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.

Periaqueductal gray activates antipredatory neural responses in the amygdala of foraging rats.

Pavlovian fear conditioning research suggests that the interaction between the dorsal periaqueductal gray (dPAG) and basolateral amygdala (BLA) acts as a prediction error mechanism in the formation of associative fear memories. However, their roles in responding to naturalistic predatory threats, characterized by less explicit cues and the absence of reiterative trial-and-error learning events, remain unexplored. In this study, we conducted single-unit recordings in rats during an 'approach food-avoid predator' task, focusing on the responsiveness of dPAG and BLA neurons to a rapidly approaching robot predator. Optogenetic stimulation of the dPAG triggered fleeing behaviors and increased BLA activity in naive rats. Notably, BLA neurons activated by dPAG stimulation displayed immediate responses to the robot, demonstrating heightened synchronous activity compared to BLA neurons that did not respond to dPAG stimulation. Additionally, the use of anterograde and retrograde tracer injections into the dPAG and BLA, respectively, coupled with c-Fos activation in response to predatory threats, indicates that the midline thalamus may play an intermediary role in innate antipredatory defensive functioning.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment.

We show that nocturnal aversive stimuli presented to mice while they are eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We also show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus, the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our findings support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders are, in part, the output of a fear-entrained clock, and provide a mechanistic insight into this clock.

The central amygdala modulates distinctive conflict-like behaviors in a naturalistic foraging task.

Conflict situations elicit a diverse range of behaviors that extend beyond the simplistic approach or avoidance dichotomy. However, many conflict-related studies have primarily focused on approach suppression, neglecting the complexity of these behaviors. In our study, we exposed rats to a semi-naturalistic foraging task, presenting them with a trade-off between a food reward and a predatory threat posed by a robotic agent. We observed that rats displayed two conflict-like behaviors (CLBs)-diagonal approach and stretched posture-when facing a robotic predator guarding a food pellet. After electrolytic lesions to the central amygdala (CeA), both conflict behaviors were significantly reduced, accompanied by a decrease in avoidance behavior (hiding) and an increase in approach behavior (frequency of interactions with the robot). A significant negative correlation between avoidance and approach behaviors emerged after the CeA lesion; however, our data suggest that CLBs are not tightly coupled with either approach or avoidance behaviors, showing no significant correlation to those behaviors. Our findings indicate that the CeA plays a crucial role in modulating conflict behaviors, competing with approach suppression in risky situations.

The circadian molecular clock in the suprachiasmatic nucleus is necessary but not sufficient for fear entrainment in the mouse.

Nocturnal aversive stimuli presented to mice during eating and drinking outside of their safe nest can entrain circadian behaviors, leading to a shift toward daytime activity. We show that the canonical molecular circadian clock is necessary for fear entrainment and that an intact molecular clockwork in the suprachiasmatic nucleus (SCN), the site of the central circadian pacemaker, is necessary but not sufficient to sustain fear entrainment of circadian rhythms. Our results demonstrate that entrainment of a circadian clock by cyclic fearful stimuli can lead to severely mistimed circadian behavior that persists even after the aversive stimulus is removed. Together, our results support the interpretation that circadian and sleep symptoms associated with fear and anxiety disorders may represent the output of a fear-entrained clock. One-Sentence Summary: Cyclic fearful stimuli can entrain circadian rhythms in mice, and the molecular clock within the central circadian pacemaker is necessary but not sufficient for fear-entrainment.

The nature and neurobiology of fear and anxiety: State of the science and opportunities for accelerating discovery.

Fear and anxiety play a central role in mammalian life, and there is considerable interest in clarifying their nature, identifying their biological underpinnings, and determining their consequences for health and disease. Here we provide a roundtable discussion on the nature and biological bases of fear- and anxiety-related states, traits, and disorders. The discussants include scientists familiar with a wide variety of populations and a broad spectrum of techniques. The goal of the roundtable was to take stock of the state of the science and provide a roadmap to the next generation of fear and anxiety research. Much of the discussion centered on the key challenges facing the field, the most fruitful avenues for future research, and emerging opportunities for accelerating discovery, with implications for scientists, funders, and other stakeholders. Understanding fear and anxiety is a matter of practical importance. Anxiety disorders are a leading burden on public health and existing treatments are far from curative, underscoring the urgency of developing a deeper understanding of the factors governing threat-related emotions.

Neurocognitive effects of stress: a metaparadigm perspective.

