Fear-related Behaviors Research Articles

Breathing modulates network activity in frontal brain regions during anxiety.

Anxiety elicits various physiological responses, including changes in respiratory rate and neuronal activity within specific brain regions such as the medial prefrontal cortex (mPFC). Previous research suggests that the olfactory bulb (OB) modulates the mPFC through respiration-coupled neuronal oscillations (RCOs), which have been linked to fear-related freezing behavior. Nevertheless, the impact of breathing on frontal brain networks during other negative emotional responses, such as anxiety-related states characterized by higher breathing rates, remains unclear. To address this, we subjected rats to the elevated plus maze (EPM) paradigm while simultaneously recording respiration and local field potentials in the OB and mPFC. Our findings demonstrate distinct respiratory patterns during EPM exploration: slower breathing frequencies prevailed in the closed arms, whereas faster frequencies were observed in the open arms, independent of locomotor activity, indicating that anxiety-like states are associated with increased respiratory rates. Additionally, we identified RCOs at different frequencies, mirroring the bimodal distribution of respiratory frequencies. RCOs exhibited higher power during open arm exploration, when they showed greater coherence with breathing at faster frequencies. Furthermore, we confirmed that nasal respiration drives RCOs in frontal brain regions, and found a stronger effect during faster breathing. Interestingly, we observed that the frequency of prefrontal gamma oscillations modulated by respiration increased with breathing frequency. Overall, our study provides evidence for a significant influence of breathing on prefrontal cortex networks during anxious states, shedding light on the complex interplay between respiratory physiology and emotional processing.Significance Statement Understanding how breathing influences brain activity during anxious states could pave the way for novel therapeutic interventions targeting respiratory control to alleviate anxiety symptoms. Our study uncovers a crucial link between respiratory patterns and anxiety-related neural activity in the brain. By investigating the interplay between breathing, neuronal oscillations, and emotional states, we reveal that anxiety induces distinct respiratory patterns, with higher breathing rates correlating with anxious behavior. Importantly, we demonstrate that respiration drives oscillatory activity in the prefrontal cortex, and this effect is potentiated during the fast breathing associated with anxiety. Furthermore, faster breathing modulates the emergence of faster prefrontal gamma oscillations. This discovery sheds new light on the intricate relationship between respiratory physiology and emotional processing.

In adult dogs is supplementary tryptophan in the diet effective in reducing signs of anxiety?

Question In adult dogs, is dietary supplementation with tryptophan compared with no dietary tryptophan supplement effective in reducing signs of anxiety? Clinical bottom line The category of research question was: Treatment. The number and type of study designs that were critically appraised were: Two studies were found to be considered appropriate in the level of hierarchy of evidence pyramid. One study was a randomised double-blinded, placebo-controlled study and the other was a single-blind, placebo-controlled study. Critical appraisal of the selected papers meeting the inclusion criteria collectively provide zero/weak/moderate/strong evidence in terms of their experimental design and implementation: Moderate. The outcomes reported are summarised as follows… One study found no overall significant influence of tryptophan in the diet as an aid in reducing anxiety and fear-related behaviour in anxious dogs in household conditions. In contrast, the second study reported lower anxiety-related behaviour in dogs by owners but did not find significant differences in cortisol levels based on urine cortisol-to-creatinine ratio. In view of the strength of evidence and the outcomes from the studies the following conclusion is made… Current evidence reveals two studies that have evaluated the effectiveness of tryptophan as a supplement to the diets of anxious canines for pet owners. One study conducted did not find a significant effect or interaction in reducing anxiety-related behaviour whilst the second study reported lower anxiety-related behaviour in dogs by owners but did not find significant differences in cortisol levels based on urine cortisol-to-creatinine ratios. The use of tryptophan as a supplementary component in the diet is thus not enough as a sole treatment to assist in reducing anxiety in anxious adult dogs.

