SUPPRESSION OF CAMP-DEPENDENT GENE EXPRESSION BY CHOLECYSTOKININ IN THE HIPPOCAMPUS N. H. NAKAMURA, a,b * K. AKIYAMA a AND T. NAITO a a Okinawa Institute of Science and Technology, Okinawa 904-2234, Japan b Mercator Research Group, Faculty of Medicine, Ruhr University Bo- chum, Bochum 44801, Germany Abstract—Our previous study suggests that “the neuropepti- dergic system” might promote a diversity of the mechanisms that regulate signal transmission in the hippocampus. Chole- cystokinin (CCK) is the mostly expressed neuropeptide gene in the hippocampus. Here, we investigated whether CCK regulates immediate-early genes (Egr1/zif268 and Fos), critical indicators of cortical neuronal activity. We showed that CCK increased Egr1/zif268 promoter activity in a neuronal cell line, which is transfected with CCK B receptor. Unexpectedly, in living hip- pocampal slices, CCK significantly suppressed cAMP-induced expression of Egr1/zif268 and Fos through CCK B receptor acti- vation. This suppression was involved in activating GABA B and cannabinoid 1 receptors. In addition to transient CCK modula- tion of action potentials on hippocampal principal neurons, we suggest that release of endogenous CCK might indirectly pro- duce the suppression of cAMP-dependent gene expression in the hippocampus. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: cholecystokinin octapeptide sulfated, immediate- early gene, zif268, c-Fos, organotypic hippocampal slice. Locally produced neuromodulators in the hippocampus (e.g. brain-derived neurotrophic factor, 17-estradiol, so- matostatin, and neuropeptide Y) have an important role in regulating neuronal activity and synaptic plasticity of the principal neurons (reviewed by Colmers and Bleakman, 1994; Baraban and Tallent, 2004; Scharfman and Ma- cLusky, 2006). As suggested in our previous findings, “the neuropeptidergic system” might promote a diversity of the mechanisms that regulate signal transmission in the hip- pocampus (Nakamura et al., in press). In particular, cho- lecystokinin (CCK) is the mostly expressed neuropeptide gene and represents 66% of almost all neuropeptides expressed in the CA1 and CA3 regions. It is known that CCK octapeptide sulfated, the active form of CCK, tran- siently modulates electrophysiological properties on hip- pocampal neurons in different fashions (Breukel et al., 1997; Shinohara and Kawasaki, 1997; Miller et al., 1997; Deng and Lei, 2006): (i) CCK facilitates depolarization on parvalbumin-expressing basket interneurons (Foldy et al., 2007); and (ii) CCK inhibits depolarization on CCK-ex- pressing basket interneurons via retrograde endocannabi- noid (eCB) actions from the principal neurons (Ali, 2007; Neu et al., 2007; Foldy et al., 2007; Karson et al., 2008). Thus, CCK may be a transient modulator having two op- posite aspects of excitement and inhibition in the hip- pocampus. Besides an action potential on the membrane of neu- rons, gene expression is another major output signal by integrating input signals on neurons (Clayton, 2000). Ac- tion potentials are immediate ion transmissions on a time scale of milliseconds, whereas activity-induced genes are subsequent modulators underlying neuronal activity and synaptic plasticity on a time scale of minutes or even hours and days. The inducible genes on the time scale of min- utes, called immediate-early genes (IEGs; e.g. Fos, Egr1/ zif268, and Arc), are identified as critical indicators of a neuronal activity and useful for a functional mapping in the cerebral cortex (Worley et al., 1993; Guzowski et al., 1999, 2001; Nakamura et al., 2010; reviewed by Kubik et al., 2007). Using IEG expression analysis, we have investi- gated the regulation by CCK in the hippocampus. We show that CCK-CCK B receptor (CCKBR) activation increases IEG expression via the signaling activity in a neuronal cell line, as previously mentioned (Wank, 1995; Noble et al., 1999). Then, we extraneously apply CCK into living hip- pocampal slices in different active conditions to under- stand CCK regulatory function in hippocampal networks. Unexpectedly, CCK suppresses cAMP-induced IEG ex- pression in living hippocampal slices. Our findings using gene expression analysis suggest potential regulatory ma- chinery in hippocampal networks, and offer an approach complementary to electrophysiological studies. EXPERIMENTAL PROCEDURES Constructs of CCKBR and Egr1/zif268 promoter CCKBR expression vector and Egr1 promoter-luciferase reporter gene construct were prepared for reporter assay. As for CCKBR expression vector, total RNA was isolated from organotypic hip- pocampal slices of postnatal day 7 (P7) C57BL/6J mouse pups (Japan SLC, Shizuoka, Japan). PCR primers were designed to amplify the full-length coding sequence, corresponding to bases 206 –1567, in the mouse CCKBR cDNA clones (GenBank acces- sion No: NM_007627). Reverse transcription-PCR was per- formed, and the amplified product of the CCKBR cDNA fragment *Corresponding author: N. H. Nakamura, Functional Architecture of Mem- ory Unit, Mercator Research Group, Faculty of Medicine, Ruhr University Bochum, Universitatsstrabe 150, 44801 Bochum, Germany. Tel: +49-32- 26739; fax: +49-234-32-14463. E-mail address: nakamun@gmail.com (N. H. Nakamura). Abbreviations: ANOVA, analysis of variance; CB1R, cannabinoid 1 receptor; CCK, cholecystokinin; CCKAR, cholecystokinin A receptor; CCKBR, cholecystokinin B receptor; DG, dentate gyrus; DIV, days in vitro; eCB, endocannabinoid; FSK, forskolin; GABA B R, GABA B re- ceptor; GiPCR, Gi protein-coupled receptor; IEG, immediate-early gene; PKC, protein kinase C; PMA, phorbol 12-myristate 13-acetate; P7, postnatal day 7. Neuroscience 187 (2011) 15–23 0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.04.031 15