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Background Excessive processing of aversive life events is a major pathology in stress-related anxiety and depressive disorders. Current pharmacological treatments have rather non-specific mechanisms of action. Somatostatin is synthesized and released as an inhibitory co-neurotransmitter by specific GABA interneurons and one of its receptors, SSTR4, is localized in brain regions involved in adaptive aversion processing and implicated in negative valence neuropathology, including the amygdala. Methods Rat and mouse experiments were conducted to investigate effects of specific SSTR4 agonism on neurobehavioral aversion processing including any normalization of stress-related hyper-responsiveness. A mouse experiment to investigate stress and SSTR4 agonism effects on reward processing was also conducted. Results In male rats (N=5-10/group) fitted with glutamate biosensors in basolateral amygdala, SSTR4 agonism attenuated glutamate release to restraint stress in control rats and particularly in rats previously exposed to chronic corticosterone. In male mice (N=10-18/group), SSTR4 agonism dose-dependently attenuated Pavlovian tone-footshock learning and memory measured as freezing behavior, both in controls (CON) and following exposure to chronic social stress (CSS) which induces excessive Pavlovian aversion learning-memory. Specificity of SSTR4 agonism effects to aversion learning-memory was demonstrated by absence of effects on discriminative reward (sucrose) learning-memory in both CON and CSS mice; SSTR4 agonism did increase reward-to-effort valuation in a dose-dependent manner, and in both CON mice and mice exposed to CSS which attenuates reward motivation. Conclusions These neuropsychopharmacological findings add substantially to the preclinical proof-of-concept evidence for SSTR4 agonism as a treatment in anxiety and depressive disorders.

Non-human primate (NHP) models are essential for developing and translating new treatments that target neural circuit dysfunction underlying human psychopathology. As a proof-of-concept for treating neuropsychiatric disorders, we used a NHP model of pathological anxiety to investigate the feasibility of decreasing anxiety by chemogenetically (DREADDs [designer receptors exclusively activated by designer drugs]) reducing amygdala neuronal activity. Intraoperative MRI surgery was used to infect dorsal amygdala neurons with AAV5-hSyn-HA-hM4Di in young rhesus monkeys. Invivo microPET studies with [11C]-deschloroclozapine and postmortem autoradiography with [3H]-clozapine demonstrated selective hM4Di binding in the amygdala, and neuronal expression of hM4Di was confirmed with immunohistochemistry. Additionally, because of its high affinity for DREADDs, and its approved use in humans, we developed an individualized, low-dose clozapine administration strategy to induce DREADD-mediated amygdala inhibition. Compared to controls, clozapine selectively decreased anxiety-related freezing behavior in the human intruder paradigm in hM4Di-expressing monkeys, while coo vocalizations and locomotion were unaffected. These results are an important step in establishing chemogenetic strategies for patients with refractory neuropsychiatric disorders in which amygdala alterations are central to disease pathophysiology.

In civilian and military settings, mild traumatic brain injury (mTBI) is a common consequence of impacts to the head, sudden blows to the body, and exposure to high-energy atmospheric shockwaves from blast. In some cases, mTBI from blast exposure results in long-term emotional and cognitive deficits and an elevated risk for certain neuropsychiatric diseases. Here, we tested the effects of mTBI on various forms of auditory-cued fear learning and other measures of cognition in male C57BL/6J mice after single or repeated blast exposure (blast TBI; bTBI). bTBI produced an abnormality in the temporal organization of cue-induced freezing behavior in a conditioned trace fear test. Spatial working memory, evaluated by the Y-maze task performance, was also deleteriously affected by bTBI. Reverse-transcription quantitative real-time polymerase chain reaction (RT-qPCR) analysis for glial markers indicated an alteration in the expression of myelin-related genes in the hippocampus and corpus callosum 1–8 weeks after bTBI. Immunohistochemical and ultrastructural analyses detected bTBI-related myelin and axonal damage in the hippocampus and corpus callosum. Together, these data suggest a possible link between blast-induced mTBI, myelin/axonal injury, and cognitive dysfunction.

The neuronal circuitry for pain signals has been intensively studied for decades. The external lateral parabrachial nucleus (PB) was shown to play a crucial role in nociceptive information processing. Previous work, including ours, has demonstrated that stimulating the neuronal pathway from the PB to the central region of the amygdala (CeA) can substitute for an actual pain signal to drive an associative form of threat/fear memory formation. However, it is still unknown whether activation of the PB–CeA pathway can directly drive avoidance behavior, escape behavior, or only acts as strategic freezing behavior for later memory retrieval. To directly address this issue, we have developed a real-time Y-maze conditioning behavioral paradigm to examine avoidance behavior induced by optogenetic stimulation of the PB–CeA pathway. In this current study, we have demonstrated that the PB–CeA pathway carries aversive information that can directly trigger avoidance behavior and thereby serve as an alarm signal to induce adaptive behaviors for later decision-making.

The GABAergic system is thought to play an important role in the control of cognition and emotion, such as fear, and is related to the pathophysiology of psychiatric disorders. For example, the expression of the 67-kDa isoform of glutamate decarboxylase (GAD67), a GABA-producing enzyme, is downregulated in the postmortem brains of patients with major depressive disorder and schizophrenia. However, knocking out the Gad1 gene, which encodes GAD67, is lethal in mice, and thus, the association between Gad1 and cognitive/emotional functions is unclear. We recently developed Gad1 knockout rats and found that some of them can grow into adulthood. Here, we performed fear-conditioning tests in adult Gad1 knockout rats to assess the impact of the loss of Gad1 on fear-related behaviors and the formation of fear memory. In a protocol assessing both cued and contextual memory, Gad1 knockout rats showed a partial antiphase pattern of freezing during training and significantly excessive freezing during the contextual test compared with wild-type rats. However, Gad1 knockout rats did not show any synchronous increase in freezing with auditory tones in the cued test. On the other hand, in a contextual memory specialized protocol, Gad1 knockout rats exhibited comparable freezing behavior to wild-type rats, while their fear extinction was markedly impaired. These results suggest that GABA synthesis by GAD67 has differential roles in cued and contextual fear memory.

