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Early life maltreatment by the caregiver constitutes a major risk factor for the development of later-life psychopathologies, including fear-related pathologies. Here, we used an animal model of early life maltreatment induced by the Scarcity-Adversity Model of low bedding (LB) where the mother is given insufficient bedding for nest building while rat pups were postnatal days (PN) 8–12. To assess effects of maltreatment on the expression of threat-elicited defensive behaviors, animals underwent odor-shock threat conditioning at three developmental stages: late infancy (PN18), adolescence (PN45) or adulthood (>PN75) and tested the next day with odor only presentations (cue test). Results showed that in typically developing rats, the response to threat increases with maturation, although experience with maltreatment in early infancy produced enhanced responding to threat in infancy and adulthood, but a decrease in maltreated adolescents. To better understand the unique features of this decreased threat responding in adolescence, c-Fos expression was assessed within the amygdala and ventromedial prefrontal cortex (vmPFC) associated with the cued expression of threat learning. Fos counts across amygdala subregions were lower in LB rats compared to controls, while enhanced c-Fos expression was observed in the vmPFC prelimbic cortex (PL). Correlational analysis between freezing behavior and Fos revealed freezing levels were correlated with CeA in controls, although more global correlations were detected in LB-reared rats, including the BA, LA, and CeA. Functional connectivity analysis between brain regions showed that LB reared rats exhibited more diffuse interconnectivity across amygdala subnuclei, compared the more heterogeneous patterns observed in controls. In addition, functional connectivity between the IL and LA switched from positive to negative in abused adolescents. Overall, these results suggest that in adolescence, the unique developmental decrease in fear expression following trauma is associated with distinct changes in regional function and long-range connectivity, reminiscent of pathological brain function. These results suggest that early life maltreatment from the caregiver perturbs the developmental trajectory of threat-elicited behavior. Indeed, it is possible that this form of trauma, where the infant’s safety signal or “safe haven” (the caregiver) is actually the source of the threat, produces distinct outcomes across development.

Brain serotonin (5-hydroxytryptamine, 5-HT) system dysfunction is implicated in exaggerated fear responses triggering various anxiety-, stress-, and trauma-related disorders. However, the underlying mechanisms are not well understood. Here, we investigated the impact of constitutively inactivated 5-HT synthesis on context-dependent fear learning and extinction using tryptophan hydroxylase 2 (Tph2) knockout mice. Fear conditioning and context-dependent fear memory extinction paradigms were combined with c-Fos imaging and electrophysiological recordings in the dorsal hippocampus (dHip). Tph2 mutant mice, completely devoid of 5-HT synthesis in brain, displayed accelerated fear memory formation and increased locomotor responses to foot shock. Furthermore, recall of context-dependent fear memory was increased. The behavioral responses were associated with increased c-Fos expression in the dHip and resistance to foot shock-induced impairment of hippocampal long-term potentiation (LTP). In conclusion, increased context-dependent fear memory resulting from brain 5-HT deficiency involves dysfunction of the hippocampal circuitry controlling contextual representation of fear-related behavioral responses.

The genetic and molecular basis underlying fear memory formation is a key theme in anxiety disorder research. Because activating transcription factor 3 (ATF3) is induced under stress conditions and is highly expressed in the hippocampus, we hypothesize that ATF3 plays a role in fear memory formation. We used fear conditioning and various other paradigms to test Atf3 knockout mice and study the role of ATF3 in processing fear memory. The results demonstrated that the lack of ATF3 specifically enhanced the expression of fear memory, which was indicated by a higher incidence of the freeze response after fear conditioning, whereas the occurrence of spatial memory including Morris Water Maze and radial arm maze remained unchanged. The enhanced freezing behavior and normal spatial memory of the Atf3 knockout mice resembles the fear response and numbing symptoms often exhibited by patients affected with posttraumatic stress disorder. Additionally, we determined that after fear conditioning, dendritic spine density was increased, and expression of Gelsolin, the gene encoding a severing protein for actin polymerization, was down-regulated in the bilateral hippocampi of the Atf3 knockout mice. Taken together, our results suggest that ATF3 may suppress fear memory formation in mice directly or indirectly through mechanisms involving modulation of actin polymerization.

