Attempts to bridge behavioral and molecular genetics through brain imaging has provided a convenient link between abnormal patterns of brain activity seen in certain patient populations and anatomical abnormalities seen in knockout/transgenic mouse models. Genetically modified mice provide useful model systems for testing the role of candidate genes in behavior and imaging studies provide neuroanatomical evidence to validate cross-species translation. The extent to which such genetic manipulations in the mouse and the resulting phenotype can be translated across species, from mouse to human, is beginning to be assessed more directly. A new direction for genetic research is to exploit behavioral paradigms being used with mice, and adapt them for use with humans in the imaging environment. Such an approach provides a direct move toward translating the function of genes in the context of human behavior. In highlighting the promise of imaging genetics, we provide a concrete example of how parallel human and mouse genetic studies can address many of the challenges of genetic studies.
We describe research that focuses on the impact of a polymorphism in the brain-derived neurotrophic factor (BDNF) gene. BDNF is a member of the neurotrophins a unique family of polypeptide growth factors that influence differentiation and survival of neurons in the developing nervous system. In adults, BDNF is important in regulating synaptic plasticity and connectivity in the brain. Recently, a common single nucleotide polymorphism in the human BDNF gene, resulting in a valine (Met) to methionine (Met) substitution in the prodomain of the peptide (Val66Met), has been shown to lead to memory impairment and risk for psychiatric illness. An understanding of how this naturally occurring polymorphism affects behavior and neuroanatomy is an important first step in linking genetic alterations in the neurotrophin system to definable biological outcomes in humans.
BDNF Val66Met is common in human populations with a prevalence of 20–30% [Shimizu et al., 2004] and because it codes for an amino acid substitution has a high likelihood of affecting the biological properties of the BDNF peptide. Indeed, it has been demonstrated that the Met allele leads to reduced activity-dependent secretion of BDNF from hippocampal neurons in culture [Egan et al., 2003; see Fig. 3A]. This effect is due to a reduced affinity of the variant (Met) BDNF for sortilin an intracellular trafficking molecule [Chen et al., 2005].
Figure 3. Model of impact of BDNF across development. (A) The genetic variant BDNF Val66Met leads to an amino acid substitution in the BDNF prodomain (Val to Met at position 66) that results in decreased activity-dependent secretion of BDNF from neurons. Thus, this trafficking defect leads to a decrease in the availability of biologically active BDNF. (B) This model predicts that BDNF levels will have different functional consequences across development. As the variant BDNF (Val66Met) has decreased secretion throughout this period, we anticipate that there will be functional deficits, evident even in childhood, but (C) these deficits will become diminished by adolescence when BDNF levels peak. In addition, BDNF levels will be modulated by environmental stressors. Carriers of the Met allele will have decreased secretion and less neurotrophic support for plasticity and change, whereas Val allele carries will show greater change, including both positive and negative effects on hippocampal structure and function, but potentially greater neurotrophic support for plasticity and resilience once a stressor is removed [Casey et al., 2009].
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The BDNFMet allele has been associated with impairment in select forms of learning and memory [Egan et al., 2003] and susceptibility to psychiatric disorders [Neves-Pereira et al., 2002; Ribases et al., 2003, 2004; Sen et al., 2003; Sklar et al., 2002]. Given the established role of BDNF in promoting learning and memory [Desai et al., 1999; Korte et al., 1995; Patterson et al., 1996], it is likely that impaired BDNF secretion, due to expression of the BDNFMet allele, may have pleiotropic effects (e.g., a single gene impacting multiple phenotypic traits) in a variety of BDNF-dependent processes.
Recently, a unique inbred genetic knock-in mouse strain was developed [Chen et al., 2006] that expresses the variant BDNF allele to recapitulate the specific phenotypic properties of the human polymorphism in vivo. All inbred mouse strains contain a Valine 66 residue in BDNF. The BDNFMet mouse is a transgenic knock-in of a methionine residue at this position that mimics the human polymorphism. This model is unique in that it is the only animal model that fully recapitulates the established phenotypic effects of a common human polymorphism expressed in the brain. Unlike traditional transgenic mouse models which alter the quantitative expression of targeted genes throughout development or at selected times, this model introduces the single polymorphic amino acid into the murine genome, thereby providing a precise physiologic model of the polymorphic effect of human BDNF Val66Met. Such testable mechanistic approaches cannot be applied to other frequent polymorphisms related to behavior. For example, the 5-HTTLPR is postulated to be a regulatory polymorphism but its activity has not been consistently identified. Furthermore, the 5-HTTLPR genetic alteration cannot be fully recapitulated in transgenic mice because the regulatory element that is polymorphic in humans does not exist in nonprimate species [Lesch et al., 1997]. The mouse model of BDNF Val66Met has been validated by studies that have found that animals carrying the Met allele manifested phenotypes (hippocampal size and hippocampal-dependent learning) that matched differences in humans expressing the BDNFMet allele, as compared to individuals with the Val/Val genotype [Chen et al., 2006].
