Identifying Molecular Substrates in a Mouse Model of the Serotonin Transporter  Environment Risk Factor for Anxiety and Depression Valeria Carola, Giovanni Frazzetto, Tiziana Pascucci, Enrica Audero, Stefano Puglisi-Allegra, Simona Cabib, Klaus-Peter Lesch, and Cornelius Gross Background: A polymorphism in the serotonin transporter (5-HTT) gene modulates the association between adverse early experiences and risk for major depression in adulthood. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT  environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse. Methods: We established a mouse model in which a heterozygous null mutation in 5-HTT moderates the effects of poor maternal care on adult anxiety and depression-related behavior. Biochemical analysis of brains from these animals was performed to identify molecular substrates of the gene, environment, and gene  environment effects. Results: Mice experiencing low maternal care showed deficient -aminobutyric acid–A receptor binding in the amygdala and 5-HTT heterozygous null mice showed decreased serotonin turnover in hippocampus and striatum. Strikingly, levels of brain-derived neurotrophic factor (BDNF) messenger RNA in hippocampus were elevated exclusively in 5-HTT heterozygous null mice experiencing poor maternal care, suggesting that developmental programming of hippocampal circuits might underlie the 5-HTT  environment risk factor. Conclusions: These findings demonstrate that serotonin plays a similar role in modifying the long-term behavioral effects of rearing environment in diverse mammalian species and identifies BDNF as a molecular substrate of this risk factor. Key Words: 5-HTT, anxiety, depression, gene  environment, maternal behavior R isk for depression is determined by both genetic and environmental factors (1), particularly adverse childhood experiences (2). The low-functioning allele of a common functional polymorphism (5-HTTLPR short-variant) in the pro- moter of the serotonin transporter gene (5-HTT, SLC6A4) has been associated with increased neuroticism (3) and increased incidence of major depression selectively in persons experienc- ing childhood maltreatment or other life stress events (4). Functional magnetic resonance imaging (fMRI) studies have shown that the 5-HTTLPR short-variant confers increased base- line neural activity in a wide range of forebrain structures (5), increased neural responses to the presentation of aversive versus neutral stimuli in structures associated with affective processing such as amygdala and hippocampus (6,7), as well as increased functional coupling between amygdala and ventromedial pre- frontal cortex during similar tasks (8). Moreover, a recent fMRI study has shown that baseline neural activity in both amygdala and hippocampus correlates positively with the number of self-reported life stress events in short-variant carriers but nega- tively in long-variant carriers (5). These findings suggest that 5-HTT influences the long-term impact of stressful life experi- ences on brain activity that, in turn, influences the response to aversive stimuli and contributes to altered behavioral patterns that are risk factors for depression. Although human imaging studies have begun to elucidate the neural circuits involved in the 5-HTT  environment risk factor, a molecular understanding of this phenomenon is lacking. Such an understanding might help to identify novel targets for the diagnosis and therapy of mood disorders. To address this need, we developed a gene-environment screening paradigm in the mouse (9). In rodents, low maternal care is associated with high anxiety-related behavior and exaggerated stress response in adulthood (10,11). This maternal programming phenomenon in rodents has been proposed to serve as a model for the increased psychopathology and stress hormone responses associated with poor maternal care and adverse childhood environment in humans (12). Thus, we set out to ask whether the 5-HTT  environment risk factor reported for depression in humans could be modeled with maternal programming of anxiety and depres- sion-related behavior in mouse. To study maternal programming in a genetically controlled manner in mice, we established offspring that were exposed to different levels of maternal care but were nevertheless derived from genetically identical parents. We performed reciprocal inter-crosses between two inbred strains, C57BL/6J (B6) and BALB/cByJ (C) that are known to exhibit large differences in maternal care. Females derived from these crosses (B6xC and CxB6) are genetically identical but provide different levels of maternal care, owing to the inter- generational transmission of maternal behavior from mother to daughter (13). Offspring of B6xC mothers are exposed to high maternal care, whereas offspring of CxB6 mothers are exposed to low maternal care (9,10). Low maternal care in our paradigm is associated with increased anxiety-related behavior, consistent From the Mouse Biology Unit (VC, GF, EA, CG), European Molecular Biology Laboratory (EMBL), Monterotondo; Department of Psychology and Cen- ter Daniel Bovet (TP, SP-A, SC), University of Rome La Sapienza; Santa Lucia Foundation (SP-A, SC), European Centre for Brain Research (CERC), Rome, Italy; and Molecular and Clinical Psychobiology (K-PL), Depart- ment of Psychiatry and Psychotherapy, University of Würzburg, Germany. Address reprint requests to Cornelius Gross, Ph.D., European Molecular Biology Laboratory (EMBL) Mouse Biology Unit, Via Ramarini 32, 00015 Monterotondo (Rome), Italy; E-mail: gross@embl.it. Received April 7, 2007; revised July 19, 2007; accepted August 24, 2007. BIOL PSYCHIATRY 2007;xx:xxx 0006-3223/07/$32.00 doi:10.1016/j.biopsych.2007.08.013 © 2007 Society of Biological Psychiatry ARTICLE IN PRESS