Brian G. Dias

Parental olfactory experience influences behavior and neural structure in subsequent generations

Using olfactory molecular specificity, we examined the inheritance of parental traumatic exposure, a phenomenon that has been frequently observed, but not understood. We subjected F0 mice to odor fear conditioning before conception and found that subsequently conceived F1 and F2 generations had an increased behavioral sensitivity to the F0-conditioned odor, but not to other odors. When an odor (acetophenone) that activates a known odorant receptor (Olfr151) was used to condition F0 mice, the behavioral sensitivity of the F1 and F2 generations to acetophenone was complemented by an enhanced neuroanatomical representation of the Olfr151 pathway. Bisulfite sequencing of sperm DNA from conditioned F0 males and F1 naive offspring revealed CpG hypomethylation in the Olfr151 gene. In addition, in vitro fertilization, F2 inheritance and cross-fostering revealed that these transgenerational effects are inherited via parental gametes. Our findings provide a framework for addressing how environmental information may be inherited transgenerationally at behavioral, neuroanatomical and epigenetic levels.

Stressor-Specific Regulation of Distinct Brain-Derived Neurotrophic Factor Transcripts and Cyclic AMP Response Element-Binding Protein Expression in the Postnatal and Adult Rat Hippocampus

Stress regulation of brain-derived neurotrophic factor (BDNF) is implicated in the hippocampal damage observed in depression. BDNF has a complex gene structure with four 5′ untranslated exons (I–IV) with unique promoters, and a common 3′ coding exon (V). To better understand the stress regulation of BDNF, we addressed whether distinct stressors differentially regulate exon-specific BDNF transcripts in the postnatal and adult hippocampus. The early life stress of maternal separation (MS) resulted in a time point-dependent differential upregulation of BDNF transcripts restricted to early postnatal life (P14-BDNF II, P21-BDNF IV, V). In adulthood, distinct stressors regulated BDNF transcripts in a signature manner. Immobilization stress, administered once, decreased all BDNF splice variants but had differing effects on BDNF I/II (increase) and III/IV (decrease) when administered chronically. Although immobilization stress reduced BDNF (V) mRNA, chronic unpredictable stress did not influence total BDNF despite altering specific BDNF transcripts. Furthermore, a prior history of MS altered the signature pattern in which adult-onset stress regulated specific BDNF transcripts. We also examined the expression of cyclic AMP response element-binding protein (CREB), an upstream transcriptional activator of BDNF, and observed a CREB induction in the postnatal hippocampus following MS. As a possible consequence of enhanced CREB and BDNF expression following MS, we examined hippocampal progenitor proliferation and observed a significant increase restricted to early life. These results suggest that alterations in CREB/BDNF may contribute to the generation of individual differences in stress neurocircuitry, providing a substrate for altered vulnerability to depressive disorders.

Amygdala-Dependent Fear Memory Consolidation via miR-34a and Notch Signaling

UNLABELLED Using an array-based approach after auditory fear conditioning and microRNA (miRNA) sponge-mediated inhibition, we identified a role for miR-34a within the basolateral amygdala (BLA) in fear memory consolidation. Luciferase assays and bioinformatics suggested the Notch pathway as a target of miR-34a. mRNA and protein levels of Notch receptors and ligands are downregulated in a time- and learning-specific manner after fear conditioning in the amygdala. Systemic and stereotaxic manipulations of the Notch pathway indicated that Notch signaling in the BLA suppresses fear memory consolidation. Impairment of fear memory consolidation after inhibition of miR-34a within the BLA is rescued by inhibiting Notch signaling. Together, these data suggest that within the BLA, a transient decrease in Notch signaling, via miR-34a regulation, is important for the consolidation of fear memory. This work expands the idea that developmental molecules have roles in adult behavior and that existing interventions targeting them hold promise for treating neuropsychiatric disorders. VIDEO ABSTRACT

Epigenetic mechanisms underlying learning and the inheritance of learned behaviors

Gene expression and regulation is an important sculptor of the behavior of organisms. Epigenetic mechanisms regulate gene expression not by altering the genetic alphabet but rather by the addition of chemical modifications to proteins associated with the alphabet or of methyl marks to the alphabet itself. Being dynamic, epigenetic mechanisms of gene regulation serve as an important bridge between environmental stimuli and genotype. In this review, we outline epigenetic mechanisms by which gene expression is regulated in animals and humans. Using fear learning as a framework, we then delineate how such mechanisms underlie learning and stress responsiveness. Finally, we discuss how epigenetic mechanisms might inform us about the transgenerational inheritance of behavioral traits that are being increasingly reported.

