Uncertainty and anxiety: Evolution and neurobiology.

Anxiety is a complex phenomenon: Its eliciting stimuli and circumstances, component behaviors, and functional consequences are only slowly coming to be understood. Here, we examine defense systems from field studies; laboratory studies focusing on experimental analyses of behavior; and, the fear conditioning literature, with a focus on the role of uncertainty in promoting an anxiety pattern that involves high rates of stimulus generalization and resistance to extinction. Respectively, these different areas provide information on evolved elicitors of defense (field studies); outline a defense system focused on obtaining information about uncertain threat (ethoexperimental analyses); and, provide a simple, well-researched, easily measured paradigm for analysis of nonassociative stress-enhanced fear conditioning (the SEFL). Results suggest that all of these-each of which is responsive to uncertainty-play multiple and interactive roles in anxiety. Brain system findings for some relevant models are reviewed, with suggestions that further analyses of current models may be capable of providing a great deal of additional information about these complex interactions and their underlying biology.

Sex, defense, and risk assessment: Who could ask for anything more?

Over the 30 years since IBNS was founded, a central theme of "Translation" has emerged. This reflects increasing realization that mental disorders such as anxiety and depression are extremely widespread, expensive and painful to societies and individuals across the world. The Blanchard lab has been particularly involved in attempts to understand the evolutionary and functional mechanisms underlying defensive behaviors as a focal component of these disorders. This involved analysis of the relationships between threatening situations/stimuli, and the behaviors (flight, freezing, fight, and risk assessment) that respond to them, for rodents; and also attempts to link these relationships to human responsivity to similar threatening events: Linkages that are complicated by factors such as domestication and sex. In particular it is important to describe and characterize the organization of defensive patterns in people as well as nonhuman animals, and to understand how these patterns can become nonfunctional and pathological.

Are cognitive aspects of defense a core feature of anxiety and depression?

Anxiety and depression are highly prevalent behavior disorders, particularly in women. Recent preclinical work using animal models has been suboptimal in predicting the efficacy of drugs targeted at these conditions, suggesting a potential discrepancy between such models and the human disorders. Notably female animals tend to be equal to, or less responsive than, males in these tasks. A number of analyses suggest that mammalian defense patterns are complex: In addition to relatively discrete and immediate fight, flight, and freezing responses, a risk assessment pattern may occur in response to threat stimuli or situations with ambiguous elements. This pattern combines defensiveness with a number of cognition-linked behaviors such as sensory attention and orientation, approach, contact, and investigation of the potential threat. Studies measuring elements of this pattern suggest that female rats, and perhaps female mice, show higher levels than equivalent males. Higher female involvement may also occur in tasks involving learning/generalization/extinction of defensiveness to conditioned stimuli. Such findings are consonant with recent analyses of "female survival strategies" based on differential adaptiveness of cognitive components of defensiveness in females, due to the necessity of female care of offspring until they are independent. These data suggest the value of additional behavioral and functional analyses of cognitive aspects of defensive behavior; contributing to both an understanding of their underlying mechanisms, and providing more sensitive measures of drug responsivity for use with animal models.

Dissecting the brain's fear systems responding to snake threats.

We examined the behavioural responses and Fos expression pattern of rats that were exposed to snake threats from shed snakeskin and a live snake. We differentiated the behavioural responses and the pattern of Fos expression in response to the odour cues and mild threat from a live snake. Animals exposed to the snake odour alone or to the confined snake showed a great deal of risk assessment. Conversely, the intensification of odour during exposure to the live snake decreased the threat ambiguity, and the animals froze for a significantly longer period. Our Fos analysis showed that a pathway formed by the posteroventral part of the medial amygdalar nucleus to the central part of the ventromedial hypothalamic nucleus appeared to be solely responsive to odour cues. In addition, we showed increased Fos expression in a parallel circuit comprising the lateral amygdalar nucleus, ventral subiculum, lateral septum, and juxtadorsomedial region of the lateral hypothalamic area that is responsive to both the odour and mild threat from a live snake. This path is likely to process the environmental boundaries of the threat to be avoided. Both paths merge into the dorsal premammillary nucleus and periaqueductal grey sites, which all increase Fos expression in response to the snake threats and are likely to organize the defensive responses. Moreover, we found that the snake threat mobilized the Edinger-Westphal and supraoculomotor nuclei, which are involved in stress adaptation and attentional mechanisms.

