Conspecific interactions predict social transmission of fear in female rats.

Social transmission of fear occurs in a subset of individuals, where an Observer displays a fear response to a previously neutral stimulus after witnessing or interacting with a conspecific Demonstrator during memory retrieval. The conditions under which fear can be acquired socially in rats have received attention in recent years, and suggest that social factors modulate social transmission of information. We previously found that one such factor, social rank, impacts fear conditioning by proxy in male rats. Here, we aimed to investigate whether social roles as determined by nape contacts in females, might also have an influence on social transmission of fear. In-line with previous findings in males, we found that social interactions in the home cage can provide insight into the social relationship between female rats and that these relationships predict the degree of fear acquired by-proxy. These results suggest that play behavior affects the social transfer/transmission of information in female rats.

A distinct cortical code for socially learned threat.

Animals can learn about sources of danger while minimizing their own risk by observing how others respond to threats. However, the distinct neural mechanisms by which threats are learned through social observation (known as observational fear learning1-4 (OFL)) to generate behavioural responses specific to such threats remain poorly understood. The dorsomedial prefrontal cortex (dmPFC) performs several key functions that may underlie OFL, including processing of social information and disambiguation of threat cues5-11. Here we show that dmPFC is recruited and required for OFL in mice. Using cellular-resolution microendoscopic calcium imaging, we demonstrate that dmPFC neurons code for observational fear and do so in a manner that is distinct from direct experience. We find that dmPFC neuronal activity predicts upcoming switches between freezing and moving state elicited by threat. By combining neuronal circuit mapping, calcium imaging, electrophysiological recordings and optogenetics, we show that dmPFC projections to the midbrain periaqueductal grey (PAG) constrain observer freezing, and that amygdalar and hippocampal inputs to dmPFC opposingly modulate observer freezing. Together our findings reveal that dmPFC neurons compute a distinct code for observational fear and coordinate long-range neural circuits to select behavioural responses.

Sex Differences In Avoidance Extinction After Contextual Fear Conditioning: Anxioescapic Behavior In Female Rats.

Fear memories are important for survival and are implicated in the etiology of fear disorders such as Post Traumatic Stress Disorder (PTSD). Fear memories are well studied pre-clinically and sex differences in rodent fear expression have been reported: females tend to freeze less than males. Whether this is a difference in fear learning or expression is debated. We aimed to differentiate between these possibilities with a task that allowed female rats to express fear memory by moving, rather than freezing. We assessed fear extinction after contextual fear conditioning in the isolated Shock Arm of a Y-maze in female and male rats by either placing them back in the isolated Shock Arm (Fear Extinction in the Shock Context) or allowing them to move freely in the Y-maze during extinction training and enter/avoid the Shock Arm (Avoidance Extinction). We confirmed that female rats freeze less than males during fear extinction in both settings. During Avoidance Extinction, however, both sexes had similar avoidance of the Shock Context, showing comparable fear memory and extinction. Additionally, female rats made more entries into the non-shock arms. Thus, female and male rats have similar fear learning but females express it with an active motor response. Furthermore, female rats also exhibited an active motor response under other anxiogenic conditions (Elevated Plus Maze) and had higher reactivity (Acoustic Startle Response) but not when fear-eliciting stimuli were present: cat hair and foot-shock. In summary, female rats have an active motor response to anxiogenic stimuli which we termed 'Anxioescapic' behavior strategy.

Formation of False Context Fear Memory Is Regulated by Hypothalamic Corticotropin-Releasing Factor in Mice.