Constrained model of genotypic effects
We have developed a model with testable hypotheses for how gene- or environment-related alterations in BDNF levels will have a significant impact on behavioral and neuroanatomic changes that vary with age. Such an approach can move the field away from simplistic notions of risk alleles, recognizing that an allele may be a risk factor during one period of development and a protective factor during another. Because the variant BDNFMet allele shows decreased regulated secretion, we predict that there will be functional deficits or biases in BDNF dependent forms of learning when physiologic levels of BDNF are low (Fig. 3B,C). However, when BDNF levels peak [e.g., during adolescence, Katoh-Semba et al., 1997] this trafficking deficit may yield only minor differences in these measures in stable enviornments. During periods of increased physiologic expression of BDNF, the lower secretion conferred by the BDNFMet allele may actually be protective and lead to risk for individuals in adolescence without this allele [e.g., BDNFVal/Val in substance abuse; see meta-analysis by Gratacos et al., 2007].
This model also encompasses nongenetic factors. Early environmental risk factors including physiological or psychological stress result in decreased neurotrophic support to certain BDNF-rich regions like the hippocampus [Smith et al., 1995]. The additional deficit in neurotrophic support in carriers of the Met allele may result in increased vulnerability to stress, and thus put them at greater risk for psychiatric disorders (e.g., anxiety, depression, and schizophrenia) that have been associated with stress. During other developmental windows when BDNF levels are high, carriers of the Val allele may be at greater risk for other psychiatric disorders given that stress can increase BDNF in the amygdala and ventral striatum, areas implicated in bipolar disorder [e.g., Geller et al., 2004] and substance abuse [Liu, 2005; Matsushita et al., 2004]. Thus, it is important to consider changes in the level of BDNF across development and the opposing effects that stress has on BDNF levels in brain regions that support very different forms of learning.
Promise for development
This model distinguishes itself by underscoring the importance of development in examination of genetic effects on behavior. First, BDNF is a molecule that is essential for developmental processes including, neuronal plasticity [Barde et al., 1987; Bramham and Messaoudi, 2005; Leibrock et al., 1989; Liao et al., 2007; Lu, 2003; Rattiner et al., 2005; Thoenen, 1995; Tongiorgi et al., 2006; Yamamoto and Hanamura, 2005]; regulation of both short-term synaptic function and long-term activity-dependent synaptic consolidation [Barco et al., 2005; Black, 1999; Katz and Shatz, 1996; Lohof et al., 1993; Lu and Chow, 1999; McAllister et al., 1999; Patterson et al., 1996; Poo, 2001; Thoenen, 1995]; potentiation of synaptic transmission [Kang and Schuman, 1995; Levine et al., 1995; Lohof et al., 1993]; modulation of long-term potentiation (LTP) in vitro and in vivo [Korte et al., 1995; Messaoudi et al., 2002; Patterson et al., 1996]; and induction of morphological changes in dendritic spines [Gomes et al., 2006; McAllister et al., 1995]. Thus, BDNF has a role in (1) synaptic plasticity; (2) inducing changes in synaptic morphology; and (3) mediating cell survival and cell proliferation during development. These functions serve to underscore the importance of considering BDNF in any neurodevelopmental disorder of learning.
Second, BDNF availability changes across development (Fig. 1B,C). Although these changes have been shown to differ by region [Hofer et al., 1990; Katoh-Semba et al., 1997; Maisonpierre et al., 1990; Webster et al., 2006], rodent studies suggest that changes in BDNF levels across development approximate an inverted U-shape function [Ivanova and Beyer, 2001; Silhol et al., 2005]. In humans, BDNF mRNA levels in cortical regions increase approximately one-third from infancy to adulthood. They are relatively low during infancy and childhood, peak during young adulthood, and are maintained at a constant level throughout adulthood. The increase in BDNF at this critical time in human development may have important implications for the etiology and treatment of the severe mental disorders that tend to present during this time [Webster et al., 2002]. The BDNF Val66Met mouse model is able to recapitulate this regional and temporal complexity as the single nucleotide polymorphism occurs in the protein coding sequence and leaves the regulatory elements of the gene unaffected, thus maintaining the normal regional and temporal expression of this gene.