Increased level of kynurenic acid is thought to contribute to the development of cognitive deficits in schizophrenia through an α7nAChR-mediated mechanism in the prefrontal cortex (PFC). However, it remains unclear to what extent disruption of PFC α7nAChR signaling impacts afferent transmission and its modulation of behavior. Using male rats, we found that PFC infusion of methyllycaconitine (MLA; α7nAChR antagonist) shifts ventral hippocampal-induced local field potential (LFP) suppression to LFP facilitation, an effect only observed in adults. Hippocampal stimulation can also elicit a GluN2B-mediated LFP potentiation (when PFC GABAAR is blocked) that is insensitive to MLA. Conversely, PFC infusion of MLA diminished the gain of amygdalar transmission, which is already enabled by postnatal day (P)30. Behaviorally, the impact of prefrontal MLA on trace fear-conditioning and extinction was also age related. While freezing behavior during conditioning was reduced by MLA only in adults, it elicited opposite effects in adolescent and adult rats during extinction as revealed by the level of reduced and increased freezing response, respectively. We next asked whether the late-adolescent onset of α7nAChR modulation of hippocampal inputs contributes to the age-dependent effect of MLA during extinction. Data revealed that the increased freezing behavior elicited by MLA in adult rats could be driven by a dysregulation of the GluN2B transmission in the PFC. Collectively, these results indicate that distinct neural circuits are recruited during the extinction of trace fear memory in adolescents and adults, likely because of the late-adolescent maturation of the ventral hippocampal-PFC functional connectivity and its modulation by α7nAChR signaling.SIGNIFICANCE STATEMENTAbnormal elevation of the astrocyte-derived metabolite kynurenic acid in the prefrontal cortex (PFC) is thought to impair cognitive functions in schizophrenia through an α7nAChR-mediated mechanism. Here, we found that prefrontal α7nAChR signaling is recruited to control the gain of hippocampal and amygdalar afferent transmission in an input-specific, age-related manner during the adolescent transition to adulthood. Behaviorally, prefrontal α7nAChR modulation of trace fear memory was also age-related, likely because of the late-adolescent maturation of the ventral hippocampal pathway and its recruitment of PFC GABAergic transmission enabled by local α7nAChR signaling. Collectively, these results reveal that distinct α7nAChR-sensitive neural circuits contribute to regulate behavior responses in adolescents and adults, particularly those requiring proper integration of hippocampal and amygdalar inputs by the PFC.

Aim Fear conditioning tests are intended to elucidate a subject's ability to associate a conditioned stimulus with an aversive, unconditioned stimulus, such as footshock. Among these tests, a paradigm related to precise cortical functions would be increasingly important in drug screening for disorders such as schizophrenia and dementia. Therefore, we established a new fear conditioning paradigm using a visual cue in mice. In addition, the validity of the test was evaluated using a genetically engineered mouse, heterozygous deficient in Mdga1 (Mdga1+/‐), which is related to schizophrenia. Results Mice were given footshocks associated with a visual cue of moving gratings at training in 25‐minute sessions. The mice showed the conditioned response of freezing behavior to the visual stimulus at testing 24 hours after the footshocks. In the test for validation, the Mdga1+/‐ deficient mice showed significantly less freezing than wild‐type mice. Conclusion The visually cued fear conditioning paradigm with moving gratings has been established, which is experimentally useful to evaluate animal cortical functions. The validity of the test was confirmed for Mdga1‐deficient mice with possible deficiency in cortical functions., The visual stimulus of moving gratings could be distinguished from still gratings by mice, and moving gratings were related to footshock. The defect obtained here by low‐frequency grating in MDGA1 deficient mice might reflect that the coarse gratings are less detectable in visual perceptions of the schizophrenic brain.

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.

Living in a group allows individuals to decrease their defenses, enabling other beneficial behaviors such as foraging. The detection of a threat through social cues is widely reported, however, the safety cues that guide animals to break away from a defensive behavior and resume alternate activities remain elusive. Here we show that fruit flies display a graded decrease in freezing behavior, triggered by an inescapable threat, with increasing group sizes. Furthermore, flies use the cessation of movement of other flies as a cue of threat and its resumption as a cue of safety. Finally, we find that lobula columnar neurons, LC11, mediate the propensity for freezing flies to resume moving in response to the movement of others. By identifying visual motion cues, and the neurons involved in their processing, as the basis of a social safety cue this study brings new insights into the neuronal basis of safety in numbers., Living in groups allows animals to decrease defenses, enabling other behaviors, but the mechanisms of safety in numbers are unknown. The authors show that fruit flies regulate freezing behavior as a function of group size and identify motion by others, and neurons that detect it, as key to this process.