Traumatic events frequently produce false fear memories. We investigated the effect of hypothalamic corticotropin-releasing factor (CRF) knockdown (Hy-Crf-KD) or overexpression (Hy-CRF-OE) on contextual fear memory, as fear stress-released CRF and hypothalamic-pituitary-adrenal axis activation affects the memory system. Mice were placed in a chamber with an electric footshock as a conditioning stimulus (CS) in Context A, then exposed to a novel chamber without CS, as Context B, at 3 h (B-3h) or 24 h (B-24h). The freezing response in B-3h was intensified in the experimental mice, compared to control mice not exposed to CS, indicating that a false fear memory was formed at 3 h. The within-group freezing level at B-24h was higher than that at B-3h, indicating that false context fear memory was enhanced at B-24h. The difference in freezing levels between B-3h and B-24h in Hy-Crf-KD mice was larger than that of controls. In Hy-CRF-OE mice, the freezing level at B-3h was higher than that of control and Hy-Crf-KD mice, while the freezing level in B-24h was similar to that in B-3h. Locomotor activity before CS and freezing level during CS were similar among the groups. Therefore, we hypothesized that Hy-Crf-KD potentiates the induction of false context fear memory, while Hy-CRF-OE enhances the onset of false fear memory formation.

High-sugar/high-fat diet modulates the effects of chronic stress in cariocas high- and low-conditioned freezing rats.

Stress and eating disorders are closely related and are a topic of major concern due to their burden on human health. Engaging in unhealthy eating habits may come as a result of stress, and it often serves to alleviate the symptoms of anxiety or as a distraction from the stressor itself or self-awareness. However, it can also lead to negative feelings of a person's body figure, guilty, or shame. As diverse as these consequences are in humans, so are the effects of the combined administration of stress and hypercaloric food in animals' models. In this study, we assessed the influence of individual innate behavioral predisposition on the effects of chronic unpredictable mild stress and the dietary supplementation with high-sugar/high-fat food. These conditions were applied to male Carioca low- and high-conditioned freezing (CLF and CHF) rats for 21 days. Behavioral results show that the hypercaloric supplement had a protective effect over the alterations caused by the stress. Notably, it was more strongly observed in CHF rather than CLF animals. As the chronic stress led to an impaired behavior in the contextual fear conditioning and the forced swimming tests in the CLF line, animals fed with the HSHF pellet scored responses similar to their untreated control. On CHF rats, these effects also were seen to a broader extent on the open field test, where the locomotor behavior was also increased. No major effects of the diet were seen in the unstressed groups. Overall, our results show that the influences of both chronic stress and hypercaloric feeding depend on innate differences in fear response traits of male Carioca rats.

Understanding sex differences in zebrafish pain- and fear-related behaviors.

Sex is an important variable in translational biomedical research. While overt sex differences have been reported for pain and fear-like behaviors in humans and rodents, these differences in other popular model organisms, such as zebrafish, remain poorly understood. Here, we evaluate potential sex differences in zebrafish behavioral responses to pain (intraperitoneal administration of 5% acetic acid) and fear stimuli (exposure to alarm substance). Overall, both male and female zebrafish exposed to pain (acetic acid injection) show lesser distance traveled, fewer top entries and more writhing-like pain-related behavior vs. controls, whereas female fish more robustly (than males) altered some other pain-like behaviors (e.g., increasing freezing episodes and time in top) in this model. In contrast, zebrafish of both sexes responded equally strongly to fear evoked by acute alarm substance exposure. Collectively, these findings emphasize the growing importance of studying sex differences in zebrafish behavioral and pain models.

Comparative metabolic profiling of posterior parietal cortex, amygdala, and hippocampus in conditioned fear memory.

