Modelling social fear in mice: advancements in social fear conditioning and social defeat paradigms.

Social anxiety disorder (SAD) is highly comorbid with alcohol use disorders, but the complex relationship between social fear and alcohol drinking is poorly understood due to the lack of specific animal models. We investigated whether social fear alters ethanol drinking under both stress-free and stress-inducing conditions and whether ethanol alleviates symptoms of social fear. We used the social fear conditioning (SFC) paradigm, an animal model with face and predictive validity to SAD, to induce specific social fear in male CD1 mice, i.e., without comorbid depression or anxiety-like behavior. Plasma corticosterone (CORT) levels were measured in conditioned (SFC+) and unconditioned (SFC-) mice after exposure to non-social or social stimuli. Ethanol drinking was assessed in the two-bottle free-choice paradigm (1) for 16days under stress-free conditions and (2) for 6h after exposure to social stimuli. The effects of ethanol drinking and social fear on anxiety-like behavior and taste preference were tested in the elevated plus-maze and sucrose and quinine preference tests. We show that exposure to social but not non-social stimuli leads to higher plasma CORT levels in SFC+ compared with SFC- mice. We also show that social fear decreases voluntary ethanol consumption under stress-free conditions, but increases ethanol consumption after exposure to social stimuli. Ethanol drinking, on the other hand, reduces social fear without altering anxiety-like behavior, locomotor activity, and taste preference. These results have important clinical connotations as they suggest that voluntary ethanol drinking might specifically reverse symptoms of social fear in a SAD-relevant animal model.

Social fear and avoidance of social situations represent the main behavioral symptoms of social anxiety disorder (SAD), a highly prevalent anxiety disorder that is poorly elucidated and has rather unsatisfactory therapeutic options. Therefore, animal models are needed to study the underlying etiology and pathophysiology of SAD and to verify the efficacy of possible novel treatment approaches. In this review, we describe and discuss the most important paradigms that have been shown to induce social avoidance and fear in rodents, including foot shock exposure, restraint stress, social isolation, social instability, social defeat, conditioned defeat, social defeat/overcrowding, chronic subordinate colony housing, chronic mild stress, maternal separation and social fear conditioning. We also describe some of the behavioral paradigms used to assess social avoidance and fear in rodents, including the social interaction test, the social preference-avoidance test, the social approach-avoidance test, the three-chambered social approach test, the partition test and the modified Y-maze test. We focus on the behavioral alterations these paradigms induce, especially on social interaction, general anxiety and depressive-like behavior given that SAD is strongly comorbid with anxiety and affective disorders.

Adolescence is a critical developmental period with heightened stress susceptibility. Traumatic experiences during this phase are highly predictive of future affective disorders, such as social anxiety disorder (SAD), which may manifest during early adolescence. Social avoidance, a major symptom of SAD, can be robustly generated in adult male and female mice using the social fear conditioning (SFC) paradigm. Using the SFC paradigm in adolescent mice, we analyze behavioral and neuroendocrine responses after adolescent social trauma. Here, we demonstrate that social fear elicited by SFC in early adolescent (EA) male mice (SFC+EA/29d) persists until adulthood (SFC+EA/57d). We further compared neuroendocrine responses to a heterotypic (elevated platform) or homotypic (exposure to a conspecific) stressor after SFC performed either in EA (SFC+EA/29d, SFC+EA/57d) or adulthood (SFC+AD). While in non‐conditioned SFC−EA/29d mice plasma corticosterone concentrations remained unchanged after social exposure in adolescence, SFC+EA/29d resulted in a hyper‐response of the HPA axis to the social, but not heterotypic stressor, with a negative correlation of plasma corticosterone concentrations and social investigation times. This effect of SFC+EA/29d on plasma corticosterone response was absent in SFC+EA/57d and SFC+AD mice indicating a higher sensitivity to social trauma in EA. We further revealed a rise in plasma oxytocin (OXT) levels in adult SFC− mice in response to the social challenge, whereas the OXT system of SFC−EA/29d mice still seems to be unresponsive to the social stimulus. Importantly, after SFC+ either in EA or AD, the OXT response to social exposure found in SFC−AD controls was completely abolished, whereas in SFC−EA/57d mice OXT levels positively correlated with social investigation times, indicating social trauma‐induced acute and long‐lasting dysfunctions of the OXT system. In summary, we show that exposure to social trauma (SFC+) in early adolescence exerts both short‐term as well as long‐term effects on social behavior. We further reveal that SFC+EA/29d prevents the corticosterone hypo‐response to social stimuli characteristic for early adolescence. Moreover, SFC+/AD and SFC+EA/57d impair the plasma OXT response to a social, but not heterotypic, stressor.