Background: Posttraumatic stress disorder (PTSD), a mental disorder induced by traumatic stress and often accompanied by depression and/or anxiety, may involve an imbalance in the neurotransmitters associated with the fear response. Korean Red Ginseng (KRG) has long been used as a traditional medicine and is known to be involved in a variety of pharmacological activities. We used the open field test and forced swimming test to examine the effects of KRG on the depression-like response of rats after exposure to single prolonged stress (SPS), leading to activation of the serotonergic system. Methods: Male rats received KRG (30, 50, and 100 mg/kg, intraperitoneal injection) once daily for 14 days after exposure to SPS. Results: Daily KRG administration significantly improved depression-like behaviors in the forced swimming test, increased the number of lines crossed and time spent in the central zone in the open field test, and decreased freezing behavior in contextual and cued fear conditioning. KRG treatment attenuated SPS-induced decreases in serotonin (5-HT) tissue concentrations in the hippocampus and medial prefrontal cortex. The increased 5-HT concentration during KRG treatment may be partially attributable to the 5-hydroxyindoleacetic acid/5-HT ratio in the hippocampus of rats with PTSD. These effects may be caused by the activation of hippocampal genes encoding tryptophan hydroxylase-1 and 2 mRNA levels. Conclusion: Our findings suggest that KRG has an antidepressant effect in rats subjected to SPS and may represent an effective use of traditional medicine for the treatment of PTSD.

Molecular and cellular mechanisms underlying the role of the prelimbic cortex in contextual fear memory remain elusive. Here we examined the kinesin family of molecular motor proteins (KIFs) in the prelimbic cortex for their role in mediating contextual fear, a form of associative memory. KIFs function as critical mediators of synaptic transmission and plasticity by their ability to modulate microtubule function and transport of gene products. However, the regulation and function of KIFs in the prelimbic cortex insofar as mediating memory consolidation is not known. We find that within one hour of contextual fear conditioning, the expression of KIF3B is upregulated in the prelimbic but not the infralimbic cortex. Importantly, lentiviral-mediated knockdown of KIF3B in the prelimbic cortex produces deficits in consolidation while reducing freezing behavior during extinction of contextual fear. We also find that the depletion of KIF3B increases spine density within prelimbic neurons. Taken together, these results illuminate a key role for KIF3B in the prelimbic cortex as far as mediating contextual fear memory. Supplementary Information The online version contains supplementary material available at 10.1186/s13041-021-00873-9.

Neurons expressing the neuropeptide hypocretin/orexin (Hcrt) in the hypothalamus promote reward-related behaviors including alcohol consumption and are shown in rodents and zebrafish to be stimulated by embryonic exposure to ethanol (EtOH). We used here in zebrafish three-dimensional analyses of the entire population of Hcrt neurons to examine how embryonic EtOH exposure at low-moderate concentrations (0.1% or 0.5% v/v) alters these neurons in relation to behavior. We found that EtOH in the water for 2 h (22–24 h post fertilization) increases the number of Hcrt neurons on the left but not right side of the brain through a stimulation of cell proliferation, this is accompanied by a decrease in locomotor activity under novel conditions but not after habituation, and these effects are evident in both larvae and adults indicating they are long lasting. Our analyses in adults revealed sexually dimorphic effects, with females consuming more EtOH-gelatin and exhibiting more freezing behavior along with an asymmetric increase in Hcrt neurons and males exhibiting increased aggression with no change in Hcrt. These findings suggest that a long lasting, asymmetric increase in Hcrt neurons induced by EtOH results from an asymmetric increase in proliferation specific to Hcrt and contributes to behavioral changes in females.

Freezing of gait (FOG), a devastating symptom of Parkinson’s disease (PD), can be refractory to current treatments such as medication and open-loop deep brain stimulation (olDBS). Recent evidence suggests that closed-loop DBS (clDBS), using beta local field potential power from the subthalamic nucleus (STN) as the control variable, can improve tremor and bradykinesia; however, no study has investigated the use of clDBS for the treatment of FOG. In this study, we provide preliminary evidence that clDBS was superior to olDBS in reducing percent time freezing and in reducing freezing behavior (gait arrhythmicity) in a person with PD and FOG. These findings warrant further investigation into the use of clDBS to treat FOG while also minimizing the total energy delivered to maintain a therapeutic effect.

Accepted manuscript, licensed CC BY-NC-ND 4.0. Published version available at https://doi.org/10.1016/j.neuropharm.2019.03.037. The use of selective serotonin reuptake inhibitors (SSRI) during pregnancy has increased tremendously, but the consequences for the offspring remain largely unclear. Several studies have described potential effects of perinatal SSRI-exposure on neurobehavioral outcomes using simplified rodent test set-ups, however these set-ups only assess a small fraction of the behavior. For translational purposes it is important to take the environmental influences into account which children are exposed to in real life. By using a seminatural environmental set-up, this study is the first to assess behavioral outcomes in offspring exposed to perinatal SSRI exposure under seminatural circumstances. Mothers received daily the SSRI fluoxetine (FLX, 10 mg/kg p.o.) or vehicle (CTR) from gestational day 1 until postnatal day 21. To assess the effect of FLX exposure during early development, female and male offspring were behaviorally tested in the seminatural environment at adulthood. Baseline behavior was measured in addition to responses during and after stressful white-noise events. Behavior was observed on two days, day 4 on which females were sexually non-receptive, and day 7, on which females were sexual receptive. Perinatal FLX exposure reduced general activity in females and increased behavior related to a social context in both males and females. After a stressful white-noise event some behaviors switched. Whereas FLX-females switch from resting socially to resting more solitarily, FLX-males show an increase in self-grooming behavior after the stressor and showed more freezing behavior in the open area. We conclude that perinatal FLX exposure leads to alterations in social and stress-coping behaviors in adulthood, when observed in a seminatural environment. Whether these adaptations in behavior are advantageous or disadvantageous remains to be established.