Fear conditioning and retrieval are suitable models to investigate the biological basis of various mental disorders. Hippocampus and amygdala neurons consolidate conditioned stimulus (CS)-dependent fear memory. Posterior parietal cortex is considered important for the CS-dependent conditioning and retrieval of fear memory. Metabolomic screening among functionally related brain areas provides molecular signatures and biomarkers to improve the treatment of psychopathologies. Herein, we analyzed and compared changes of metabolites in the hippocampus, amygdala, and posterior parietal cortex under the fear retrieval condition. Metabolite profiles of posterior parietal cortex and amygdala were similarly changed after fear memory retrieval. While the retrieval of fear memory perturbed various metabolic pathways, most metabolic pathways that overlapped among the three brain regions had high ranks in the enrichment analysis of posterior parietal cortex. In posterior parietal cortex, the most perturbed pathways were pantothenate and CoA biosynthesis, purine metabolism, glutathione metabolism, and NAD+ dependent signaling. Metabolites of posterior parietal cortex including 4'-phosphopantetheine, xanthine, glutathione, ADP-ribose, ADP-ribose 2'-phosphate, and cyclic ADP-ribose were significantly regulated in these metabolic pathways. These results point to the importance of metabolites of posterior parietal cortex in conditioned fear memory retrieval and may provide potential biomarker candidates for traumatic memory-related mental disorders.

Functional organization of the midbrain periaqueductal gray for regulating aversive memory formation.

Innately aversive experiences produce rapid defensive responses and powerful emotional memories. The midbrain periaqueductal gray (PAG) drives defensive behaviors through projections to brainstem motor control centers, but the PAG has also been implicated in aversive learning, receives information from aversive-signaling sensory systems and sends ascending projections to the thalamus as well as other forebrain structures which could control learning and memory. Here we sought to identify PAG subregions and cell types which instruct memory formation in response to aversive events. We found that optogenetic inhibition of neurons in the dorsolateral subregion of the PAG (dlPAG), but not the ventrolateral PAG (vlPAG), during an aversive event reduced memory formation. Furthermore, inhibition of a specific population of thalamus projecting dlPAG neurons projecting to the anterior paraventricular thalamus (aPVT) reduced aversive learning, but had no effect on the expression of previously learned defensive behaviors. By contrast, inactivation of dlPAG neurons which project to the posterior PVT (pPVT) or centromedial intralaminar thalamic nucleus (CM) had no effect on learning. These results reveal specific subregions and cell types within PAG responsible for its learning related functions.

Ventral hippocampus mediates the context-dependence of two-way signaled avoidance in male rats.

Considerable work indicates that instrumental responding is context-dependent, but the neural mechanisms underlying this phenomenon are poorly understood. Given the important role for the hippocampal formation in contextual processing, we hypothesized that reversible inactivation of the hippocampus would impair the context-dependence of active avoidance. To test this hypothesis, we used a two-way signaled active avoidance (SAA) task that requires rats to shuttle across a divided chamber during a tone CS in order to avoid a footshock US. After training, avoidance responding was assessed in an extinction test in both the training context and a novel context in a counterbalanced order. Rats performed significantly more avoidance responses in the training context than in the novel context, demonstrating the context-dependence of shuttle avoidance behavior. To examine the role of the hippocampus in the context-dependence of SAA, we reversibly inactivated either the dorsal (DH) or ventral hippocampus (VH) prior to testing. Inactivation of the VH eliminated the context-dependence of SAA and elevated avoidance responding in the novel context to levels similar to that expressed in the training context. In contrast, DH inactivation had no effect on avoidance in either context, and neither manipulation affected freezing behavior. Therefore, the integrity of the VH, but not DH, is required for the expression of the context-dependence of avoidance behavior.

OFF-transient alpha RGCs mediate looming triggered innate defensive response.

Animals respond to visual threats, such as a looming object, with innate defensive behaviors. Here, we report that a specific type of retinal ganglion cell (RGC), the OFF-transient alpha RGC, is critical for the detection of looming objects. We identified Kcnip2 as its molecular marker. The activity of the Kcnip2-expressing RGCs encodes the size of the looming object. Ablation or suppression of these RGCs abolished or severely impaired the escape and freezing behaviors of mice in response to a looming object, while activation of their somas in the retina, or their axon terminals in the superior colliculus, triggered immediate escape behavior. Our results link the activity of a single type of RGC to visually triggered innate defensive behaviors and underscore that ethologically significant visual information is encoded by a labeled line strategy as early as in the retina.

