Leptin Counteracts the Hypoxia-Induced Inhibition of Spontaneously Firing Hippocampal Neurons: A Microelectrode Array Study Daniela Gavello, Jonathan Rojo-Ruiz¤a, Andrea Marcantoni¤b, Claudio Franchino, Emilio Carbone, Valentina Carabelli* Department of Drug Science and Technology, NIS Center, CNISM, University of Torino, Torino, Italy Abstract Besides regulating energy balance and reducing body-weight, the adipokine leptin has been recently shown to be neuroprotective and antiapoptotic by promoting neuronal survival after excitotoxic and oxidative insults. Here, we investigated the firing properties of mouse hippocampal neurons and the effects of leptin pretreatment on hypoxic damage (2 hours, 3% O2). Experiments were carried out by means of the microelectrode array (MEA) technology, monitoring hippocampal neurons activity from 11 to 18 days in vitro (DIV). Under normoxic conditions, hippocampal neurons were spontaneously firing, either with prevailing isolated and randomly distributed spikes (11 DIV), or with patterns characterized by synchronized bursts (18 DIV). Exposure to hypoxia severely impaired the spontaneous activity of hippocampal neurons, reducing their firing frequency by 54% and 69%, at 11 and 18 DIV respectively, and synchronized their firing activity. Pretreatment with 50 nM leptin reduced the firing frequency of normoxic neurons and contrasted the hypoxia-induced depressive action, either by limiting the firing frequency reduction (at both ages) or by increasing it to 126% (in younger neurons). In order to find out whether leptin exerts its effect by activating large conductance Ca2+-activated K+ channels (BK), as shown on rat hippocampal neurons, we applied the BK channel blocker paxilline (1 mM). Our data show that paxilline reversed the effects of leptin, both on normoxic and hypoxic neurons, suggesting that the adipokine counteracts hypoxia through BK channels activation in mouse hippocampal neurons. Citation: Gavello D, Rojo-Ruiz J, Marcantoni A, Franchino C, Carbone E, et al. (2012) Leptin Counteracts the Hypoxia-Induced Inhibition of Spontaneously Firing Hippocampal Neurons: A Microelectrode Array Study. PLoS ONE 7(7): e41530. doi:10.1371/journal.pone.0041530 Editor: Jon T. Brown, University of Bristol, United Kingdom Received February 20, 2012; Accepted June 26, 2012; Published July 25, 2012 Copyright: 2012 Gavello et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the San Paolo Foundation, Grant # 2008.1155 to V.C. (Neuroscience Programme), and by the Marie Curie Research Training Network ‘‘CavNET’’ Contract MRTN-CT-2006-035367. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: valentina.carabelli@unito.it ¤a Current address: Instituto de Biolog´ıa y Gene´tica Molecular (IBGM), Unidad de Fisiolog´ıa Celular y Molecular, Universidad de Valladolid, Valladolid, Spain ¤b Current address: Institut de Pharmacologie Mole´culaire et Cellulaire, CNRS/UNSA UMR6097, Sophia Antipolis, Valbonne, France Introduction Leptin is an adipokine produced by fat tissue and encoded by the Ob (obese) gene. It is characterized by a receptor-mediated action exerted through a class I cytokine receptor [1] associated with JAKs (Janus Tyrosine Kinases) and it activates a signalling cascade mediated by PI3K (phosphoinositide 3-kinase) and the pathway of Ras-Raf-MAPK [2,3]. Besides its body-weight re- ducing effects [4], increasing evidence has recently shown that leptin may reduce neuronal activity [5] and may promote neuronal survival from different injuries, such as excitotoxic and oxidative insults [6]. Leptin has also clear therapeutical effects, as demonstrated by its ability to reverse the loss of dopaminergic neurons in a model of Parkinson’s disease [7]. Moreover, leptin exerts a neuroprotective action against ischemic injury in rat cortical and hippocampal neurons, reducing neuronal cell death [8,3]. There is also evidence for a role of leptin in opposing cell death mechanisms, through a ERK1/2, MAPK and STAT3 signalling pathway [9,3]. In the past ten years, several works pointed out the correlation between hypoxia and the increased risk of AD (Alzheimer’s Disease): a period of reduced oxygen supply, occurring during chronic cerebral hypoperfusion (CCH) or cerebral ischemia, which may lead to the up-regulation of b-amyloid precursor protein cleavage enzyme 1 (BACE-1) and downstream neurotoxic plaques formation [10,11]. So far, most studies on leptin’s neuroprotective effects were performed on the leptin-mediated neuronal survival by means of cell viability assays, while only few reports focused on the effects on neuronal activity [12]. A functional characterization of neuronal firing properties under normoxic and hypoxic conditions would be of great interest in this regard, since malfunctioning in signal transduction has been shown to be related to the first symptoms of neurodegenerative disorders, while cell death occurs at later stages of the disease [13]. In this work we investigated this critical issue by taking advantage of the MEA devices, which allow repeated non-invasive multisite recordings at different days in vitro [14,15]. We compared the firing properties of mouse hippocampal neurons at 11 and 18 DIV, and monitored the effects of leptin, both under normoxic and hypoxic conditions. Our data demonstrate that spontaneous firing at 11 and 18 DIV is severely compromised by PLoS ONE | www.plosone.org 1 July 2012 | Volume 7 | Issue 7 | e41530