Fear Behavior Research Articles

Age-associated alteration of innate defensive response to a looming stimulus and brain functional connectivity pattern in mice

Innate defensive behaviors are essential for species survival. While these behaviors start to develop early in an individual’s life, there is still much to be understood about how they evolve with advancing age. Considering that aging is often accompanied by various cognitive and physical declines, we tested the hypothesis that innate fear behaviors and underlying cerebral mechanisms are modified by aging. In our study we investigated this hypothesis by examining how aged mice respond to a looming visual threat compared to their younger counterparts. Our findings indicate that aged mice exhibit a different fear response than young mice when facing this imminent threat. Specifically, unlike young mice, aged mice tend to predominantly display freezing behavior without seeking shelter. Interestingly, this altered behavioral response in aged mice is linked to a distinct pattern of functional brain connectivity compared to young mice. Notably, our data highlights a lack of a consistent brain activation following the fear response in aged mice, suggesting that innate defensive behaviors undergo changes with aging.

Effect of chronic alcohol exposure and single-prolonged stress on conditioned fear behavior

The present study investigated the impact of chronic intermittent ethanol (CIE) exposure and single-prolonged stress (SPS) on the acquisition of fear memories in both male and female Wistar rats. Adult rats were first subjected to CIE by vapor inhalation followed by SPS. Following a subsequent 8-day incubation period, the rats underwent a Pavlovian fear conditioning procedure (tone-shock pairings) followed by cued-tone extinction training, and then testing of extinction recall memory and fear renewal memory. In control animals that had not been exposed to either CIE or SPS, female rats exhibited significantly lower levels of freezing compared to male rats during tone-shock pairings. This lower level of freezing in female rats during conditioning was associated with an increased speed of movement compared to males. Also compared to males, female rats exhibited lower levels of fear extinction, recall, and renewal. Exposure to CIE, SPS, or CIE+SPS had no effect on freezing during the cued-conditioning, extinction, or extinction recall phases of the testing procedure in either sex. In fear renewal, CIE exposure decreased freezing in male but not female rats, while SPS increased freezing in female but not male rats. CIE exposure significantly reduced freezing during the fear renewal phase. Taken together, these results provide further evidence that male and female rats adopt different avoidance strategies for threat responding. These results also revealed that prior exposure to CIE, SPS, or CIE+SPS had minimal effects on threat responding using conditioned freezing as an indicator of fear responsivity.

How can ethology inform the neuroscience of fear, aggression and dominance?

The study of behaviour is dominated by two approaches. On the one hand, ethologists aim to understand how behaviour promotes adaptation to natural contexts. On the other, neuroscientists aim to understand the molecular, cellular, circuit and psychological origins of behaviour. These two complementary approaches must be combined to arrive at a full understanding of behaviour in its natural setting. However, methodological limitations have restricted most neuroscientific research to the study of how discrete sensory stimuli elicit simple behavioural responses under controlled laboratory conditions that are only distantly related to those encountered in real life. Fortunately, the recent advent of neural monitoring and manipulation tools adapted for use in freely behaving animals has enabled neuroscientists to incorporate naturalistic behaviours into their studies and to begin to consider ethological questions. Here, we examine the promises and pitfalls of this trend by describing how investigations of rodent fear, aggression and dominance behaviours are changing to take advantage of an ethological appreciation of behaviour. We lay out current impediments to this approach and propose a framework for the evolution of the field that will allow us to take maximal advantage of an ethological approach to neuroscience and to increase its relevance for understanding human behaviour.

Zebrafish Maintenance Conditions Affect Behavioral and Biochemical Biomarkers: A Possible Interfering Factor on the Research Results.

Over the years, scientific research with fish models has grown at a rapid pace, and issues such as animal welfare are becoming increasingly important in various areas of animal husbandry and experimentation. Here, we evaluated whether Danio rerio behavior is affected by long-term maintenance (75 days) in an enriched environment or a chronic stress (CS) situation. In addition, we evaluated some biochemical parameters related to redox status. We concluded that long-term maintenance of zebrafish in enriched environment might induce an anxiety-like behavior pattern when these fish are faced with an acute subsequent stressor. These anxiety results, the increased school cohesion, and the absence of oxidative damage allow us to hypothesize that the fish maintained in environmental enrichment (EE) situation is more reactive, showing a strong protective reaction to the stress. From an applicable perspective, we show that both too much stress and too little stress are not ideal for zebrafish stocks. In CS situations, fish can habituate and might not respond optimally to test conditions. In opposite, the low stress promoted by environmental enrichment also renders the fish incapable of dealing with occasional stressors optimally, because now even normal conditions appear stressful to them and may elicit fear behaviors they normally would not exhibit.