Dissecting the brain’s fear systems responding to snake threats

We examined the behavioral responses and Fos expression pattern of rats that were exposed to snake threats from shed snakeskin and a live snake. We differentiated the behavioral responses and the pattern of Fos expression in response to the odor cues and mild threat from a live snake. Animals exposed to the snake odor alone or to the confined snake showed a great deal of risk assessment. Conversely, the intensification of odor during exposure to the live snake decreased the threat ambiguity, and the animals froze for a significantly longer period. Our Fos analysis showed that a pathway formed by the posteroventral part of the medial amygdalar nucleus to the central part of the ventromedial hypothalamic nucleus appeared to be solely responsive to odor cues. In addition, we showed increased Fos expression in a parallel circuit comprising the lateral amygdalar nucleus, ventral subiculum, lateral septum and juxtadorsomedial region of the lateral hypothalamic area that is responsive to both the odor and mild threat from a live snake. This path is likely to process the environmental boundaries of the threat to be avoided. Both paths merge into the dorsal premammillary nucleus and periaqueductal gray sites, which all increase Fos expression in response to the snake threats and are likely to organize the defensive responses. Moreover, we found that the snake threat mobilized the Edinger-Westphal and supraoculomotor nuclei, which are involved in stress adaptation and attentional mechanisms.

Repeated exposure of naïve and peripheral nerve-injured mice to a snake as an experimental model of post-traumatic stress disorder and its co-morbidity with neuropathic pain.

Confrontation of rodents by natural predators provides a number of advantages as a model for traumatic or stressful experience. Using this approach, one of the aims of this study was to investigate a model for the study of post-traumatic stress disorder (PTSD)-related behaviour in mice. Moreover, because PTSD can facilitate the establishment of chronic pain (CP), and in the same way, patients with CP have an increased tendency to develop PTSD when exposed to a traumatic event, our second aim was to analyse whether this comorbidity can be verified in the new paradigm. C57BL/6 male mice underwent chronic constriction injury of the sciatic nerve (CCI), a model of neuropathic CP, or not (sham groups) and were submitted to different threatening situations. Threatened mice exhibited enhanced defensive behaviours, as well as significantly enhanced risk assessment and escape behaviours during context reexposure. Previous snake exposure reduced open-arm time in the elevated plus-maze test, suggesting an increase in anxiety levels. Sham mice showed fear-induced antinociception immediately after a second exposure to the snake, but 1 week later, they exhibited allodynia, suggesting that multiple exposures to the snake led to increased nociceptive responses. Moreover, after reexposure to the aversive environment, allodynia was maintained. CCI alone produced intense allodynia, which was unaltered by exposure to either the snake stimuli or reexposure to the experimental context. Together, these results specifically parallel the behavioural symptoms of PTSD, suggesting that the snake/exuvia/reexposure procedure may constitute a useful animal model to study PTSD.

Defensive behaviors and brain regional activation changes in rats confronting a snake.

