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Conditional Calcineurin Knockout Mice Exhibit Multiple Abnormal Behaviors Related to Schizophrenia

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Conditional Calcineurin Knockout Mice Exhibit Multiple Abnormal Behaviors Related to Schizophrenia Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to schizophrenia Tsuyoshi Miyakawa*†, Lorene M. Leiter*, David J. Gerber*, Raul R. Gainetdinov‡, Tatyana D. Sotnikova‡, Hongkui Zeng*§, Marc G. Caron‡, and Susumu Tonegawa*¶ *Howard Hughes Medical Institute, The Picower Center for Learning and Memory and RIKEN͞Massachusetts Institute of Technology Neuroscience Research Center, Departments of Biology and Brain and Cognitive Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139; and ‡Howard Hughes Medical Institute, Departments of Cell Biology and Medicine, Duke University Medical Center, Durham, NC 27710 Contributed by Susumu Tonegawa, May 15, 2003 Calcineurin (CN), a calcium- and calmodulin-dependent protein phos- (lights on at 7:00 a.m.) with access to food and water ad libitum. phatase, plays a significant role in the central nervous system. Pre- Behavioral testing was performed between 9:00 a.m. and 5:00 p.m. viously, we reported that forebrain-specific CN knockout mice (CN except for the home cage social interaction test. All procedures mutant mice) have impaired working memory. To further analyze the relating to animal care and treatment conformed to Massachusetts behavioral effects of CN deficiency, we subjected CN mutant mice to Institute of Technology and National Institutes of Health guide- a comprehensive behavioral test battery. Mutant mice showed in- lines. Sequences of tests, housing conditions, and the number of creased locomotor activity, decreased social interaction, and impair- animals used are shown in Table 1, which is published as supporting ments in prepulse inhibition and latent inhibition. In addition, CN information on the PNAS web site, www.pnas.org. The methods for mutant mice displayed an increased response to the locomotor rotarod, hot plate, light͞dark transition, elevated plus maze, object stimulating effects of MK-801. Collectively, the abnormalities of CN exploration, social interaction (in a novel environment), Porsolt mutant mice are strikingly similar to those described for schizophre- forced swim tests, in vivo microdialysis, and HPLC assessment of nia. We propose that alterations affecting CN signaling could com- brain content of monoamines and metabolites are described in prise a contributing factor in schizophrenia pathogenesis. Supporting Text, which is published as supporting information on the PNAS web site. alcineurin (CN), also called protein phosphatase 2B, is a heterodimeric Ca2ϩ͞calmodulin-dependent serine͞threonine Open Field Test. Each subject was placed in the center of an open C ϫ ϫ protein phosphatase composed of CNB regulatory and CNA field apparatus (40 40 30 cm; Accuscan Instruments, Colum- catalytic subunits (1). Originally identified in the brain, CN was later bus, OH). Total distance traveled (in cm), vertical activity, time found to play a critical role in T cell function, through activation of spent in the center, and the beam–break counts for stereotyped nuclear factor of activated T cell-mediated transcription of cytokine behaviors were recorded. Data were collected over a 60-min period. genes, including the IL-2 gene (2, 3). This action of CN comprises a target for the immunosuppressants, cyclosporin A and FK506, Social Interaction in Home Cage. To monitor social behavior between which associate with immunophilins and bind to and inactivate CN. two mice in a familiar environment, a system that automatically More recently, CN has been shown to play an important role in CNS analyzes social behavior in home cages of mice was developed (Fig. 7, which is published as supporting information on the PNAS web functions, including neurite extension, synaptic plasticity and learn- ϫ ϫ ing and memory (4, 5). site). The system contains a home cage (29 18 12 cm) and a We previously reported a severe and specific working memory filtered cage top, separated by a 13-cm-high metal stand containing deficit of forebrain specific CNB-deficient mice (CN mutant mice) an infrared video camera, which is attached at the top of the stand. as assessed by delayed matching to place Morris water maze and Two genetically identical mice that had been housed separately were placed together in a home cage. Their social behavior was then eight-arm radial maze paradigms (5). To further investigate the NEUROSCIENCE monitored for 3 days. Outputs from the video cameras were fed into behavioral significance of CN, CN mutant mice were subjected to a Macintosh computer. Images from each cage were captured at a a comprehensive behavioral test battery (6, 7). CN mutant mice rate of one frame per second. Social interaction was measured by display a spectrum of abnormalities that is strikingly similar to those counting the number of particles in each frame: two particles observed in schizophrenia patients. In addition, a number of indicated the mice were not in contact with each other; and one supporting lines