Increased c-Fos Expression of Lateral Habenula during Social Transmission of Negative Valence in Prairie Voles

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Horn, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7443708/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Mar, 2026 Read the published version in Molecular Autism → Version 1 posted 12 You are reading this latest preprint version Abstract Background Social learning is the process of acquiring social skills, new information, or associating negative or positive valence to a context through the observation of others and through direct social interaction with others. Neurodevelopmental disorders such as autism spectrum disorder or ASD show deficits in social salience and reciprocal affective responses. Social learning is known to implicate brain areas that relate to both aspects of social salience and affective empathy such as basolateral amygdala (BLA), anterior cingulate cortex (ACC), and anterior insula (AI). Lateral Habenula (LHb), a brain area renowned for its role in negative reinforcement learning and reward prediction error has not been yet extensively studied in the domain of social learning. Methods We developed an adapted version of fear conditioning by proxy paradigm called “Social Transmission of Negative Valence” or STNV and tested social rodent species prairie voles on the task. Observers experienced negative social conditioning through a proxy cage mate that served as the demonstrator during retrieval of a cued fear memory. Observers went through a social memory recall session 24 hours after observation. We measured observers’ freezing time, self-grooming, rearing, and the range of frequency of ultrasonic vocalizations emitted as sign of distress. We also quantified immediate early gene translation as a proxy for neural activity using c-Fos immunochemistry 80 min after observing demonstrators going through memory recall. Results Socially conditioned observers that were exposed to the fear-conditioned demonstrators displayed increased freezing time, self-grooming, and rearing during social recall sessions compared to control observers. They also displayed higher USVs frequency on average compared to controls. Socially conditioned observers showed increased c-Fos expression in the LHb and BLA, ACC and AI, in comparison to controls. Conclusions Prairie voles can be conditioned to threat through social transmission of negative valence. They activate brain areas known to be involved in affective processes and social salience. LHb can be another area of interest for neural correlates of social learning and may further be investigated as a part of a Social Affect Salience Network. Social salience lateral habenula anterior cingulate cortex prairie voles social transmission of fear or threat affective empathy anterior insula basolateral amygdala autism negative valence Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Social learning plays a crucial role in our understanding of neurodevelopmental disorders where social dysfunction is characteristic, such as autism spectrum disorder (ASD). Although prevalence of ASD has increased substantially in the past decade with recent CDC estimates of 1 in 31 children in the United States having autism [ 1 ] , research on treatment has been hindered, in part, by a lack of knowledge in the brain and molecular underpinnings of the complexity and heterogeneity of social dysfunctions [ 2 , 3 ] . In addition, there are relatively few paradigms that measure social learning via an interaction between partners in translational animal models. Prior research has used social learning paradigms that hold significant translational relevance for ASD, and other disorders characterized by empathy impairments, mainly supporting the concept of social transmission of aversive stimuli via observational learning. Observational fear learning (OFL) or social fear learning (SFL) are well-established, across-species, paradigms that seek to measure transmission of stress between demonstrators (receiving aversive stimuli such as a shock, paired with conditioned stimuli such as a tone) and observers watching this classical fear conditioning. During OFL paradigms in rodents, social stress transmission is depicted by increased freezing behavior in observers witnessing demonstrators receiving aversive stimuli [ 4 – 10 ] and increased freezing behavior in a recall session 24h after transmission of stress [ 7 ] . OFL studies regarding social transmission of pain and analgesia in rodents [ 11 , 12 ] have also resulted in similar distressed phenotypes, such as increased freezing levels, displayed by both the affected demonstrator and observer. Rearing, self-grooming, and ultrasonic vocalizations (USVs) are classic behavioral markers of acute stress in rodents but are underexplored in OFL or STF experiments [ 13 – 17 ] . The social transmission of fear by proxy (STF) paradigm in rats [ 10 , 18 – 22 ] also measures social learning but through indirect exposure by the transmission of subtle social signals, where observers watch their partner freeze to the tone during memory recall instead of observing them during shock reception and fear acquisition. During a socially acquired threat recall session the following day, observer rats displayed increased freezing to the conditioned stimulus without demonstrators present as a sign of social transmission of the acquired threat. In terms of neural correlates implicated in observational social learning or social transmission of threat or pain, the anterior cingulate cortex (ACC), anterior insula (AI), and basolateral amygdala (BLA) are among the main areas of interest that showed increased activation and connectivity in response to the observation of stressed conspecifics in rodents [ 23 – 44 ] . Pharmacological manipulation and inactivation of the ACC showed its essential role in the acquisition phase of OFL and BLA in acquisition and recall [ 7 , 45 , 46 ] . We propose that these areas, the ACC, AI, and BLA are part of a “Social Affect Salience Network” (SAS) and found to be activated in response to the pain or stress of others in humans [ 47 , 48 ] . These areas are involved in detecting salient social events and in generating and regulating emotional responses and associated values to these events. They are also part of the salience network at resting-state functional connectivity [ 11 , 49 – 55 ] . Another area of particular interest that has significant connections with both the AI and ACC, is the Lateral Habenula or LHb that plays an important role in processing negative motivational information [ 56 , 57 ] . LHb has been extensively studied in the context of anti-reward processing and negative reward prediction [ 58 ] . Studies in macaques [ 59 ] and rodents [ 60 ] have identified activation of the habenula in the absence of a reward, or presence of a punishment, confirming its role in learning and motivation [ 61 – 65 ] . Additional involvement has been suggested in the pathophysiology of conditions such as major depressive disorder [ 62 ] , substance use disorder, schizophrenia, and bipolar disorder due to dysregulated reward circuitry [ 66 ] . Despite its known role in learning and emotional processing and its connection to the SAS network, there is less investigation of its role in social learning. We hypothesize that LHb will be activated during social learning in negative valence contexts in rodent species along with the other known SAS brain areas, and specifically during the observation of social distress or negative valence in others. Understanding these pathways and behavioral phenotypes associated with subtle social learning advances our understanding of the neural basis of social deficits in animal models of ASD . Inspired by the fear by proxy paradigm, here we present an adapted version termed Social Transmission of Negative Valence (STNV) and threat in line with the nomenclature of NIMH RDoC domain criteria [ 67 ] that aims to assess social transmission of negative valence (NV) in observers through witnessing the conditioned partner going through stress during a NV memory recall session. We decided to test STNV in prairie voles ( Microtus ochrogaster ), a highly sophisticated social species, due to their display of highly complex, species-specific social behaviors which include pair bond formation/maintenance, bi-parental care, social loss, and consolation [ 68 – 74 ] . We predicted that prairie vole observers would show increased freezing, self-grooming, rearing, and modified USV frequencies during NV social recall as a demonstration of social transmission of negative valence. Notably, socially conditioned observers exhibited increased freezing, self-grooming, rearing and higher ultrasonic vocalizations as well as higher neuronal activation in the LHb, ACC, BLA, and AI. Our study demonstrates that prairie voles as highly translational animal models for neurodevelopmental disorders such as ASD and suggests that the lateral habenula plays a valuable role in Social Affect Salience processing. Methods Animals Prairie voles ( Microtus ochrogaster ) used in this experiment were sexually naïve wild-type males and females, nine weeks of age. Voles were raised in a breeding colony at the University of Toledo, weaned 21 days after birth, and socially housed in same-sex duos or trios on a 12:12 light: dark fcycle. Water and food were provided and libitum during their course of life. All breeder animals and experimental subjects were less than 5 generations from the wild. All the housing, breeding, handling, and experimental procedures were approved by the University of Toledo Institutional Animal Care and Use Committee (IACUC) and were conducted following the University of Toledo Department of Laboratory Animal Resources guidelines (DLAR). Social experiments used same-sex adult pairs, all housed together since weaning. In trio cages, the third animal was removed from the enclosure at least one week before testing, allowing the remaining couple to habituate to the housing change. Adult subjects tested as solo subjects for validation studies were individually housed since weaning. An equal number of male and female subjects were used for all experiments. Experimental procedure: Social transmission of negative valence Social transmission of negative valence (STNV) is a behavioral assay that aims to measure the transmission of threat and anxiety in “observers” from the observation of a stressed conspecific, or “demonstrator” (Fig. 1 ). We developed this adapted version of a previously validated social transmission of threat paradigm used in rats [ 18 ] . Social transmission of negative valence is a two-day paradigm that assesses social learning in the prairie voles by measuring anxiety phenotypes such as freezing levels, grooming, rearing, and USVs in observers. For each of two experiments we used co-housed same-sex pairs of male and female prairie voles (9 weeks old) in two groups (shocked demonstrators and no-shock controls) to measure STNV. Day 1, session 1: Classic Threat Conditioning . Pair housed siblings with individual ear tags were brought into the testing room at least 30 minutes before testing, and the home cage was placed in a sound-attenuating isolation booth (ROOM, Brooklyn, NY). One animal was randomly designated as the observer while the other was designated as the demonstrator for the remainder of testing. The demonstrator was transferred to the shock-capable side of a two-sided modular threat conditioning cage in a sound-attenuating box (Coulbourn, Harvard Apparatus) located within a sound-attenuating booth (ROOM), separated by a transparent barrier from the shock-incapable side. Demonstrators in the experimental group were first exposed to 300 seconds of habituation with no tones and no shocks, followed by 15 consecutive tones (30s, 6KHz, 80–84 dB) each preceding a mild foot shock (1s, 1 mA) with an inter-tone interval of 120 seconds. Control demonstrators underwent the same protocol with no shocks delivered. For context, we used a clear barrier, cinnamon scent, bar grid floor, and house lights (Context A). The primary outcome measure was freezing during the tones, with freezing during habituation as a secondary measure. We recorded and analyzed freezing time using the automated program FreezeFrame (Harvard Apparatus, Holliston, MA). Freezing was counted when no animal movement was detected above threshold for more than 1 sec. To eliminate demonstrators not responding normally to test conditions, a planned outlier test was performed on average freezing across 15 tones. Demonstrators with an