In the present study, we examined behavioral and brain regional activation changes of rats). To a nonmammalian predator, a wild rattler snake (Crotalus durissus terrificus). Accordingly, during snake threat, rat subjects showed a striking and highly significant behavioral response of freezing, stretch attend, and, especially, spatial avoidance of this threat. The brain regional activation patterns for these rats were in broad outline similar to those of rats encountering other predator threats, showing Fos activation of sites in the amygdala, hypothalamus, and periaqueductal gray matter. In the amygdala, only the lateral nucleus showed significant activation, although the medial nucleus, highly responsive to olfaction, also showed higher activation. Importantly, the hypothalamus, in particular, was somewhat different, with significant Fos increases in the anterior and central parts of the ventromedial hypothalamic nucleus (VMH), in contrast to patterns of enhanced Fos expression in the dorsomedial VMH to cat predators, and in the ventrolateral VMH to an attacking conspecific. In addition, the juxtodorsalmedial region of the lateral hypothalamus showed enhanced Fos activation, where inputs from the septo-hippocampal system may suggest the potential involvement of hippocampal boundary cells in the very strong spatial avoidance of the snake and the area it occupied. Notably, these two hypothalamic paths appear to merge into the dorsomedial part of the dorsal premammillary nucleus and dorsomedial and lateral parts of the periaqueductal gray, all of which present significant increases in Fos expression and are likely to be critical for the expression of defensive behaviors in responses to the snake threat.

Epigenetic Delay in the Neurodevelopmental Trajectory of DNA Methylation States in Autism Spectrum Disorders.

Autism spectrum disorders (ASD) are hypothesized to originate in utero from perturbations in neural stem cell niche regions of the developing brain. Dynamic epigenetic processes including DNA methylation are integral to coordinating typical brain development. However, the extent and consequences of alterations to DNA methylation states in neural stem cell compartments in ASD are unknown. Here, we report significant DNA methylation defects in the subventricular zone of the lateral ventricles from postmortem brain of 17 autism diagnosed compared to 17 age- and gender-matched typically developing individuals. Both array- and sequencing-based genome-wide methylome analyses independently revealed that these alterations were preferentially targeted to intragenic and bivalently modified chromatin domains of genes predominately involved in neurodevelopment, which associated with aberrant precursor messenger RNA splicing events of ASD-relevant genes. Integrative analysis of our ASD and typically developing postmortem brain methylome datasets with that from fetal brain at different neurodevelopmental stages revealed that the methylation states of differentially methylated loci associated with ASD remarkably resemble the methylation states at earlier time points in fetal brain development. This observation was confirmed using additional methylome datasets from three other brain regions. Altogether, these findings implicate an epigenetic delay in the trajectory of normal DNA methylation states during the course of brain development that may consequently lead to deleterious transcriptomic events in ASD and support the hypothesis of an early developmental origin of ASD.

Risk assessment and serotonin: Animal models and human psychopathologies.

Risk assessment (RA) is an evolved, generally adaptive, mechanism comprising focused attention and appraisal of potential threat stimuli and situations. Initially characterized in animal models, it provides a number of behavioral and functional parallels to patterns of rumination, gaze biases, and other forms of affective cognition that appear to be disregulated in depression and anxiety. Serotonergic mechanisms are involved in these mood disorders, and an emerging body of evidence suggests that they may modulate the affective cognitive changes common to such psychopathologies. Findings of parallel effects of serotonin systems in RA would support a view that it may provide a useful behavioral endophenotype for translational research on mood disorders. This review examines the involvement of serotonergic mechanisms in both animal models of RA, and in an array of tasks focusing on affective cognitive changes in individuals with depression or anxiety. Results suggest substantial serotonin involvement in both RA behaviors measured in rats or mice, and in the "intersection of emotional and cognitive processes" [43] in people.

What we imagine is what we do? A critical overview about mental imagery as a strategy to study human defensive responses.

There is not a single and perfect instinctive behavior to react to threatening situations. However, the study of particular features of these situations suggests the existence of prototypical emotional reactions and associated defensive behaviors. Since all living beings are subjected to common evolutionary pressures, such as predation and conspecific competition, it is plausible that there is conservation of some basic defensive responses in their behavioral repertoire. The choice for approaching or withdrawing from a given situation depends, among others things, on environmental features, including the threat intensity and the distance from the source of the threat. If these basic responses were conserved in humans, they should be expressed in ways similar to those observed in non-human animals. Due to ethical reasons and easy application, mental imagery has been used to test this hypothesis. The studies included in this review point to the validity of this method, with both self-report and neurophysiological findings corroborating the hypothesis under scrutiny. Despite the need for additional investigation to deal with some limitations, the information obtained with this method can help to a better understanding of the conditions that provoke specific defensive behaviors and related emotions. This knowledge may also contribute to identify vulnerability factors for fear/anxiety-related disorders.