Precision of (endo)phenotype
Genetically influenced forms of learning that lie at the core of neurodevelopmental disorders include those that capture the difficulties some individuals have in: (1) recognizing signals of safety or danger (cued learning); and (2) learning to adjust behavior when actual associations no longer exist (extinction). Unlike disease states, the tasks that examine these types of learning can be assessed equivalently in typically and atypically developing humans and mice. Although most studies have emphasized the role of BDNF in learning and memory processes supported by the hippocampus, high levels of BDNF mRNA and protein are expressed in the amygdala [Conner et al., 1997; Yan et al., 1997] suggesting another important potential site for BNDF-mediated plasticity. In studies focusing on the hippocampus, BDNF has been shown to facilitate long term potentiation (LTP) at hippocampal CA1 synapses [Figurov et al., 1996; Korte et al., 1995; Patterson et al., 1996] and BDNF mRNA levels have been found to increase following induction of LTP [Barco et al., 2005; Bramham et al., 1996; Castren et al., 1993; Pang and Lu, 2004; Patterson et al., 1992, 1996; Radecki et al., 2005; Zakharenko et al., 2003]. The activity-dependent secretion of BDNF enhances the molecular mechanisms of synaptic restructuring needed to support LTP. We have shown [Chen et al., 2005] that the Val66Met mutation in the BDNF gene leads to a decrease in this regulated secretion of BDNF, suggesting that carriers of this allele would have compromised BDNF-dependent synaptic modulation. In humans, Val/Met individuals have repeatedly been shown to have a smaller hippocampal volume relative to individuals who are homozygous for the Val allele (Val/Val) [Bueller et al., 2006; Pezawas et al., 2004; Szeszko et al., 2005].
In this context, we provide data that focus on simple measures that reflect adaptation to environmental change/stress (e.g., fear conditioning) and that appear to lie at the very core of a number of clinical disorders [Charney and Manji, 2004; Duman et al., 1997; Nestler et al., 2002; Pine, 2007]. Importantly, these measures can be tested across species and throughout development and have known underlying biological substrates. Using such measures across development and under varying degrees of stress, will ultimately allow us to examine vulnerability and protection of each BDNF allele (Val and Met), in an attempt to understand gene X environment interactions across development. Mouse and human data are presented to illustrate this multilevel approach for understanding gene function. The objective of this study was to test if the Val66Met genotype could impact extinction learning in our mouse model, and if such findings could be generalized to human populations. For preliminary gene X development and gene X environment findings see Casey et al., [ 2009].
Cross species validation of genetic results
We examined the impact of the variant BDNF on classic fear conditioning and extinction paradigms [Soliman et al., 2010]. Approximately 70 mice and 70 humans were tested. The mice include 17 BDNFVal/Val, 33 BDNFVal/Met, and 18 BDNFMet/Met. The human sample included 36 Met allele carriers (31 BDNFVal/Met and 5 BDNFMet/Met) and 36 nonMet allele carriers group-matched on age, gender, and ethnic background. To avoid spurious allelic associations, we balanced demographic factors, including age, gender, and ethnicity across genotype categories [Soliman et al., 2010; Supporting Information Table S1]. We also performed ethnicity-specific analyses and found that the effect of the Met allele on extinction and conditioning, as measured by change in SCR with time, was not driven by any single ethnic group (extinction: F(3,64) = 0.32, P < 0.81) or (conditioning: F(3,64) = 0.69, P < 0.56]. Fear conditioning consisted of pairing a neutral cue with an unconditioned aversive stimulus until the cue itself took on properties of the unconditioned stimulus (US) of an impending aversive event. The extinction procedure consisted of repeated presentations of the cue (i.e., conditioned stimulus or CS) alone.
There were no effects of BDNF genotype on fear conditioning in mice or humans as measured by freezing behavior to the conditioned stimulus in the mice (F(2,65) = 1.58, P < 0.22) and by skin conductance response in humans to the cue predicting the aversive stimulus relative to a neutral cue (F(1,70) = 0.67, P < 0.42). However, both the mice and humans showed slower extinction in Met allele carriers than in nonMet allele carriers as shown in Figure 4A,B below. Moreover, human functional magnetic resonance imaging data provide neuroanatomical validation of the cross-species translation. Specifically, we find alterations in frontoamygdala circuitry, known to support fear conditioning and extinction in previous rodent [LeDoux, 2000; Milad and Quirk, 2002; Myers and Davis, 2002; Quirk et al., 2003] and human [Delgado et al., 2008; Gottfried and Dolan 2004; Kalisch et al., 2006; LaBar et al., 1998; Phelps et al., 2004; Schiller et al., 2008] studies, as a function of BDNF genotype. Met allele carriers show less ventromedial prefrontal cortical (vmPFC) activity during extinction relative to nonMet allele carriers (Fig. 4C), but greater amygdala activity relative to nonMet allele carriers (Fig. 4D). These findings suggest that cortical regions essential for extinction in animals and humans [Milad and Quirk, 2002; Milad et al., 2007; Phelps et al., 2004] are less responsive in Met allele carriers during extinction. Morover, amygdala recruitment, which should show diminished activity during the extinction [Phelps et al., 2004] remains elevated in Met allele carriers.