average freezing greater than two standard deviations away from the mean were eliminated from subsequent analyses along with their matched observers. Day 1, session 2: Social Transmission of Negative Valence. The demonstrator was kept in the experimental cage following threat conditioning. The observer was brought into the same cage on the shock-incapable side of the transparent barrier to exclude physical contact and to allow sensory observations. The observer and demonstrator were then exposed to a 300-second habituation period followed by five consecutive tones (30s, 6KHz, 80–84 dB) with an inter-tone interval of 120 seconds, for a total of 930 seconds. Freezing time was recorded and analyzed as before for the observer and demonstrator, with freezing during the tones as the primary outcome measure. Following the STNV session, observers were returned to their home cage, and demonstrators were isolated overnight in a separate cage (Experiment 1- Primary Experiment (Fig. 2 )) or both demonstrators and observers were returned to their home cage (Experiment 2-Consoling Control (Fig. 3 )). We separated the observer and demonstrator to prevent social buffering resulting from consoling behavior in the home cage [ 75 ] , which we hypothesized would reduce stress in subsequent recall sessions and attenuate social memory. Day 2: Social Threat Memory. Twenty-four hours after Day 1 testing, the observer was re-introduced to the “safe” side of the experimental cage with a metal barrier separating the cage in half. Context B consisted of a metal barrier, peppermint scent, and infrared lights. The observer was exposed to a 300s habituation period followed by five consecutive tones (30s, 6KHz, 80–84 dB) with an inter-tone interval of 120 sec. Freezing was recorded and analyzed as before, with freezing during the tones as a primary outcome measure. The session was video recorded and self-grooming duration and rearing frequency were quantified as primary outcome measures of negative valence. These behaviors were manually rated by a blinded experimenter using a behavioral coding system (The Observer XT, Noldus, Wageningen, the Netherlands). Self-grooming bouts were counted if the animal groomed itself for more than 3 sec. Rearing consisted of an animal standing on its rear limbs, extending its forelimbs. Finally, ultrasonic vocalizations (USVs) were recorded during this session in a randomly selected subset of observers using an Ultravox microphone (Noldus, Wageningen, the Netherlands). The microphone was placed on the top of the experimental chamber. The microphone's gain was reduced to 54% of its maximum capacity. Vocalizations were analyzed using the DeepSqueak software using a rat matrix with frequency cut-offs between 20–120 Hz and a score threshold of 0.5. Based on a validation study (below), the primary outcome measure was principal frequency (Hz). USV Classical Threat Conditioning (CTC) Validation study A validation experiment was conducted to better characterize vocalizations during stress and fear learning in prairie voles [ 76 – 78 ] . Singly housed voles (N = 10 experimental, N = 6 controls) underwent classical threat conditioning exactly as described above for Day 1 but without the second STNV session. Subjects were tested for threat memory as described for Day 2. Vocalizations were recorded during the threat memory test as described above. Study design, sample size, and eliminations This study includes data from two experiments on social transmission of negative valence. The experimental unit for social experiments was the pair, which consisted of two co-housed animals, a “demonstrator” and an “observer.” In Experiment 1, observers and demonstrators were housed separately following testing on Day 1; in Experiment 2, observers and demonstrators were housed together following testing on Day 1. Experiment 1 included two groups: pairs with a shocked demonstrator (N = 32) and no-shock controls (N = 30). Following the first session of Day 1, one pair from each group was eliminated as outliers based on the demonstrator’s response to threat conditioning, leaving N = 31 pairs in the shock group and N = 29 control pairs for subsequent sessions. Experiment 2 also included pairs with a shock demonstrator (N = 22) and no shock controls (N = 21). One pair was eliminated as an outlier following the first session of Day 1, and an additional two pairs were eliminated following a technical error during Session 2 that prevented the collection of data, leaving N = 20 pairs in the shock group and N = 20 control pairs for subsequent sessions. In the USV validation study, N = 10 single prairie voles experienced tone-shock pairings along with N = 6 no-shock controls. The following day, USVs were recorded from all subjects during a recall test. Subsequently, as part of Experiment 2, N = 9 observers per group were selected at random to have USVs recorded during the recall test on Day 2. Blinding Fully automated scoring was used for measures of freezing behavior and ultrasonic vocalizations. For manual scoring of self-grooming and rearing, video files were renamed by research staff not involved in testing, and behaviors were manually rated by a blinded experimenter using pre-defined criteria. Immunohistochemistry Aiming to explore the brain correlates of social learning, we quantified immediate early gene translation as a proxy for neural activity using c-Fos immunochemistry. Exactly 80 minutes following the end of the first day of STNV, observers (control demonstrator N = 10; shocked demonstrator N = 10) were euthanized by isoflurane overdose and immediately perfused transcranially with 30mL of sterile PBS, followed by 20mL of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in PBS then 50mL of 4% paraformaldehyde with 0.125% glutaraldehyde in PBS. Perfusions were done using a homemade perfusion pump set to a constant rate of 6mL/minute. Brains were dissected and submerged in a 4% paraformaldehyde solution in PBS for 24 hours and then transferred to a 30% sucrose solution in PBS for cryoprotection. Following the cryoprotection, perfused brains were cut into 40 µm sections using a cryostat (Leica CM3050 S, Deer Park, IL) and stored in PBS at 4°C until stained. Immunochemistry : Brain sections were washed 5 x 5 min in TBS, incubated for 10 min in 1% Sodium Borohydride (NaBH4), and washed 3 x 5 min in TBS. Slices were incubated in a 0.2% TritonX-100 solution (Sigma-Aldrich, St. Louis, MO) in TBS containing 10% bovine albumin serum (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature. Sections were then incubated overnight in a TBS solution containing 0.05% TritonX,- 2% BSA, and a primary rabbit polyclonal anti-fos antibody (Cell Signaling, Cat#: 2250, 1:1000 dilution) while being rocked at 4°C. The next day, sections were washed 3 X 5 min in 0.05%TPBS. Then they were incubated in TBS, 0.05% TritonX, and 2% BSA containing an AlexaFlour 488 goat anti-rabbit secondary antibody (Sigma-Aldrich, St. Louis, MO) for 2 hours at room temperature, protected from the light. Sections were washed 5 x 5 min in 1X TBS before being mounted and cover slipped using VectaShield media with DAPI (Sigma-Aldrich, St. Louis, MO). Imaging and counting : Sections were imaged at 4x magnification (Cytation5, Agilent, Santa Clara, CA) in the Integrated Core Facilities at the University of Toledo. Images were then analyzed for fluorescence intensity bilaterally at five target areas in the brain (anterior insula (AI), anterior cingulate cortex (ACC), basolateral amygdala (BLA), medial habenula (MHb), and lateral habenula (LHb) using the MCID software (GE Healthcare Life Sciences, Marlborough, MA). Brain regions were identified and manually outlined bilaterally by matching the DAPI image to the mouse brain atlas. The optical density (OD) of fluorescence within the outlined area was then quantified for both the fluorescent antibody (Channel 1) and DAPI (Channel 2). We then calculated the ratio of these ODs for each outlined area, representing c-Fos immunofluorescence per nuclear area. From 1–5 measurements of OD ratio per brain region and side were collected from serial sections; these measurements were analyzed for coefficient of variation, and measurements outside of 30% coefficient of variation were removed. The average OD ratio measurements across sections was used as the primary outcome measure of interest. Statistics and Reproducibility: For both observers and demonstrators, threat acquisition was tested using a repeated measures ANOVA with post-hoc t-tests, with time as a within-subject factor and group as a between-subject factor (demonstrators, day 1 session 1, 2x15 ANOVA with 15 tones; observers, day 1 session 2, 2x5 ANOVA with 5 tones). Threat recall was assessed in demonstrators and observers using t-tests on average freezing across 5 tones (demonstrators, day 1 session 2; observers, day 2). We used Pearson correlations to examine the correlation in freezing time between observers and demonstrators in each group separately. We used t-tests to compare self-grooming duration and rearing frequency between the stress and control groups. The ultrasonic vocalization studies also used t-tests to primary frequencies between stress and control groups. For immunohistochemistry, we used separate two-factor ANOVAs (factors of group (between) and side (within) with post-hoc t-tests to compare c-Fos immunofluorescence per nuclear area in the left and right ACC, AI, BLA, MHb, and LHb. Results i. Social Transmission of Negative Valence Experiment Classical fear learning in demonstrators . In the first session of STNV, demonstrators underwent classic fear conditioning, following a habituation period, in which they received 15 consecutive presentations of a tone paired with mild foot shocks (Fig. 1 ). Control demonstrators underwent an identical procedure without the presence of shocks. Demonstrators in the shock group (N = 32) and the no-shock control group (N = 30) did not differ in baseline freezing during habituation (t-test, t(60) = 0.251, d = 0.06, p = 0.80) signaling that both groups had similar stress levels. Demonstrators in the shock group successfully acquired a within-session conditioned response over the course of fifteen tones relative to no-shock controls, as evidenced by increased total freezing during fifteen tones (ANOVA, main effect of group, F(1,60) = 42.8, η2 = 0.42, p < 0.001) and a difference in the 15-tone freezing response curve (ANOVA, group-time interaction, F(14,47) = 4.4, η 2 = 0.57, p < 0.001, Fig. 2 a), findings that corroborate the growing literature evidence of classical fear learning in rodent species. Social threat acquisition in observers . During the second session of Day 1, observers were placed across a transparent barrier (permitting sight and sound) from demonstrators during a habituation period followed by 5 presentations of the conditioned tone with no shocks. Observers did not differ in their baseline freezing during habituation in both groups (t-test, t(58) = 0.074, d = 0.02, p = 0.94), suggesting similar baseline levels of stress between the two groups. Demonstrators in the shock group responded to the conditioned tone with elevated freezing relative to controls (t-test, t(58) = 2.7, d = 0.70, p = 0.009; Fig. 2 b). During the threat recall session and while demonstrators in the shock group responded to the tone, observers did not show elevated freezing responses in comparison to observers of no-shock control demonstrators (t-test, t(58) = 1.2, d = 0.31, p = 0.23; Fig. 2 c). Nonetheless, we observed an increased correlation between freezing in observers and demonstrators in the experimental condition. Freezing in observers and demonstrators in the shock group went from uncorrelated during tone 1 (Pearson’s r=-0.054, p = 0.77) to correlated during tone 5 (Pearson’s r = 0.45, p = 0.011) and these two correlations were significantly different (Fisher’s transformation, p = 0.04). This change in behavior in observers suggests a degree of emotional contagion during the social threat acquisition session. Social transmission of negative valence (STNV) in observers . Observers were tested 24 hours after witnessing the shocked or unshocked demonstrators to measure their responses to the conditioned tone, including freezing, self-grooming, and rearing behaviors as a potential indicators of negative valence systems. Observers in the shock group responded to the conditioned tone with elevated freezing compared to controls (t-test, t(58) = 2.0, d = 0.52, p = 0.049; Fig. 2 d), demonstrating the recall of a socially transmitted threat response. Observers in the shock group also