CANNABINOID AND NITRIC OXIDE SIGNALING INTERPLAY IN THE MODULATION OF HIPPOCAMPAL HYPEREXCITABILITY: STUDY ON ELECTROPHYSIOLOGICAL AND BEHAVIORAL MODELS OF TEMPORAL LOBE EPILEPSY IN THE RAT F. CARLETTI, * G. GAMBINO, V. RIZZO, G. FERRARO AND P. SARDO Department of ‘‘Biomedicina Sperimentale e Neuroscienze Cliniche’’ (Bio.Ne.C.), ‘‘Sezione di Fisiologia umana G. Pagano’’, University of Palermo, Corso Tukory, 129, 90134 Palermo, Italy Abstract—A growing bulk of evidence suggests that cannabinoid system plays a pivotal role in the control of hyperexcitability phenomena. Notwithstanding, the anticonvulsant action of cannabinoids has not been fully addressed, in particular the involvement of potential cellular neuromodulators, for instance nitric oxide. In the current study, we focused on two distinct rat models of temporal lobe epilepsy, the Maximal Dentate Activation and the pilocarpine-induced acute seizures, providing both electro- physiological and behavioral data on cannabinoid and nitrergic system interplay. We evaluated the antiepileptic effects of WIN 55,212-2, (R)-(+)-[2,3-dihydro-5-methyl-3-(4- morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]-1 -naphthalenylmethanone (WIN), a CB agonist, and of 7-Nitroindazole (7NI), a preferential neuronal nitric oxide synthase (nNOS) inhibitor, at different doses, alone and in combination. MDA study showed that these drugs protected animals in a dose-dependent manner from electrically induced epileptiform discharges. In pilocarpine model, a dose-related activity of 7NI and WIN: a) decreased the behavioral scoring, used to describe the severity of chemi- cally induced acute seizures; b) affected latency of the onset of acute convulsions; c) dampened mortality rate. Interestingly, the combination of the treatments brought to light that individually ineffective doses of WIN turn into effective when nNOS activity is pharmacologically inhibited in both experimental conditions. This effect is mediated by CB 1 receptor since the co-administration of N-(piperidin- 1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H- pyrazole-3-carboxamide (AM251), a CB 1 receptor specific antagonist, thwarted the 7NI–WIN convergent action. In the light of this, our findings suggest a putative antagonism between CBr-activated pathway and NO signaling in the context of neuronal hyperexcitability and contribute to eluci- date possible synaptic processes underlying neuroprotec- tive properties of cannabinoids, with a view to better integrate antiepileptic therapy. Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hippocampus, temporal lobe epilepsy, cannabinoids, behavior, percentage of protection, electro- physiology. INTRODUCTION Epilepsy is a neurological disorder characterized by the appearance of spontaneously recurrent seizures (Hauser and Hesdorffer, 1990). In particular, temporal lobe epilepsy (TLE) is the most common type of partial complex seizure in adulthood (Hauser et al., 1996; Wieser, 2004), known for an unsatisfactory response to current therapy. Nowadays, many studies have been con- ducted on neuronal processes underlying epileptic states, particularly about the modulation of synaptic transmis- sion. Over the last decades, growing interest has been rising on the role of cannabinoids (CB) as endogenous antiepileptic agents in the brain (Wallace et al., 2001; Hofmann and Frazier, 2013), despite the therapeutic application of their exogenous analogs may expose to acute and chronic side effects (Gerra et al., 2010; Hill et al., 2012). Cannabinoid receptors (CBr) contribute to the synaptic function modulating neurotransmitter signal- ing via a feedback mechanism since they couple to pre- dominantly presynaptic Gi/o proteins (Howlett et al., 2002). In the hippocampus, the on-demand production of endocannabinoids from over-activated postsynaptic cells inhibits neurotransmitter release, hence protecting against excitotoxicity (Marsicano et al., 2003). Our previ- ous experiments using the Maximal Dentate Activation (MDA) model of TLE revealed that (R)-(+) WIN 55,212- 2 (hereafter, WIN), a CB non-selective agonist, exerts antiepileptic effects (Rizzo et al., 2009), in agreement with other authors (Wallace et al., 2001, 2003; Monory et al., 2006). Moreover, we highlighted that the endogenous cannabinoid receptor type 1 (CB 1 ) receptors, rather than http://dx.doi.org/10.1016/j.neuroscience.2015.06.047 0306-4522/Ó 2015 IBRO. Published by Elsevier Ltd. All rights reserved. * Corresponding author. Tel: +39-091-655-58-13; fax: +39-091-655- 58-16. E-mail addresses: fabio.carletti@unipa.it (F. Carletti), giuditta. gambino@unipa.it (G. Gambino), valerio.rizzo@unipa.it (V. Rizzo), giuseppe.ferraro@unipa.it (G. Ferraro), pierangelo.sardo@unipa.it (P. Sardo). Abbreviations: 7NI, 7-Nitroindazole; AB, angular bundle; AD, after discharge; AED, antiepileptic drugs; AM251, N-(piperidin-1-yl)-5-(4- iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxa mide; ANOVA, analysis of variance; CBr, cannabinoid receptors; DG, dentate gyrus; mAChr, muscarinic acetylcholine receptor; MDA, Maximal Dentate Activation; NMDAr, N-methyl-D-aspartate receptor; nNOS, neuronal nitric oxide synthase; sGC, soluble Guanylyl Cyclase; TLE, temporal lobe epilepsy; WIN 55,212-2, (R)-(+)-[2,3-dihydro-5- methyl-3-(4-morpholinylmethyl) pyrrolo[1,2,3-de]-1,4-benzoxazin-6-Yl]- 1-naphthalenylmethanone. Neuroscience 303 (2015) 149–159 149