Figure 4. Altered behavior and neural circuitry underlying extinction in mice and humans with BDNF Val66Met. Impaired extinction in Met allele carriers (Val/Met and Met/Met) as a function of time in 68 mice (A) and 72 humans (B) as indexed by percent time freezing in mice and skin conductance response (SCR) in humans to the conditioned stimulus when it was no longer paired with the aversive stimulus. (C) Brain activity as indexed by percent change in MR signal during extinction in the ventromedial prefrontal cortex (vmPFC) by genotype (xyz = −4, 24, 3), with Met allele carriers having significantly less activity than Val/Val homozygotes [VM < VV = blue], image threshold P < 0.05, corrected. (D) Genotypic differences in left amygdala activity during extinction (xyz = −25, 2, −20) in 70 humans, with Met allele carriers having significantly greater activity than Val/Val homozygotes [VM > VV = orange], image threshold P < 0.05, corrected. *P < 0.05. **MM were included in the analysis with VM, but plotted separately to see dose response. All results are presented as a mean ± SEM. VV = Val/Val; VM = Val/Met; MM = Met/Met [Soliman et al., 2010].
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The mouse model provides the ability to directly test for dose-dependent effects of the Met allele (Fig. 4A) that is problematic in human studies given the rarity of humans homozygous for the Met allele. Moreover, the mouse model provides the ability to test the effects of the Met allele in both a controlled genetic and environmental background not feasible in humans. Thus, any behavioral differences observed in the mouse can be reliably assigned to the effects of the Val66Met polymorphism. Together with the human behavioral findings, these data provide confidence of the effects of BDNF Val66Met across species. With the added human imaging data, the effects are shown to be biologically valid as they directly map onto known circuits involved in fear conditioning and extinction.
The findings are exciting as they provide an example of bridging human behavioral and imaging genetics with a molecular mouse model. Each of these approaches alone, provide limited information on gene function in complex human behavior, but together, they are forming bridges between animal models and human psychiatric disorders. Specifically, the findings of impaired extinction in mice and humans with the BDNF Met allele, suggests a role in anxiety disorders showing impaired learning of cues that signal safety versus threat, and in the efficacy of treatments that rely on extinction mechanisms such as exposure therapy.
Mouse strain effects
As noted, previous reports have reported strain differences in the efficacy of standard fear conditioning protocols and the rate of extinction Balogh and Wehner, 2003; Bolivar et al., 2001; Brinks et al., 2008; Waddell et al., 2004]. These differences may be due to strain differences in pain sensitivity, learning, or other variables. The results above are based on data collected from C57BL/6J mice. To directly compare the contribution of the Val66Met polymorphism to fear extinction across genetic backgrounds, we used identical fear conditioning and extinction paradigms in a sample of Val66Met Swiss Webster mice. Fear extinction occurred more rapidly in Swiss Webster mice compared to C57BL/6J mice. Despite these differences, the Swiss Webster homozygous BDNF Met allele mice, like the C57BL/6J Val66Met mice, showed slower extinction to the condition stimulus to than wildtype (Val/Val) mice [see Soliman et al., 2010; Supporting Information].
Future genetic approaches
Although the majority of imaging genetic imaging studies have focused on candidate genes, comparisons of gene expression between individuals or experimental groups has been greatly facilitated in the past decade by the introduction of genome-wide microarrays or “gene-chips.” A genome-wide association study is defined as any study of genetic variation across the entire human genome that is designed to identify genetic associations with observable traits or the presence or absence of a disease or condition. Whole genome information, when combined with clinical and other phenotype data, offers the potential for increased understanding of basic biological processes and those affecting human health, improvement in the prediction of disease and treatment, and ultimately the realization of the promise of personalized medicine.
A potential use for combining imaging genetics with genome-wide microarray methods is to use a biological signature in much the same way that behavioral signatures such as social responsiveness have been used in genetic research on autism [Duvall et al., 2007]. A neural signature specific to a disorder such as functional coupling between ventromedial prefrontal cortex and amygdala may serve to identify risk genes involved in distinct disorders but with a common pathway in frontolimbic circuitry. Numerous cross-site imaging initiatives are underway to begin to provide sufficiently large samples of scans needed to pursue this approach.