A role for Tac2, NkB, and Nk3 receptor in normal and dysregulated fear memory consolidation.

The centromedial amygdala (CeM), a subdivision of the central amygdala (CeA), is believed to be the main output station of the amygdala for fear expression. We provide evidence that the Tac2 gene, expressed by neurons specifically within the CeM, is required for modulating fear memories. Tac2 is colocalized with GAD65 and CaMKIIα but not with PKCd and Enk neurons in the CeM. Moreover, the Tac2 product, NkB, and its specific receptor, Nk3R, are also involved in the consolidation of fear memories. Increased Tac2 expression, through a stress-induced PTSD-like model, or following lentiviral CeA overexpression, are sufficient to enhance fear consolidation. This effect is blocked by the Nk3R antagonist osanetant. Concordantly, silencing of Tac2-expressing neurons in CeA with DREADDs impairs fear consolidation. Together, these studies further our understanding of the role of the Tac2 gene and CeM in fear processing and may provide approaches to intervention for fear-related disorders.

Towards new approaches to disorders of fear and anxiety

Fear and anxiety are debilitating conditions that affect a significant number of individuals in their lifetimes. Understanding underlying mechanisms of these disorders affords us the possibility of therapeutic intervention. Such clarity in terms of mechanism and intervention can only come from an amalgamation of research from human to animal studies that attempt to mimic the human condition, both of which are discussed in this review. We begin by presenting an outline of our current understanding of the neurobiological basis of fear and anxiety. This outline spans various levels of organization that include the circuitry, molecular pathways, genetic and epigenetic components of fear and anxiety. Using these organizational levels as a scaffold, we then discuss strategies that are currently used to ameliorate these disorders, and forecast future interventions that hold therapeutic promise. Among these newer promising treatments, we include, optogenetic, pharmacological, and extinction-based approaches, as well as lifestyle modifications, with combinatorial treatment regimens of these holding the most promise.

Models of Intergenerational and Transgenerational Transmission of Risk for Psychopathology in Mice.

Trajectories toward risk or resilience in psychiatric disorders are influenced by acquired and inherited factors. More recently, evidence from rodent studies suggest that acquired risk factors can be transmitted through non-genomic, epigenetic mechanisms to subsequent generations, potentially contributing to a cycle of disease and disease risk. Here, we review examples of transmission of environmental factors across generations and illustrate the difference between behavioral transmission and epigenetic inheritance. We highlight essential definitions of intergenerational and transgenerational transmission of disease risk with corresponding examples. We then explore how these phenomena may influence our understanding of psychiatric disorders leading toward new prevention and therapeutic approaches.

PACAP and the PAC1 receptor in post-traumatic stress disorder.

