Knowing when and where to express fear is essential to survival. Recent work in fear extinction paradigms reveals that the contextual regulation of fear involves a neural network involving the hippocampus, medial prefrontal cortex, and amygdala. The amygdaloid basal nuclei (BA) receive convergent input from the ventral hippocampus (VH) and prelimbic (PL) prefrontal cortex and may integrate VH and PL input to regulate fear expression. To examine the functional organization of this neural circuit, we used cellular imaging of c-fos expression in anatomically defined neuronal populations and circuit disconnections to identify the pathways involved in the contextual control of extinguished fear. Before behavioral testing, we infused a retrograde tracer into the amygdala to label BA-projecting neurons in VH and PL. Rats then underwent fear conditioning and extinction and were tested for their fear to the extinguished conditioned stimulus (CS) in either the extinction context or in another context; freezing behavior served as the index of conditional fear. CS presentation outside the extinction context renewed conditional freezing and was associated with significantly more c-fos expression in BA-projecting neurons in the VH and PL than that induced by CS presentation in the extinction context. We next examined whether direct or indirect projections of VH to BA mediate fear renewal. Interestingly, disconnections of the VH from either the BA or PL eliminated renewal. These findings suggest that convergent inputs from both the VH and PL in the BA mediate the contextual control of fear after extinction.

Recent studies have suggested that physiological and behavioral traits may be transgenerationally inherited through the paternal lineage, possibly via non-genomic signals derived from the sperm. To investigate how paternal stress might influence offspring behavioral phenotypes, a model of hypothalamic–pituitary–adrenal (HPA) axis dysregulation was used. Male breeders were administered water supplemented with corticosterone (CORT) for 4 weeks before mating with untreated female mice. Female, but not male, F1 offspring of CORT-treated fathers displayed altered fear extinction at 2 weeks of age. Only male F1 offspring exhibited altered patterns of ultrasonic vocalization at postnatal day 3 and, as adults, showed decreased time in open on the elevated-plus maze and time in light on the light–dark apparatus, suggesting a hyperanxiety-like behavioral phenotype due to paternal CORT treatment. Interestingly, expression of the paternally imprinted gene Igf2 was increased in the hippocampus of F1 male offspring but downregulated in female offspring. Male and female F2 offspring displayed increased time spent in the open arm of the elevated-plus maze, suggesting lower levels of anxiety compared with control animals. Only male F2 offspring showed increased immobility time on the forced-swim test and increased latency to feed on the novelty-supressed feeding test, suggesting a depression-like phenotype in these animals. Collectively, these data provide evidence that paternal CORT treatment alters anxiety and depression-related behaviors across multiple generations. Analysis of the small RNA profile in sperm from CORT-treated males revealed marked effects on the expression of small noncoding RNAs. Sperm from CORT-treated males contained elevated levels of three microRNAs, miR-98, miR-144 and miR-190b, which are predicted to interact with multiple growth factors, including Igf2 and Bdnf. Sustained elevation of glucocorticoids is therefore involved in the transmission of paternal stress-induced traits across generations in a process involving small noncoding RNA signals transmitted by the male germline.

There is growing evidence that the preconceptual lifestyle and other environmental exposures of a father can significantly alter the physiological and behavioral phenotypes of their children. We and others have shown that paternal preconception stress, regardless of whether the stress was experienced during early-life or adulthood, results in offspring with altered anxiety and depression-related behaviors, attributed to hypothalamic–pituitary–adrenal axis dysregulation. The transgenerational response to paternal preconceptual stress is believed to be mediated by sperm-borne small noncoding RNAs, specifically microRNAs. As physical activity confers physical and mental health benefits for the individual, we used a model of voluntary wheel-running and investigated the transgenerational response to paternal exercise. We found that male offspring of runners had suppressed reinstatement of juvenile fear memory, and reduced anxiety in the light–dark apparatus during adulthood. No changes in these affective behaviors were observed in female offspring. We were surprised to find that running had a limited impact on sperm-borne microRNAs. The levels of three unique microRNAs (miR-19b, miR-455 and miR-133a) were found to be altered in the sperm of runners. In addition, we discovered that the levels of two species of tRNA-derived RNAs (tDRs)—tRNA-Gly and tRNA-Pro—were also altered by running. Taken together, we believe this is the first evidence that paternal exercise is associated with an anxiolytic behavioral phenotype of male offspring and altered levels of small noncoding RNAs in sperm. These small noncoding RNAs are known to have an impact on post-transcriptional gene regulation and can thus change the developmental trajectory of offspring brains and associated affective behaviors.

Extinction in adult animals, including humans, appears to involve the medial prefrontal cortex (mPFC). However, the role of mPFC in extinction across development has not yet been studied. Given several recent demonstrations of developmental differences in extinction of conditioned fear at a behavioral level, different neural circuitries may mediate fear extinction across development. In all experiments, noise conditioned stimulus (CS) and shock unconditioned stimulus (US) were used. In experiment 1A, temporary unilateral inactivation of the mPFC during extinction training impaired long-term extinction the following day in postnatal day 24 (P24) rats but not in P17 rats. In experiment 1B, bilateral inactivation of the mPFC again failed to disrupt long-term extinction in P17 rats. In experiment 2, extinction training increased phosphorylated mitogen-activated protein kinase (pMAPK) in the mPFC for P24 rats but not for P17 rats, whereas rats of both ages displayed elevated pMAPK in the amygdala. Across both ages, “not trained,” “reactivated,” and “no extinction” control groups expressed very low numbers of pMAPK-immunoreactive (IR) neurons across both neural structures. This result indicates that the mere conditioning experience, the exposure to the CS, or the expression of CS-elicited fear in and of itself is not sufficient to explain the observed increase in pMAPK-IR neurons in the mPFC and/or the amygdala after extinction. Together, these findings show that extinction in P17 rats does not involve the mPFC, which has important theoretical and clinical implications for the treatment of anxiety disorders in humans.