Interpeduncular GABAergic neuron function controls threat processing and innate defensive adaptive learning.

The selection of appropriate defensive behaviors in the face of potential threat is fundamental to survival. However, after repeated exposures to threatening stimuli that did not signal real danger, an animal must learn to adjust and optimize defensive behaviors. Despite extensive research on innate threat processing, little is known how individuals change their defensive behaviors when presented with recurrent threat exposures without evidence of a real risk. Insight into this process is critical as its dysregulation may contribute to neuropsychiatric conditions, such as anxiety disorders. Here, we used the visual looming stimulus (VLS) paradigm in mice to investigate innate threat processing and adaptive defensive learning. Repeated exposure to VLS over consecutive sessions reduced immediate freezing responses and time spent inside a sheltered area upon VLS events, leading to an increase in foraging behaviors. Fiber photometry recordings and optogenetic manipulations revealed that VLS innate adaptive defensive learning is associated with reduced recruitment of the midbrain interpeduncular nucleus (IPN), a structure associated with fear and anxiety-related behaviors. Functional circuit-mapping identified a role for select IPN projections to the laterodorsal tegmental nucleus in gating defensive learning. Finally, we uncovered a subpopulation of IPN neurons that express the neuropeptide somatostatin and encode safety- and avoidance signals in response to VLS. These results identify critical behavioral signatures of innate defensive responses and a circuit that regulates the essential features of threat processing.

Distinct ventral hippocampal inhibitory microcircuits regulating anxiety and fear behaviors

In emotion research, anxiety and fear have always been interconnected, sharing overlapping brain structures and neural circuitry. Recent investigations, however, have unveiled parallel long-range projection pathways originating from the ventral hippocampus, shedding light on their distinct roles in anxiety and fear. Yet, the mechanisms governing the emergence of projection-specific activity patterns to mediate different negative emotions remain elusive. Here, we show a division of labor in local GABAergic inhibitory microcircuits of the ventral hippocampus, orchestrating the activity of subpopulations of pyramidal neurons to shape anxiety and fear behaviors in mice. These findings offer a comprehensive insight into how distinct inhibitory microcircuits are dynamically engaged to encode different emotional states.

484 Exploring fear in client-owned dogs: Insights from behavioral testing, owner surveys, and biomarker validation

Abstract Since the SARS-CoV-2 pandemic, pet professionals and owners have observed an increase in anxiety-related behaviors, including fearfulness, contributing to behavioral problems, reduction in pet and owner welfare, and increased rates of shelter relinquishment. The objectives of this study were to evaluate canine demographics and owner-identified fearfulness compared with fear-related behaviors during in-person behavior tests, with further verification using fear-related biomarkers. Client-owned dogs [n = 102; 25.3 ± 11.6 kg body weight (BW)] were included between 1 to 10 yr of age, of varying breeds, sex and neuter statuses, and home dynamics. Dogs had no known health issues, were not receiving medication, and were fed a standardized diet for 4 wk prior to testing. Owners provided information via a questionnaire regarding fear, anxiety, excitability, and separation behaviors, as well as information on the lifestyle and background of the dog. Behavior tests, which consisted of a novel human, novel object, and an open field test, were performed indoors in a constructed 3 x 3 m arena at the University of Guelph. Fecal samples were provided by owners and blood samples were collected immediately following the behavior assessment. Commercial ELISA kits were used to quantify fecal IgA, serum serotonin (5-HT), and 5-hydroxyindoleacetic acid (5-HIAA), then the 5-HIAA:5-HT ratio was calculated. Behavior tests were recorded on a GoPro camera, and behaviors were coded using BORIS. For preliminary analysis, owner questionnaire data relating to human-directed fear was separated into frequency of human-directed fear (HFF), intensity of human-directed fear (HFI), frequency of fear-related behaviors during grooming or veterinary visits (VHF), and intensity of fear-related behaviors during grooming or veterinary visits (VHI) section. Regression analyses were conducted using SAS, with outcomes derived from behavioral tests serving as dependent variables, and predictor variables encompassing sex, neuter status, age, BW, biomarker concentrations, and owner survey scores. Significance was determined at P ≤ 0.05 and trends at P ≤ 0.10. Preliminary data analysis using data from the novel human test of 50 dogs indicates that the duration of reduced body posture during the test was positively correlated with VHF (P = 0.0201) and VHI (P = 0.0226) scores, and negatively correlated with HIAA concentrations (P = 0.0134). The duration of the “side” ear position was also positively associated with HFF (P = 0.0219), HFI (P = 0.0471), and VHI (P = 0.0170) scores, with a trend observed in VHF (P = 0.0818) scores. Duration of time with ears pinned back was similarly associated with HFF (P = 0.0064), HFI (P = 0.0076), and VHI (0.0192) scores. These results suggest that behavior may be reliably predicted from owner-completed surveys and can be a valuable tool in gathering information for both professionals and the public. Further data analysis from the present study will be conducted to further understand the relationship between canine demographics, owner identified fear scores, physiological biomarkers and behavior testing results.