Corticostriatal control of defense behavior in mice induced by auditory looming cues.

Animals exhibit innate defense behaviors in response to approaching threats cued by the dynamics of sensory inputs of various modalities. The underlying neural circuits have been mostly studied in the visual system, but remain unclear for other modalities. Here, by utilizing sounds with increasing (vs. decreasing) loudness to mimic looming (vs. receding) objects, we find that looming sounds elicit stereotypical sequential defensive reactions: freezing followed by flight. Both behaviors require the activity of auditory cortex, in particular the sustained type of responses, but are differentially mediated by corticostriatal projections primarily innervating D2 neurons in the tail of the striatum and corticocollicular projections to the superior colliculus, respectively. The behavioral transition from freezing to flight can be attributed to the differential temporal dynamics of the striatal and collicular neurons in their responses to looming sound stimuli. Our results reveal an essential role of the striatum in the innate defense control.

A Novel Pavlovian Fear Conditioning Paradigm to Study Freezing and Flight Behavior.

Fear- and anxiety-related behaviors significantly contribute to an organism's survival. However, exaggerated defensive responses to perceived threat are characteristic of various anxiety disorders, which are the most prevalent form of mental illness in the United States. Discovering the neurobiological mechanisms responsible for defensive behaviors will aid in the development of novel therapeutic interventions. Pavlovian fear conditioning is a widely used laboratory paradigm to study fear-related learning and memory. A major limitation of traditional Pavlovian fear conditioning paradigms is that freezing is the only defensive behavior monitored. We recently developed a modified Pavlovian fear conditioning paradigm that allows us to study both conditioned freezing and flight (also known as escape) behavior within individual subjects. This model employs higher intensity footshocks and a greater number of pairings between the conditioned stimulus and unconditioned stimulus. Additionally, this conditioned flight paradigm utilizes serial presentation of pure tone and white noise auditory stimuli as the conditioned stimulus. Following conditioning in this paradigm, mice exhibit freezing behavior in response to the tone stimulus, and flight responses during the white noise. This conditioning model can be applied to the study of rapid and flexible transitions between behavioral responses necessary for survival.

Fear response-based prediction for stress susceptibility to PTSD-like phenotypes.

Most individuals undergo traumatic stresses at some points in their life, but only a small proportion develop stress-related disorders such as anxiety diseases and posttraumatic stress disorder (PTSD). Although stress susceptibility is one determinant of mental disorders, the underlying mechanisms and functional implication remain unclear yet. We found that an increased amount of freezing that animals exhibited in the intertrial interval (ITI) of a stress-enhanced fear learning paradigm, predicts ensuing PTSD-like symptoms whereas resilient mice show ITI freezing comparable to that of unstressed mice. To examine the behavioral features, we developed a systematic analytical approach for ITI freezing and stress susceptibility. Thus, we provide a behavioral parameter for prognosis to stress susceptibility of individuals in the development of PTSD-like symptoms as well as a new mathematical means to scrutinize freezing behavior.

Involvement of the lateral habenula in fear memory.

Increasing evidence points to the engagement of the lateral habenula (LHb) in the selection of appropriate behavioral responses in aversive situations. However, very few data have been gathered with respect to its role in fear memory formation, especially in learning paradigms in which brain areas involved in cognitive processes like the hippocampus (HPC) and the medial prefrontal cortex (mPFC) are required. A paradigm of this sort is trace fear conditioning, in which an aversive event is preceded by a discrete stimulus, generally a tone, but without the close temporal contiguity allowing for their association based on amygdala-dependent information processing. In a first experiment, we analyzed cellular activations (c-Fos expression) induced by trace fear conditioning in subregions of the habenular complex, HPC, mPFC and amygdala using a factorial analysis to unravel functional networks through correlational analysis of data. This analysis suggested that distinct LHb subregions engaged in different aspects of conditioning, e.g. associative processes and onset of fear responses. In a second experiment, we performed chemogenetic LHb inactivation during the conditioning phase of the trace fear conditioning paradigm and subsequently assessed contextual and tone fear memories. Whereas LHb inactivation did not modify rat's behavior during conditioning, it induced contextual memory deficits and enhanced fear to the tone. These results demonstrate the involvement of the LHb in fear memory. They further suggest that the LHb is engaged in learning about threatening environments through the selection of relevant information predictive of a danger.