This paper evaluates a methodology that enables one-dimensional freezing to make a homogeneous frozen soil specimen. Numerical analyses that simulate one-dimensional freezing are conducted, considering the phase change of pore water. After evaluating the applicability of the numerical analysis model, the effects of geotechnical characteristics on the freezing behavior of ice-rich sandy soil are evaluated through parametric studies. In addition, a nonlinear regression model is proposed that can predict the time required for complete freezing of saturated soil. According to the results from nonlinear regression analysis, the model demonstrates reliable accuracy (R2 = 0.998) in predicting the amount of time required for complete freezing, even with data from testing datasets not used in the developing stage.

The central nervous system modulates heart function on a beat-to-beat basis via increasingly understood mechanisms. Conversely, whether and how humoral/functional cardiac variations shape brain activity and adaptive behavior remains unclear. This study shows that mice overexpressing adenylyl cyclase type 8 in myocytes (TGAC8), characterized by persistently elevated heart rate/contractility, also display increased locomotion. This effect is sustained by enhanced gamma rhythms, as evidenced by simultaneous behavioral and EEG/ECG monitoring. These changes are specific because they are not paralleled by other modifications, such as heightened anxiety-like behavior. In unison, TGAC8 mice hippocampus exhibits upregulated GABA-A receptors, whose activation chiefly accounts for gamma activity generation. Moreover, the Granger causality analysis between ECG and EEG attests to the causal involvement of the autonomic component of the heartbeat in shaping EEG gamma oscillations in a bottom-up modality. Mechanistically, TGAC8 harbors elevated circulating dopamine/DOPA levels of cardiac origin and upregulated hippocampal D5 dopamine receptor levels. In synergy with the GABA-A receptor, D5 activation favors hippocampal inhibitory currents that drive EEG gamma oscillations. These studies, therefore, inform how heart-initiated functional and/or humoral modifications reverberate back to the brain to modulate specific primary adaptive responses, such as locomotion.SignificanceThe brain is continuously aware of the functional status of many bodily organs, modulating, for instance, the heart’s activity beat-by-beat. Conversely, how cardiac activity modifications impact brain function and behavior is less understood. We disclose that augmenting myocyte adenyl cyclase 8 (AC8) activity in mice increases their locomotion. Elevated cardiac AC8 levels lead to higher circulating dopamine and DOPA, hormones crucially involved in movement control, and increased expression of the hippocampus’s GABA-A and D5 receptors; the activation of the latter modifies hippocampal gamma oscillations shaping locomotor activity. Thus, the brain interprets changes in myocardial AC8 activity as a “sustained exercise-like” situation and responds by activating areas commanding to increase locomotion.

The inability to extinguish a traumatic memory is a key aspect of post-traumatic stress disorder (PTSD). While PTSD affects 10–20% of individuals who experience a trauma, women are particularly susceptible to developing the disorder. Despite this notable female vulnerability, few studies have investigated this particular resistance to fear extinction observed in females. Similar to humans, rodent models of Pavlovian fear learning and extinction show a wide range of individual differences in fear learning and extinction, although female rodents are considerably understudied. Therefore, the present study examined individual differences in fear responses, including freezing behavior and ultrasonic vocalizations (USVs), of female Long–Evans rats during acquisition of fear conditioning and cued fear extinction. Similar to prior studies in males, female rats displayed individual variation in freezing during cued fear extinction and were divided into extinction competent (EC) and extinction resistant (ER) phenotypes. Differences in freezing between ER and EC females were accompanied by shifts in rearing during extinction, but no darting was seen in any trial. Freezing behavior during fear learning did not differ between the EC and ER females. Vocalizations emitted in the 22 and 50 kHz ranges during fear learning and extinction were also examined. Unlike vocalizations seen in previous studies in males, very few 22 kHz distress vocalizations were emitted by female rats during fear acquisition and extinction, with no difference between ER and EC groups. Interestingly, all female rats produced significant levels of 50 kHz USVs, and EC females emitted significantly more 50 kHz USVs than ER rats. This difference in 50 kHz USVs was most apparent during initial exposure to the testing environment. These results suggest that like males, female rodents show individual differences in both freezing and USVs during fear extinction, although females appear to vocalize more in the 50 kHz range, especially during initial periods of exposure to the testing environment, and emit very few of the 22 kHz distress calls that are typically observed in males during fear learning or extinction paradigms. Overall, these findings show that female rodents display fear behavior repertoires divergent from males.

Plasticity in the dentate gyrus (DG) is strongly influenced by ethanol, and ethanol experience alters long-term memory consolidation dependent on the DG. However, it is unclear if DG plasticity plays a role in dysregulation of long-term memory consolidation during abstinence from chronic ethanol experience. Outbred male Wistar rats experienced 7 weeks of chronic intermittent ethanol vapor exposure (CIE). Seventy-two hours after CIE cessation, CIE and age-matched ethanol-naive Air controls experienced auditory trace fear conditioning (TFC). Rats were tested for cue-mediated retrieval in the fear context either twenty-four hours (24 hr), ten days (10 days), or twenty-one days (21 days) later. CIE rats showed enhanced freezing behavior during TFC acquisition compared to Air rats. Air rats showed significant fear retrieval, and this behavior did not differ at the three time points. In CIE rats, fear retrieval increased over time during abstinence, indicating an incubation in fear responses. Enhanced retrieval at 21 days was associated with reduced structural and functional plasticity of ventral granule cell neurons (GCNs) and reduced expression of synaptic proteins important for neuronal plasticity. Systemic treatment with the drug Isoxazole-9 (Isx-9; small molecule that stimulates DG plasticity) during the last week and a half of CIE blocked altered acquisition and retrieval of fear memories in CIE rats during abstinence. Concurrently, Isx-9 modulated the structural and functional plasticity of ventral GCNs and the expression of synaptic proteins in the ventral DG. These findings identify that abstinence-induced disruption of fear memory consolidation occurs via altered plasticity within the ventral DG, and that Isx-9 prevented these effects.