Stressful experiences, both physical and psychological, that are overwhelming (i.e., inescapable and unpredictable), can measurably affect subsequent neuronal properties and cognitive functioning of the hippocampus. At the cellular level, stress has been shown to alter hippocampal synaptic plasticity, spike and local field potential activity, dendritic morphology, neurogenesis, and neurodegeneration. At the behavioral level, stress has been found to impair learning and memory for declarative (or explicit) tasks that are based on cognition, such as verbal recall memory in humans and spatial memory in rodents, while facilitating those that are based on emotion, such as differential fear conditioning in humans and contextual fear conditioning in rodents. These vertically related alterations in the hippocampus, procedurally observed after subjects have undergone stress, are generally believed to be mediated by recurrently elevated circulating hypothalamic-pituitary-adrenal (HPA) axis effector hormones, glucocorticoids, directly acting on hippocampal neurons densely populated with corticosteroid receptors. The main purposes of this review are to (i) provide a synopsis of the neurocognitive effects of stress in a historical context that led to the contemporary HPA axis dogma of basic and translational stress research, (ii) critically reappraise the necessity and sufficiency of the glucocorticoid hypothesis of stress, and (iii) suggest an alternative metaparadigm approach to monitor and manipulate the progression of stress effects at the neural coding level. Real-time analyses can reveal neural activity markers of stress in the hippocampus that can be used to extrapolate neurocognitive effects across a range of stress paradigms (i.e., resolve scaling and dichotomous memory effects issues) and understand individual differences, thereby providing a novel neurophysiological scaffold for advancing future stress research.

Chemogenetic modulation of the medial prefrontal cortex regulates resistance to acute stress-induced cognitive impairments.

The medial prefrontal cortex (mPFC) has been implicated in regulating resistance to the effects of acute uncontrollable stress. We previously showed that mPFC-lesioned animals exhibit impaired object recognition memory after acute exposure to a brief stress that had no effect in normal animals. Here, we used designer receptors exclusively activated by designer drugs to determine how modulating mPFC activity affects recognition-memory performance under stressful conditions. Specifically, animals with chemogenetic excitation or inhibition of the mPFC underwent either a brief ineffective stress (20-min restraint + 20 tail shocks) or a prolonged effective stress (60-min restraint + 60 tail shocks). Subsequent recognition memory tests showed that animals with chemogenetic mPFC inhibition exposed to brief stress showed impairment in an object recognition memory task, whereas those with chemogenetic mPFC excitation exposed to prolonged stress did not. Thus, the present findings the decreased mPFC activity exacerbates acute stress effects on memory function whereas increased mPFC activity counters these stress effects provide evidence that the mPFC bidirectionally modulates stress resistance.

Ecological analysis of Pavlovian fear conditioning in rats.

Pavlovian fear conditioning, which offers the advantage of simplicity in both the control of conditional and unconditional stimuli (CS, US) presentation and the analysis of specific conditional and unconditional responses (CR, UR) in a controlled laboratory setting, has been the standard model in basic and translational fear research. Despite 100 years of experiments, the utility of fear conditioning has not been trans-situationally validated in real-life contexts. We thus investigated whether fear conditioning readily occurs and guides the animal's future behavior in an ecologically-relevant environment. To do so, Long-Evans rats foraging for food in an open arena were presented with a tone CS paired with electric shock US to their dorsal neck/body that instinctively elicited escape UR to the safe nest. On subsequent test days, the tone-shock paired animals failed to exhibit fear CR to the CS. In contrast, animals that encountered a realistic agent of danger (a looming artificial owl) paired with a shock, simulating a plausible predatory strike, instantly fled to the nest when presented with a tone for the first time. These results highlight the possibility of a nonassociative, rather than standard associative, fear process providing survival function in life-threatening situations that animals are likely to encounter in nature.

A role of anterior cingulate cortex in the emergence of worker-parasite relationship.

We studied the brain mechanisms underlying action selection in a social dilemma setting in which individuals' effortful gains are unfairly distributed among group members. A stable "worker-parasite" relationship developed when three individually operant-conditioned rats were placed together in a Skinner box equipped with response lever and food dispenser on opposite sides. Specifically, one rat, the "worker," engaged in lever-pressing while the other two "parasitic" rats profited from the worker's effort by crowding the feeder in anticipation of food. Anatomically, c-Fos expression in the anterior cingulate cortex (ACC) was significantly higher in worker rats than in parasite rats. Functionally, ACC inactivation suppressed the worker's lever-press behavior drastically under social, but only mildly under individual, settings. Transcriptionally, GABAA receptor- and potassium channel-related messenger RNA expressions were reliably lower in the worker's, relative to parasite's, ACC. These findings indicate the requirement of ACC activation for the expression of exploitable, effortful behavior, which could be mediated by molecular pathways involving GABAA receptor/potassium channel proteins.