Analysis of the impact of coronavirus infection on human health: epidemiological data, vaccination strategies, psychological aspect, consequences (literature review)

The COVID-19 (COronaVIrus Disease 2019) pandemic has caused widespread morbidity, mortality, and socioeconomic crisis around the world. Since its first outbreak in 2019, COVID-19 has evolved continuously, leading to the emergence of several variants that have gained greater transmission efficiency, severity, and immune evasion properties. Vaccination has proven to be an important strategy to control the spread of the virus and mitigate its impact. The development of multiple COVID-19 vaccines within 1 year of the virus's emergence was unprecedented. The purpose of the article was to analyze and summarize the scientific literature on the impact of COVID-19 on global health, including epidemiological data, vaccination strategies, psychological impact of the pandemic, and human health outcomes. The analysis found that COVID-19 vaccination has been very effective in reducing morbidity, hospitalizations and intensive care unit admissions. However, ongoing surveillance, monitoring of options and adaptive vaccination strategies are crucial to maximize the benefits of vaccination and effectively control the spread of the virus. Globally, the dramatic and rapid spread of COVID-19 has triggered social changes that have led to an increased burden of mental health and fear-related behaviors. The physical and social distancing imposed in many parts of the world has had a significant impact on how the general population communicates and interacts with each other. Extreme avoidance of the lack of social contact has created a sense of "disconnection" that has been further exacerbated by the inability to meet friends and family at social gatherings and supportive places such as churches, restaurants, workplaces, and sports facilities. The closure of schools and many businesses, along with rising unemployment, further exacerbated feelings of isolation, financial hardship, anxiety, and depression. The article provides information on the mental impact of COVID-19 on patients, namely, the virus contributes to mental distress, anxiety, depression, suicide risk, post-traumatic stress disorder, cognitive impairment and sleep disorders. Keywords: COVID, spread, vaccines, anxiety, depression.

Dissociable dorsal medial prefrontal cortex ensembles are necessary for cocaine seeking and fear conditioning in mice

The dorsal medial prefrontal cortex (dmPFC) plays a dual role in modulating drug seeking and fear-related behaviors. Learned associations between cues and drug seeking are encoded by a specific ensemble of neurons. This study explored the stability of a dmPFC cocaine seeking ensemble over 2 weeks and its influence on persistent cocaine seeking and fear memory retrieval. In the first series of experiments, we trained TetTag c-fos-driven-EGFP mice in cocaine self-administration and tagged strongly activated neurons with EGFP during the initial day 7 cocaine seeking session. Subsequently, a follow-up seeking test was conducted 2 weeks later to examine ensemble reactivation between two seeking sessions via c-Fos immunostaining. In the second series of experiments, we co-injected viruses expressing TRE-cre and a cre-dependent inhibitory PSAM-GlyR into the dmPFC of male and female c-fos-tTA mice to enable “tagging” of cocaine seeking ensemble or cued fear ensemble neurons with inhibitory chemogenetic receptors. These c-fos-tTA mice have the c-fos promoter that drives expression of the tetracycline transactivator (tTA). The tTA can bind to the tetracycline response element (TRE) site on the viral construct, resulting in the expression of cre-recombinase, which enables the expression of cre-dependent inhibitory chemogenetic receptors and fluorescent reporters. Then we investigated ensemble contribution to subsequent cocaine seeking and fear recall during inhibition of the tagged ensemble by administering uPSEM792s (0.3 mg/kg), a selective ligand for PSAM-GlyR. In both sexes, there was a positive association between the persistence of cocaine seeking and the proportion of reactivated EGFP+ neurons within the dmPFC. More importantly, inhibition of the cocaine seeking ensemble suppressed cocaine seeking, but not recall of fear memory, while inhibition of the fear ensemble reduced conditioned freezing but not cocaine seeking. The results demonstrate that cocaine and fear recall ensembles in the dmPFC are stable, but largely exclusive from one another.

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

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

Does the stress axis mediate behavioural flexibility in a social cichlid, Neolamprologus pulcher?