of evidence are consistent with the possibility that particle demonstrated contact between the two mice. We also alterations in CN function occur in schizophrenia. Furthermore, in measured locomotor activity during these experiments by quanti- our accompanying paper, we investigated the potential involvement fying the number of pixels changed between each pair of successive of altered CN signaling in genetic susceptibility to schizophrenia, frames. For details of the image analysis, see Supporting Text. and we report evidence supporting association of the PPP3CC gene encoding the CNA␥ catalytic subunit with disease (8). Based on Latent Inhibition Test. This experiment was performed in a similar these findings, we propose that alterations affecting CN signaling manner to that reported by others (9) with the equipment used by could comprise an important contributing factor in schizophrenia Zeng et al. (5). On the first day, each mouse was placed in a pathogenesis. conditioning chamber (Coulbourn Instruments, Allentown, PA). Materials and Methods The mice were divided into two groups: preexposed (P) group and non-preexposed (NP) group. The P group received 40 white noise Animals and Experimental Design. The generation of the CN mutants tones (68 dB, 5-sec duration, 25-sec interstimulus interval), whereas is detailed elsewhere (5). The background strain used to generate the mutation was C57BL͞6J. All mutant and control mice were in ͞ a pure C57BL 6J background. All CN mutant mice consisted of Abbreviations: CN, calcineurin; NMDA, N-methyl-D-aspartate; P, preexposed; NP, non- homozygous floxed, heterozygous Cre recombinase transgenic preexposed. mice. All control mice consisted of homozygous or heterozygous †Present address: Horizontal Medical Research Organization, Faculty of Medicine, Kyoto floxed, Cre transgene negative littermates. All behavioral tests were University, Kyoto 606-8501, Japan. carried out with male mice that were 10 weeks old at the start of the §Present address: Primal Incorporated, 1124 Columbia Street, Seattle, WA 98104. testing. Mice were housed in a room with a 12-hr light͞dark cycle ¶To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.1432926100 PNAS ͉ July 22, 2003 ͉ vol. 100 ͉ no. 15 ͉ 8987–8992 the NP group received no stimulus during an equivalent period. Immediately after the tone preexposure or the exposure to the chamber, tone–shock pairs consisting of a 5-sec tone coterminating with a 2-sec foot shock at 0.40 mA were delivered to both groups with a 25-sec interstimulus interval. Afterward, mice remained in the chamber for 25 sec before being returned to the home cage. On day 2, the mice were placed back in the conditioning chamber for 5 min for the measurement of freezing to the context. On day 3, the mice were put in a white Plexiglas chamber scented with vanilla essence, and after 180 sec, a 180-sec tone was delivered to measure cued freezing. Prepulse Inhibition Task. A startle reflex measurement system was used (MED Associates, St. Albans, VT) and the experiment was performed in a manner identical to that of Miyakawa et al. (7). See Supporting Text for details. Sensitivity to MK-801 and Amphetamine. To test the locomotor activating effects of MK-801 or amphetamine (Sigma), mice were habituated to an open field for 1 h, and then drugs were adminis- tered i.p. Amphetamine or MK-801 were dissolved in saline and administered at 0.1 ml per 10 g of body weight. Because there was a difference in baseline locomotor activity between genotypes, the ratio of activity during the hour after injection to the activity during the hour before injection was used as an index of the locomotor Fig. 1. Increased locomotor activity of CN mutant mice in the open field test. activating effects of the drugs. Distance traveled (cm) (A), vertical activity (B), and stereotypic behavior counts (C) were significantly greater in CN mutants than in controls. Time spent in center (D) Quantification of Nesting. One piece of nesting material (Nestlets, was shorter during the first 30 min in CN mutants relative to controls. Ancare, Bellmore, NY), made of cotton fiber, was introduced into a cage in which a mouse was individually housed. Pictures of the in a number of behavioral tasks including the hidden platform nests were taken by a digital camera (Olympus, Melville, NY) and version of the Morris water maze and cued and contextual fear exported into a computer. The number of scattered particles of the conditioning (5). Thus, it is unlikely that specific behavioral abnor- nestlets was counted for each cage by the NIH IMAGE program. malities in these mice are a reflection of general health effects. In Vivo Microdialysis. In vivo microdialysis measurements of extra- Increased Locomotor Activity. CN mutants consistently showed a cellular dopamine and metabolites were performed in freely mov- pronounced increase in locomotor activity in several different tests. ing mice in an identical manner to that described in Mohn et al. (10). Total distance traveled by CN mutants was significantly greater See Supporting Text for details. than that of controls during an open field test (Fig.
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