The study of fear conditioning has led to a better understanding of fear and anxiety-based disorders such as post-traumatic stress disorder (PTSD). Despite the fact many of these disorders are more common in women than in men, the vast majority of work investigating fear conditioning in rodents has been conducted in males. The goal of the work presented here was to better understand how biological sex affects contextual fear conditioning and expression. To this end, rats of both sexes were trained to fear a specific context and fear responses were measured upon re-exposure to the conditioning context. In the first experiment, male and female rats were given context fear conditioning and tested the next day during which freezing behavior was measured. In the second experiment, rats were trained and tested in a similar fashion while fear-potentiated startle and defecation were measured. We found that males showed more freezing behavior than females during a fear expression test. The expression of fear-potentiated startle did not differ between sexes, while males exhibited more defecation during a test in a novel context. These data suggest that the expression of defensive behavior differs between sexes and highlight the importance of using multiple measures of fear when comparing between sexes.

The amygdala plays a critical role in the acquisition and consolidation of fear-related memories. Recent studies have demonstrated that ADP-ribosylation of histones, accelerated by PARPs, affects the chromatin structure and the binding of chromatin remodeling complexes with transcription factors. Inhibition of PARP-1 activity during the labile phase of re-consolidation may erase memory. Accordingly, we investigated the possibility of interfering with fear conditioning by PARP-1 inhibition. Herein, we demonstrate that injection of PARP-1 inhibitors, specifically into the CeA or i.p., in different time windows post-retrieval, attenuates freezing behavior. Moreover, the association of memory with pharmacokinetic timing of PARP inhibitor arrival to the brain enabled/achieved attenuation of a specific cue-associated memory of fear but did not hinder other memories (even traumatic events) associated with other cues. Our results suggest using PARP-1 inhibitors as a new avenue for future treatment of PTSD by disrupting specific traumatic memories in a broad time window, even long after the traumatic event. The safety of using these PARP inhibitors, that is, not interfering with other natural memories, is an added value.

Freezing behavior is used as a measure of a rodent’s ability to learn during fear conditioning. However, it is possible that the expression of other behaviors may compete with freezing, particularly in rodent populations that have not been thoroughly studied in this context. Rearing and grooming are complex behaviors that are frequently exhibited by mice during fear conditioning. Both behaviors have been shown to be stress-sensitive, and the expression of these behaviors is dependent upon strain background. To better understand how genetic background impacts behavioral responses during fear conditioning, we examined freezing, rearing, and grooming frequencies prior to fear conditioning training and across different stages of fear conditioning testing in male mice from eight inbred mouse strains (C57BL/6J, DBA/2J, FVB/NJ, SWR/J, BTBR T + ltpr3Tf/J, SM/J, LP/J, 129S1/SvlmJ) that exhibited diverse freezing responses. We found that genetic background determined rearing and grooming expression throughout fear conditioning, and their patterns of expression across stages of fear conditioning were strain dependent. Using publicly available SNP data, we found that polymorphisms in Dab1, a gene that is implicated in both grooming and learning phenotypes, separated the strains with high contextual grooming from the others using a hierarchical clustering analysis. This suggested a potential genetic mechanism for the observed behavioral differences. These findings demonstrate that genetic background determines behavioral responses during fear conditioning and suggest that shared genetic substrates underlie fear conditioning behaviors.

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 context-dependence of avoidance behavior.

Defensive behavioral responses are contingent upon threat intensity, proximity, and context of exposure. Based on these factors, we developed a classical conditioning paradigm that elicits clear transitions between conditioned freezing and flight behavior within individual subjects. This model is crucial for the understanding the pathologies involved in anxiety, panic, and post-traumatic stress disorders. 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.

Introduction Post‐traumatic stress disorder (PTSD) is an anxiety disorder induced by psychologically traumatic events. Using a rat model, this study aimed to determine whether psychological trauma alters relative expression between pro‐inflammatory and anti‐inflammatory markers in microglia. To meet this goal, expression of genes encoding i‐NOS, arginase, TNF‐α, interleukin‐10, CD74, and Mannose Receptor C was analyzed on multiple days following trauma exposure. Methods Single‐prolonged stress (SPS) was used to model PTSD in male Sprague‐Dawley rats. Twenty‐four rats (12 Controls and 12 SPS‐exposed) were sacrificed on Days 1, 3, and 7 post‐SPS. Twenty‐four (12 Controls and 12 SPS‐exposed) additional rats were exposed to classical fear conditioning on Day 7, and fear extinction on Days 8, 9, 10, 15, 16, and 17. Freezing behavior was measured to assess fear resolution. Microglial isolates were collected from the frontal cortex, and RNA was extracted. Changes in relative expression of target genes were quantified via RT‐PCR. Results SPS rats showed significant decreases in IL‐10 and TNF‐α expression and increases in the i‐NOS:Arginase and TNF‐α:IL‐10 ratios compared to Controls on Day 1, but not on Day 3 or Day 7 for any of the dependent variables. Day 17 SPS rats showed a significant decrease in IL‐10 expression and an increase in the TNF‐α:IL‐10 ratio, further characterized by a significant inverse relationship between IL‐10 expression and fear persistence. Conclusion Psychological trauma impacts the immunological phenotype of microglia of the frontal cortex. Consequently, future studies should further evaluate the mechanistic role of microglia in PTSD pathology., This study tested the hypothesis that exposure to psychological trauma would result in increased expression of pro‐inflammatory markers relative to anti‐inflammatory markers by microglia of the frontal cortex. To do this, frontal cortices of rats were collected at various time points after exposure to a psychological trauma in the form of Single‐Prolonged Stress. A significant change in microglial gene expression was found that correlated with key behavioral changes, indicating a possible immunological basis for post‐traumatic stress disorder.

