GENETIC DELETION OF MT 1 /MT 2 MELATONIN RECEPTORS ENHANCES MURINE COGNITIVE AND MOTOR PERFORMANCE G. O’NEAL-MOFFITT, * J. PILLI, S. S. KUMAR AND J. OLCESE Florida State University Program in Neuroscience, College of Medicine, Department of Biomedical Sciences, 1115 West Call Street, Tallahassee, FL 32306, USA Abstract—Melatonin, an indoleamine hormone secreted into circulation at night primarily by the brain’s pineal gland, has been shown to have a wide variety of actions on the devel- opment and physiology of neurons in the CNS. Acting via two G-protein-coupled membrane receptors (MT 1 and MT 2 ), melatonin modulates neurogenesis, synaptic functions, neuronal cytoskeleton and gene expression. In the present studies, we sought to characterize the behavior and neuro- nal biology of transgenic mice lacking both of these melato- nin receptors as a way to understand the hormone’s receptor versus non-receptor-mediated actions in CNS- dependent activities, such as learning and memory, anxiety, general motor performance and circadian rhythmicity. Assessment of these behaviors was complemented by molecular analyses of gene expression in the brain. Our results demonstrate mild behavioral hyperactivity and a lengthened circadian period of free-running motor activity in melatonin receptor-deficient mice as compared to recep- tor-intact control mice beginning at an early age. Significant improvement in cognitive performance was found using the Barnes Maze and the Y-Maze. No behavioral changes in anxiety levels were found. Electrophysiological measures in hippocampal slices revealed a clear enhancement of long-term potentiation in mice lacking melatonin receptors with no significant differences in paired-pulse facilitation. Quantitative analysis of brain protein expression levels of phosphoCREB and phosphoERK1/2 and key markers of synaptic activity (synapsin, glutamate receptor 1, spinophi- lin, and glutamic acid decarboxylase 1) revealed significant differences between the double-knockout and wild-type ani- mals, consistent with the behavioral findings. Thus, genetic deletion of melatonin receptors produces mice with enhanced cognitive and motor performance, supporting the view that these receptors play an important role in neurobehavioral development. Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. Key words: melatonin, learning, memory, activity, LTP, mice. INTRODUCTION Knockout mice lacking both the MT 1 and the MT 2 (encoded by the MTNR1a and MTNR1b genes respectively) melatonin receptors have been used in a variety of studies to examine the mechanisms of melatonin action in specific disorders and conditions, including focal cerebral ischemia (Kilic et al., 2012), methamphetamine-induced locomotor sensitization (Hutchinson et al., 2012), sleep–wake characterization (Comai et al., 2013), nicotine sensitivity (Mexal et al., 2012), and blood glucose regulation (Mu¨hlbauer et al., 2009); however, no comprehensive characterization of baseline differences between these transgenic animals and their wild-type counterparts has been published. Sim- ilarly, aspects of neurodevelopment have not been addressed before, despite the fact that these animals’ brains develop not only without the receptor-mediated influence of melatonin, but they also lack the impact of the normally constituently active MT 1 receptor (Dubocovich et al., 2010). The genetic absence of specific hormone receptors can result in a variety of neurobiological disorders, e.g., oxytocin receptors and social behavior (cf. Donaldson and Young, 2008), vasoactive intestinal peptide receptors and circadian behavior (Harmar et al., 2002), androgen receptors and reproductive systems (Chang et al., 2013). Functionally significant polymorphisms in the cod- ing sequences of both the human MT 1 and the human MT 2 melatonin receptors have been identified and found to be associated with some diseases and disorders (Li et al., 2013; Comai and Gobbi, 2014). A few of the http://dx.doi.org/10.1016/j.neuroscience.2014.07.018 0306-4522/Ó 2014 Published by Elsevier Ltd. on behalf of IBRO. * Corresponding author. Address: 1115 West Call Street, Florida State University, College of Medicine, Tallahassee, FL 32306, USA. Tel: +1-850-645-1479. E-mail addresses: gmoffitt@neuro.fsu.edu (G. O’Neal-Moffitt), jyotsna. pilli@med.fsu.edu (J. Pilli), sanjay.kumar@med.fsu.edu (S. S. Kumar), james.olcese@med.fsu.edu (J. Olcese). Abbreviations: aCSF, artificial cerebrospinal fluid; ADHD, attention deficit/hyperactivity disorder; ANOVA, analysis of variance; C3B6, mice with a combination C3H/He and C57BL/6 background; cDNA, complementary deoxyribonucleic acid; CAT, catalase; CREB, cAMP response element-binding protein; DBL-KO, double-knockout; DD, dark–dark or constant dark; DNA, deoxyribonucleic acid; EDTA, ethylenediaminetetraacetic acid; EPM, elevated plus maze; EPSP, excitatory post-synaptic potential; ERK, extracellular signal-regulated kinases; GAD1, glutamic acid decarboxylase 1; GluR1, glutamate receptor subunit 1; GPx-1, glutathione peroxidase 1; HEPES, 2-[4-(2- hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; HPC, hippocampus; LD, light–dark; LTP, long-term potentiation; MAPKs, mitogen-activated protein kinases; mRNA, messenger ribonucleic acid; MTNR, melatonin receptor; MT 1 , melatonin receptor type 1; MT 2 , melatonin receptor type 2; MTNR1a, gene encoding the MT 1 receptor; MTNR1b, gene encoding the MT 2 receptor; NIR, near-infrared; Nrf2, nuclear factor erythroid 2-related factor 2; NT, non-transgenic; PGK, phosphoglycerate kinase promoter; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; SCN, suprachiasmatic nuclei; SOD1, superoxide dismutase 1; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis. Neuroscience 277 (2014) 506–521 506