showed a significant increase in time spent self-grooming (t-test, t = 2.2, d = 0.56, p = 0.035) and rearing bouts (t-test, t = 2.4, d = 0.63, p = 0.019) compared to observers in the control group, suggesting an increase in negatively valenced behaviors [ 61 – 64 ] (Figs. 2 e-f) Ultrasonic vocalization (USVs) in observers as another indicator of STNV . USVs were measured as an additional marker of stress response in observers. Given that this task was not performed previously in prairie voles and that stress-responses USVs are not well established in these species, we conducted a control experiment during which we measured USVs during classical fear learning in a new subset of prairie voles to examine the type of calls and frequencies prairie voles exhibit during general stress and to examine whether it corroborates with what was found during STNV. In this control experiment, we exposed prairie voles to tone-shock pairs (or tone-only controls) and, 24 hours later, recorded USVs produced during a fear memory recall session. Prairie voles conditioned to the tone-shock produced USVs at a higher principal frequency (t-test, t(14) = 2.6, d = 1.3, p = 0.022 Fig. 3 d), suggesting that higher call frequencies are associated with negative valence and may represent generalized stressed calls. No other call features were reported as statistically significant during this CTC experiment (Fig. 3 e-f). We subsequently recorded USVs in a subset of observers during social threat recall on Day 2. Observers previously paired with demonstrators in the shock group produced USVs at a higher principal frequency (one-tailed t-test, t(14) = 2.0, d = 0.99, p = 0.035) than those paired with naïve demonstrators (Fig. 3 a). This increase in principal frequency may reflect stress in observers from the shock group, consistent with changes in fear-conditioned demonstrators. Exploratory analysis of other call features also revealed shortening of average call length (t(14) = 2.8, p = 0.015) and a decrease in average power (t(14) = 2.9, p = 0.012) in observers paired with conditioned demonstrators (Fig. 3 b-c), differences that were not observed when recording from directly fear conditioned voles. This may suggest that observers in the shock group produce different types of calls that are selective to socially transmitted negative valence. Further replication is needed to characterize USVs frequencies and calls during social transmission of negative valence. ii. Control Experiment for STNV Impact of consoling behavior on social transmission of negative valence . In a separate consoling control (CC) experiment, sibling voles went through the same STNV paradigm as described above, except that they were both returned to the home cage following the 2 sessions on Day 1. We predicted that this housing condition would allow for social buffering behaviors in the home cage, including consoling behavior [ 75 ] and subsequently reducing the stress in demonstrators and the social transmission of negative valence in observers. As before, demonstrators in the shock group (N = 22) did not differ from controls (N = 21) in baseline freezing during habituation (t-test, t(41) = 1.15, d = 0.35, p = 0.26), they successfully acquired a within-session conditioned freezing response (ANOVA, main effect of group, F(1,41) = 17.9, η2 = 0.30, p < 0.001; group-time interaction, F(14,28) = 3.1, η2 = 0.61, p = 0.006, Fig. 4 a), and they demonstrated the conditioned freezing response during session 2 (t-test, t(38) = 3.4, d = 1.1, p = 0.002; Fig. 4 b). As predicted, observers did not show any differences in within-session freezing response (ANOVA, main effect of group, F(1,38) = 0.93, η2 = 0.024, p = 0.34; group-time interaction, F(4,35) = 0.79, η2 = 0.0.047, p = 0.79; Fig. 4 c) or freezing to the tone on Day 2 (t-test, t(38) = 0.45, d = 0.14, p = 0.66; Fig. 4 d), suggesting that reunion with the partner did enhance social buffering and reduced subsequent social learning . iii Neural correlates of STNV. We examined variations in c-Fos expression between observers of control (N = 10) and shocked (N = 10) demonstrators following STNV on Day 1, focusing on five key areas: the anterior cingulate cortex (ACC), anterior insula (AI), basolateral amygdala (BLA), and the medial and lateral habenula (MHb and LHb) (Fig. 5 ). Observers in the shock group showed an increase in c-Fos immunofluorescence in the ACC (ANOVA, main effect of group, F(1,18) = 0.049, η2 = 0.20, p = 0.049), AI (ANOVA, main effect of group, F(1,17) = 7.3, η2 = 0.30, p = 0.015), BLA (ANOVA, main effect of group, F(1,16) = 9.1, η2 = 0.36, p = 0.008), and LHb (ANOVA, main effect of group, F(1,17) = 5.5, η2 = 0.24, p = 0.031), but not the MHb (ANOVA, main effect of group, F(1,17) = 0.006, p = 0.94) (Fig. 5 ), corroborating the hypothesis that LHb can be part of the SAS network . Exploratory analysis on laterality in each region found that only the AI showed significant lateralization (ANOVA, group x side interaction, F(1,17) = 5.27, η2 = 0.24, p = 0.035), with the right AI activated more than the left in observers in the shock group (t-test, left vs. right AI in shock group, p = 0.026) but not different than the left in observers in the control group (p > 0.05). The right AI was more activated in observers in the shock group than in control observers (t-test, control vs. shock in right AI, p = 0.0061). The left AI was not different between the two groups (p > 0.05) Discussion Here, we used a translational and a highly social animal model, prairie voles, to investigate the behavioral and neuronal correlates of social learning, a phenomenon that is deficient in autism spectrum disorder and other neurodevelopmental disorders. We first found that prairie voles can acquire threat and associate a neutral stimulus to aversive experience based on the observation of conditioned demonstrators undergoing distress (exhibiting freezing behavior) during a threat recall session. Prairie vole observers also showed increases in classic negatively valenced responses, including increased rearing, increased self-grooming, and altered USVs. These results show that both the threat value and the negative valence of a cue can be socially transmitted through the observation of subtle social cues from a prairie vole demonstrator. Self-grooming has been linked to the hypothalamus-amygdala axis where studies have found a correlation between increased activation of the amygdala and increased self-grooming in rodents exhibiting anxious responses [ 79 – 82 ] . Rearing has also been shown to be stimulus sensitive and increases in response to stress [ 13 ] . Therefore, the significant increases in rearing and self-grooming behaviors by the observers paired with conditioned demonstrators seen in STNV are indicative of higher threat acquisition and transmission of negative valence from their partners. In addition to behavioral markers, social transmission of negative valence was also demonstrated by differential ultrasonic vocalizations (USVs) in observers paired with demonstrators under distress in comparison to observers paired with non-stressed demonstrators. During social threat recall on day 2, socially conditioned observers exhibited higher frequency USVs on average compared to controls. This higher USV frequency was also exhibited in a separate group of prairie voles (in a separate experiment) that underwent themselves, as demonstrators, a classical fear learning task, during fear recall session in comparison to controls, signaling that this elevated frequency is an indicator of elevated stress levels. Changes in USVs have been documented in prairie voles previously as an indicator of distress. Previous research has found that infant’s stress in prairie voles related to social separation caused hormonal responses that correlated with increased vocal emission rates in pups [ 83 , 84 ] . Heart rate in voles has also been found to be linked to social distress [ 85 ] and to vocal emissions where an average range of 27-35kHz was recorded during a period of social isolation with access of transmission of olfactory cues to a familiar conspecific housed nearby [ 86 ] . Although USVs were shown to decrease in number as the vole gets older [ 83 ] potentially due to less dependence on mother, they are still highly vocal during cries of distress. In addition to higher principal frequency of USVs, socially conditioned observers exhibited shorter USV call lengths and reduced average power as compared to controls. These differences in length calls and power were not observed in prairie voles undergoing classical fear learning and foot shocks. It is possible that these indicators are specific to distress in social context and maybe selective indicators of stress for others instead of stress for self. Further replications are necessary to conclude if these exploratory findings on shortness in call lengths and a reduction in call power are generalized markers of stress for others in prairie voles and in other species with highly translational values. To corroborate further that these changes in behavioral and ultrasonic vocalizations are specific markers of negative valence triggered by the distress of others and not by random factors, we conducted an additional control experiment in which we manipulated the distress levels in demonstrators by adding a social buffering condition. We showed that by reuniting observers and their distressed partners in their home cage after classical fear learning and threat recall on day 1, socially conditioned observers did not show increased freezing behavior in response to the conditioned context on day 2 in comparison to controls. Reunion which is well documented in the literature to be associated with consoling behavior, is likely to have acted as social buffering and therefore reduced the levels of distress in the partner, which have led to a more neutral response during social threat recall on the second day. Lack of direct consoling data (due to logistic difficulties) is one limitation for the study and future studies using STNV in voles can provide further validation to the role of social buffering in altering social transmission of negative valence. At the neuronal level, we conducted c-Fos immunofluorescence after observation of the stressed partner to assess the brain activity of key areas of interest as stated in the hypothesis including LHb, BLA, AI and ACC, as part of the SAS network. In line with the hypothesis, we found elevated neuronal activity in these areas in socially conditioned observers in comparison to control observers, indicating that LHb can play an important role in social learning. In accordance with our hypothesis, results demonstrated an activation of the LHb neurons in response to social stress. Most literature focuses on the habenula in non-social reinforcement learning and negative valence [ 58 , 87 – 89 ] where the lateral habenula, specifically, has shown to be the central hub for aversive and impulsive action integration [ 59 , 90 ] . The LHbs associated neurons receive afferent input from the limbic system and basal ganglia, helping to modulate motivation and emotional information [ 91 ] . It then exerts indirect inhibitory control over midbrain dopaminergic (ventral tegmental area, substantia nigra pars compacta) and serotonergic (raphe nuclei) centers through activating GABAergic neurons. Due to this, LHb stimulation is typically associated with behavioral responses to aversive stimuli and suppression of reward-related activity [ 58 , 90 , 92 – 96 ] . This leads to the performance of avoidance responses such as social withdrawal and reduced motivation where in fact overactivation has been correlated with depressive-like phenotypes: anhedonia, behavioral despair, and heightened anxiety [ 87 , 88 ] . On the contrary, suppression of the LHb increases dopamine turnover, leading to rewarding phenotypes, facilitating approach behaviors, reduced anxiety, and diminished avoidance responses [ 90 , 95 ] . Disruptions within the LHb circuitry are noted to contribute to the dysregulation of underlying impairments in learning, decision-making, and affective processing; all of which are critical for socially based behaviors [ 92 , 94 , 97 ] . Although a more detailed exploration of this connectivity is necessary for application across diverse psychiatric disorders, a study comparing magnetic resonance imaging in humans found that across all ages included, the habenula was larger in those diagnosed with ASD when compared to controls [ 98 ] . Other literature shows experimental models in adolescent rats that have shown the exclusion from social