Effects of estrus cycle stage on defensive behavior in female Long-Evans hooded rats.

This study investigated the influence of the estrus cycle in mediating cat odor-induced unconditioned and conditioned defensive behaviors in female Long-Evans hooded rats. Unconditioned defensive behaviors were assessed during predatory cue exposure; conditioned defensive behaviors were examined 24 h after threat exposure. Estrus phases were determined by microscopic examination of vaginal smears within 10 min of completing the behavioral tests. Compared to no-odor controls, female rats exposed to cat odor exhibited both unconditioned and conditioned defensive behaviors, including elevated levels of freezing, risk assessment and avoidance. Rats in proestrus and estrus exhibited reduced levels of defensive behavior during the unconditioned test trial compared to subjects in diestrus and metestrus. Specifically, estrus stages characterized by high levels of circulating estrogens and progesterone were associated with reduced immobility (i.e. freezing) and enhanced active defense (i.e. risk assessment), profiles that may enable mate seeking and subsequent reproduction in potentially dangerous or novel environments. These results suggest a specific role for ovarian hormone fluctuations in mediating unconditioned fear- and anxiety-like defensive behaviors during exposure to predatory odors.

Translating dynamic defense patterns from rodents to people.

Specific defensive behaviors of rodents are shaped by features of the eliciting threat stimuli and situation. Threat scenarios confirmed these relationships in people, with results substantially replicated in 4 additional scenario studies. Subsequent human studies involve computer games measuring fear as flight from threat stimuli and anxiety as alternation between two threats. Stabilometric studies have shown reduction in sway (freezing) to inescapable (e.g. with gun pointed at subject) threatening photographs; but enhanced lateral sway (flight attempts) to escapable threats; (gun pointed away from subject). Relationships between threat ambiguity, risk assessment, and anxiety have been validated by identification of videos of facial expressions to ambiguous threats, as anxiety; and systematic biases toward threat stimuli by anxious individuals. Enhanced rumination, interpretable as unsuccessful risk assessment, is a dynamic component of both anxiety and depression, particularly in women. While there is less experimental work on defensive threat/attack, a transdiagnostic "Fear of Harm" phenotype of aggression associated with fear suggests that this is a component of pathological as well as normal human defensive behavior.

Neuronal correlates of asocial behavior in a BTBR T (+) Itpr3(tf)/J mouse model of autism.

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized, in part, by an inability to adequately respond to social cues. Patients diagnosed with ASD are often devoid of empathy and impaired in understanding other people's emotional perspective. The neuronal correlates of this impairment are not fully understood. Replicating such a behavioral phenotype in a mouse model of autism would allow us insight into the neuronal background of the problem. Here we tested BTBR T(+)Itpr3(tf)/J (BTBR) and c57BL/6J (B6) mice in two behavioral paradigms: the Transfer of Emotional Information test and the Social Proximity test. In both tests BTBR mice displayed asocial behavior. We analyzed c-Fos protein expression in several brain regions after each of these tests, and found that, unlike B6 mice, BTBR mice react to a stressed cagemate exposure in the Transfer of Emotional Information test with no increase of c-Fos expression in either the prefrontal cortex or the amygdala. However, after Social Proximity exposure we observed a strong increase in c-Fos expression in the CA3 field of the hippocampus and two hypothalamic regions of BTBR brains. This response was accompanied by a strong activation of periaqueductal regions related to defensiveness, which suggests that BTBR mice find unavoidable social interaction highly aversive.