GluN2B on Adult-Born Granule Cells Modulates (R,S)-Ketamine's Rapid-Acting Effects in Mice.

Standard antidepressant treatments often take weeks to reach efficacy and are ineffective for many patients. (R,S)-ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, has been shown to be a rapid-acting antidepressant and to decrease depressive symptoms within hours of administration. While previous studies have shown the importance of the GluN2B subunit of the NMDA receptor on interneurons in the medial prefrontal cortex, no study to our knowledge has investigated the influence of GluN2B-expressing adult-born granule cells. Here, we examined whether (R,S)-ketamine's efficacy depends on adult-born hippocampal neurons using a genetic strategy to selectively ablate the GluN2B subunit of the NMDA receptor from Nestin+ cells in male and female mice, tested across an array of standard behavioral assays. We report that in male mice, GluN2B expression on 6-week-old adult-born neurons is necessary for (R,S)-ketamine's effects on behavioral despair in the forced swim test and on hyponeophagia in the novelty suppressed feeding paradigm, as well on fear behavior following contextual fear conditioning. In female mice, GluN2B expression is necessary for effects on hyponeophagia in novelty suppressed feeding. These effects were not replicated when ablating GluN2B from 2-week-old adult-born neurons. We also find that ablating neurogenesis increases fear expression in contextual fear conditioning, which is buffered by (R,S)-ketamine administration. In line with previous studies, these results suggest that 6-week-old adult-born hippocampal neurons expressing GluN2B partially modulate (R,S)-ketamine's rapid-acting effects. Future work targeting these 6-week-old adult-born neurons may prove beneficial for increasing the efficacy of (R,S)-ketamine.

Identification of a stress-responsive subregion of the basolateral amygdala in male rats.

The basolateral amygdala (BLA) is reliably activated by psychological stress and hyperactive in conditions of pathological stress or trauma; however, subsets of BLA neurons are also readily activated by rewarding stimuli and can suppress fear and avoidance behaviours. The BLA is highly heterogeneous anatomically, exhibiting continuous molecular and connectivity gradients throughout the entire structure. A critical gap remains in understanding the anatomical specificity of amygdala subregions, circuits, and cell types explicitly activated by acute stress and how they are dynamically activated throughout stimulus exposure. Using a combination of topographical mapping for the activity-responsive protein FOS and fiber photometry to measure calcium transients in real-time, we sought to characterize the spatial and temporal patterns of BLA activation in response to a range of novel stressors (shock, swim, restraint, predator odour) and non-aversive, but novel stimuli (crackers, citral odour). We report four main findings: (1) the BLA exhibits clear spatial activation gradients in response to novel stimuli throughout the medial-lateral and dorsal-ventral axes, with aversive stimuli strongly biasing activation towards medial aspects of the BLA; (2) novel stimuli elicit distinct temporal activation patterns, with stressful stimuli exhibiting particularly enhanced or prolonged temporal activation patterns; (3) changes in BLA activity are associated with changes in behavioural state; and (4) norepinephrine enhances stress-induced activation of BLA neurons via the ß-noradrenergic receptor. Moving forward, it will be imperative to combine our understanding of activation gradients with molecular and circuit-specificity.