play induces anxiety-like phenotypes and increases c-Fos expression within the LHb, implicating its role in emotional consequences of early social stress [ 99 ] . A more recent study in 2024 study on social fear conditioning in mice found that neurons projecting from the lateral habenula (LHb) to the medial prefrontal cortex (mPFC) were highly activated during social fear, and that inhibiting this LHb–mPFC pathway significantly reduced fear responses [ 100 ] . These results align with evidence that core symptoms of ASD, such as communication deficits, repetitive behaviors, sensory dysregulation, and mood disorders, overlap with domains in which the habenula has been implicated. Additionally, the higher activation of the ACC and AI in observers of stressed voles is in accordance with previous literature involving these areas of social learning and emotion contagion. Shank3 mutant mice, an animal model of autism, display structural and functional impairments in the ACC, associated with deficits in social interaction [ 101 , 102 ] . Studies in humans, macaques, and rodents show that the ACC is essential for the social success of an individual in a group environment [ 103 – 106 ] . They have also shown that the ACC, AI, and BLA are jointly activated in empathy-related or emotion-contagion studies [ 48 , 107 – 109 ] , reinforcing the idea of complementary motor and sensory system feedback [ 110 – 112 ] as well as connections through the SAS network. These findings contribute to our understanding of stress-induced c-Fos activation patterns in specific brain regions, shedding light on neural responses to social stressors and implications in those diagnosed with autism. Limitations We lack explicit consoling data in the control experiment to show that social buffering (duration of consoling behavior or allogrooming) is the cause of lack of freezing during social threat recall in the behavioral control experiment. Future experiments should include strangers studying the behavioral and physiological responses in prairie voles to the stress of unfamiliar others. Inhibition lateral habenula and studying its effects on social learning can be crucial to better characterize its role in this process. Conclusions Our findings suggest that prairie voles are capable of socially transmitting information and can acquire new information through observation of subtle social cues. We discovered that lateral habenula area plays an important role in social transmission of negative valence. Further molecular and pharmacological manipulations confirming the essential role of this area in Social Affect Salience Network can be crucial. In humans, investigating the role of lateral habenula in social learning and in neurodevelopmental disorders such as autism can be also critical as it can shed the light to new targeted brain circuitry or areas implicated in social affective and social salience processes. Abbreviations Lateral habenula (LHb) Basolateral amygdala (BLA) Anterior insula (AI) Anterior cingulate cortex (ACC) Social transmission of negative valence (STNV) Autism spectrum disorder (ASD) Medial habenula (MHb) Ultrasonic vocalizations (USVs) Negative valence (NV) Social Affect Salience Network (SAS) Declarations Acknowledgements We would like to acknowledge the integral contributions of Daniella Gamboa Pabón, who passed away prior to the publication of this manuscript. We honor her legacy and are grateful for the opportunity to continue and share the work she fostered. To read more of her story, please visit: https://www.utoledo.edu/med/research/andari/daniella.html. Funding Declaration E.A. discloses support for the research of this work through a gift from ProMedica Health System Foundation to The University of Toledo [Autism and Social Neuroscience, index number 207007. Author contributions D.G-P. conceptualized the study, conducted the investigation and data curation, performed the formal analysis and co-wrote the manuscript. J.H-K. conducted the visualization, organization, and formatting of data and results, co-wrote the manuscript, and contributed to the interpretation of findings. S.P. and B.A.H. conducted the investigation, data curation, and data analysis. J.P.B. conducted/supervised the investigation, conducted analysis, and co-wrote the manuscript. E.A. conceptualized the study design, supervised the study investigation, conducted analysis, supervised visualization, and co-wrote the final version of the manuscript. Competing interests The authors declare no competing interests. Data availability All raw data has been included in supplemental files. Additional information (Supplementary information) Supplementary figures and tables can be found in the supplemental file. References Shaw KA, et al. 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Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 26 Mar, 2026 Read the published version in Molecular Autism → Version 1 posted Editorial decision: Revision requested 27 Oct, 2025 Reviews received at journal 09 Oct, 2025 Reviews received at journal 06 Oct, 2025 Reviews received at journal 04 Oct, 2025 Reviewers agreed at journal 28 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers agreed at journal 17 Sep, 2025 Reviewers invited by journal 17 Sep, 2025 Editor assigned by journal 27 Aug, 2025 Submission checks completed at journal 26 Aug, 2025 First submitted to journal 23 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7443708","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":518295586,"identity":"6f8769ea-b585-4bbd-9e14-4f31fecf8439","order_by":0,"name":"Daniella Gamboa Pabón","email":"","orcid":"","institution":"The University of Toledo","correspondingAuthor":false,"prefix":"","firstName":"Daniella","middleName":"Gamboa","lastName":"Pabón","suffix":""},{"id":518295587,"identity":"a642acc0-6a19-42b6-93d1-0885c272bc65","order_by":1,"name":"Jolee Hatfield-King","email":"","orcid":"","institution":"The University of Toledo","correspondingAuthor":false,"prefix":"","firstName":"Jolee","middleName":"","lastName":"Hatfield-King","suffix":""},{"id":518295588,"identity":"7084a503-deb1-468e-8985-15e8169262e2","order_by":2,"name":"Shivangi Patel","email":"","orcid":"","institution":"MetroHealth System","correspondingAuthor":false,"prefix":"","firstName":"Shivangi","middleName":"","lastName":"Patel","suffix":""},{"id":518295589,"identity":"a1a10807-3fac-4d4e-92d0-331410933a2e","order_by":3,"name":"Brandon A. 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18:21:37","extension":"html","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":204966,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/26ec0b051d00421bd50d29bf.html"},{"id":92205380,"identity":"cee1a1bd-d7a6-4044-8b77-8676cfcfd80c","added_by":"auto","created_at":"2025-09-25 18:21:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":110188,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSocial transmission of negative valence (Primary Experiment).\u003c/strong\u003e Experimental design of the task.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/2010650d7bd9ebbb62dd0bdf.png"},{"id":92205382,"identity":"fe2df521-ccdf-4e0f-b622-738650f31896","added_by":"auto","created_at":"2025-09-25 18:21:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":222437,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSocial Transmission of Negative Valence (Primary Experiment)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-b: Threat conditioning in demonstrators. (a)\u003c/strong\u003e Demonstrators exposed to tone-shock pairings on Day 1, session 1, developed a conditioned freezing response over the course of 15 tones, as compared to no-shock controls. \u003cstrong\u003e(b)\u003c/strong\u003e Demonstrators exposed to tone-shock pairings demonstrated a conditioned freezing response to tones while across a transparent barrier from an observer. Error bars show SEM. *p \u0026lt; .05 **p \u0026lt; 0.01.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ec-d: Social threat conditioning in observers. (c)\u003c/strong\u003e Observers across a transparent barrier from fear-conditioned demonstrators on Day 1, session 2 did not acquire a within-session freezing response to tones. \u003cstrong\u003e(d)\u003c/strong\u003eOn Day 2, observers of fear-conditioned demonstrators showed a conditioned freezing response to the tone. Error bars show SEM. * p \u0026lt; .05 **p \u0026lt; 0.01\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ee- f: Negatively valenced behaviors in observers\u003c/strong\u003e. During the threat recall session on Day 2, observers of fear-conditioned demonstrators showed an increase in negatively valenced behaviors, including \u003cstrong\u003e(e)\u003c/strong\u003e self-grooming and \u003cstrong\u003e(f)\u003c/strong\u003e rearing. Error bars show SEM. *p \u0026lt; .05 **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/e96256132b0d5fc391ec82fb.png"},{"id":92205384,"identity":"db46b8a6-5407-4cc2-bcf9-74fb26c42c79","added_by":"auto","created_at":"2025-09-25 18:21:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":217171,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCharacteristics of Ultrasonic Vocalizations in STNV and CTC\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-c: Social Transmission of Negative Valence in Observers (Primary Experiment):\u003c/strong\u003e During the threat recall session on Day 2, observers of fear-conditioned demonstrators showed varying USV characteristics, including \u003cstrong\u003e(a)\u003c/strong\u003e higher principal frequency, \u003cstrong\u003e(b)\u003c/strong\u003e increased average call length, \u003cstrong\u003e(c)\u003c/strong\u003e decreased average power. Error bars show SEM. *p \u0026lt; .05 **p \u0026lt; 0.01\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ed-f: Classical Threat Conditioning in Prairie voles (Validation Experiment):\u003c/strong\u003e During the validation experiment, conditioned prairie voles exhibited \u003cstrong\u003e(d)\u003c/strong\u003e higher principal frequency but did not display change in \u003cstrong\u003e(e)\u003c/strong\u003e average call length or \u003cstrong\u003e(f)\u003c/strong\u003eaverage power. Error bars show SEM. *p \u0026lt; .05 **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/722ae7d2ae9c15840bd96528.png"},{"id":92205388,"identity":"a25b0883-1019-446f-a942-54a81a1545b9","added_by":"auto","created_at":"2025-09-25 18:21:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":262236,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eConsoling Control for STNV\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ea-d: Social buffering and social threat conditioning. (a)\u003c/strong\u003e As before, demonstrators exposed to tone-shock pairings (A) developed a conditioned freezing response and \u003cstrong\u003e(b)\u003c/strong\u003edemonstrated that conditioned freezing response while across a transparent barrier from an observer. \u003cstrong\u003e(c) \u003c/strong\u003eThe observer did not acquire a within-session freezing response. \u003cstrong\u003e(d)\u003c/strong\u003e On Day 2, following unhindered overnight cohabitation between demonstrators and observers, observers of fear-conditioned demonstrators did not show a conditioned freezing response to the tone. Error bars show SEM. *p \u0026lt; .05 **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/50f2e1eaec38e6df7ce91cfd.png"},{"id":92205385,"identity":"5c9475f4-32c7-49d6-8f7b-6bb2b35ed619","added_by":"auto","created_at":"2025-09-25 18:21:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":63944,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eImmunohistochemistry.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eGFP (c-Fos) immunofluorescence in Habenula, lateral (left) and medial (right), Basolateral Amygdala, Anterior Insula, and Anterior Cingulate Cortex. Dashed lines represent the quantified area for each region for both the blue and green channels. (\u003cstrong\u003eB)\u003c/strong\u003e Relative differences in brain region-specific activity in observers of shocked demonstrators as compared to control observers. Bars show the quantitative differences in region-specific cellular activity, as measured by c-Fos immunofluorescence per nuclear area. *p \u0026lt; .05 **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Picture5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/6519bd5aab902810205468db.jpg"},{"id":105755834,"identity":"88cb1f6d-c934-42af-8784-a1a9f9052866","added_by":"auto","created_at":"2026-03-30 16:31:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1873684,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7443708/v1/da67d637-1465-47ce-afed-13546285066e.