Central oxytocin regulates social familiarity and scent marking behavior that involves amicable odor signals between male mice.

The effect of oxytocin on social behavior and odor communication was investigated in male C57BL/6J mice. In three-male colonies, in visible burrow systems, icv oxytocin (OT) infusion before colony formation substantially increased huddling together over the initial 8 h of grouping, accompanied by decreased expression of a number of social approaches associated with conspecific aggression and defense. OT antagonist infusion had little impact on expression of social approaches but decreased time engaging in social components including huddle over the initial 8 h. These results demonstrate a linkage of social familiarity to OT availability in the brain. In a scent marking paradigm central infusion of OT reduced territorial marking towards male conspecifics, and this in turn reduced the scent marking of untreated stimulus males to OT-infused subjects. Infusion of an OT antagonist into stimulus mice who were confronted with OT-infused subjects prevented the reduction/suppression of scent marking that was normally seen following exposure of social odors released from OT-injected mice. Odor of pair-housed mice also induced a suppression of territorial scent marking in odor recipients, but OT antagonist administration into pair-housed mice blocked this suppressive effect of odor cue. These results indicate that central OT modulates release as well as detection of amicable signals facilitating/maintaining familiar relationships and suppressing territorial behavior between male mice. Overall, these findings suggest that OT plays a significant role in regulating social familiarity via changing qualities of conspecific odor cues.

Applying the ethoexperimental approach to neurodevelopmental syndrome research reveals exaggerated defensive behavior in Mecp2 mutant mice.

Rett syndrome is a Pervasive Developmental Disorder (PDD) associated with de novo mutations of the methyl CpG-binding protein 2 (MECP2) gene. Mecp2 functions as a transcription factor that regulates the expression of hundreds of genes. Identification of the role of Mecp2 in specific neurodevelopmental symptoms remains an important research aim. We previously demonstrated that male mice possessing a truncation mutation in Mecp2 are hyper-social. We predicted that reduced fear or anxiety might underlie this enhanced affiliation. In order to probe risk assessment and anxiety-like behavior, we compared Mecp2 truncation mutants to their wild-type littermates in the elevated plus maze and elevated zero maze. Additionally, subjects were administered the mouse defense test battery to evaluate unconditioned fear- and panic-like behavior to a graded set of threat scenarios and a predator stimulus. Mutant mice showed no significant changes in anxiety-like behavior. Yet, they displayed hyper-reactive escape and defensive behaviors to an animate predatory threat stimulus. Notably, mutant mice engaged in exaggerated active defense responding to threat stimuli at nearly all phases of the fear battery. These results reveal abnormalities in emotion regulation in Mecp2 mutants particularly in response to ecologically relevant threats. This hyper-responsivity suggests that transcriptional targets of Mecp2 are critical to emotion regulation. Moreover, we suggest that detailed analysis of defensive behavior and aggression with ethologically relevant tasks provides an avenue to interrogate gene-behavior mechanisms of neurodevelopmental and other psychiatric conditions.

Cortagine infused into the medial prefrontal cortex attenuates predator-induced defensive behaviors and Fos protein production in selective nuclei of the amygdala in male CD1 mice.

Corticotropin-releasing factor (CRF) plays an essential role in coordinating the autonomic, endocrine and behavioral responses to stressors. In this study, we investigated the role of CRF within the medial prefrontal cortex (mPFC) in modulating unconditioned defensive behaviors, by examining the effects of microinfusing cortagine a selective type-1 CRF receptor (CRF1) agonist, or acidic-astressin a preferential CRF1 antagonist, into the mPFC in male CD-1 mice exposed to a live predator (rat exposure test--RET). Cortagine microinfusions significantly reduced several indices of defense, including avoidance and freezing, suggesting a specific role for CRF1 within the infralimbic and prelimbic regions of the mPFC in modulating unconditioned behavioral responsivity to a predator. In contrast, microinfusions of acidic-astressin failed to alter defensive behaviors during predator exposure in the RET. Cortagine microinfusions also reduced Fos protein production in the medial, central and basomedial, but not basolateral subnuclei of the amygdala in mice exposed to the rat predatory threat stimulus. These results suggest that CRF1 activation within the mPFC attenuates predator-induced unconditioned anxiety-like defensive behaviors, likely via inhibition of specific amygdalar nuclei. Furthermore, the present findings suggest that the mPFC represents a unique neural region whereby activation of CRF1 produces behavioral effects that contrast with those elicited following systemic administration of CRF1 agonists.