Post-traumatic stress disorder (PTSD) occurs following life-threatening events. The activity of the hypothalamic-pituitary-adrenal (HPA) axis, which serves as the first line of defense against stress, is dysfunctional in this disorder. The current study aimed to investigate the role of Crocin in normalizing HPA function in an animal model of PTSD induced by electric foot shock. Rats were treated with Crocin 5 min prior to stress induction. The stimulus was re-introduced after 21 days, and we measured individual behaviors such as sniffing, rearing, grooming, and freezing. Enzyme-linked immunosorbent assays were performed to measure plasma levels of Corticosterone. On day 28, after rats were weighed and sacrificed, the adrenal and thymus glands were removed and subjected to real-time polymerase chain reaction to quantify the gene expression of corticotrophin-releasing hormone (CRH), glucocorticoid receptor (GluR), and arginine vasopressin (AVP). Our results demonstrate that rats re-exposed to a stressor developed characteristic symptoms of PTSD, but these were attenuated by Crocin. Treated rats showed significant changes in CRH expression in the hypothalamus, GluR expression in the pituitary, plasma Corticosterone levels, and freezing behavior. Together, these findings suggest that Crocin can regulate HPA axis activity in PTSD. It may serve an appropriate treatment for subjects who experience a traumatic event.

Freezing of gait (FOG) is a common, disabling gait disturbance in Parkinson’s disease (PD) and other Parkinsonian syndromes. Freezing also occurs during non-gait movements involving the upper limbs. The mechanisms underlying freezing are complex, likely involving motor, cognitive, and sensory systems that contribute to the episodes. Here, we reported a 60-year-old female with a 24-year history of parkinsonism who experienced significant FOG when ambulatory. Disease progression resulted in her permanent use of a powered wheelchair. While using the power chair, the patient experiences apparent paroxysmal freezing in the hand and arm used to steer and propel the chair. These episodes, some lasting up to several minutes, occur only in circumstances (e.g., entering and leaving an elevator) that are similar to environments known to elicit and exacerbate FOG. Episodes are transient and can be volitionally interrupted by the patient but sometimes require external assistance. Therapeutic intervention for this type of potential freezing has yet to be determined. This case may provide insight into the complex nature of freezing behavior and suggests a need for new approaches to treating non-traditional freezing behavior.

Trauma- and stress-related disorders are among the most common types of mental illness affecting the U.S. population. For many of these disorders, there is a striking sex difference in lifetime prevalence; for instance, women are twice as likely as men to be affected by posttraumatic stress disorder (PTSD). Gonadal steroids and their metabolites have been implicated in sex differences in fear and anxiety. One example, allopregnanolone (ALLO), is a neuroactive metabolite of progesterone that allosterically enhances GABAA receptor activity and has anxiolytic effects. Like other ovarian hormones, it not only occurs at different levels in males and females but also fluctuates over the female reproductive cycle. One brain structure that may be involved in neuroactive steroid regulation of fear and anxiety is the bed nucleus of the stria terminalis (BNST). To explore this question, we examined the consequences of augmenting or reducing ALLO activity in the BNST on the expression of Pavlovian fear conditioning in rats. In Experiment 1, intra-BNST infusions of ALLO in male rats suppressed freezing behavior (a fear response) to the conditioned context, but did not influence freezing to a discrete tone conditioned stimulus (CS). In Experiment 2, intra-BNST infusion of either finasteride, an inhibitor of ALLO synthesis, or 17-phenyl-(3α,5α)-androst-16-en-3-ol, an ALLO antagonist, in female rats enhanced contextual freezing; neither treatment affected freezing to the tone CS. These findings support a role for ALLO in modulating contextual fear via the BNST and suggest that sex differences in fear and anxiety could arise from differential steroid regulation of BNST function. The susceptibility of women to disorders such as PTSD may be linked to cyclic declines in neuroactive steroid activity within fear circuitry.