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Increased c-Fos Expression of Lateral Habenula during Social Transmission of Negative Valence in Prairie Voles","fulltext":[{"header":"Background","content":"\u003cp\u003eSocial learning plays a crucial role in our understanding of neurodevelopmental disorders where social dysfunction is characteristic, such as autism spectrum disorder (ASD). Although prevalence of ASD has increased substantially in the past decade with recent CDC estimates of 1 in 31 children in the United States having autism\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e, research on treatment has been hindered, in part, by a lack of knowledge in the brain and molecular underpinnings of the complexity and heterogeneity of social dysfunctions \u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. In addition, there are relatively few paradigms that measure social learning via an interaction between partners in translational animal models.\u003c/p\u003e\u003cp\u003ePrior research has used social learning paradigms that hold significant translational relevance for ASD, and other disorders characterized by empathy impairments, mainly supporting the concept of social transmission of aversive stimuli via observational learning. Observational fear learning (OFL) or social fear learning (SFL) are well-established, across-species, paradigms that seek to measure transmission of stress between demonstrators (receiving aversive stimuli such as a shock, paired with conditioned stimuli such as a tone) and observers watching this classical fear conditioning. During OFL paradigms in rodents, social stress transmission is depicted by increased freezing behavior in observers witnessing demonstrators receiving aversive stimuli \u003csup\u003e[\u003cspan additionalcitationids=\"CR5 CR6 CR7 CR8 CR9\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e and increased freezing behavior in a recall session 24h after transmission of stress \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. OFL studies regarding social transmission of \u003cem\u003epain and analgesia\u003c/em\u003e in rodents \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e have also resulted in similar distressed phenotypes, such as increased freezing levels, displayed by both the affected demonstrator and observer. Rearing, self-grooming, and ultrasonic vocalizations (USVs) are classic behavioral markers of acute stress in rodents but are underexplored in OFL or STF experiments \u003csup\u003e[\u003cspan additionalcitationids=\"CR14 CR15 CR16\" citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe social transmission of fear by proxy (STF) paradigm in rats \u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e also measures social learning but through indirect exposure by the transmission of subtle social signals, where observers watch their partner freeze to the tone during memory recall instead of observing them during shock reception and fear acquisition. During a socially acquired threat recall session the following day, observer rats displayed increased freezing to the conditioned stimulus without demonstrators present as a sign of social transmission of the acquired threat.\u003c/p\u003e\u003cp\u003eIn terms of neural correlates implicated in observational social learning or social transmission of threat or pain, the anterior cingulate cortex (ACC), anterior insula (AI), and basolateral amygdala (BLA) are among the main areas of interest that showed increased activation and connectivity in response to the observation of stressed conspecifics in rodents \u003csup\u003e[\u003cspan additionalcitationids=\"CR24 CR25 CR26 CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34 CR35 CR36 CR37 CR38 CR39 CR40 CR41 CR42 CR43\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]\u003c/sup\u003e. Pharmacological manipulation and inactivation of the ACC showed its essential role in the acquisition phase of OFL and BLA in acquisition and recall \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe propose that these areas, the ACC, AI, and BLA are part of a \u0026ldquo;Social Affect Salience Network\u0026rdquo; (SAS) and found to be activated in response to the pain or stress of others in humans \u003csup\u003e[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]\u003c/sup\u003e. These areas are involved in detecting salient social events and in generating and regulating emotional responses and associated values to these events. They are also part of the salience network at resting-state functional connectivity \u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan additionalcitationids=\"CR50 CR51 CR52 CR53 CR54\" citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eAnother area of particular interest that has significant connections with both the AI and ACC, is the \u003cspan type=\"ItalicUnderline\" class=\"ItalicUnderline\" name=\"Emphasis\"\u003eLateral Habenula or LHb\u003c/span\u003e that plays an important role in processing negative motivational information \u003csup\u003e[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]\u003c/sup\u003e. LHb has been extensively studied in the context of anti-reward processing and negative reward prediction \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]\u003c/sup\u003e. Studies in macaques \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]\u003c/sup\u003e and rodents \u003csup\u003e[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]\u003c/sup\u003e have identified activation of the habenula in the absence of a reward, or presence of a punishment, confirming its role in learning and motivation \u003csup\u003e[\u003cspan additionalcitationids=\"CR62 CR63 CR64\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]\u003c/sup\u003e. Additional involvement has been suggested in the pathophysiology of conditions such as major depressive disorder \u003csup\u003e[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]\u003c/sup\u003e, substance use disorder, schizophrenia, and bipolar disorder due to dysregulated reward circuitry \u003csup\u003e[\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDespite its known role in learning and emotional processing and its connection to the SAS network, there is less investigation of its role in social learning. We hypothesize that LHb will be activated during social learning in negative valence contexts in rodent species along with the other known SAS brain areas, and specifically during the observation of social distress or negative valence in others. \u003cem\u003eUnderstanding these pathways and behavioral phenotypes associated with subtle social learning advances our understanding of the neural basis of social deficits in animal models of ASD\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eInspired by the fear by proxy paradigm, here we present an adapted version termed Social Transmission of Negative Valence (STNV) and threat in line with the nomenclature of NIMH RDoC domain criteria \u003csup\u003e[\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]\u003c/sup\u003e that aims to assess social transmission of negative valence (NV) in observers through witnessing the conditioned partner going through stress during a NV memory recall session. We decided to test STNV in prairie voles (\u003cem\u003eMicrotus ochrogaster\u003c/em\u003e), a highly sophisticated social species, due to their display of highly complex, species-specific social behaviors which include pair bond formation/maintenance, bi-parental care, social loss, and consolation \u003csup\u003e[\u003cspan additionalcitationids=\"CR69 CR70 CR71 CR72 CR73\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]\u003c/sup\u003e. We predicted that prairie vole observers would show increased freezing, self-grooming, rearing, and modified USV frequencies during NV social recall as a demonstration of social transmission of negative valence.\u003c/p\u003e\u003cp\u003eNotably, socially conditioned observers exhibited increased freezing, self-grooming, rearing and higher ultrasonic vocalizations as well as higher neuronal activation in the LHb, ACC, BLA, and AI. Our study demonstrates that prairie voles as highly translational animal models for neurodevelopmental disorders such as ASD and suggests that the lateral habenula plays a valuable role in Social Affect Salience processing.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eAnimals\u003c/h2\u003e\u003cp\u003ePrairie voles (\u003cem\u003eMicrotus ochrogaster\u003c/em\u003e) used in this experiment were sexually na\u0026iuml;ve wild-type males and females, nine weeks of age. Voles were raised in a breeding colony at the University of Toledo, weaned 21 days after birth, and socially housed in same-sex duos or trios on a 12:12 light: dark fcycle. Water and food were provided and libitum during their course of life. All breeder animals and experimental subjects were less than 5 generations from the wild. All the housing, breeding, handling, and experimental procedures were approved by the University of Toledo Institutional Animal Care and Use Committee (IACUC) and were conducted following the University of Toledo Department of Laboratory Animal Resources guidelines (DLAR).\u003c/p\u003e\u003cp\u003eSocial experiments used same-sex adult pairs, all housed together since weaning. In trio cages, the third animal was removed from the enclosure at least one week before testing, allowing the remaining couple to habituate to the housing change. Adult subjects tested as solo subjects for validation studies were individually housed since weaning. An equal number of male and female subjects were used for all experiments.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental procedure: Social transmission of negative valence\u003c/h3\u003e\n\u003cp\u003eSocial transmission of negative valence (STNV) is a behavioral assay that aims to measure the transmission of threat and anxiety in \u0026ldquo;observers\u0026rdquo; from the observation of a stressed conspecific, or \u0026ldquo;demonstrator\u0026rdquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We developed this adapted version of a previously validated social transmission of threat paradigm used in rats \u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Social transmission of negative valence is a two-day paradigm that assesses social learning in the prairie voles by measuring anxiety phenotypes such as freezing levels, grooming, rearing, and USVs in observers. For each of two experiments we used co-housed same-sex pairs of male and female prairie voles (9 weeks old) in two groups (shocked demonstrators and no-shock controls) to measure STNV.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDay 1, session 1: Classic Threat Conditioning\u003c/span\u003e. Pair housed siblings with individual ear tags were brought into the testing room at least 30 minutes before testing, and the home cage was placed in a sound-attenuating isolation booth (ROOM, Brooklyn, NY). One animal was randomly designated as the observer while the other was designated as the demonstrator for the remainder of testing. The demonstrator was transferred to the shock-capable side of a two-sided modular threat conditioning cage in a sound-attenuating box (Coulbourn, Harvard Apparatus) located within a sound-attenuating booth (ROOM), separated by a transparent barrier from the shock-incapable side. Demonstrators in the experimental group were first exposed to 300 seconds of habituation with no tones and no shocks, followed by 15 consecutive tones (30s, 6KHz, 80\u0026ndash;84 dB) each preceding a mild foot shock (1s, 1 mA) with an inter-tone interval of 120 seconds. Control demonstrators underwent the same protocol with no shocks delivered. For context, we used a clear barrier, cinnamon scent, bar grid floor, and house lights (Context A).\u003c/p\u003e\u003cp\u003eThe primary outcome measure was freezing during the tones, with freezing during habituation as a secondary measure. We recorded and analyzed freezing time using the automated program FreezeFrame (Harvard Apparatus, Holliston, MA). Freezing was counted when no animal movement was detected above threshold for more than 1 sec. To eliminate demonstrators not responding normally to test conditions, a planned outlier test was performed on average freezing across 15 tones. Demonstrators with an average freezing greater than two standard deviations away from the mean were eliminated from subsequent analyses along with their matched observers.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDay 1, session 2: Social Transmission of Negative Valence.