The role of behavior in translational models for psychopathology: functionality and dysfunctional behaviors.

The history of science has frequently included a problem-based impetus toward research that can be translated expeditiously into solutions. A current problem is that psychopathologies, typically chronic, contribute hugely to the economic and social burden of medical care, especially in the United States. For behavioral neuroscientists a psychopathology-aimed translational research emphasis particularly involves animal models to facilitate the experimental and invasive work necessary to an understanding of the biology of normal and aberrant behavior. When the etiology of a particular psychopathology is unknown, and there are no specific biomarkers, behavioral parallels between the focal disorder and its putative models become crucial elements in assessing model validity. Evaluation of these parallels is frequently neglected, reflecting in part the lack of a systematic conceptualization of the organization of behavior and how this may be conserved across species. Recent work specifically attempting to bridge this gap suggests that analysis of behaviors that are functional - adaptive in crucial situations such as danger or social contexts - can facilitate an understanding of the parallels between behaviors of human and nonhuman species, including the dysfunctional behaviors of psycho pathologies. As research with animal models comes to provide a more systematic analysis of particular behaviors and their adaptive functions, cross-talk between model and focal psychopathology may be advantageous to understanding both.

Heparan sulfate deficiency in autistic postmortem brain tissue from the subventricular zone of the lateral ventricles.

Abnormal cellular growth and organization have been characterized in postmortem tissue from brains of autistic individuals, suggestive of pathology in a critical neurogenic niche, the subventricular zone (SVZ) of the brain lateral ventricles (LV). We examined cellular organization, cell proliferation, and constituents of the extracellular matrix such as N-sulfated heparan sulfate (HS) and laminin (LAM) in postmortem brain tissue from the LV-SVZ of young to elderly individuals with autism (n=4) and age-matched typically developing (TD) individuals (n=4) using immunofluorescence techniques. Strong and systematic reductions in HS immunofluorescence were observed in the LV-SVZ of the TD individuals with increasing age. For young through mature, but not elderly, autistic pair members, HS was reduced compared to their matched TDs. Cellular proliferation (Ki67+) was higher in the autistic individual of the youngest age-matched pair. These preliminary data suggesting that HS may be reduced in young to mature autistic individuals are in agreement with previous findings from the BTBR T+tf/J mouse, an animal model of autism; from mice with genetic modifications reducing HS; and with genetic variants in HS-related genes in autism. They suggest that aberrant extracellular matrix glycosaminoglycan function localized to the subventricular zone of the lateral ventricles may be a biomarker for autism, and potentially involved in the etiology of the disorder.

The BTBR T+ tf/J mouse model for autism spectrum disorders-in search of biomarkers.

Autism spectrum disorders (ASD) form a common group of neurodevelopmental disorders appearing to be under polygenic control, but also strongly influenced by multiple environmental factors. The brain mechanisms responsible for ASD are not understood and animal models paralleling related emotional and cognitive impairments may prove helpful in unraveling them. BTBR T+ tf/J (BTBR) mice display behaviors consistent with the three diagnostic categories for ASD. They show impaired social interaction and communication as well as increased repetitive behaviors. This review covers much of the data available to date on BTBR behavior, neuroanatomy and physiology in search for candidate biomarkers, which could both serve as diagnostic tools and help to design effective treatments for the behavioral symptoms of ASD.