Central oxytocin (OXT) has anxiolytic and pro-social properties both in humans and rodents, and has been proposed as a therapeutic option for anxiety and social dysfunctions. Here, we utilized a mouse model of social fear conditioning (SFC) to study the effects of OXT on social fear, and to determine whether SFC causes alterations in central OXT receptor (OXTR) binding and local OXT release. Central infusion of OXT, but not arginine vasopressin, prior to social fear extinction training completely abolished social fear expression in an OXTR-mediated fashion without affecting general anxiety or locomotion. SFC caused increased OXTR binding in the dorso-lateral septum (DLS), central amygdala, dentate gyrus, and cornu ammunis 1, which normalized after social fear extinction, suggesting that these areas form part of a brain network involved in the development and neural support of social fear. Microdialysis revealed that the increase in OXT release observed in unconditioned mice within the DLS during social fear extinction training was attenuated in conditioned mice. Consequently, increasing the availability of local OXT by infusion of OXT into the DLS reversed social fear. Thus, alterations in the brain OXT system, including altered OXTR binding and OXT release within the DLS, play an important role in SFC and social fear extinction. Thus, we suggest that the OXT system is adversely affected in disorders associated with social fear, such as social anxiety disorder and reinstalling an appropriate balance of the OXT system may alleviate some of the symptoms.

Social anxiety disorder (SAD) is a highly prevalent and comorbid anxiety disorder with rather unclear underlying mechanisms. Here, we aimed to characterize neurobiological changes occurring in mice expressing symptoms of social fear and to identify possible therapeutic targets for SAD. Social fear was induced via social fear conditioning (SFC), a validated animal model of SAD. We assessed the expression levels of the immediate early genes (IEGs) cFos, Fosl2 and Arc as markers of neuronal activity and the expression levels of several genes of the GABAergic, serotoninergic, oxytocinergic, vasopressinergic and neuropeptide Y (NPY)-ergic systems in brain regions involved in social behavior or fear-related behavior in SFC+ and SFC− mice 2 h after exposure to a conspecific. SFC+ mice showed a decreased number and density of cFos-positive cells and decreased expression levels of IEGs in the dorsal hippocampus. SFC+ mice also showed alterations in the expression of NPY and serotonin system-related genes in the paraventricular nucleus of the hypothalamus, basolateral amygdala, septum and dorsal raphe nucleus, but not in the dorsal hippocampus. Our results describe neuronal alterations occurring during the expression of social fear and identify the NPY and serotonergic systems as possible targets in the treatment of SAD.