BackgroundMaladaptive defensive responses such as excessive avoidance behavior have received increasing attention as a main mechanism for the development and maintenance of chronic pain complaints. However, another defensive response which is commonly studied in animals as a proxy for fear is freezing behavior. No research to date has investigated human freezing behavior in the context of pain. In addition, there is an increasing realization that social context can affect pain-relevant processes such as pain experience and pain behavior but less is known about the effects of social context on defensive responses to pain. Hence, this study investigated freezing behavior and facial pain expression in the context of pain, and their modulation by social context.MethodsHealthy, pain-free participants (N = 39) stood on a stabilometric force platform in a threatening or safe social context, which was manipulated using angry or happy facial stimuli. In some trials, an auditory cue (conditioned stimulus; CS) predicted the occurrence of painful electrocutaneous stimulus (unconditioned stimulus; pain-US). We assessed body sway (an index of freezing), heart rate, facial pain expression, self-reported pain intensity, unpleasantness, and pain-US expectancy during the CS and the context alone (no CS).ResultsThe results were mixed. Neither the anticipation of pain, nor social context affected body sway. Heart rate and painful facial expression were reduced in the threatening social context at high anxiety levels. A threatening social context also elicited higher pain-US expectancy ratings. In sum, a threatening social context increases the expectation of pain, but reduces the facial expression of pain and lowers heart rate in highly anxious individuals.

The rates of dehardening and rehardening in response to rapid temperature changes in winter are important traits that affect the survival, growth and productivity of the European pear (Pyrus communis [L.]) cultivars in northern countries. The frost hardiness (FH) of shoots of three pear cultivars were studied by a series of freezing tests, after sampling in natural conditions, after dehardening in a growth chamber at 5 degrees C for 3-4 days (D1) and 16 days (D2), and then after rehardening at -7 degrees C for 5-7 days (R1 and R2). The FH was assessed by a differential thermal analysis (DTA) to measure the low temperature exotherm (LTE) of shoots, by relative electrolyte leakage (REL) of shoots and by visual damage scoring (VD) of shoots and buds. According to the DTA, the FH of the cultivars varied between -38 degrees C ('Conference' in D2) and -41 degrees C ('Pepi' in R2). The shoots of the cultivar 'Pepi' and 'Conference' had the highest and the lowest FH, respectively, in all conditions and methods. All the cultivars had the lowest shoot FH after dehardening in either D1 (between -26 degrees C and -30 degrees C by REL and between -28 degrees C and -30 degrees C by VD) or D2 (between -38 degrees C and -40 degrees C by DTA), and the highest FH after rehardening (R1) preceded by D1 (between -30 degrees C and -34 degrees C by REL, and between -29 degrees C and -32 degrees C by VD). After the dehardening in D1, the buds did not reharden but continued to deharden (the average FH by VD - 24.5 degrees C). In the forcing conditions, bud growth was resumed most rapidly in 'Conference', indicating a shallower dormancy in this cultivar than in 'Pepi' or 'Clara Frijs'. We conclude that the pear cultivars responded to temperature changes in mid-winter, but less than expected, and the responses were similar in all cultivars.

Background: The study of the biological basis of fear in animal models has progressed considerably because of the energy and space that the brain devotes to this basic emotion. Electrical stimulation targets several structures of the brain to examine its behavioral effects and to understand the role of different regions in underlying mechanisms of fear processing and anxiety in preclinical models. Objectives: In this study, the effects of high-frequency deep brain stimulation (DBS) of the basolateral amygdala (BLA) and prelimbic (PL) sub-region of the prefrontal cortex were evaluated on the extinction process of conditioned fear. Methods: This study was performed on 35 male Wistar rats in the weight range of 220 – 250 g. After selecting the animals, they were separated into five groups. Then, we did stereotactic surgery on rats for electrode implantation. After recovery, some rats were conditioned, followed by a 10-day treatment schedule via high-frequency DBS in the BLA or PL. Next, freezing behavior was measured as a predicted response dedicated to extinction, without shock (re-exposure). In addition, we used ELISA and Western blot to estimate blood serum corticosterone levels and c-Fos protein expression. Results: The mean freezing time recorded for the PL group was significantly lower than that of both the BLA group and the PC group (P < 0.01). The BLA group and PC group were also significantly different (P < 0.001). Corticosterone results indicated that the PL group had significantly higher serum corticosterone levels compared with both the BLA group and the PC group (P < 0.01). In addition, the BLA group revealed a significant reduction in c-Fos expression compared with the PC (P < 0.001). Conclusions: This study provides further evidence for the contribution of the prelimbic cortex and amygdala both in acquisition and extinction processes during contextual fear conditioning. However, the PL stimulation by high-frequency DBS might be more involved in the extinction process and play a more important role as an enhancer.