Translational approaches to the neurobiological study of conditional discrimination and inhibition: Implications for psychiatric disease.

There is a growing number of studies investigating discriminatory fear conditioning and conditioned inhibition of fear to assess safety learning, in addition to extinction of cued fear. Despite all of these paradigms resulting in a reduction in fear expression, there are nuanced differences among them, which could be mediated through distinct behavioral and neural mechanisms. These differences could impact how we approach potential treatment options in clinical disorders with dysregulated fear responses. The objective of this review is to give an overview of the conditional discrimination and inhibition findings reported in both animal models and human neuropsychiatric disorders. Both behavioral and neural findings are reviewed among human and rodent studies that include conditional fear discrimination via conditional stimuli with and without reinforcement (CS+ vs. CS-, respectively) and/or conditional inhibition of fear through assessment of the fear response to a compound CS-/CS+ cue versus CS+. There are several parallels across species in behavioral fear expression as well as neural circuits promoting fear reduction in response to a CS- and/or CS-/CS+ compound cue. Continued and increased efforts to compare similar behavioral fear inhibition paradigms across species are needed to make breakthrough advances in our understanding and treatment approaches to individuals with fear disorders. (PsycInfo Database Record (c) 2024 APA, all rights reserved).

Bidirectional fear modulation by discrete anterior insular circuits in male mice.

The brain's ability to appraise threats and execute appropriate defensive responses is essential for survival in a dynamic environment. Humans studies have implicated the anterior insular cortex (aIC) in subjective fear regulation and its abnormal activity in fear/anxiety disorders. However, the complex aIC connectivity patterns involved in regulating fear remain under investigated. To address this, we recorded single units in the aIC of freely moving male mice that had previously undergone auditory fear conditioning, assessed the effect of optogenetically activating specific aIC output structures in fear, and examined the organization of aIC neurons projecting to the specific structures with retrograde tracing. Single-unit recordings revealed that a balanced number of aIC pyramidal neurons' activity either positively or negatively correlated with a conditioned tone-induced freezing (fear) response. Optogenetic manipulations of aIC pyramidal neuronal activity during conditioned tone presentation altered the expression of conditioned freezing. Neural tracing showed that non-overlapping populations of aIC neurons project to the amygdala or the medial thalamus, and the pathway bidirectionally modulated conditioned fear. Specifically, optogenetic stimulation of the aIC-amygdala pathway increased conditioned freezing, while optogenetic stimulation of the aIC-medial thalamus pathway decreased it. Our findings suggest that the balance of freezing-excited and freezing-inhibited neuronal activity in the aIC and the distinct efferent circuits interact collectively to modulate fear behavior.

How to reduce fear in a snail: Take an aspirin, call me in the morning

Aspirin (Acetylsalicylic acid, ASA), one of the widely used non-steroid anti-inflammatory drugs can easily end up in sewage effluents and thus it becomes necessary to investigate the effects of aspirin on behaviour of aquatic organisms. Previous studies in mammals have shown ASA to alter fear and anxiety-like behaviours. In the great pond snail Lymnaea stagnalis, ASA has been shown to block a ‘sickness state’ induced by lipopolysaccharide injection which upregulates immune and stress-related genes thus altering behavioural responses. In Lymnaea, eliciting physiological stress may enhance memory formation or block its retrieval depending on the stimulus type and intensity. Here we examine whether ASA will alter two forms of associative-learning memory in crayfish predator-experienced Lymnaea when ASA exposure accompanies predator-cue-induced stress during the learning procedure. The two trainings procedures are: 1) operant conditioning of aerial respiration; and 2) a higher form of learning, called configural learning, which here is dependent on evoking a fear response. We show here that ASA alone does not alter homeostatic aerial respiration, feeding behaviour or long-term memory (LTM) formation of operantly conditioned aerial respiration. However, ASA blocked the enhancement of LTM formation normally elicited by training snails in predator cue. ASA also blocked configural learning, which makes use of the fear response elicited by the predator cue. Thus, ASA alters how Lymnaea responds cognitively to predator detection.

