Convergent effects of mouse Pet-1 deletion and human PET-1 variation on amygdala fear and threat processing
Introduction
The serotonin (5-hydroxytryptamine) neurotransmitter system plays a key role in regulating emotion, and genetic variations in the serotonin system influence individual differences in emotion and risk for emotional disorders (Holmes, 2008). Genetically-driven variation in regulators of serotonin signaling (e.g., tryptophan hydroxylase-2, Tph2; the serotonin transporter, 5-HTT) is associated with higher levels of anxiety-like behavior (Zhang et al., 2004) and deficient fear extinction (Wellman et al., 2007), as well as increased dendritic arborization in ventromedial prefrontal cortex (vmPFC) and higher spine density in basolateral amygdala (BLA) neurons (Nietzer et al., 2011, Wellman et al., 2007). In humans, a polymorphism in the 5-HTT promoter region is associated with functional uncoupling of prefrontal cortex (PFC) and amygdala and amygdala hyperactivity in response to threat (Hariri et al., 2002, Pezawas et al., 2005). Individuals with this polymorphism exhibit impaired fear extinction (Hartley et al., 2012) and are at increased risk for depression after a history of stressful life events (Caspi et al., 2010).
Given serotonin's role in brain development (Gaspar et al., 2003), these effects may be driven by malformation of corticolimbic circuits mediating anxiety and fear (Ansorge et al., 2007, Esaki et al., 2005, Holmes et al., 2003). In this context, a major candidate for serotonergic influences on brain development is the ETS domain transcription factor Pet-1 (Pheochromocytoma 12 ets) aka FEV (Deneris, 2011). Pet-1 plays a critical role in differentiation and forebrain targeting of serotonin neurons, and expression of regulatory serotonin receptors on these neurons (Hendricks et al., 2003, Liu et al., 2010). Pet-1 knockout (KO) dramatically reduces the number of serotonin-immunoreactive neurons from embryonic development onwards, resulting in an ~ 80% reduction of serotonin in forebrain target regions (Deneris, 2011, Hendricks et al., 2003).
Increased anxiety-like behavior has been reported in mice with either constitutive Pet-1 KO (Hendricks et al., 2003) or Pet-1 KO restricted to adulthood (Kiyasova et al., 2011, Liu et al., 2010, Schaefer et al., 2009). Intriguingly, a preliminary report found that Pet-1 KO had enhanced conditioned fear behavior (Kiyasova et al., 2011). Serotonergic effects on fear extinction are of particular clinical relevance because deficits in fear extinction characterize anxiety disorders such as posttraumatic stress disorder (PTSD) (Milad et al., 2009). Indeed, disruption of serotonin genes produces morphological abnormalities in brain regions mediating fear extinction, notably the BLA (Herry et al., 2010) and vmPFC (Burgos-Robles et al., 2009, Graybeal et al., 2011, Wilber et al., 2011). However, the critical question of how lifelong loss of serotonin affects extinction of learned fear behavior remains unanswered.
Given the key role for the serotonergic systems in regulating emotional behavior, here we assessed the consequences of Pet-1 deletion for fear extinction as well as anxiety-like behaviors and stress responses. Further, emotional disorders are highly comorbid with alcohol abuse and the serotonin system modulates EtOH's effects on behavior. For example, disruption of serotonin signaling, via 5-HTT KO, leads to exaggerated sensitivity to acute intoxicating effects of EtOH (Boyce-Rustay et al., 2006, Daws et al., 2006). Therefore, we also examined responses on an EtOH test battery. In addition, in a separate cohort of behaviorally naïve mice, we examined potential neural mechanisms at the level of dendritic arborization in BLA and vmPFC. We hypothesized that mice with genetic inactivation of Pet-1 would show alterations in emotional behavior and corticolimbic dendritic morphology relative to wild-type mice. We then interrogated the potential translational impact of our preclinical analyses by conducting a human neuroimaging genetics study of the association between a common PET-1 (aka FEV) single nucleotide polymorphism (rs860573) and threat-related amygdala reactivity, a human intermediate neural phenotype that reliably varies as a function of polymorphisms in serotonergic genes (Hariri and Holmes, 2006). The use of non-human animal models allows for explicit manipulation of Pet-1, while the extension in humans interrogating genetic variation within the PET-1 gene (FEV) allows for preliminary translational evidence for the importance of PET-1 in the emergence of individual differences in clinically relevant brain function and the related risk for psychopathology (Hariri, 2010). Thus, we hypothesized that genetic variation within the PET-1 gene (FEV) would associate with differences in amygdala reactivity; and these differences would parallel differences in emotional and fear behaviors in the Pet-1 knockout mice.
Section snippets
Subjects
Pet-1 null mutant mice were generated as previously described (Hendricks et al., 2003) and repeatedly backcrossed into the C57BL/6J strain for 10 generations. Wild-type (WT), heterozygous (HET), and KO mice were littermates generated from HET × HET matings (Lerch-Haner et al., 2008, Millstein et al., 2006). Mice were bred and maintained at The Jackson Laboratory (Bar Harbor, ME) and shipped to NIH at 7–9 weeks of age, or bred and maintained at NIH. Testing began when mice were ≥ 10 weeks old. Mice
Participants
Genetic and neuroimaging data were available from 375 participants who completed the Duke Neurogenetics Study (Nikolova and Hariri, 2012), an ongoing protocol assessing a range of behavioral, experiential, and biological phenotypes among young adult volunteers. All participants provided written informed consent in accordance with Duke University guidelines and were in good general health. For completing the study, each participant received $120 remuneration. Study exclusion criteria included:
Pet-1 KO show increased acquisition, expression and post-extinction recovery of fear
There were significant effects of genotype (F2,67 = 3.31, p < .05) and trial (F3,201 = 135.51, p < .01) and a borderline genotype × trial interaction (F6,201 = 2.10, p = .0546) for freezing during conditioning. Post hoc tests showed that freezing was not different between genotypes at baseline or during the first tone exposure prior to pairing with the first shock, but was significantly higher in Pet-1 KO mice than WT controls during the second and third tone exposures (Fig. 3). There was a genotype × trial
Discussion
The major findings of the current study were that 1) in mice, Pet-1 deletion produced amygdala dendritic hypertrophy and an augmented, extinction-resistant fear memory, and, 2) in humans, a PET-1 gene (FEV) variant associated with threat-driven amygdala reactivity and risk for psychopathology. Importantly, particularly in light of the importance of replication even with intermediate biological phenotypes (but see also Goldman and Ducci, 2007, Hart et al., 2013), our human imaging genetics
Conclusions
Loss of brain serotonin via gene deletion of Pet-1 produced an increased fear memory that was liable to recover following extinction, as well as dendritic hypertrophy in a key brain region mediating fear and extinction. While previous studies have demonstrated how variation in genes regulating other major components of the serotonergic system alter corticolimbic morphology and associated fear extinction processing, they have focused on effects likely to produce hyperserotonergia — for instance,
Statement of interest
This work was supported by the US-Israel Binational Science Foundation (grant number 2007096 to AH, CLW, MM); the Intramural Research Program of the National Institute on Alcoholism and Alcohol Abuse (Z01-AA000411 to AH), and Duke University.
Acknowledgments
We are very grateful to Guoxiang Luo for genotyping and Drew Rosenbarger for technical assistance.
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