The importance of catecholamines in post-traumatic stress disorder (PTSD) still needs to be explored. We aimed to evaluate epinephrine’s (EPI) causal role and molecular mechanism for the persistence of PTSD traumatic memories. Wild-type (WT) and EPI-deficient mice (phenylethanolamine-N-methyltransferase-knockout mice, Pnmt-KO) were induced with PTSD and behavioral tests were performed. Some Pnmt-KO mice were administered with EPI or vehicle. Catecholamines were quantified by HPLC-ED. Nr4a1, Nr4a2, and Nr4a3 mRNA expression were evaluated by real-time PCR in hippocampus samples. It was observed an increase in EPI and freezing behavior, and a decrease in open arm entries in the elevated plus-maze test and time spent in the light in the light–dark test in WT mice in the PTSD-induction group compared to control. After induction of PTSD, Pnmt-KO mice showed a decrease in freezing, as well as an increase in open arm entries and transitions between compartments compared to WT. After PTSD induction, Pnmt-KO mice administered with EPI showed an increase in freezing compared with the vehicle. On day 0 of PTSD induction, it was observed an increase in mRNA expression of Nr4a2 and Nr4a3 genes in the hippocampus of WT mice compared to control, contrary to Pnmt-KO mice. In conclusion, our data suggest that EPI may be involved in the persistence of traumatic memories in PTSD, possibly through enhancement of the expression of Nr4a2 and Nr4a3 genes in the hippocampus. Peripheral administration of EPI restored contextual traumatic memories in Pnmt-KO mice, which suggests a causal role for EPI. The persistence of contextual traumatic memories may contribute to anxiety-like behavior and resistance of traumatic memory extinction in this PTSD mice model.

Animals can use social information to detect threat in the environment. In particular, social learning allows animals to learn about dangers without incurring in the costs of trial-and-error learning. In zebrafish, both chemical and visual social cues elicit an innate alarm response, which consists of erratic movement followed by freezing behavior. Injured zebrafish release an alarm substance from their skin that elicits the alarm response. Similarly, the sight of conspecifics displaying the alarm response can also elicit the expression of this response in observers. In this study, we investigated if these social cues of danger can also be used by zebrafish as unconditioned stimulus (US) in learning. We found that only the chemical cue was effective in the social fear conditioning. We suggest that this differential efficacy of social cues results from the fact that the alarm cue is a more reliable indicator of threat, than the sight of an alarmed conspecific. Therefore, although multiple social cues may elicit innate responses not all have been evolutionarily co-opted to act as US in associative learning. Furthermore, the use of the expression of the immediate early genes as markers of neuronal activity showed that chemical social fear conditioning is paralleled by a differential activation of the olfactory bulbs and by a different pattern of functional connectivity across brain regions involved in olfactory processing. FCT - Fundação para a Ciência e Tecnologia info:eu-repo/semantics/publishedVersion

Background Avoiding danger and accessing environmental resources are two fundamental, yet conflicting, survival instincts across species. To maintain a balance between these instincts, animals must efficiently distinguish approaching threats from low-threat cues. However, little is known about the key visual features that animals use to promptly detect such imminent danger and thus facilitate an immediate and appropriate behavioral response. Results We used an automatic behavior detection system in mice to quantify innate defensive behaviors, including freezing, flight, and rearing, to a series of looming visual stimuli with varying expanding speeds and varying initial and final sizes. Looming visual stimuli within a specific “alert range” induced flight behavior in mice. Looming stimuli with an angular size of 10–40° and an expanding speed of 57–320°/s were in this range. Stimuli with relatively low expanding speeds tended to trigger freezing behavior, while those with relatively high expanding speeds tended to trigger rearing behavior. If approaching objects are in this “alert range,” their visual features will trigger a flight response; however, non-threatening objects, based on object size and speed, will not. Conclusions These results indicate a simple strategy in mice that is used to detect predators and suggest countermeasures that predators may have taken to overcome these defensive strategies.

Posttraumatic stress disorder (PTSD) is triggered by traumatic events in 10–20% of exposed subjects. N-linked glycosylation, by modifying protein functions, may provide an important environmental link predicting vulnerability. Our goals were (1) to find alterations in plasma N- glycome predicting stress-vulnerability ; (2) to investigate how trauma affects N-glycome in the plasma (PGP) and in three PTSD-related brain regions (prefrontal cortex, hippocampus and amygdala ; BGP), hence, uncover specific targets for PTSD treatment. We examined male (1) controls, (2) traumatized vulnerable and (3) traumatized resilient rats both before and several weeks after electric footshock. Vulnerable and resilient groups were separated by z-score analysis of behavior. Higher freezing behavior and decreased social interest were detected in vulnerable groups compared to control and resilient rats. Innate anxiety did not predict vulnerability, but pretrauma levels of PGP10(FA1G1Ga1), PGP11(FA2G2), and PGP15(FA3G2) correlated positively with it, the last one being the most sensitive. Traumatic stress induced a shift from large, elaborate N- glycans toward simpler neutral structures in the plasma of all traumatized animals and specifically in the prefrontal cortex of vulnerable rats. In plasma trauma increased PGP17(A2G2S) level in vulnerable animals. In all three brain regions, BGP11(F(6)A2B) was more abundant in vulnerable rats, while most behavioral correlations occurred in the prefrontal cortex. In conclusion, we found N-glycans (especially PGP15(FA3G2)) in plasma as possible biomarkers of vulnerability to trauma that warrants further investigation. Posttrauma PGP17(A2G2S1) increase showed overlap with human results highlighting the utility and relevance of this animal model. Prefrontal cortex is a key site of trauma-induced glycosylation changes that could modulate the behavioral outcome.