Behavioural flexibility plays a major role in the way animals cope with novel situations, and physiological stress responses are adaptive and highly efficient mechanisms to cope with unpredictable events. Previous studies investigating the role of stress responses in mediating behavioural flexibility were mostly done in laboratory rodents using stressors and cognitive challenges unrelated to the ecology of the species. To better understand how stress mediates behavioural flexibility in a natural context, direct manipulations of the stress response and cognitive tests in ecologically relevant contexts are needed. To this aim, we pharmacologically blocked glucocorticoid receptors (GR) in adult Neolamprologus pulcher using a minimally invasive application of a GR antagonist. GR blockade prevents the recovery after a stressful event, which we predicted to impair behavioural flexibility. After the application of the GR antagonist, we repeatedly exposed fish to a predator and tested their behavioural flexibility using a detour task, i.e. fish had to find a new, longer route to the shelter when the shortest route was blocked. While the latencies to find the shelter were not different between treatments, GR blocked fish showed more failed attempts during the detour tasks than control fish. Furthermore, weak performance during the detour tasks was accompanied by an increase of fear related behaviours. This suggests that blocking GR changed the perception of fear and resulted in an impaired behavioural flexibility. Therefore, our results support a potential link between the capacity to recover from stressors and behavioural flexibility in N. pulcher with potential consequences for an effective and adaptive coping with changing environments.

Affective Implications of Human-Animal Relationship on Pig Welfare: Integrating Non-Linear Heart Rate Variability Measures.

The human-animal relationship is crucial for animal welfare. Gentle handling enhances pigs' comfort while rough handling causes fear and stress. This study examined how different human-animal relationship qualities affect the behavior and heart rate variability (linear and non-linear parameters) of 36 nursery pigs. Over six weeks, pigs experienced positive (n = 12), minimal (n = 12), or negative (n = 12) human handling. Their responses to handlers were then assessed in an experimental arena with four phases: habituation, exposure to the handler standing and sitting, and forced interaction. Pigs subjected to negative handling exhibited increased fear-related behaviors, spending less time in contact with the handler. They also exhibited heightened stress responses, with greater LF/HF ratio and Lmean values compared with positively handled pigs. Conversely, gently handled pigs displayed affiliative behaviors, accepting more strokes, and higher parasympathetic activation, indicated by greater RMSSD/SDNN and SampEn values, suggesting a more positive affective state. Minimally handled pigs exhibited some behavioral similarities to gently handled pigs, although physiological data indicated that the interaction was likely more rewarding for the gently handled pigs. These results emphasize the impact of human-animal relationships on pig welfare and highlight the value of incorporating non-linear heart rate variability parameters in such evaluations.

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

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

Contextual fear conditioning in zebrafish: Influence of different shock frequencies, context, and pharmacological modulation on behavior

Contextual fear conditioning is a protocol used to assess associative learning across species, including fish. Here, our goal was to expand the analysis of behavioral parameters that may reflect aversive behaviors in a contextual fear conditioning protocol using adult zebrafish (Danio rerio) and to verify how such parameters can be modulated. First, we analyzed the influence of an aversive stimulus (3 mild electric shocks for 5 s each at frequencies of 10, 100 or 1000 Hz) on fish behavior, and their ability to elicit fear responses in the absence of shock during a test session. To confirm whether the aversive responses are context-dependent, behaviors were also measured in a different experimental environment in a test session. Furthermore, we investigated the effects of dizocilpine (MK-801, 2 mg/kg, i.p.) on fear-related responses. Zebrafish showed significant changes in baseline activity immediately after shock exposure in the training session, in which 100 Hz induced robust contextual fear responses during the test session. Importantly, when introduced to a different environment, animals exposed to the aversive stimulus did not show any differences in locomotion and immobility-related parameters. MK-801 administered after the training session reduced fear responses during the test, indicating that glutamate NMDA-receptors play a key role in the consolidation of contextual fear-related memory in zebrafish. In conclusion, by further exploring fear-related behaviors in a contextual fear conditioning task, we show the effects of different shock frequencies and confirm the importance of context on aversive responses for associative learning in zebrafish. Additionally, our data support the use of zebrafish in contextual fear conditioning tasks, as well as for advancing pharmacological studies related to associative learning in translational neurobehavioral research.