\u003c/span\u003e The demonstrator was kept in the experimental cage following threat conditioning. The observer was brought into the same cage on the shock-incapable side of the transparent barrier to exclude physical contact and to allow sensory observations. The observer and demonstrator were then exposed to a 300-second habituation period followed by five consecutive tones (30s, 6KHz, 80\u0026ndash;84 dB) with an inter-tone interval of 120 seconds, for a total of 930 seconds. Freezing time was recorded and analyzed as before for the observer and demonstrator, with freezing during the tones as the primary outcome measure.\u003c/p\u003e\u003cp\u003eFollowing the STNV session, observers were returned to their home cage, and demonstrators were isolated overnight in a separate cage (Experiment 1- Primary Experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e)) or both demonstrators and observers were returned to their home cage (Experiment 2-Consoling Control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e)). We separated the observer and demonstrator to prevent social buffering resulting from consoling behavior in the home cage \u003csup\u003e[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e, which we hypothesized would reduce stress in subsequent recall sessions and attenuate social memory.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eDay 2: Social Threat Memory.\u003c/span\u003e Twenty-four hours after Day 1 testing, the observer was re-introduced to the \u0026ldquo;safe\u0026rdquo; side of the experimental cage with a metal barrier separating the cage in half. Context B consisted of a metal barrier, peppermint scent, and infrared lights. The observer was exposed to a 300s habituation period followed by five consecutive tones (30s, 6KHz, 80\u0026ndash;84 dB) with an inter-tone interval of 120 sec. Freezing was recorded and analyzed as before, with freezing during the tones as a primary outcome measure.\u003c/p\u003e\u003cp\u003eThe session was video recorded and self-grooming duration and rearing frequency were quantified as primary outcome measures of negative valence. These behaviors were manually rated by a blinded experimenter using a behavioral coding system (The Observer XT, Noldus, Wageningen, the Netherlands). Self-grooming bouts were counted if the animal groomed itself for more than 3 sec. Rearing consisted of an animal standing on its rear limbs, extending its forelimbs.\u003c/p\u003e\u003cp\u003eFinally, ultrasonic vocalizations (USVs) were recorded during this session in a randomly selected subset of observers using an Ultravox microphone (Noldus, Wageningen, the Netherlands). The microphone was placed on the top of the experimental chamber. The microphone's gain was reduced to 54% of its maximum capacity. Vocalizations were analyzed using the DeepSqueak software using a rat matrix with frequency cut-offs between 20\u0026ndash;120 Hz and a score threshold of 0.5. Based on a validation study (below), the primary outcome measure was principal frequency (Hz).\u003c/p\u003e\n\u003ch3\u003eUSV Classical Threat Conditioning (CTC) Validation study\u003c/h3\u003e\n\u003cp\u003eA validation experiment was conducted to better characterize vocalizations during stress and fear learning in prairie voles \u003csup\u003e[\u003cspan additionalcitationids=\"CR77\" citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]\u003c/sup\u003e. Singly housed voles (N\u0026thinsp;=\u0026thinsp;10 experimental, N\u0026thinsp;=\u0026thinsp;6 controls) underwent classical threat conditioning exactly as described above for Day 1 but without the second STNV session. Subjects were tested for threat memory as described for Day 2. Vocalizations were recorded during the threat memory test as described above.\u003c/p\u003e\n\u003ch3\u003eStudy design, sample size, and eliminations\u003c/h3\u003e\n\u003cp\u003eThis study includes data from two experiments on social transmission of negative valence. The experimental unit for social experiments was the pair, which consisted of two co-housed animals, a \u0026ldquo;demonstrator\u0026rdquo; and an \u0026ldquo;observer.\u0026rdquo; In Experiment 1, observers and demonstrators were housed separately following testing on Day 1; in Experiment 2, observers and demonstrators were housed together following testing on Day 1. Experiment 1 included two groups: pairs with a shocked demonstrator (N\u0026thinsp;=\u0026thinsp;32) and no-shock controls (N\u0026thinsp;=\u0026thinsp;30). Following the first session of Day 1, one pair from each group was eliminated as outliers based on the demonstrator\u0026rsquo;s response to threat conditioning, leaving N\u0026thinsp;=\u0026thinsp;31 pairs in the shock group and N\u0026thinsp;=\u0026thinsp;29 control pairs for subsequent sessions. Experiment 2 also included pairs with a shock demonstrator (N\u0026thinsp;=\u0026thinsp;22) and no shock controls (N\u0026thinsp;=\u0026thinsp;21). One pair was eliminated as an outlier following the first session of Day 1, and an additional two pairs were eliminated following a technical error during Session 2 that prevented the collection of data, leaving N\u0026thinsp;=\u0026thinsp;20 pairs in the shock group and N\u0026thinsp;=\u0026thinsp;20 control pairs for subsequent sessions.\u003c/p\u003e\u003cp\u003eIn the USV validation study, N\u0026thinsp;=\u0026thinsp;10 single prairie voles experienced tone-shock pairings along with N\u0026thinsp;=\u0026thinsp;6 no-shock controls. The following day, USVs were recorded from all subjects during a recall test. Subsequently, as part of Experiment 2, N\u0026thinsp;=\u0026thinsp;9 observers per group were selected at random to have USVs recorded during the recall test on Day 2.\u003c/p\u003e\n\u003ch3\u003eBlinding\u003c/h3\u003e\n\u003cp\u003eFully automated scoring was used for measures of freezing behavior and ultrasonic vocalizations. For manual scoring of self-grooming and rearing, video files were renamed by research staff not involved in testing, and behaviors were manually rated by a blinded experimenter using pre-defined criteria.\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eImmunohistochemistry\u003c/h2\u003e\u003cp\u003eAiming to explore the brain correlates of social learning, we quantified immediate early gene translation as a proxy for neural activity using c-Fos immunochemistry. Exactly 80 minutes following the end of the first day of STNV, observers (control demonstrator N\u0026thinsp;=\u0026thinsp;10; shocked demonstrator N\u0026thinsp;=\u0026thinsp;10) were euthanized by isoflurane overdose and immediately perfused transcranially with 30mL of sterile PBS, followed by 20mL of 1% paraformaldehyde (Sigma-Aldrich, St. Louis, MO) in PBS then 50mL of 4% paraformaldehyde with 0.125% glutaraldehyde in PBS. Perfusions were done using a homemade perfusion pump set to a constant rate of 6mL/minute. Brains were dissected and submerged in a 4% paraformaldehyde solution in PBS for 24 hours and then transferred to a 30% sucrose solution in PBS for cryoprotection. Following the cryoprotection, perfused brains were cut into 40 \u0026micro;m sections using a cryostat (Leica CM3050 S, Deer Park, IL) and stored in PBS at 4\u0026deg;C until stained.\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImmunochemistry\u003c/span\u003e: Brain sections were washed 5 x 5 min in TBS, incubated for 10 min in 1% Sodium Borohydride (NaBH4), and washed 3 x 5 min in TBS. Slices were incubated in a 0.2% TritonX-100 solution (Sigma-Aldrich, St. Louis, MO) in TBS containing 10% bovine albumin serum (Sigma-Aldrich, St. Louis, MO) for 1 hour at room temperature. Sections were then incubated overnight in a TBS solution containing 0.05% TritonX,- 2% BSA, and a primary rabbit polyclonal anti-fos antibody (Cell Signaling, Cat#: 2250, 1:1000 dilution) while being rocked at 4\u0026deg;C. The next day, sections were washed 3 X 5 min in 0.05%TPBS. Then they were incubated in TBS, 0.05% TritonX, and 2% BSA containing an AlexaFlour 488 goat anti-rabbit secondary antibody (Sigma-Aldrich, St. Louis, MO) for 2 hours at room temperature, protected from the light. Sections were washed 5 x 5 min in 1X TBS before being mounted and cover slipped using VectaShield media with DAPI (Sigma-Aldrich, St. Louis, MO).\u003c/p\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImaging and counting\u003c/span\u003e: Sections were imaged at 4x magnification (Cytation5, Agilent, Santa Clara, CA) in the Integrated Core Facilities at the University of Toledo. Images were then analyzed for fluorescence intensity bilaterally at five target areas in the brain (anterior insula (AI), anterior cingulate cortex (ACC), basolateral amygdala (BLA), medial habenula (MHb), and lateral habenula (LHb) using the MCID software (GE Healthcare Life Sciences, Marlborough, MA). Brain regions were identified and manually outlined bilaterally by matching the DAPI image to the mouse brain atlas. The optical density (OD) of fluorescence within the outlined area was then quantified for both the fluorescent antibody (Channel 1) and DAPI (Channel 2). We then calculated the ratio of these ODs for each outlined area, representing c-Fos immunofluorescence per nuclear area. From 1\u0026ndash;5 measurements of OD ratio per brain region and side were collected from serial sections; these measurements were analyzed for coefficient of variation, and measurements outside of 30% coefficient of variation were removed. The average OD ratio measurements across sections was used as the primary outcome measure of interest.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eStatistics and Reproducibility:\u003c/h3\u003e\n\u003cp\u003eFor both observers and demonstrators, threat acquisition was tested using a repeated measures ANOVA with post-hoc t-tests, with time as a within-subject factor and group as a between-subject factor (demonstrators, day 1 session 1, 2x15 ANOVA with 15 tones; observers, day 1 session 2, 2x5 ANOVA with 5 tones). Threat recall was assessed in demonstrators and observers using t-tests on average freezing across 5 tones (demonstrators, day 1 session 2; observers, day 2). We used Pearson correlations to examine the correlation in freezing time between observers and demonstrators in each group separately. We used t-tests to compare self-grooming duration and rearing frequency between the stress and control groups. The ultrasonic vocalization studies also used t-tests to primary frequencies between stress and control groups. For immunohistochemistry, we used separate two-factor ANOVAs (factors of group (between) and side (within) with post-hoc t-tests to compare c-Fos immunofluorescence per nuclear area in the left and right ACC, AI, BLA, MHb, and LHb.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003ei. Social Transmission of Negative Valence Experiment\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eClassical fear learning in demonstrators\u003c/span\u003e.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eIn the first session of STNV, demonstrators underwent classic fear conditioning, following a habituation period, in which they received 15 consecutive presentations of a tone paired with mild foot shocks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Control demonstrators underwent an identical procedure without the presence of shocks. Demonstrators in the shock group (N\u0026thinsp;=\u0026thinsp;32) and the no-shock control group (N\u0026thinsp;=\u0026thinsp;30) did not differ in baseline freezing during habituation (t-test, t(60)\u0026thinsp;=\u0026thinsp;0.251, d\u0026thinsp;=\u0026thinsp;0.06, p\u0026thinsp;=\u0026thinsp;0.80) signaling that both groups had similar stress levels. Demonstrators in the shock group successfully acquired a within-session conditioned response over the course of fifteen tones relative to no-shock controls, as evidenced by increased total freezing during fifteen tones (ANOVA, main effect of group, F(1,60)\u0026thinsp;=\u0026thinsp;42.8, η2\u0026thinsp;=\u0026thinsp;0.42, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and a difference in the 15-tone freezing response curve (ANOVA, group-time interaction, F(14,47)\u0026thinsp;=\u0026thinsp;4.4, η\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.57, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea), findings that corroborate the growing literature evidence of classical fear learning in rodent species.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSocial threat acquisition in observers\u003c/span\u003e.