Maternal immune activation during pregnancy is an environmental risk factor for psychiatric illnesses such as schizophrenia and autism in the offspring. Hence, changes in an array of behaviors, including behavioral flexibility, consistent with altered functioning of cortico-limbic circuits have been reported in rodent models of maternal immune activation. Surprisingly, previous studies have not examined the effect of maternal immune activation on the extinction of fear conditioning which depends on cortico-limbic circuits. Thus, we tested the effects of treating pregnant Long Evans rats with the viral mimetic polyI:C (gestational day 15; 4 mg/kg; i.v.) on fear conditioning and extinction in the male offspring using two different tasks. In the first experiment, we observed no effect of polyI:C treatment on the acquisition or extinction of a classically conditioned fear memory in a non-discriminative auditory cue paradigm. However, polyI:C-treated offspring did increase contextual freezing during the recall of fear extinction in this non-discriminative paradigm. The second experiment utilized a recently developed task to explicitly test the ability of rats to discriminate among cues signifying fear, reward, and safety; a task that requires behavioral flexibility. To our surprise, polyI:C-treated rats acquired the task in a manner similar to saline-treated rats. However, upon subsequent extinction training, they showed significantly faster extinction of the freezing response to the fear cue. In contrast, during the extinction recall test, polyI:C-treated offspring showed enhanced freezing behavior before and after presentation of the fear cue, suggesting an impairment in their ability to regulate fear behavior. These behavioral results are integrated into the literature suggesting impairments in cortico-limbic brain function in the offspring of rats treated with polyI:C during pregnancy.

Freezing of gait (FOG) is a disabling symptom of advanced Parkinson’s disease (PD) that leads to an increased risk of falls and nursing home placement. Interestingly, multiple lines of evidence suggest that the manifestation of FOG is related to specific deficits in cognition, such as set shifting and the ability to process conflict-related signals. These findings are consistent with the specific patterns of abnormal cortical processing seen during functional neuroimaging experiments of FOG, implicating increased neural activation within cortical structures underlying cognition, such as the Cognitive Control Network. In addition, these studies show that freezing episodes are associated with abnormalities in the BOLD response within key structures of the basal ganglia, such as the striatum and the subthalamic nucleus. In this article, we discuss the implications of these findings on current models of freezing behaviour and propose an updated model of basal ganglia impairment during FOG episodes that integrates the neural substrates of freezing from the cortex and the basal ganglia to the cognitive dysfunctions inherent in the condition.

Pavlovian threat (fear) conditioning (PTC) is an experimental paradigm that couples innate aversive stimuli with neutral cues to elicit learned defensive behavior in response to the neutral cue. PTC is commonly used as a translational model to study neurobiological and behavioral aspects of fear and anxiety disorders, including Posttraumatic Stress Disorder (PTSD). Though PTSD is a complex, multi-faceted construct that cannot be fully captured in animals, PTC is a conceptually valid model for studying the development and maintenance of learned threat responses. Thus it can inform the understanding of PTSD symptomatology. However, there are significant individual differences in posttraumatic stress that are not as of yet accounted for in studies of PTC. Individuals exposed to danger have been shown to follow distinct patterns: some adapt rapidly and completely (resilience), others adapt slowly (recovery), and others failure to adapt (chronic stress response). Identifying similar behavioral outcomes in PTC increases the translatability of this model. In this report, we present a flexible methodology for identifying individual differences in PTC by modeling latent subpopulations, or classes, characterized by defensive behavior during training. We provide evidence from a reanalysis of previously examined PTC learning and extinction data in rats to demonstrate the effectiveness of this methodology in identifying outcomes analogous to those observed in humans exposed to threat. By utilizing Latent Class Growth Analysis to test for heterogeneity in freezing behavior during threat conditioning and extinction learning in adult male outbred rats (n =58), three outcomes were identified: rapid extinction (57.3%), slow extinction (32.3%), and failure to extinguish (10.3%), indicating that heterogeneity analogous to that in naturalistic human studies is present in experimental animal studies, strengthening their translatability in understanding stress responses in humans.

