia, References 1. Tartaglia LA, Dembski M, Weng X, Deng N, Culpepper J, et al. (1995) Identification and expression cloning of a leptin receptor, OB-R. Cell 83(7): 1263–71. 2. Shanley LJ, O’Malley D, Irving AJ, Ashford MLJ, Harvey J (2002) Leptin inhibits epileptiform-like activity in rat hippocampal neurons via PI 3-kinase- driven activation of BK channels. Journal of Physiology 545(Pt 3): 933–44. 3. Zhang F, Chen J (2008) Leptin protects hippocampal CA1 neurons against ischemic injury. Journal of Neurochemistry 107(2): 578–87. 4. Halaas JL, Gajiwala KS, Maffei M, Cohen SL, Chait BT, et al. (1995) Weight- reducing effects of the plasma protein encoded by the obese gene. Science 269(5223): 543–6. 5. Shanley LJ, Irving AJ, Rae MG, Ashford MLJ, Harvey J (2002) Leptin inhibits rat hippocampal neurons via activation of large conductance calcium-activated K+ channels. Nature Neuroscience Brief communications 5(4): 299–300. 6. Guo Z, Jiang H, Xu X, Duan W, Mattson MP (2008) Leptin-mediated cell survival signaling in hippocampal neurons mediated by JAK STAT3 and mitochondrial stabilization. Journal of Biological Chemistry 283(3): 1754–63. 7. Weng Z, Signore AP, Gao Y, Wang S, Zhang F, et al. (2007) Leptin protects against 6-hydroxydopamine-induced dopaminergic cell death via mitogen- activated protein kinase signaling. Journal of Biological Chemistry 282(47): 34479–91. 8. Zhang F, Wang S, Signore AP, Chen J (2007) Neuroprotective effects of leptin against ischemic injury induced by oxygen-glucose deprivation and transient cerebral ischemia. Stroke 38(8): 2329–36. 9. Russo VC, Metaxas S, Kobayashi K, Harris M, Werther GA (2004) Antiapoptotic effects of leptin in human neuroblastoma cells. Endocrinology 145(9): 4103–12. 10. Guglielmotto M, Aragno M, Autelli R, Giliberto L, Novo E, et al. (2009) The up-regulation of BACE1 mediated by hypoxia and ischemic injury: role of oxidative stress and HIF1alpha. Journal of Neurochemistry 108(4): 1045–56. 11. Zhiyou C, Yong Y, Shanquan S, Jun Z, Liangguo H, et al. (2009) Upregulation of BACE1 and beta-amyloid protein mediated by chronic cerebral hypoperfu- sion contributes to cognitive impairment and pathogenesis of Alzheimer’s disease. Neurochemical Research 34(7): 1226–35. 12. Wahl AS, Buchthal B, Rode F, Bomholt SF, Freitag HE, et al. (2009) Hypoxic/ ischemic conditions induce expression of the putative pro-death gene Clca1 via activation of extrasynaptic N-methyl-D-aspartate receptors. Neuroscience 158(1): 344–52. Hypoxia, Leptin and Hippocampal Neurons Firing which reveal the involvement of BK channels in promoting neuronal survival [49,50]. As proposed by Hu [51], activation of BK channels may serve as an ‘‘emergency brake’’, preventing cell damage or apoptosis under pathophysiological conditions that result in a large Ca2+ transient, such as hypoxic/ischemic injury. Thus, the activation of BK channels by leptin could counteract the hypoxic depolarization [12,52], repolarizing the cells, so protect- ing the neurons from the post-hypoxic reduction of firing frequency. In accordance with this concept, BK channel openers are reported to provide significant cortical neuroprotection during acute brain ischemia [53]. Still more pronounced effects, induced by leptin, have been found on DIV 11 hippocampal neurons, since leptin completely reverses the effects of hypoxia even by potentiating their firing activity. On average, leptin counteracts hypoxia on younger neurons both by reducing the number of inhibited channels and by inducing a potentiation of the firing frequency in 76% of the channels. Similarly to elder neurons, the addition of paxilline causes leptin to become ineffective against hypoxia, thus restoring the inhibition of spontaneous activity. Thus, from a functional point of view, we can conclude that leptin acts by effectively counteracting the depressive effects of chronic hypoxia on neuronal activity. Future works will be addressed to reveal whether leptin may preferentially affect specific neuronal populations. Author Contributions Conceived and designed the experiments: DG VC EC. Performed the experiments: DG CF. Analyzed the data: DG AM. Contributed reagents/ materials/analysis tools: AM JR-R CF. Wrote the paper: VC DG EC. 13. Klein WL (2002) Ab toxicity in Alzheimer’s disease: globular oligomers (ADDLs) as new vaccine and drug targets. Neurochemistry International 41: 5, 345–352. 14. Arnold FJ, Hofmann F, Bengtson CP, Wittmann M, Vanhoutte P, et al. (2005). Microelectrode array recordings of cultured hippocampal networks reveal a simple model for transcription and protein synthesis-dependent plasticity. Journal of Physiology 564(Pt 1): 3–19. 15. Berretta N, Bernardi G, Mercuri NB (2011) Firing properties and functional connectivity of substantia nigra pars compacta neurones recorded with a multi- electrode array in vitro. Journal of Physiology 588(Pt 10): 1719–35. 16. Bertinaria M, Rolando B, Giorgis M, Montanaro G, Guglielmo S, et al. (2010) Synthesis, physicochemical characterization, and biological activities of new carnosine derivatives stable in human serum as potential neuroprotective agents. Journal of Medicinal Chemistry 54(2): 611–21. 17. Baldelli P, Hernandez-Guijo JM, Carabelli V, Carbone E (2005) Brain-derived neurotrophic factor enhances GABA release probability and nonuniform distribution of N- and P/Q-type channels on release sites of hippocampal inhibitory synapses. Journal of Neuroscience 25(13): 3358–68. 18. MacLaren EJ, Charlesworth P, Coba MP, Grant SG (2011) Knockdown of mental disorder susceptibility genes disrupts neuronal network physiology in vitro. Molecular and Cellular Neuroscience 47(2): 93–9. 19. Marcantoni A, Baldelli P, Hernandez-Guijo JM, Comunanza V, Carabelli, et al. (2007) L-type calcium channels in adrenal chromaffin cells: Role in pace-making and secretion. Cell Calcium 42: 397–408. 20. Harris KD, Hirase H, Leinekugel X, Henze DA, Buzsa´ki G (2011) Temporal interaction between single spikes and complex spike bursts in hippocampal pyramidal cells. Neuron. 32(1): 141–9. 21. Vandael DH, Marcantoni A, Mahapatra S, Caro A, Ruth P, et al. (2010) Cav1.3 and BK channels for timing and regulating cell firing. Molecular Neurobiology 42(3): 185–98. 22. Henze DA, Borhegyi Z, Csicsvari J, Mamiya A, Harris KD, et al. (2000) Intracellular features predicted by extracellular recordings in the hippocampus in vivo. Journal of Neurophysiology 84: 390–400. 23. Stett U, Egert E, Guenther F, Hofmann T, Meyer W, et al. (2003) Biological application of microelectrode arrays in drug discovery and basic research. Analitycal and Bioanalitycal Chemistry 377: 486–495. 24. Li X, Zhou W, Zeng S, Liu M, Luo Q (2007) Long-term recording on multi- electrode array reveals degraded inhibitory connection in neuronal network development. Biosensors and Bioelectronics 22(7): 1538–43. PLoS ONE | www.plosone.org 16 July 2012 | Volume 7 | Issue 7 | e41530