Social anxiety disorder (SAD) is a major health concern with high lifetime prevalence. The current medication is rather unspecific and, despite considerable efforts, its efficacy is still unsatisfactory. However, there are no appropriate and specific animal models available to study the underlying etiology of the disorder. Therefore, we aimed to establish a model of specific social fear in mice and use this social fear conditioning (SFC) model to assess the therapeutic efficacy of the benzodiazepine diazepam and of the antidepressant paroxetine; treatments currently used for SAD patients. We show that by administering electric foot shocks (2-5, 1 s, 0.7 mA) during the investigation of a con-specific, the investigation of unfamiliar con-specifics was reduced for both the short- and long-term, indicating lasting social fear. The induced fear was specific to social stimuli and did not lead to other behavioral alterations, such as fear of novelty, general anxiety, depression, and impaired locomotion. We show that social fear was dose-dependently reversed by acute diazepam, at doses that were not anxiolytic in a non-social context, such as the elevated plus maze. Finally, we show that chronic paroxetine treatment reversed social fear. All in all, we demonstrated robust social fear after exposure to SFC in mice, which was reversed with both acute benzodiazepine and chronic antidepressant treatment. We propose the SFC model as an appropriate animal model to identify the underlying etiology of SAD and possible novel treatment approaches.

Social anxiety disorder (SAD) is caused by traumatic social experiences. It is characterized by intense fear and avoidance of social contexts, which can be robustly mimicked by the social fear conditioning (SFC) paradigm. The extinction phase of the SFC paradigm is akin to exposure therapy for SAD and requires learning to disassociate the trauma with the social context. Learning-induced acetylation of histones is critical for extinction memory formation and its endurance. Although class I histone deacetylases (HDACs) regulate the abovementioned learning process, there is a lack of clarity in isoforms and spatial specificity in HDAC function in social learning. Utilizing the SFC paradigm, we functionally characterized the role of HDAC1, specifically in the lateral septum (LS), in regulating the formation of long-term social fear extinction memory. We measured a local increase in activity-inducing HDAC1 phosphorylation at serine residues of social fear-conditioned (SFC+) mice in response to the extinction of social fear. We also found that LS-HDAC1 function negatively correlates with acute social fear extinction learning using pharmacological and viral approaches. Further, inhibition of LS-HDAC1 enhanced the expression of the GABA-A receptor β1 subunit (Gabrb1) in SFC+ mice, and activation of GABA-A receptors facilitated acute extinction learning. Finally, the facilitation of extinction learning by HDAC1 inhibition or GABA-A receptor activation within the LS led to the formation of long-lasting extinction memory, which persisted even 30 days after extinction. Our results show that HDAC1-mediated regulation of GABA signaling in the LS is crucial for the formation of long-lasting social fear extinction memory.

Women nearly twice as often develop social anxiety disorder (SAD) compared to men. The reason for this difference is still being debated. The present study investigates gender differences and the effect of male versus female agents in low (LSA) and high socially anxious (HSA) participants regarding the acquisition and extinction of social fear in virtual reality (VR). In a social fear conditioning (SFC) paradigm, 60 participants actively approached several agents, some of which were paired with an aversive unconditioned stimulus (US) consisting of a verbal rejection and spitting simulated by an aversive air blast (CS+), or without an US (CS−). Primary outcome variables were defined for each of the 4 levels of emotional reactions including experience (fear ratings), psychophysiology (fear-potentiated startle), behavior (avoidance), and cognition (recognition task). Secondary outcome variables were personality traits, contingency ratings, heart rate (HR), and skin conductance response (SCR). As hypothesized, fear ratings for CS+ increased significantly during acquisition and the differentiation between CS+ and CS− vanished during extinction. Additionally, women reported higher fear compared to men. Furthermore, a clear difference in the fear-potentiated startle response between male CS+ and CS− at the end of acquisition indicates successful SFC to male agents in both groups. Concerning behavior, results exhibited successful SFC in both groups and a general larger distance to agents in HSA than LSA participants. Furthermore, HSA women maintained a larger distance to male compared to female agents. No such differences were found for HSA men. Regarding recognition, participants responded with higher sensitivity to agent than object stimuli, suggesting a higher ability to distinguish the target from the distractor for social cues, which were on focus during SFC. Regarding the secondary physiological outcome variables, we detected an activation in HR response during acquisition, but there were no differences between stimuli or groups. Moreover, we observed a gender but no CS+/CS− differences in SCR. SFC was successfully induced and extinguished according to the primary outcome variables. VR is an interesting tool to measure emotional learning processes on different outcome levels with enhanced ecological validity. Future research should further investigate social fear learning mechanisms for developing more efficient treatments of SAD.