Facial blushing and feather fluffing are indicators of emotions in domestic fowl (Gallus gallus domesticus).

The study of facial expressions in mammals provided great advances in the identification of their emotions and then in the comprehension of their sentience. So far, this area of research has excluded birds. With a naturalist approach, we analysed facial blushing and feather displays in domestic fowl. Hens were filmed in situations contrasting in emotional valence and arousal level: situations known to indicate calm states (positive valence / low arousal), have rewarding effects (positive valence / high arousal) or induce fear-related behaviour (negative valence / high arousal). Head feather position as well as skin redness of comb, wattles, ear lobes and cheeks varied across these situations. Skin of all four areas was less red in situations with low arousal compared to situations with higher arousal. Furthermore, skin redness of the cheeks and ear lobes also varied depending on the valence of the situation: redness was higher in situations with negative valence compared to situations with positive valence. Feather position also varied with the situations. Feather fluffing was mostly observed in positively valenced situations, except when hens were eating. We conclude that hens have facial displays that reveal their emotions and that blushing is not exclusive to humans. This opens a promising way to explore the emotional lives of birds, which is a critical step when trying to improve poultry welfare.

Climbing Fiber Activation Induced by Footshock in the Cerebellar Vermis Lobule IV/V of Freely Moving Mice.

Parallel fibers (PFs) in the cerebellar cortex are involved in a series of coordinated responses in the fear conditioning paradigm induced by footshock. However, whether footshock can activate cerebellar climbing fibers (CFs) remains unclear. In this study, we recorded calcium (Ca2+) activity in CFs by optical fiber photometry in the cerebellar vermis lobule IV/V of freely moving mice with footshock stimulation. We found that the activation of CFs in the lobule IV/V was highly correlated with footshock stimulation but not with the sound stimulation used as a control. This result suggests that afferent information from CFs might be associated with the motor initiation of fear-related behaviors or fear emotion itself. Thus, our results suggest that a characteristic CF signal in the cerebellar cortex might be related to fear processing or footshock-related behaviors (such as startle responses or pain sensation).

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

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

Sex-specific role of high-fat diet and stress on behavior, energy metabolism, and the ventromedial hypothalamus

BackgroundScientific evidence highlights the influence of biological sex on the relationship between stress and metabolic dysfunctions. However, there is limited understanding of how diet and stress concurrently contribute to metabolic dysregulation in both males and females. Our study aimed to investigate the combined effects of high-fat diet (HFD) induced obesity and repeated stress on fear-related behaviors, metabolic, immune, and hypothalamic outcomes in male and female mice.MethodsTo investigate this, we used a highly reliable rodent behavioral model that faithfully recapitulates key aspects of post-traumatic stress disorder (PTSD)-like fear. We subjected mice to footshock stressor followed by a weekly singular footshock stressor or no stressor for 14 weeks while on either an HFD or chow diet. At weeks 10 and 14 we conducted glucose tolerance and insulin sensitivity measurements. Additionally, we placed the mice in metabolic chambers to perform indirect calorimetric measurements. Finally, we collected brain and peripheral tissues for cellular analysis.ResultsWe observed that HFD-induced obesity disrupted fear memory extinction, increased glucose intolerance, and affected energy expenditure specifically in male mice. Conversely, female mice on HFD exhibited reduced respiratory exchange ratio (RER), and a significant defect in glucose tolerance only when subjected to repeated stress. Furthermore, the combination of repeated stress and HFD led to sex-specific alterations in proinflammatory markers and hematopoietic stem cells across various peripheral metabolic tissues. Single-nuclei RNA sequencing (snRNAseq) analysis of the ventromedial hypothalamus (VMH) revealed microglial activation in female mice on HFD, while male mice on HFD exhibited astrocytic activation under repeated stress.ConclusionsOverall, our findings provide insights into complex interplay between repeated stress, high-fat diet regimen, and their cumulative effects on health, including their potential contribution to the development of PTSD-like stress and metabolic dysfunctions, emphasizing the need for further research to fully understand these interconnected pathways and their implications for health.