A commentary on A zebrafish model of glucocorticoid resistance shows serotonergic modulation of the stress response by Griffiths, B. B., Schoonheim, P. J., Ziv, L., Voelker, L., Baier, H., and Gahtan, E. (2012). Front. Behav. Neurosci. 6:68. doi: 10.3389/fnbeh.2012.00068 The significance of the glucocorticoid receptor (GR) in stress response regulation and mood disorders has been demonstrated by many studies in animal and human models (Anacker et al., 2011a). The paper by Griffiths' and colleagues adds to this evidence, by investigating GR function and its role in stress response regulation and antidepressant action in an exciting new zebrafish model. The main finding of this study is that mutant zebrafish larvae with impaired GR transactivation, due to a single nucleotide substitution in a region essential for DNA binding (grs357 mutants), exhibit behavioral abnormalities and neuroendocrine dysfunction. Specifically, grs357 mutants show elevated startle responses, a surrogate measure of anxiety-like behavior in fish, as well as increased cortisol levels and impaired HPA axis feedback control, demonstrated by reduced suppression of pituitary pomc upon treatment with the synthetic glucocorticoid, betamethasone. The authors also test whether the selective serotonin reuptake inhibitor (SSRI) antidepressant, fluoxetine, is able to reverse the neuroendocrine and behavioral phenotype of grs357 mutants. Their findings suggest that fluoxetine does not reverse neuroendocrine abnormalities resulting from impaired GR transactivation, while it still effectively counteracts anxiety-like behavior. These findings confirm the crucial role of the GR in neuroendocrine regulation and behavioral stress responses, and are in line with the authors' previous work, in which they have identified stress axis abnormalities and increased freezing behavior upon exposure to a novel environment in the same mutant fish (Ziv et al., 2012). Their new findings thus consolidate the use of the zebrafish model to investigate GR-dependent regulation of the stress response. The data suggest that some of the behavioral effects of fluoxetine on reversing startle response abnormalities may be independent of GR transactivation in fish. However, it is important to note that many GR-mediated effects are indeed independent of direct GR binding to the DNA, particularly the glucocorticoid-mediated regulation of pomc expression (Tuckermann et al., 1999; Bilodeau et al., 2006). It thus remains to be elucidated whether GR protein-protein interactions, such as transrepression of the transcription factors NFkB, AP-1, or cyclic AMP response element binding protein (CREB), may indeed account for some of the SSRI effects on anxiety-like behavior in this study. Moreover, an increasing body of work suggests that a membrane-bound GR may mediate some of the rapid, non-genomic effects of glucocorticoids, particularly glucocorticoid-induced changes in neuronal excitability and memory consolidation (for comprehensive reviews, please see: Haller et al., 2008 and Groeneweg et al., 2012). While previous studies have shown a crucial role for genomic GR effects in antidepressant action (Anacker et al., 2011b), the involvement of the membrane-bound form of the GR in the mechanism of action of antidepressants remains elusive and may possibly account for some of the behavioral changes upon antidepressant treatment in grs357 mutant fish, in which only GR transactivation is abolished. As the authors suggest, the role of serotonergic signaling on the neuroendocrine system, behavior, and the regulation of GR function is likely to be of great importance for the effects of SSRI antidepressants (Laplante et al., 2002). At the same time, potential serotonin-independent molecular mechanisms of antidepressant action, such as liberation of G-proteins from membrane associated lipid rafts, have been suggested (Donati and Rasenick, 2005), and may represent possible alternative pathways involved in stress response regulation and GR activation, which warrant further investigation. Of course, when modeling psychiatric disorders in fish, it needs to be carefully considered that the behavioral repertoire, the neural networks and the psychological responses to stress in teleosts are largely different from respective responses in the mammalian brain. Behavioral studies in higher animals, such as rodents and primates, as well as cellular and molecular studies in human tissues, will therefore indeed remain indispensable for our understanding of stress-induced brain abnormalities, depression pathophysiology, and antidepressant treatment response. To this end, it will be interesting for future investigations to examine if other well established neurobiological correlates of depression, such as alterations in neuronal gene expression or adult neurogenesis, can also be modeled in comparable regions of the fish brain (Kizil et al., 2012). In addition, evolutionary differences in corticosteroid receptor function have been demonstrated and need to be considered when investigating GR function across different phylogenetic taxa (Bury and Sturm, 2007). Nevertheless, the zebrafish is a useful model to investigate principle mechanisms underlying stress response regulation, and it offers a unique possibility for high-throughput drug screening in vivo, facilitating the discovery of new targets that alter neuroendocrine responses and proxy measures of stress-induced behavioral abnormalities. In order to identify new treatment targets and to elucidate the sophisticated heterogeneous mechanisms underlying depression pathogenesis, a cross-species investigation into the several layers of the complex nature of stress-induced biological abnormalities will be essential to our success. The paper by Griffiths' and colleagues, together with previous work on GR function in zebrafish (Ziv et al., 2012), is a great example for such an approach, and offers a novel high-throughput platform in live fish to investigate drug responses and GR function in an in vivo system.