Currently, there are no animal models for studying both specific social fear and social fear with comorbidities. Here, we investigated whether social fear conditioning (SFC), an animal model with face, predictive and construct validity for social anxiety disorder (SAD), leads to the development of comorbidities at a later stage over the course of the disease and how this affects the brain sphingolipid metabolism. SFC altered both the emotional behavior and the brain sphingolipid metabolism in a time-point-dependent manner. While social fear was not accompanied by changes in non-social anxiety-like and depressive-like behavior for at least two to three weeks, a comorbid depressive-like behavior developed five weeks after SFC. These different pathologies were accompanied by different alterations in the brain sphingolipid metabolism. Specific social fear was accompanied by increased activity of ceramidases in the ventral hippocampus and ventral mesencephalon and by small changes in sphingolipid levels in the dorsal hippocampus. Social fear with comorbid depression, however, altered the activity of sphingomyelinases and ceramidases as well as the sphingolipid levels and sphingolipid ratios in most of the investigated brain regions. This suggests that changes in the brain sphingolipid metabolism might be related to the short- and long-term pathophysiology of SAD.

Neuropeptide S reduces fear and avoidance of con-specifics induced by social fear conditioning and social defeat, respectively

In many social anxiety disorder (SAD) patients, the efficacy of antidepressant therapy is unsatisfactory. Here, we investigated whether mice deficient for the lysosomal glycoprotein acid sphingomyelinase (ASM−/−) represent an appropriate tool to study antidepressant-resistant social fear. We also investigated whether neuropeptide Y (NPY) reduces this antidepressant-resistant social fear in ASM−/− mice, given that NPY reduced social fear in a mouse model of SAD, namely social fear conditioning (SFC). We show that neither chronic paroxetine nor chronic amitriptyline administration via drinking water were successful in reducing SFC-induced social fear in ASM−/− mice, while the same treatment reduced social fear in ASM+/− mice and completely reversed social fear in ASM+/+ mice. This indicates that the antidepressants paroxetine and amitriptyline reduce social fear via the ASM-ceramide system and that ASM−/− mice represent an appropriate tool to study antidepressant-resistant social fear. The intracerebroventricular administration of NPY, on the other hand, reduced social fear in ASM−/− mice, suggesting that NPY might represent an alternative pharmacotherapy for antidepressant-resistant social fear. These results suggest that medication strategies aimed at increasing brain NPY concentrations might improve symptoms of social fear in SAD patients who fail to respond to antidepressant treatments.

Social interactions are critical for mammalian survival and evolution. Dysregulation of social behavior often leads to psychopathologies such as social anxiety disorder, denoted by intense fear and avoidance of social situations. Using the social fear conditioning (SFC) paradigm, we analyzed expression levels of miR-132-3p and miR-124-3p within the septum, a brain region essential for social preference and avoidance behavior, after acquisition and extinction of social fear. Here, we found that SFC dynamically altered both microRNAs. Functional in vivo approaches using pharmacological strategies, inhibition of miR-132-3p, viral overexpression of miR-132-3p, and shRNA-mediated knockdown of miR-132-3p specifically within oxytocin receptor-positive neurons confirmed septal miR-132-3p to be critically involved not only in social fear extinction, but also in oxytocin-induced reversal of social fear. Moreover, Argonaute-RNA-co-immunoprecipitation-microarray analysis and further in vitro and in vivo quantification of target mRNA and protein, revealed growth differentiation factor-5 (Gdf-5) as a target of miR-132-3p. Septal application of GDF-5 impaired social fear extinction suggesting its functional involvement in the reversal of social fear. In summary, we show that septal miR-132-3p and its downstream target Gdf-5 regulate social fear expression and potentially mediate oxytocin-induced reversal of social fear.