Oxytocin receptor control social information about fear expression of others in mice

The social functions of oxytocin are diverse, and the specific aspects of information processing involved in emotional contagion remain unclear. We compared some fear-related behaviors among oxytocin receptor knockout mice and oxytocin-receptor-reduced mice with that of wild-type mice. In the observational fear assay, which reflects fear emotional contagion, mice that observed other individuals receiving electric shocks exhibited vicarious freezing. Mice with reduced or knockout oxytocin receptor expression showed reduced vicarious freezing. In the emotional discrimination assay, which reflects the ability to perceive others’ emotional cues, we compared approach and scent-sniffing behaviors toward fear and emotionally neutral individuals. While wild-type mice were able to detect the fear emotion of others, mice with reduced or knocked-out oxytocin receptors showed reduced discrimination ability. In the fear behavior assays, which do not present social cues, we did not find these differences in oxytocin receptor expression in the brain. These findings indicate that oxytocin plays a role in emotional contagion by perceiving the emotions of others.

Oral care interventions for autistic individuals: A systematic review.

Oral care-related challenges are well documented in the autistic community; dental care remains one of the most prevalent unmet health needs among autistic individuals. This review examined interventions designed to improve oral health in autistic individuals from children and adult populations. Through a systematic process, 36 studies were identified. These studies focused on improving home-based oral care skills and routines and reducing fear, anxiety, and/or negative behaviors in the dental clinic. Studies incorporated different types of techniques for facilitating oral care practice, including preparatory interventions to support home-based hygiene activities or improve an approaching dental encounter (n = 29), most often using visual aids, and/or strategies to manage behavioral difficulties exhibited in the dental office (n = 17). Some studies used both approaches (n = 10), combining visual aids prior to a visit with behavior management. Using an evidence-based rubric, we reviewed the methodological quality of the studies and found that most were only "adequate" (n = 8) or "weak" (n = 23) in reporting their evidence. This review has two key findings: (1) there is support for preparatory home-based visual interventions to improve toothbrushing and/or ready patients for dental visits; and (2) distraction or sensory-reducing interventions may also improve experiences in the dental clinic. Only one study purposefully recruited autistic adults, and no studies included intervention elements tailored to race/ethnicity, culture, and/or socioeconomic status. This review highlights the need for more studies investigating the impact of oral care-related interventions for autistic individuals of all ages and identifies a gap in interventions for autistic adults and those from minoritized populations.

Exploring the role of environmental enrichment and early life adversity on emotional development

Early life adversity has been linked with a higher probability of developing behavioral impairments and environmental manipulation is a strategy that may reduce the negative effects of exposure to adversity in early life. Here, we focused on exploring the influence of environmental enrichment (EE) as a protective factor in the context of early life adversity. We hypothesized that 24 hours of maternal deprivation (MD), in the second week of life, could induce anxiety-like behavior alterations and that exposure to EE could induce resilience to these behaviors due to alterations in the serotonergic system. Male Wistar rats were exposed to MD, on postnatal days 11 and 13, and to EE, after weaning. In adulthood, we performed a series of behavioral tests for fear, anxiety, and locomotor activity. We also measured the levels of serotonin in the amygdala and dorsal raphe nucleus. Our results revealed that MD does not impact fear behavior or the levels of serotonin, while EE decreases locomotor activity in a novel environment and enhances exploration in the predator odor test. EE also decreases serotonin in the amygdala and increases its turnover rate levels. Our findings provide insights into the critical timeframe during which stress exposure impacts the development and confirm that exposure to EE has an independent and protective effect for anxiety-like behaviors later in life.

A survey investigating owner perceptions and management of firework-associated fear in dogs in the Greater Sydney area.

To investigate the prevalence of firework-associated fear in dogs in Sydney, owner perception of their dog's response to fireworks, perceived efficacy of interventions to manage fearful behaviours and the frequency of dog owners seeking professional advice for these behaviours. Dog owners in the Greater Sydney area were invited to complete an anonymous online survey. From 387 valid responses, 44.4% (171 of 385) reported their dogs were fearful of fireworks. The most common fear-related behaviour was seeking an owner or caretaker (120 of 161, 74.5%). Most owners responded by bringing their dog inside or trying to comfort or reassure their dog. Only 22.5% of owners sought professional advice for their dog's fear of fireworks, but of these, 65.5% considered that advice to be effective. Source and breed group were significantly associated with fear of fireworks (P = 0.011, P = 0.036 respectively). Fear of fireworks was also significantly associated with fear of thunder (P < 0.0001), gunshots (P < 0.0001) and vehicles (P = 0.0009). Fear of fireworks and other loud noises negatively impacts canine welfare, yet only a small percentage of owners sought professional advice. There is scope for veterinarians to educate owners and raise awareness about the identification and management of noise-associated fear and reduce the risk of escalation of fearful behaviours.

