Type-II cadherins modulate neural activity in cultured rat hippocampal neurons Eiji Matsunaga a,b , Tohru Kurotani c , Kenta Suzuki a,d and Kazuo Okanoya a,c,e Cadherins, cell adhesion molecules widely expressed in the nervous system, are thought to be involved in synapse formation and function. To explore the role of cadherins in neuronal activity, we performed electrophysiological and morphological analyses of rat hippocampal cultured neurons overexpressing type-II cadherins, such as cadherin-6B and cadherin-7. We found that cadherin-6B increased but cadherin-7 decreased the number of protrusions of dendritic spines, and affected the frequency of miniature excitatory postsynaptic currents. Our results suggest that type-II cadherins may modulate neural activity by regulating neuronal morphology. NeuroReport 22:629–632  c 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins. NeuroReport 2011, 22:629–632 Keywords: cadherin, electrophysiology, hippocampal culture neuron, miniature excitatory postsynaptic current a Laboratory for Biolinguistics, b Laboratory for Symbolic Cognitive Development, RIKEN Brain Science Institute, Wako, c JST, ERATO, Okanoya Emotional Information Project, Wako, d Division of Life Science, Graduate School of Science and Engineering, Saitama University, Saitama and e Department of Life Sciences, Graduate School of Arts and Sciences, University of Tokyo, Tokyo, Japan Correspondence to Tohru Kurotani, JST, ERATO, Okanoya Emotional Information Project, Hirosawa 2-1, Wako, 351-0198 Japan and Kazuo Okanoya, RIKEN Brain Science Institute, Hirosawa 2-1, Wako, 351-0198 Japan Tel: +81 48 462 1111 ¾ 6823; fax: + 81 48 467 7503; e-mail: kurotani@brain.riken.jp; kazuookanoya@gmail.com Received 13 May 2011 accepted 23 May 2011 Introduction Cadherins are cell adhesion molecules that belong to the cadherin superfamily and are widely expressed in a variety of tissues [1]. In the nervous system, cadherins play various roles not only in synapse formation, but also in axon guidance [2,3], dendritic spine formation [4,5], or interaction with neurotransmitter receptors [6]. Among the superfamily, type-II cadherins show neural circuit- related expressions (each type-II cadherins is expressed in some restricted population of neurons that are synaptically connected with each other) [7]. In studies using mutant mice, it was proposed that type-II cadherins are involved in synaptic activity. For example, cadherin-11 (Cad11) mutant mice show enhanced long-term potentia- tion in CA1 neurons and defects in fear-condition learning [8], whereas Cad8 mutant mice have a reduced miniature excitatory postsynaptic current (mEPSC) in temperature-sensitive neurons [9]. Thus, type-II cadher- ins might regulate neuronal activity and get involved in higher brain function, although the detailed mechanism still remains unknown at molecular and behavioral levels. Previously, we performed in-situ hybridization analysis of gene expressions in the brain of a songbird, Bengalese finch, and found that type-II cadherin expressions are changed during the development [10]. Songbirds acquire their vocalizations through imitation from their fathers during the juvenile stage [11]. They first listen to their tutors’ songs (sensory learning stage) and then start to practice singing by themselves (sensorimotor learning stage) to copy the songs. In-situ hybridization analysis revealed that downregulation of cad7 expression and upregulation of cad6B occurred between postnatal days 30 and 60. In the case of B. finch, sensorimotor learning stage is started around postnatal days 40–50, therefore, the timing of changes in those cadherin expressions corresponds to the transitional period from sensory to sensorimotor learning stage [10]. Thus, we suspected that Cad6B and Cad7 have different activities in morphological and electrophysiolo- gical properties, and that change of these cadherin expressions control neuronal plasticity. Herein, to examine such a possibility, we performed electrophysiological and morphological analyses of Cad6B-overexpressing/Cad7- overexpressing cultured rat hippocampal neurons, as the culture system is well-established and is easy to analyze electrophysiological properties and neuronal morphology. Materials and methods Plasmid preparation Full-length chicken cad6B or cad7 cDNA [12] was inserted into the EcoRI–NheI site of pCL20-MSCV-GFP expres- sion vector to obtain pCL20-MSCV-Cad6GFP or pCL20- MSCV-Cad7-GFP , respectively [13]. Preparation of hippocampal culture neurons Primary rat hippocampal neurons were cultured as described previously [14]. We dissected the hippocampal region of E19 rat embryos and cultured dissociated cells (5.0  10 4 cells/well) on cover slips (Fischer, Pittsburgh, Pennsylvania, USA) coated with poly-L lysine (Sigma- Aldrich, Deisenhofen, Germany) in 24-well plates. At 7 days in vitro, 0.5 mg of pCL20-MSCV-GFP , pCL20-MSCV- Cad6GFP , or pCL20-MSCV-Cad7-GFP was transfected Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Website (www.neuroreport.com). Neurophysiology, basic and clinical 629 0959-4965  c 2011 Wolters Kluwer Health | Lippincott Williams & Wilkins DOI: 10.1097/WNR.0b013e3283491665 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.