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eDuring the second session of Day 1, observers were placed across a transparent barrier (permitting sight and sound) from demonstrators during a habituation period followed by 5 presentations of the conditioned tone with no shocks. Observers did not differ in their baseline freezing during habituation in both groups (t-test, t(58)\u0026thinsp;=\u0026thinsp;0.074, d\u0026thinsp;=\u0026thinsp;0.02, p\u0026thinsp;=\u0026thinsp;0.94), suggesting similar baseline levels of stress between the two groups. Demonstrators in the shock group responded to the conditioned tone with elevated freezing relative to controls (t-test, t(58)\u0026thinsp;=\u0026thinsp;2.7, d\u0026thinsp;=\u0026thinsp;0.70, p\u0026thinsp;=\u0026thinsp;0.009; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). During the threat recall session and while demonstrators in the shock group responded to the tone, observers did not show elevated freezing responses in comparison to observers of no-shock control demonstrators (t-test, t(58)\u0026thinsp;=\u0026thinsp;1.2, d\u0026thinsp;=\u0026thinsp;0.31, p\u0026thinsp;=\u0026thinsp;0.23; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Nonetheless, we observed an increased correlation between freezing in observers and demonstrators in the experimental condition. Freezing in observers and demonstrators in the shock group went from uncorrelated during tone 1 (Pearson\u0026rsquo;s r=-0.054, p\u0026thinsp;=\u0026thinsp;0.77) to correlated during tone 5 (Pearson\u0026rsquo;s r\u0026thinsp;=\u0026thinsp;0.45, p\u0026thinsp;=\u0026thinsp;0.011) and these two correlations were significantly different (Fisher\u0026rsquo;s transformation, p\u0026thinsp;=\u0026thinsp;0.04). This change in behavior in observers suggests a degree of emotional contagion during the social threat acquisition session.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eSocial transmission of negative valence (STNV) in observers\u003c/span\u003e.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eObservers were tested 24 hours after witnessing the shocked or unshocked demonstrators to measure their responses to the conditioned tone, including freezing, self-grooming, and rearing behaviors as a potential indicators of negative valence systems. Observers in the shock group responded to the conditioned tone with elevated freezing compared to controls (t-test, t(58)\u0026thinsp;=\u0026thinsp;2.0, d\u0026thinsp;=\u0026thinsp;0.52, p\u0026thinsp;=\u0026thinsp;0.049; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed), demonstrating the recall of a socially transmitted threat response. Observers in the shock group also showed a significant increase in time spent self-grooming (t-test, t\u0026thinsp;=\u0026thinsp;2.2, d\u0026thinsp;=\u0026thinsp;0.56, p\u0026thinsp;=\u0026thinsp;0.035) and rearing bouts (t-test, t\u0026thinsp;=\u0026thinsp;2.4, d\u0026thinsp;=\u0026thinsp;0.63, p\u0026thinsp;=\u0026thinsp;0.019) compared to observers in the control group, suggesting an increase in negatively valenced behaviors \u003csup\u003e[\u003cspan additionalcitationids=\"CR62 CR63\" citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]\u003c/sup\u003e (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee-f)\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eUltrasonic vocalization (USVs) in observers as another indicator of STNV\u003c/span\u003e.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eUSVs were measured as an additional marker of stress response in observers. Given that this task was not performed previously in prairie voles and that stress-responses USVs are not well established in these species, we conducted a control experiment during which we measured USVs during classical fear learning in a new subset of prairie voles to examine the type of calls and frequencies prairie voles exhibit during general stress and to examine whether it corroborates with what was found during STNV. In this control experiment, we exposed prairie voles to tone-shock pairs (or tone-only controls) and, 24 hours later, recorded USVs produced during a fear memory recall session. Prairie voles conditioned to the tone-shock produced USVs at a higher principal frequency (t-test, t(14)\u0026thinsp;=\u0026thinsp;2.6, d\u0026thinsp;=\u0026thinsp;1.3, p\u0026thinsp;=\u0026thinsp;0.022 Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed), suggesting that higher call frequencies are associated with negative valence and may represent generalized stressed calls. No other call features were reported as statistically significant during this CTC experiment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ee-f).\u003c/p\u003e\u003cp\u003eWe subsequently recorded USVs in a subset of observers during social threat recall on Day 2. Observers previously paired with demonstrators in the shock group produced USVs at a higher principal frequency (one-tailed t-test, t(14)\u0026thinsp;=\u0026thinsp;2.0, d\u0026thinsp;=\u0026thinsp;0.99, p\u0026thinsp;=\u0026thinsp;0.035) than those paired with na\u0026iuml;ve demonstrators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). This increase in principal frequency may reflect stress in observers from the shock group, consistent with changes in fear-conditioned demonstrators. Exploratory analysis of other call features also revealed shortening of average call length (t(14)\u0026thinsp;=\u0026thinsp;2.8, p\u0026thinsp;=\u0026thinsp;0.015) and a decrease in average power (t(14)\u0026thinsp;=\u0026thinsp;2.9, p\u0026thinsp;=\u0026thinsp;0.012) in observers paired with conditioned demonstrators (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb-c), differences that were not observed when recording from directly fear conditioned voles. This may suggest that observers in the shock group produce different types of calls that are selective to socially transmitted negative valence. Further replication is needed to characterize USVs frequencies and calls during social transmission of negative valence.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eii. Control Experiment for STNV\u003c/h2\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003e\u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eImpact of consoling behavior on social transmission of negative valence\u003c/span\u003e.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e\u003cp\u003eIn a separate consoling control (CC) experiment, sibling voles went through the same STNV paradigm as described above, except that they were both returned to the home cage following the 2 sessions on Day 1. We predicted that this housing condition would allow for social buffering behaviors in the home cage, including consoling behavior \u003csup\u003e[\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]\u003c/sup\u003e and subsequently reducing the stress in demonstrators and the social transmission of negative valence in observers. As before, demonstrators in the shock group (N\u0026thinsp;=\u0026thinsp;22) did not differ from controls (N\u0026thinsp;=\u0026thinsp;21) in baseline freezing during habituation (t-test, t(41)\u0026thinsp;=\u0026thinsp;1.15, d\u0026thinsp;=\u0026thinsp;0.35, p\u0026thinsp;=\u0026thinsp;0.26), they successfully acquired a within-session conditioned freezing response (ANOVA, main effect of group, F(1,41)\u0026thinsp;=\u0026thinsp;17.9, η2\u0026thinsp;=\u0026thinsp;0.30, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; group-time interaction, F(14,28)\u0026thinsp;=\u0026thinsp;3.1, η2\u0026thinsp;=\u0026thinsp;0.61, p\u0026thinsp;=\u0026thinsp;0.006, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), and they demonstrated the conditioned freezing response during session 2 (t-test, t(38)\u0026thinsp;=\u0026thinsp;3.4, d\u0026thinsp;=\u0026thinsp;1.1, p\u0026thinsp;=\u0026thinsp;0.002; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). As predicted, observers did not show any differences in within-session freezing response (ANOVA, main effect of group, F(1,38)\u0026thinsp;=\u0026thinsp;0.93, η2\u0026thinsp;=\u0026thinsp;0.024, p\u0026thinsp;=\u0026thinsp;0.34; group-time interaction, F(4,35)\u0026thinsp;=\u0026thinsp;0.79, η2\u0026thinsp;=\u0026thinsp;0.0.047, p\u0026thinsp;=\u0026thinsp;0.79; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec) or freezing to the tone on Day 2 (t-test, t(38)\u0026thinsp;=\u0026thinsp;0.45, d\u0026thinsp;=\u0026thinsp;0.14, p\u0026thinsp;=\u0026thinsp;0.66; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed), \u003cem\u003esuggesting that reunion with the partner did enhance social buffering and reduced subsequent social learning\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eiii Neural correlates of STNV.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe examined variations in c-Fos expression between observers of control (N\u0026thinsp;=\u0026thinsp;10) and shocked (N\u0026thinsp;=\u0026thinsp;10) demonstrators following STNV on Day 1, focusing on five key areas: the anterior cingulate cortex (ACC), anterior insula (AI), basolateral amygdala (BLA), and the medial and lateral habenula (MHb and LHb) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eObservers in the shock group showed an increase in c-Fos immunofluorescence in the ACC (ANOVA, main effect of group, F(1,18)\u0026thinsp;=\u0026thinsp;0.049, η2\u0026thinsp;=\u0026thinsp;0.20, p\u0026thinsp;=\u0026thinsp;0.049), AI (ANOVA, main effect of group, F(1,17)\u0026thinsp;=\u0026thinsp;7.3, η2\u0026thinsp;=\u0026thinsp;0.30, p\u0026thinsp;=\u0026thinsp;0.015), BLA (ANOVA, main effect of group, F(1,16)\u0026thinsp;=\u0026thinsp;9.1, η2\u0026thinsp;=\u0026thinsp;0.36, p\u0026thinsp;=\u0026thinsp;0.008), and LHb (ANOVA, main effect of group, F(1,17)\u0026thinsp;=\u0026thinsp;5.5, η2\u0026thinsp;=\u0026thinsp;0.24, p\u0026thinsp;=\u0026thinsp;0.031), but not the MHb (ANOVA, main effect of group, F(1,17)\u0026thinsp;=\u0026thinsp;0.006, p\u0026thinsp;=\u0026thinsp;0.94) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e), \u003cem\u003ecorroborating the hypothesis that LHb can be part of the SAS network\u003c/em\u003e.\u003c/p\u003e\u003cp\u003eExploratory analysis on laterality in each region found that only the AI showed significant lateralization (ANOVA, group x side interaction, F(1,17)\u0026thinsp;=\u0026thinsp;5.27, η2\u0026thinsp;=\u0026thinsp;0.24, p\u0026thinsp;=\u0026thinsp;0.035), with the right AI activated more than the left in observers in the shock group (t-test, left vs. right AI in shock group, p\u0026thinsp;=\u0026thinsp;0.026) but not different than the left in observers in the control group (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The right AI was more activated in observers in the shock group than in control observers (t-test, control vs. shock in right AI, p\u0026thinsp;=\u0026thinsp;0.0061). The left AI was not different between the two groups (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05)\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHere, we used a translational and a highly social animal model, prairie voles, to investigate the behavioral and neuronal correlates of social learning, a phenomenon that is deficient in autism spectrum disorder and other neurodevelopmental disorders. We first found that prairie voles can acquire threat and associate a neutral stimulus to aversive experience based on the observation of conditioned demonstrators undergoing distress (exhibiting freezing behavior) during a threat recall session. Prairie vole observers also showed increases in classic negatively valenced responses, including increased rearing, increased self-grooming, and altered USVs. These results show that both the threat value and the negative valence of a cue can be socially transmitted through the observation of subtle social cues from a prairie vole demonstrator.\u003c/p\u003e\u003cp\u003eSelf-grooming has been linked to the hypothalamus-amygdala axis where studies have found a correlation between increased activation of the amygdala and increased self-grooming in rodents exhibiting anxious responses \u003csup\u003e[\u003cspan additionalcitationids=\"CR80 CR81\" citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]\u003c/sup\u003e. Rearing has also been shown to be stimulus sensitive and increases in response to stress \u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Therefore, the significant increases in rearing and self-grooming behaviors by the observers paired with conditioned demonstrators seen in STNV are indicative of higher threat acquisition and transmission of negative valence from their partners.