Stress-induced deviations in central nervous system development has long-term effects on adult mental health. Previous research in humans demonstrates that prenatal or adolescent stress increases the risk for psychiatric disorders. Animal models investigating the effects of stress during prenatal or adolescent development produces behavioral outcomes analogous to those observed in humans. However, whether adolescent stress exposure potentiates the effects of prenatal stress is currently unknown. Thus, the current study tested whether adolescent stress increases the impact of prenatal stress on contextual and cued fear memory in adulthood. Male and female Sprague Dawley rats were exposed to a chronic variable stress schedule during the last week of gestation, during adolescence, or during both developmental periods before undergoing fear conditioning training in adulthood. Our hypothesis predicted that the combined effects of prenatal and adolescent stress on contextual and cued fear memory would be greater than the effects of stress during either time period alone. In contrast to our hypothesis, however, we found independent effects of prenatal and adolescent stress on contextual and cued fear memory in both sexes, with no additional combined impact of stress exposure during both developmental phases. In males, developmental stress increased freezing behavior during contextual and cued testing regardless of whether stress exposure was prenatal, adolescent, or combined prenatal and adolescent stress exposure. In contrast, the effects of developmental stress in females were both test- and ovarian hormone status-dependent. During cued testing, nonstressed female freezing behavior depended on estrous cycle phase, whereas freezing behavior in stressed females did not, suggesting that developmental stress interferes with hormone-dependent cued fear memory. No effects of developmental stress or estrous cycle phase were observed for contextual fear memory in females. The results of the current study suggest that the effects of prenatal and adolescent stress on contextual and cued fear memory are not cumulative, but the effects of developmental stress on associative memory differ between males and females.

Parabrachial CGRP neurons receive diverse threat-related signals and contribute to multiple phases of adaptive threat responses, with their inactivation attenuating both unconditioned behavioral responses to somatic pain and fear-memory formation. Because CGRPPBN neurons respond broadly to multi-modal threats, it remains unknown how these distinct adaptive processes are individually engaged. We show that while three partially separable subsets of CGRPPBN neurons broadly collateralize to their respective downstream partners, individual projections accomplish distinct functions: hypothalamic and extended amygdalar projections elicit assorted unconditioned threat responses including autonomic arousal, anxiety, and freezing behavior, while thalamic and basal forebrain projections generate freezing behavior and, unexpectedly, contribute to associative fear learning. Moreover, the unconditioned responses generated by individual projections are complementary, with simultaneous activation of multiple sites driving profound freezing behavior and bradycardia that are not elicited by any individual projection. This semi-parallel, scalable connectivity schema likely contributes to flexible control of threat responses in unpredictable environments.

Dopamine (DA) neurons are thought to encode reward prediction error (RPE), in addition to other signals, such as salience. While RPE is known to support learning, the role of salience in learning remains less clear. To address this, we recorded and manipulated VTA DA neurons in mice during fear extinction. We applied deep learning to classify mouse freezing behavior, eliminating the need for human scoring. Our fiber photometry recordings showed DA neurons in medial and lateral VTA have distinct activity profiles during fear extinction: medial VTA activity more closely reflected RPE, while lateral VTA activity more closely reflected a salience-like signal. Optogenetic inhibition of DA neurons in either region slowed fear extinction, with the relevant time period for inhibition differing across regions. Our results indicate salience-like signals can have similar downstream consequences to RPE-like signals, although with different temporal dependencies.

Widely available low-cost electronics encourage the development of open-source tools for neuroscientific research. In recent years, many neuroscientists recognized the open science movement for its potential to stimulate and encourage science that is less focused on money, and more on robustness, validity, questioning and understanding. Here, we wanted to contribute to this global community by creating a research platform based on a common digital kitchen scale. This everyday ordinary kitchen tool is sometimes used in neuroscience research in various ways; however, its use is limited by sampling rate and inability to store and analyze data. To tackle this problem we developed a Platform for Acoustic STArtle or PASTA. This robust and simple platform enables users to obtain data from kitchen scale load cells at a high sampling rate, store it and analyze it. Here, we used it to analyze acoustic startle and prepulse inhibition sensorimotor gating in rats treated intracerebroventricularly with streptozotocin, but the system can be easily modified and upgraded for other purposes. In accordance with open science principles, we shared complete hardware design with instructions. Furthermore, we also disclose our software codes written for PASTA data acquisition (C++, Arduino) and acoustic startle experimental protocol (Python) and analysis (ratPASTA R package—R-based Awesome Toolbox for PASTA, and pastaWRAP—Python wrapper package for ratPASTA). To further encourage the development of our PASTA platform we demonstrate its sensitivity by using PASTA-gathered data to extract breathing patterns during rat freezing behavior in our experimental protocol.

We investigated how the exchange of sensory signals modulates the individual behaviors of juvenile crayfish in an anti-predatory context as well as during intraspecific agonistic encounters. We first compared crayfish housed in total sensory isolation or in pairs with access to chemical and visual cues. After one week of housing, we analyzed their individual responses to a visual danger signal while they were foraging. We found that crayfish previously housed in pairs with exchange of sensory signals responded to a simulated predator attack predominately with freezing behavior, whereas animals deprived of all sensory communication mostly responded by performing escape tail-flips. Next, we used the same housing conditions in-between repeated fights in pairs of crayfish. Aggressive and submissive behaviors increased in subsequent fights both after total isolation and after exchange of olfactory and visual signals. Thus, unlike responses to simulated predator attacks, intraspecific agonistic behavior was not modulated by exposure to the same sensory signals. However, when we tested the effects of olfactory or visual communication independently, aggression increased dramatically after the exchange of olfactory signals, which also led to a high number of rank reversals in second fights, suggesting a destabilization of the original dominance relationship. Exposure to visual cues during the one-week separation, however, produced the opposite effect reducing agonistic behaviors and rank reversals. These findings demonstrate that exchange of sensory signals modulate future anti-predatory decision-making and intraspecific agonistic behaviors discretely, suggesting that the effect of these signals on shared neural circuitry is context-dependent.