"Nobody Here Likes Her"-The Impact of Parental Verbal Threat Information on Children's Fear of Strangers.

Parental verbal threat (vs. safety) information about strangers may induce fears of these strangers in adolescents. In this multi-method experimental study, utilizing a within-subject design, parents provided standardized verbal threat or safety information to their offspring (N=77, Mage=11.62 years, 42 girls) regarding two strangers in the lab. We also explored whether the impact of parental verbal threat information differs depending on the social anxiety levels of parents or fearful temperaments of adolescents. Adolescent's fear of strangers during social interaction tasks was assessed using cognitive (fear beliefs, attention bias), behavioral (observed avoidance and anxiety), and physiological (heart rate) indices. We also explored whether the impact of parental verbal threat information differs depending on the social anxiety levels of parents or fearful temperaments of adolescents. The findings suggest that a single exposure to parental verbal threat (vs. safety) information increased adolescent's self-reported fears about the strangers but did not increase their fearful behaviors, heart rate, or attentional bias. Furthermore, adolescents of parents with higher social anxiety levels or adolescents with fearful temperaments were not more strongly impacted by parental verbal threat information. Longitudinal research and studies investigating parents' naturalistic verbal expressions of threat are needed to expand our understanding of this potential verbal fear-learning pathway.

A prefrontal-habenular circuitry regulates social fear behaviour.

The medial prefrontal cortex (mPFC) has been implicated in the pathophysiology of social impairments including social fear. However, the precise subcortical partners that mediate mPFC dysfunction on social fear behaviour have not been identified. Employing a social fear conditioning paradigm, we induced robust social fear in mice and found that the lateral habenula (LHb) neurons and LHb-projecting mPFC neurons are synchronously activated during social fear expression. Moreover, optogenetic inhibition of the mPFC-LHb projection significantly reduced social fear responses. Importantly, consistent with animal studies, we observed an elevated prefrontal-habenular functional connectivity in subclinical individuals with higher social anxiety characterized by heightened social fear. These results unravel a crucial role of the prefrontal-habenular circuitry in social fear regulation and suggest that this pathway could serve as a potential target for the treatment of social fear symptom often observed in many psychiatric disorders.

Neuroethology: Fear outside the box

Behavioral neuroscience has successfully and in great detail deconstructed circuit mechanisms underlying fear behaviors using reductionist approaches. Recent research in more naturalistic settings now reveals additional higher-level organization, where hypothalamic circuits multiplex threat detection and fear memory updating to safely navigate complex environments.

Impact of early socialisation in foster care on kitten behaviour

ObjectivesThe critical socialisation period for kittens is estimated to be 2–7 weeks of age. Kittens reared in shelters often have poorer outcomes due to high rates of infectious disease. Therefore, many shelters are reliant on foster care programs to improve kitten survival rates and behaviour. However, little is known about the impact of specific early socialisation factors on kitten behaviour and health. MethodsDuring a 23-month period, 598 kitten foster carers completed a kitten socialisation questionnaire. The relationships between kitten weight/age at intake, days in care, singleton status, and the presence or absence of the queen during care on kitten behaviour and disease incidence were explored. Associations with exposure to noise, adults, children, location where the kitten was kept in the home, time spent alone, and ringworm treatment were also investigated. ResultsOverall being kept in social areas of the house with less noise/activity and spending some time alone was associated with positive kitten behaviour. There was no difference in fear or anxiety responses for bottle fed kittens, singletons and those without the queen. Ringworm treatment was associated with more fearful behaviour in kittens. Conclusionsand relevance: Future foster carers should be encouraged to keep kittens in social areas of the house, whilst also ensuring kittens can spend some time alone. If the home is loud, carers should slowly introduce the kitten/s to the louder and busier areas of the house before being housed there all the time. Training of carers to use more positive reinforcement techniques for administering ringworm treatment may benefit kitten behaviour.