'Fearful-place' coding in the amygdala-hippocampal network.

Animals seeking survival needs must be able to assess different locations of threats in their habitat. However, the neural integration of spatial and risk information essential for guiding goal-directed behavior remains poorly understood. Thus, we investigated simultaneous activities of fear-responsive basal amygdala (BA) and place-responsive dorsal hippocampus (dHPC) neurons as rats left the safe nest to search for food in an exposed space and encountered a simulated 'predator.' In this realistic situation, BA cells increased their firing rates and dHPC place cells decreased their spatial stability near the threat. Importantly, only those dHPC cells synchronized with the predator-responsive BA cells remapped significantly as a function of escalating risk location. Moreover, optogenetic stimulation of BA neurons was sufficient to cause spatial avoidance behavior and disrupt place fields. These results suggest a dynamic interaction of BA's fear signalling cells and dHPC's spatial coding cells as animals traverse safe-danger areas of their environment.

Corrigendum: The Risky Closed Economy: A Holistic, Longitudinal Approach to Studying Fear and Anxiety in Rodents.

[This corrects the article DOI: 10.3389/fnbeh.2020.594568.].

The Risky Closed Economy: A Holistic, Longitudinal Approach to Studying Fear and Anxiety in Rodents.

Basic research of fear and anxiety in rodents has historically utilized a limited set of behavioral paradigms, for example, Pavlovian (classical) fear conditioning, the elevated plus-maze, or inhibitory (passive) avoidance. These traditional paradigms measure a limited selection of variables over a short duration, providing only a "snapshot" of fear and anxiety-related behavior. Overreliance on these paradigms and such behavioral snapshots ultimately lead to a narrow understanding of these complex motivational states. Here, we elaborate on the closed economy; a seldom-used paradigm that has been modified to comprehensively study fear and anxiety-related behavior and neurocircuitry in rodents. In this modified "Risky Closed Economy (RCE)" paradigm, animals live nearly uninterrupted in behavioral chambers where the need to acquire food and water and avoid threat is integrated into the task. Briefly, animals are free to acquire all of their food and water in a designated foraging zone. An unsignaled, unpredictable threat (footshock) is introduced into the foraging zone after a baseline activity and consumption period to model the risk of predation, which is then removed for a final extinction assessment. This longitudinal design, wherein data from a multitude of variables are collected automatically and continuously for 23 h/day over several weeks to months, affords a more holistic understanding of the effects of fear and anxiety on day-to-day behavior. Also, we discuss its general benefits relevant to other topics in neuroscience research, its limitations, and present data demonstrating for the first time The Risky Closed Economy's viability in mice.

Brief stress impairs recognition memory through amygdalar activation in animals with medial prefrontal cortex lesions.

The medial prefrontal cortex (mPFC) is thought to exert inhibitory control over stress-induced activation of the amygdala and neurocognitive effects. As evidence to support this, we examined how exposure to either a brief or prolonged stress affected on amygdalar c-Fos levels and recognition memory of animals with mPFC chemical lesions. mPFC-lesioned and sham-operated animals were subjected to either a brief 20-min restraint+20 tailshocks or a prolonged 60-min restraint+60 tailshocks. Post-stress performances in the object recognition memory and c-Fos immunoreactivity in the amygdala were then assessed. In sham-operated animals, the object recognition memory was reliably impaired following the prolonged, but not following the brief stress exposure. On the other hand, in mPFC-lesioned animals, the brief stress significantly impaired recognition memory and enhanced c-Fos expression in the amygdala. Present findings of loss of mPFC activity exacerbating stress effects provide causal evidence that the mPFC exerts inhibitory control on stress.

Amygdala, Medial Prefrontal Cortex and Glucocorticoid Interactions Produce Stress-Like Effects on Memory.

Adverse stress effects on the hippocampal memory system are generally thought to be due to the high level of circulating glucocorticoids directly modifying the properties of hippocampal neurons and, accordingly, the results should be reproducible with exogenous administration of cortisol in humans and corticosterone in rodents. However, glucocorticoid levels increased to other events, such as exercise and environment enrichment, do not impair but instead enhance hippocampal memory, indicating that cortisol/corticosterone are not invariant causal factors of stress. To better model the complex psychophysiological attributes of stress (i.e., aversiveness, lack of controllability, and glucose metabolism), we examined the functions of the amygdala, medial prefrontal cortex (mPFC), and corticosterone on a hippocampal-based one-trial novel object recognition (OR) memory task in rats. Specifically, animals were subjected to amygdala stimulation, mPFC inactivation, and corticosterone treatments separately or in combination during behavioral testing. Collective amygdala, mPFC, and corticosterone manipulations significantly impaired OR memory comparable to behavioral stress. By contrast, single and dual treatments failed to reliably decrease memory functioning. These results suggest that negative mnemonic impacts of uncontrollable stress involve the amalgamation of heightened amygdala and diminished mPFC activities, and elevated circulating corticosterone level.