Serotonergic treatment normalizes midbrain dopaminergic neuron increase after periaqueductal gray stimulation.

Electrical stimulation of the dorsolateral periaqueductal gray (dlPAG) in rats has been shown to elicit panic-like behaviour and can be a useful as an unconditioned stimulus for modelling anticipatory fear and agoraphobia in a contextual fear conditioning paradigm. In this study, we further analysed our previous data on the effects of escitalopram (a selective serotonin reuptake inhibitor, SSRI) and buspirone (a 5-HT1A receptor partial agonist) on dlPAG-induced anticipatory fear behaviour in a rat model using freezing as a measure. We then attempted to unravel some of the interactions with dopamine signalling using tyrosine hydroxylase (TH) immunohistochemistry to probe the effects on dopaminergic neurons. We showed that acute treatment of escitalopram, but not buspirone, was effective in reducing anticipatory freezing behaviour, while chronic administrations of both drugs were effective. We found that the dlPAG stimulation induced increase number of dopaminergic neurons in the ventral tegmental area (VTA) which was reversed in both chronic buspirone and escitalopram groups. We further found a strong positive correlation between the number of dopaminergic neurons and freezing in the VTA and showed positive correlations between dopaminergic neurons in the VTA and substantia nigra pars compacta (SNpc) in escitalopram and buspirone groups, respectively. Overall, we showed that chronic treatment with an SSRI and a 5-HT1A agonist reduced anticipatory freezing behaviour which seems to be associated, through correlative studies, with a reversal of dlPAG stimulation induced increase in number of dopaminergic neurons in the VTA and/or SNpc.

Prefrontal cortex projections to the nucleus reuniens suppress freezing following two-way signaled avoidance training.

Signaled active avoidance (SAA) behavior requires the suppression of defensive reactions, such as freezing, that conflict with the avoidance response. The neural mechanisms of this inhibitory process are not well understood. Here, we demonstrate that ventromedial prefrontal cortex projections to the nucleus reuniens of the thalamus are recruited following SAA training to suppress freezing in rats. This projection may serve as a crucial common pathway for the inhibition of innate defensive reactions that interfere with proactive behavior, thus facilitating adaptive coping.

Kv4.1, a Key Ion Channel For Low Frequency Firing of Dentate Granule Cells, Is Crucial for Pattern Separation.

The dentate gyrus (DG) in the hippocampus may play key roles in remembering distinct episodes through pattern separation, which may be subserved by the sparse firing properties of granule cells (GCs) in the DG. Low intrinsic excitability is characteristic of mature GCs, but ion channel mechanisms are not fully understood. Here, we investigated ionic channel mechanisms for firing frequency regulation in hippocampal GCs using male and female mice, and identified Kv4.1 as a key player. Immunofluorescence analysis showed that Kv4.1 was preferentially expressed in the DG, and its expression level determined by Western blot analysis was higher at 8-week than 3-week-old mice, suggesting a developmental regulation of Kv4.1 expression. With respect to firing frequency, GCs are categorized into two distinctive groups: low-frequency (LF) and high-frequency (HF) firing GCs. Input resistance (Rin) of most LF-GCs is lower than 200 MΩ, suggesting that LF-GCs are fully mature GCs. Kv4.1 channel inhibition by intracellular perfusion of Kv4.1 antibody increased firing rates and gain of the input-output relationship selectively in LF-GCs with no significant effect on resting membrane potential and Rin, but had no effect in HF-GCs. Importantly, mature GCs from mice depleted of Kv4.1 transcripts in the DG showed increased firing frequency, and these mice showed an impairment in contextual discrimination task. Our findings suggest that Kv4.1 expression occurring at late stage of GC maturation is essential for low excitability of DG networks and thereby contributes to pattern separation.SIGNIFICANCE STATEMENT The sparse activity of dentate granule cells (GCs), which is essential for pattern separation, is supported by high inhibitory inputs and low intrinsic excitability of GCs. Low excitability of GCs is thought to be attributable to a high K+ conductance at resting membrane potentials, but this study identifies Kv4.1, a depolarization-activated K+ channel, as a key ion channel that regulates firing of GCs without affecting resting membrane potentials. Kv4.1 expression is developmentally regulated and Kv4.1 currents are detected only in mature GCs that show low-frequency firing, but not in less mature high-frequency firing GCs. Furthermore, mice depleted of Kv4.1 transcripts in the dentate gyrus show impaired pattern separation, suggesting that Kv4.1 is crucial for sparse coding and pattern separation.