Are we afraid of the woods? – An investigation of the implicit and explicit fear reactions to forests

The beneficial effects of nature exposure have been repeatedly documented and encourage frequent and regular contact with nature and especially highlight forests. However, in human history, forests have also been associated with negative emotions such as fear and were seen as dangerous environments. While existing literature could demonstrate that natural environments can evoke fear, the focus was on the explicit perception. Given that research has shown the significance of additional implicit processes in fear-related behaviour, we aim to explore the presence of an implicit fear response to forests. Therefore, in an online study, we investigated the explicit and implicit fear reactions to forests by a Northern German sample of N = 256. Using three explicit measurements, we investigated fear and danger perception on a semantic and visual level of the stimulus category "forest" compared to the human-made urban green space "park" and the urban setting "house". Additionally, we assessed the unconscious response tendencies towards the forest within three implicit tasks: Subliminal Priming Procedure (SPP), Affect Misattribution Procedure (AMP) and Approach-Avoidance Task (AAT). Within the analyzed sample, the subliminally presented word forest evoked a stronger positive valence response compared to park (SPP). In contrast to houses, the forest showed a stronger approach and weaker avoidance tendency (AAT). At the same time, both the three explicit and one implicit measurement (AMP) showed a stronger fear perception of forests compared to parks or houses. Considering the increasingly utilised beneficial effects of nature in interventions, these findings should be acknowledged when implementing nature exposure in interventions and treatments.

Reducing FKBP51 Expression in the Ventral Hippocampus Decreases Auditory Fear Conditioning in Male Rats.

Fear conditioning evokes a physiologic release of glucocorticoids that assists learning. As a cochaperone in the glucocorticoid receptor complex, FKBP51 modulates stress-induced glucocorticoid signaling and may influence conditioned fear responses. This study combines molecular and behavioral approaches to examine whether locally reducing FKBP51 expression in the ventral hippocampus is sufficient to affect fear-related behaviors. We hypothesized that reducing FKBP51 expression in the VH would increase glucocorticoid signaling to alter auditory fear conditioning. Adult male rats were injected with an adeno-associated virus (AAV) vector expressing short hairpin - RNAs (shRNA) targeting FKBP5 into the ventral hippocampus to reduce FKBP5 levels or a control AAV. Infusion of FKBP5-shRNA into the ventral hippocampus decreased auditory fear acquisition and recall. Although animals injected with FKBP5-shRNA showed less freezing during extinction recall, the difference was due to a reduced fear recall rather than improved extinction. Reducing ventral hippocampus FKBP51 did not affect exploratory behavior in either the open field test or the elevated zero maze test but did increase passive behavior in the forced swim test, suggesting that the reduction in auditory fear recall was not due to more active responses to acute stress. Furthermore, lower ventral hippocampus FKBP51 levels did not alter corticosterone release in response to restraint stress, suggesting that the reduced fear recall was not due to lower corticosterone release. Our findings suggest FKBP51 in the ventral hippocampus plays a selective role in modulating fear-learning processes and passive behavioral responses to acute stress rather than hypothalamic-pituitary-adrenal axis reactivity or exploratory responses.

Salience signaling and stimulus scaling of ventral tegmental area glutamate neuron subtypes.