Post-traumatic stress disorder (PTSD) affects approximately 5–10% of all individuals and is more predominant in women (Breslau, 2001). Advances in treatment and prevention will require identifying biomarkers to aid early diagnosis and understanding the mechanisms underlying maladaptive responses to trauma. In a highly traumatized, urban civilian population, we have recently reported that women (but not men) that had been diagnosed with PTSD had higher blood PACAP (pituitary adenylate cyclase-activating polypeptide) levels (Ressler et al, 2011) (Figure 1a). The high PACAP levels were correlated with physiological measures of the acoustic startle reflex, which have previously also been associated with PTSD risk. In addition, to further understand the responsiveness of the PACAP system to emotionally relevant cues and ovarian hormones, we showed that mRNA levels of the receptor for PACAP–PAC1R, are increased in the extended amygdala of adult rodents following classical fear conditioning and as a function of estrogen exposure. These findings suggested that PACAP might be involved in the symptoms characteristic of women diagnosed with PTSD. Figure 1 Relationship between the pituitary adenylate cyclase-activating polypeptide (PACAP)–PAC1 receptor (PAC1R) system and post-traumatic stress disorder (PTSD). (a) Females, but not males with high plasma PACAP38 levels show more PTSD symptoms. (b) ... To further genetically probe this association, we analyzed the PACAP (Adcyap1) gene and its receptor-PAC1 (Adcyap1r1). Only the Adcyap1r1 SNP (rs2267735) was found to be associated with PTSD diagnosis in women (again not in males) (Figure 1b). Although note that this SNP association was not replicated in a less traumatized cohort (Chang et al., 2012). At the epigenetic level, we also found that differential methylation of the Adcyap1r1 gene was associated with PTSD symptoms (Figure 1c). Given the approximately 2 : 1 sex-bias in PTSD prevalence, we find it exciting that the Adcyap1r1 SNP is within a predicted estrogen response element (ERE). Furthermore, in a postmortem sample, we found that females had differential cortical expression of Adcyap1r1 mRNA levels as a function of their genotype. Notably, a recent follow-up study finds that the same genetic risk is associated with higher acoustic startle in traumatized boys and girls before puberty, suggesting that the estrogen effect may be age-dependent (Jovanovic et al, 2012). Looking to the future, the association between PTSD and the PACAP–PAC1 receptor system in traumatized populations warrants further investigation on several important levels. For example, longitudinal prospective studies are needed to ascertain whether peripheral PACAP levels would serve as a robust predictive biomarker of eventual PTSD diagnosis. The PACAP–PAC1 receptor system has been studied for its role in stress responsiveness (Vaudry et al, 2009; Stroth et al, 2011), and as such presents a convergence between the stress and learning components of PTSD that should be investigated further. The genetic observation of a predicted ERE supports other findings that hormonal mechanisms may underlie the sex-bias in PTSD prevalence (Ferree et al, 2011; Lebron-Milad and Milad, 2012), but replications of this finding are necessary. Finally, learning is accompanied by epigenetic modifications at key genetic loci (Zovkic and Sweatt, 2012), and probing the epigenetic signatures at the Adcyap1and Adcyap1r1 genes as a result of previous traumatic experience or hormonal condition presents fertile ground for empirical analysis (Figure 1). In summary, the relationship between PTSD and the PACAP–PAC1 receptor system affords us the opportunity to address PTSD from the perspectives of stress physiology, endocrinology, epigenetics, and predictive biomarkers using human samples and animal models—a truly multi-pronged attack that may be required for understanding complex neuropsychiatric disorders.

Experimental evidence needed to demonstrate inter- and trans-generational effects of ancestral experiences in mammals

Environmental factors routinely influence an organisms biology. The inheritance or transmission of such influences to descendant generations would be an efficient mode of information transfer across generations. The developmental stage at which a specific environment is encountered by the ancestral generation, and the number of generations over which information about that environment is registered, determines an inter‐ vs. trans‐generational effect of ancestral influence. This commentary will outline the distinction between these influences. While seductive in principle, inter‐ and trans‐generational inheritance in mammals is a hotly debated area of research inquiry. We present constructive criticism of such inheritance, and suggest potential experimental avenues for reconciliation. Finally, epigenetic mechanisms present an avenue for gene regulation that is dynamic. We briefly discuss how such malleability affords the potential for a reversal of any detrimental environmental influences that might have adversely impacted ancestral or descendant generations.

Estrogen-dependent association of HDAC4 with fear in female mice and women with PTSD

Women are at increased risk of developing post-traumatic stress disorder (PTSD) following a traumatic event. Recent studies suggest that this may be mediated, in part, by circulating estrogen levels. This study evaluated the hypothesis that individual variation in response to estrogen levels contributes to fear regulation and PTSD risk in women. We evaluated DNA methylation from blood of female participants in the Grady Trauma Project and found that serum estradiol levels associates with DNA methylation across the genome. For genes expressed in blood, we examined the association between each CpG site and PTSD diagnosis using linear models that adjusted for cell proportions and age. After multiple test correction, PTSD associated with methylation of CpG sites in the HDAC4 gene, which encodes histone deacetylase 4, and is involved in long-term memory formation and behavior. DNA methylation of HDAC4 CpG sites were tagged by a nearby single-nucleotide polymorphism (rs7570903), which also associated with HDAC4 expression, fear-potentiated startle and resting-state functional connectivity of the amygdala in traumatized humans. Using auditory Pavlovian fear conditioning in a rodent model, we examined the regulation of Hdac4 in the amygdala of ovariectomized (OVX) female mice. Hdac4 messenger RNA levels were higher in the amygdala 2 h after tone-shock presentations, compared with OVX-homecage control females. In naturally cycling females, tone-shock presentations increased Hdac4 expression relative to homecage controls for metestrous (low estrogen) but not the proestrous (high estrogen) group. Together, these results support an estrogenic influence of HDAC4 regulation and expression that may contribute to PTSD in women.