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.

Sleep Loss Immediately After Fear Memory Reactivation Attenuates Fear Memory Reconsolidation.

Permanently stored memories become labile through a process called reactivation. Once reactivated, these memories need reconsolidation to become permanent. Sleep is critical for memory consolidation. Is sleep necessary for memory reconsolidation? We hypothesized that sleep loss immediately after fear reactivation (FR) will prevent memory reconsolidation. To test our hypothesis, two experiments were performed in adult male C57BL/6J mice exposed to contextual fear conditioning paradigm with inescapable foot shock as unconditional stimulus (US) and contextual cage as conditional stimulus (CS). Sleep loss was achieved either by 5 h of sleep deprivation (SD; Experiment 1) or by systemic infusion of modafinil (200 mg/Kg, ip), an FDA approved wake-promoting agent (Experiment 2). One hour after light-onset, fear memory acquisition (FMA) was performed on Day 1. Mice were allowed to explore CS for 5 min followed by presentation of US (7 foot-shocks; 0.5 mA, 2.0 s duration) at pseudorandom intervals. Controls were exposed to similar CS but no shocks were delivered. On Day 2, mice were exposed to CS for 2 min (without US; for FR) followed by either sleep loss or no sleep loss. On Day 3, fear memory recall (FMR) was performed by exposing mice to CS (without US) for 12 min. Percent time spent in freezing was monitored during FC, FR and FMR. Our results suggested that as compared to sleeping controls, mice with sleep loss immediately after FR displayed a significant reduction in percent time freezing during FMR. These results suggest that sleep loss may prevent memory reconsolidation.

The amygdala differentially regulates defensive behaviors evoked by CO2.

CO2 inhalation can provoke panic attacks in humans, and the likelihood is increased in patients with panic disorder. Identifying brain sites involved could provide important mechanistic insight into the illness. In mice, the amygdala has been suggested to promote CO2 responses; however, recent studies in humans with amygdala damage indicate the amygdala is not required for CO2-induced fear and panic and might actually oppose these responses. To clarify the role of the amygdala, we produced lesions in mice paralleling the human lesions, and characterized behavioral responses to CO2. Compared to sham controls, we found that amygdala-lesioned mice froze less to 10% CO2, and unlike shams they also began to jump frenetically. At 20% CO2, controls also exhibited jumping, suggesting it is a normal response to more extreme CO2 concentrations. The effect of amygdala lesions was specific to CO2 as amygdala-lesioned mice did not jump in response to a predator odor or to an auditory conditioned stimulus. In amygdala-lesioned mice, jumping evoked by 10% CO2 was eliminated by co-lesioning the dorsal periaqueductal gray, a structure implicated in panic and escape-related behaviors. Together, these observations suggest a dual role for the amygdala in the CO2 response: promoting CO2-induced freezing, and opposing CO2-induced jumping, which may help explain the exaggerated CO2 responses in humans with amygdala lesions.