Ventral tegmental area (VTA) glutamatergic neurons participate in reward, aversion, drug-seeking, and stress. Subsets of VTA VGluT2+ neurons are capable of co-transmitting glutamate and GABA (VGluT2+VGaT+ neurons), transmitting glutamate without GABA (VGluT2+VGaT- neurons), or co-transmitting glutamate and dopamine (VGluT2+TH+ neurons), but whether these molecularly distinct subpopulations show behavior-related differences is not wholly understood. We identified that neuronal activity of each VGluT2+ subpopulation is sensitive to reward value but signaled this in different ways. The phasic maximum activity of VGluT2+VGaT+ neurons increased with sucrose concentration, whereas VGluT2+VGaT- neurons increased maximum and sustained activity with sucrose concentration, and VGluT2+TH+ neurons increased sustained but not maximum activity with sucrose concentration. Additionally, VGluT2+ subpopulations signaled consummatory preferences in different ways. VGluT2+VGaT- neurons and VGluT2+TH+ neurons showed a signaling preference for a behaviorally-preferred fat reward over sucrose, but in temporally-distinct ways. In contrast, VGluT2+VGaT+ neurons uniquely signaled a less behaviorally-preferred sucrose reward compared with fat. Further experiments suggested that VGluT2+VGaT+ consummatory reward-related activity was related to sweetness, partially modulated by hunger state, and not dependent on caloric content or behavioral preference. All VGluT2+ subtypes increased neuronal activity following aversive stimuli but VGluT2+VGaT+ neurons uniquely scaled their magnitude and sustained activity with footshock intensity. Optogenetic activation of VGluT2+VGaT+ neurons during low intensity footshock enhanced fear-related behavior without inducing place preference or aversion. We interpret these data such that VTA glutamatergic subpopulations signal different elements of rewarding and aversive experiences and highlight the unique role of VTA VGluT2+VGaT+ neurons in enhancing the salience of behavioral experiences.

Sex-Dependent Effects of Angiotensin Type 2 Receptor–Expressing Medial Prefrontal Cortex Interneurons in Fear Extinction Learning

BackgroundThe renin-angiotensin system has been identified as a potential therapeutic target for posttraumatic stress disorder, although its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC), but their function and mechanism in the mPFC remain unexplored. Therefore, we used a combination of imaging, cre/lox, and behavioral methods to investigate mPFC-AT2R–expressing neurons in fear and stess related behavior. MethodsTo characterize mPFC-AT2R–expressing neurons in the mPFC, AT2R-Cre/tdTomato male and female mice were used for immunohistochemistry. mPFC brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker were quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using a Cre-expressing adeno-associated virus. Mice then underwent Pavlovian auditory fear conditioning, elevated plus maze, and open field testing. ResultsImmunohistochemistry results revealed that AT2R was densely expressed throughout the mPFC and primarily expressed in somatostatin interneurons in a sex-dependent manner. Following fear conditioning, mPFC-AT2R Cre-lox deletion impaired extinction and increased exploratory behavior in female but not male mice, while locomotion was unaltered by mPFC-AT2R deletion in both sexes. ConclusionsThese results identify mPFC-AT2R+ neurons as a novel subgroup of somatostatin interneurons and reveal their role in regulating fear learning in a sex-dependent manner, potentially offering insights into novel therapeutic targets for posttraumatic stress disorder.

The Effects of Bacillus amyloliquefaciens SC06 on Behavior and Brain Function in Broilers Infected by Clostridium perfringens.

Poultry studies conducted on Clostridium perfringens (CP) mainly focus on the effects of intestinal health and productive performance. Notably, the probiotic Bacillus amyloliquefaciens SC06 (BaSC06) is known to play a role in preventing bacterial infection. However, whether CP could induce the changes in brain function and behaviors and whether BaSC06 could play roles in these parameters is yet to be reported. The aim of this study was to evaluate the effects of BaSC06 on stress-related behaviors and gene expression, as well as the brain morphology and mRNA sequence of the hypothalamus in broiler chickens. A total of 288 one-day-old chicks were randomly divided into four groups: (1) a control group with no treatment administered or infection; (2) birds treated with the BaSC06 group; (3) a CP group; and (4) a BaSC06 plus CP (Ba_CP) group. The results showed that stress and fear-related behaviors were significantly induced by a CP infection and decreased due to the treatment of BaSC06. CP infection caused pathological damage to the pia and cortex of the brain, while BaSC06 showed a protective effect. CP significantly inhibited hypothalamic GABA and promoted HTR1A gene expression, while BaSC06 promoted GABA and decreased HTR1A gene expression. The different genes were nearly found between the comparisons of control vs. Ba group and Ba vs. CP group, while there were a great number of different genes between the comparisons of control vs. Ba_CP as well as CP vs. Ba_CP. Several different gene expression pathways were found that were related to disease, energy metabolism, and nervous system development. Our results will help to promote poultry welfare and health, as well as provide insights into probiotics to replace antibiotics and reduce resistance in the chicken industry.