Sex Differences in Foraging Rats to Naturalistic Aerial Predator Stimuli.

Rodents in the wild are under nearly constant threat of aerial predation and thus have evolved adaptive innate defensive behaviors, such as freezing or fleeing, in response to a perceived looming threat. Here we employed an ethologically relevant paradigm to study innate fear of aerial predators in male and female rats during a goal-oriented task. Rats foraging for food in a large arena encountered either a 2D or 3D looming stimulus, to which they instinctively fled back to a safe nest. When facing a direct aerial threat, female rats exhibited a greater fear response than males and this divergence maintained when exposed to the environment on subsequent days with no predator interaction, suggesting stronger contextual fear in female rats. These results may have relevance toward exploring neurobiological mechanisms associated with higher diagnosis rates of fear and anxiety-related disorders in women as compared with men.

Fear paradigms: The times they are a-changin'.

Fear is considered an integral part of the brain's defensive mechanism that evolved to protect animals and humans from predation and other ecological threats. Hence, it is logical to study fear from the perspective of antipredator-survival behaviors and circuits by sampling a range of threatening situations that organisms are likely to encounter in the wild. In the past several decades, however, mainstream fear research has focused on the importance of associative learning; that is, how animals become frightened of innocuous cues as consequences of their contingent pairing with aversive events. While significant discoveries have been made, contemporary fear models derived from learning studies are likely to provide only a partial picture of the brain's fear system because they cannot simulate the dynamic range of risky situations in nature that require various adaptive actions and decisions. This review considers two different approaches to study fear, grounded on behaviorism and ethology and examines their contributions in revealing the naturalistic workings of fear in guiding and shaping behavior as animals make real-world choices.

Brain stimulation patterns emulating endogenous thalamocortical input to parvalbumin-expressing interneurons reduce nociception in mice.

BACKGROUND: The bursting pattern of thalamocortical (TC) pathway dampens nociception. Whether brain stimulation mimicking endogenous patterns can engage similar sensory gating processes in the cortex and reduce nociceptive behaviors remains uninvestigated. OBJECTIVE: We investigated the role of cortical parvalbumin expressing (PV) interneurons within the TC circuit in gating nociception and their selective response to TC burst patterns. We then tested if transcranial magnetic stimulation (TMS) patterned on endogenous nociceptive TC bursting modulate nociceptive behaviors. METHODS: The switching of TC neurons between tonic (single spike) and burst (high frequency spikes) firing modes may be a critical component in modulating nociceptive signals. Deep brain electrical stimulation of TC neurons and immunohistochemistry were used to examine the differential influence of each firing mode on cortical PV interneuron activity. Optogenetic stimulation of cortical PV interneurons assessed a direct role in nociceptive modulation. A new TMS protocol mimicking thalamic burst firing patterns, contrasted with conventional continuous and intermittent theta burst protocols, tested if TMS patterned on endogenous TC activity reduces nociceptive behaviors in mice. RESULTS: Immunohistochemical evidence confirmed that burst, but not tonic, deep brain stimulation of TC neurons increased the activity of PV interneurons in the cortex. Both optogenetic activation of PV interneurons and TMS protocol mimicking thalamic burst reduced nociceptive behaviors. CONCLUSIONS: Our findings suggest that burst firing of TC neurons recruits PV interneurons in the cortex to reduce nociceptive behaviors and that neuromodulation mimicking thalamic burst firing may be useful for modulating nociception.

Dynamic coding of predatory information between the prelimbic cortex and lateral amygdala in foraging rats.

Predation is considered a major selective pressure in the evolution of fear, but the neurophysiology of predator-induced fear is unknown. We simultaneously recorded lateral amygdala (LA) and prelimbic (PL) area neuronal activities as rats exited a safe nest to search for food in an open space before, during, and after encountering a "predator" robot programmed to surge from afar. Distinct populations of LA neurons transiently increased spiking as rats either advanced or fled the robot, whereas PL neurons showed longer-lasting spike trains that preceded and persisted beyond LA activity. Moreover, discrete LA-PL cell pairs displayed correlated firings only when the animals either approached or fled the robot. These results suggest a general fear function of the LA-PL circuit where the PL participates in the initial detection of potential threats, the LA signals the occurrence of real threats, and the dynamic LA-PL interaction optimizes defensive readiness for action.