Social anxiety disorder is characterized by a persistent fear and avoidance of social situations, but available treatment options are rather unspecific. Using an established mouse social fear conditioning (SFC) paradigm, we profiled gene expression and chromatin alterations after the acquisition and extinction of social fear within the septum, a brain region important for social fear and social behaviors. Here, we particularly focused on the successful versus unsuccessful outcome of social fear extinction training, which corresponds to treatment responsive versus resistant patients in the clinics. Validation of coding and non-coding RNAs revealed specific isoforms of the long non-coding RNA (lncRNA) Meg3 regulated, depending on the success of social fear extinction. Moreover, PI3K/AKT was differentially activated with extinction success in SFC-mice. In vivo knockdown of specific Meg3 isoforms increased baseline activity of PI3K/AKT signaling, and mildly delayed social fear extinction. Using ATAC-Seq and CUT&RUN, we found alterations in the chromatin structure of specific genes, which might be direct targets of lncRNA Meg3.

Social anxiety disorder (SAD) is a common anxiety disorder characterized by a marked fear of social situations. Treatments for SAD, including exposure therapy and medication, are not satisfactory for all patients. This has led to the development of several paradigms to study social fear in rodents. However, there are still some social impairments observed in SAD patients that have never been examined in rodent models. Indeed, social situations avoided by SAD patients include not only social interactions but also public performances and being observed by others. Nevertheless, tests used to assess sociability in rodents evaluate mostly social interaction in pairs. Thus, we developed a new test—a socially enriched environment test—that evaluates sociability within a group of three unfamiliar conspecifics in an enriched environment. In this study, we induced a SAD-like behavior (i.e., social fear) in male mice using social fear conditioning (SFC) and then tested social fear using the socially enriched environment test and the three-chamber test. Finally, we tested the effects of fear extinction and acute diazepam treatment in reversing social fear. Results revealed, in conditioned mice, decreased object exploration in proximity to conspecifics, social interaction, and mouse-like object exploration. Extinction training, but not acute diazepam treatment, reversed SFC-induced behavioral changes. These findings demonstrate that the socially enriched environment test provides an appropriate behavioral approach to better understand the etiology of SAD. This test may also have important implications in the exploration of new treatments.

In a previous study we could show that social fear can be induced and extinguished using virtual reality (VR). In the present study, we aimed to investigate the belongingness effect in an operant social fear conditioning (SFC) paradigm which consisted of an acquisition and an extinction phase. Forty-three participants used a joystick to approach different virtual male agents that served as conditioned stimuli. Participants were randomly allocated to one of two experimental conditions. In the electroshock condition, the unconditioned stimulus (US) used during acquisition was an electric stimulation. In the social threat condition, the US consisted of an offense: a spit in the face, mimicked by a sound and a weak air blast to the participant’s neck combined with an insult. In both groups the US was presented when participants were close to the agent (75% contingency for CS+). Outcome variables included subjective, psychophysiological and behavioral data. As expected, fear and contingency ratings increased significantly during acquisition and the differentiation between CS+ and CS- vanished during extinction. Furthermore, a clear difference in skin conductance between CS+ and CS- at the beginning of the acquisition indicated that SFC had been successful. However, a fast habituation to the US was found toward the end of the acquisition phase for the physiological response. Furthermore, participants showed avoidance behavior toward CS+ in both conditions. The results show that social fear can successfully be induced and extinguished in VR in a human sample. Thus, our paradigm can help to gain insight into learning and unlearning of social fear. Regarding the belongingness effect, the social threat condition benefits from a better differentiation between the aversive and the non-aversive stimuli. As next step we suggest comparing social-phobic patients to healthy controls in order to investigate possible differences in discrimination learning and to foster the development of more efficient treatments for social phobia.