Complementation testing identifies genes mediating effects at quantitative trait loci underlying fear-related behavior

Knowing the genes involved in quantitative traits provides an entry point to understanding the biological bases of behavior, but there are very few examples where the pathway from genetic locus to behavioral change is known. To explore the role of specific genes in fear behavior, we mapped three fear-related traits, tested fourteen genes at six quantitative trait loci (QTLs) by quantitative complementation, and identified six genes. Four genes, Lamp, Ptprd, Nptx2, and Sh3gl, have known roles in synapse function; the fifth, Psip1, was not previously implicated in behavior; and the sixth is a long non-coding RNA, 4933413L06Rik, of unknown function. Variation in transcriptome and epigenetic modalities occurred preferentially in excitatory neurons, suggesting that genetic variation is more permissible in excitatory than inhibitory neuronal circuits. Our results relieve a bottleneck in using genetic mapping of QTLs to uncover biology underlying behavior and prompt a reconsideration of expected relationships between genetic and functional variation.

Sex-Dependent Effects of Angiotensin Type 2 Receptor Expressing Medial Prefrontal Cortex (mPFC) Interneurons in Fear Extinction Learning

Background: The renin-angiotensin system (RAS) has been identified as a potential therapeutic target for PTSD, though its mechanisms are not well understood. Brain angiotensin type 2 receptors (AT2Rs) are a subtype of angiotensin II receptors located in stress and anxiety-related regions, including the medial prefrontal cortex (mPFC). Their function and mechanism in the mPFC, however, remain unexplored. We therefore used a combination of neuroanatomical, chemogenetic, and behavioral methods to investigate mPFC-AT2R-expressing neuron involvement in fear learning. Methods: To characterize mPFC-AT2R-expressing neurons in the mPFC, AT2R-Cre/td-Tomato male and female brains were perfused and used for immunohistochemistry. Brain sections were stained with glutamatergic or interneuron markers, and density of AT2R+ cells and colocalization with each marker was quantified. To assess fear-related behaviors in AT2R-flox mice, we selectively deleted AT2R from mPFC neurons using an AAV-Cre or GFP virus. Mice then underwent Pavlovian auditory fear conditioning and anxiety-like and locomotor behavior testing using open field (OF) and elevated plus maze (EPM) tests. Results: IHC revealed that AT2R is expressed throughout the mPFC in males ( 208.6±48.8 cells/mm2) and females ( 139.0±59.8 cells/mm2) and has low co-expression on glutamatergic neurons ( TBR1 co-staining, males: 7.1%±1.8%; females: 13.2%±4.2%). Of the interneuron markers tested, AT2R is primarily expressed on somatostatin interneurons, and has higher colocalization in the mPFC of females than males ( males: 16.5%±1.2%, females: 31.8%±4.2%, p=0.02). Following fear conditioning and extinction, mPFC-AT2R deletion impaired extinction in female ( p=0.03) but not male ( p=0.76) mice. Locomotion in the OF was unaltered by mPFC-AT2R deletion in males or females ( total distance travelled; males: GFP 44.11m±5.92m, Cre 56.00m±10.29m, p=0.35; females: GFP 42.86m±4.85m, Cre 42.39m±4.97m, p=0.95), while AT2R-deleted females had increased exploratory behavior in the EPM ( open arm entries; males: GFP 12.38±1.02, Cre 13.22±1.54, p=0.66; females: GFP 12.50±0.96, Cre 20.00±2.81, p=0.02). Conclusion: These results lend support for mPFC-AT2R+ neurons as a novel somatostatin subgroup that influences fear extinction in a sex-dependent manner. This furthers underscores the role of mPFC in top-down regulation and a unique role for peptidergic (ie., angiotensin) mPFC regulation of fear and sex differences, and increases the understanding of circuitry and function of the brain RAS in disordered fear learning. This may lead to improved therapeutic treatments for PTSD. This research and PJM was supported by NIH1R01HL137103-01A1 and the American Heart Association 15CSA24340001. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.