\u003c/p\u003e\u003cp\u003eIn addition to behavioral markers, social transmission of negative valence was also demonstrated by differential ultrasonic vocalizations (USVs) in observers paired with demonstrators under distress in comparison to observers paired with non-stressed demonstrators. During social threat recall on day 2, socially conditioned observers exhibited higher frequency USVs on average compared to controls. This higher USV frequency was also exhibited in a separate group of prairie voles (in a separate experiment) that underwent themselves, as demonstrators, a classical fear learning task, during fear recall session in comparison to controls, signaling that this elevated frequency is an indicator of elevated stress levels. Changes in USVs have been documented in prairie voles previously as an indicator of distress. Previous research has found that infant\u0026rsquo;s stress in prairie voles related to social separation caused hormonal responses that correlated with increased vocal emission rates in pups \u003csup\u003e[\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e, \u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]\u003c/sup\u003e. Heart rate in voles has also been found to be linked to social distress \u003csup\u003e[\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e]\u003c/sup\u003e and to vocal emissions where an average range of 27-35kHz was recorded during a period of social isolation with access of transmission of olfactory cues to a familiar conspecific housed nearby \u003csup\u003e[\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]\u003c/sup\u003e. Although USVs were shown to decrease in number as the vole gets older \u003csup\u003e[\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]\u003c/sup\u003e potentially due to less dependence on mother, they are still highly vocal during cries of distress.\u003c/p\u003e\u003cp\u003eIn addition to higher principal frequency of USVs, socially conditioned observers exhibited shorter USV call lengths and reduced average power as compared to controls. These differences in length calls and power were not observed in prairie voles undergoing classical fear learning and foot shocks. It is possible that these indicators are specific to distress in social context and maybe selective indicators of stress for others instead of stress for self. Further replications are necessary to conclude if these exploratory findings on shortness in call lengths and a reduction in call power are generalized markers of stress for others in prairie voles and in other species with highly translational values.\u003c/p\u003e\u003cp\u003eTo corroborate further that these changes in behavioral and ultrasonic vocalizations are specific markers of negative valence triggered by the distress of others and not by random factors, we conducted an additional control experiment in which we manipulated the distress levels in demonstrators by adding a social buffering condition. We showed that by reuniting observers and their distressed partners in their home cage after classical fear learning and threat recall on day 1, socially conditioned observers did not show increased freezing behavior in response to the conditioned context on day 2 in comparison to controls. Reunion which is well documented in the literature to be associated with consoling behavior, is likely to have acted as social buffering and therefore reduced the levels of distress in the partner, which have led to a more neutral response during social threat recall on the second day. Lack of direct consoling data (due to logistic difficulties) is one limitation for the study and future studies using STNV in voles can provide further validation to the role of social buffering in altering social transmission of negative valence.\u003c/p\u003e\u003cp\u003eAt the neuronal level, we conducted c-Fos immunofluorescence after observation of the stressed partner to assess the brain activity of key areas of interest as stated in the hypothesis including LHb, BLA, AI and ACC, as part of the SAS network. In line with the hypothesis, we found elevated neuronal activity in these areas in socially conditioned observers in comparison to control observers, indicating that LHb can play an important role in social learning.\u003c/p\u003e\u003cp\u003eIn accordance with our hypothesis, results demonstrated an activation of the LHb neurons in response to social stress. Most literature focuses on the habenula in non-social reinforcement learning and negative valence \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan additionalcitationids=\"CR88\" citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]\u003c/sup\u003e where the lateral habenula, specifically, has shown to be the central hub for aversive and impulsive action integration \u003csup\u003e[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]\u003c/sup\u003e. The LHbs associated neurons receive afferent input from the limbic system and basal ganglia, helping to modulate motivation and emotional information \u003csup\u003e[\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]\u003c/sup\u003e. It then exerts indirect inhibitory control over midbrain dopaminergic (ventral tegmental area, substantia nigra pars compacta) and serotonergic (raphe nuclei) centers through activating GABAergic neurons. Due to this, LHb stimulation is typically associated with behavioral responses to aversive stimuli and suppression of reward-related activity \u003csup\u003e[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan additionalcitationids=\"CR93 CR94 CR95\" citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e]\u003c/sup\u003e. This leads to the performance of avoidance responses such as social withdrawal and reduced motivation where in fact overactivation has been correlated with depressive-like phenotypes: anhedonia, behavioral despair, and heightened anxiety \u003csup\u003e[\u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e, \u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]\u003c/sup\u003e. On the contrary, suppression of the LHb increases dopamine turnover, leading to rewarding phenotypes, facilitating approach behaviors, reduced anxiety, and diminished avoidance responses \u003csup\u003e[\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e, \u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eDisruptions within the LHb circuitry are noted to contribute to the dysregulation of underlying impairments in learning, decision-making, and affective processing; all of which are critical for socially based behaviors \u003csup\u003e[\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e, \u003cspan citationid=\"CR97\" class=\"CitationRef\"\u003e97\u003c/span\u003e]\u003c/sup\u003e. Although a more detailed exploration of this connectivity is necessary for application across diverse psychiatric disorders, a study comparing magnetic resonance imaging in humans found that across all ages included, the habenula was larger in those diagnosed with ASD when compared to controls \u003csup\u003e[\u003cspan citationid=\"CR98\" class=\"CitationRef\"\u003e98\u003c/span\u003e]\u003c/sup\u003e. Other literature shows experimental models in adolescent rats that have shown the exclusion from social play induces anxiety-like phenotypes and increases c-Fos expression within the LHb, implicating its role in emotional consequences of early social stress \u003csup\u003e[\u003cspan citationid=\"CR99\" class=\"CitationRef\"\u003e99\u003c/span\u003e]\u003c/sup\u003e. A more recent study in 2024 study on social fear conditioning in mice found that neurons projecting from the lateral habenula (LHb) to the medial prefrontal cortex (mPFC) were highly activated during social fear, and that inhibiting this LHb\u0026ndash;mPFC pathway significantly reduced fear responses \u003csup\u003e[\u003cspan citationid=\"CR100\" class=\"CitationRef\"\u003e100\u003c/span\u003e]\u003c/sup\u003e. These results align with evidence that core symptoms of ASD, such as communication deficits, repetitive behaviors, sensory dysregulation, and mood disorders, overlap with domains in which the habenula has been implicated.\u003c/p\u003e\u003cp\u003eAdditionally, the higher activation of the ACC and AI in observers of stressed voles is in accordance with previous literature involving these areas of social learning and emotion contagion. Shank3 mutant mice, an animal model of autism, display structural and functional impairments in the ACC, associated with deficits in social interaction \u003csup\u003e[\u003cspan citationid=\"CR101\" class=\"CitationRef\"\u003e101\u003c/span\u003e, \u003cspan citationid=\"CR102\" class=\"CitationRef\"\u003e102\u003c/span\u003e]\u003c/sup\u003e. Studies in humans, macaques, and rodents show that the ACC is essential for the social success of an individual in a group environment \u003csup\u003e[\u003cspan additionalcitationids=\"CR104 CR105\" citationid=\"CR103\" class=\"CitationRef\"\u003e103\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR106\" class=\"CitationRef\"\u003e106\u003c/span\u003e]\u003c/sup\u003e. They have also shown that the ACC, AI, and BLA are jointly activated in empathy-related or emotion-contagion studies \u003csup\u003e[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan additionalcitationids=\"CR108\" citationid=\"CR107\" class=\"CitationRef\"\u003e107\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR109\" class=\"CitationRef\"\u003e109\u003c/span\u003e]\u003c/sup\u003e, reinforcing the idea of complementary motor and sensory system feedback \u003csup\u003e[\u003cspan additionalcitationids=\"CR111\" citationid=\"CR110\" class=\"CitationRef\"\u003e110\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR112\" class=\"CitationRef\"\u003e112\u003c/span\u003e]\u003c/sup\u003e as well as connections through the SAS network. These findings contribute to our understanding of stress-induced c-Fos activation patterns in specific brain regions, shedding light on neural responses to social stressors and implications in those diagnosed with autism.\u003c/p\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eLimitations\u003c/h2\u003e\u003cp\u003eWe lack explicit consoling data in the control experiment to show that social buffering (duration of consoling behavior or allogrooming) is the cause of lack of freezing during social threat recall in the behavioral control experiment. Future experiments should include strangers studying the behavioral and physiological responses in prairie voles to the stress of unfamiliar others. Inhibition lateral habenula and studying its effects on social learning can be crucial to better characterize its role in this process.\u003c/p\u003e\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur findings suggest that prairie voles are capable of socially transmitting information and can acquire new information through observation of subtle social cues. We discovered that lateral habenula area plays an important role in social transmission of negative valence. Further molecular and pharmacological manipulations confirming the essential role of this area in Social Affect Salience Network can be crucial. In humans, investigating the role of lateral habenula in social learning and in neurodevelopmental disorders such as autism can be also critical as it can shed the light to new targeted brain circuitry or areas implicated in social affective and social salience processes.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eLateral habenula (LHb)\u003c/p\u003e\n\u003cp\u003eBasolateral amygdala (BLA)\u003c/p\u003e\n\u003cp\u003eAnterior insula (AI)\u003c/p\u003e\n\u003cp\u003eAnterior cingulate cortex (ACC)\u003c/p\u003e\n\u003cp\u003eSocial transmission of negative valence (STNV)\u003c/p\u003e\n\u003cp\u003eAutism spectrum disorder (ASD)\u003c/p\u003e\n\u003cp\u003eMedial habenula (MHb)\u003c/p\u003e\n\u003cp\u003eUltrasonic vocalizations (USVs)\u003c/p\u003e\n\u003cp\u003eNegative valence (NV)\u003c/p\u003e\n\u003cp\u003eSocial Affect Salience Network (SAS)\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe would like to acknowledge the integral contributions of Daniella Gamboa Pabón, who passed away prior to the publication of this manuscript. We honor her legacy and are grateful for the opportunity to continue and share the work she fostered. \u0026nbsp;To read more of her story, please visit: https://www.utoledo.edu/med/research/andari/daniella.html.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eE.A. discloses support for the research of this work through a gift from\u0026nbsp;ProMedica Health System Foundation to The University of Toledo [Autism and Social Neuroscience, index number 207007.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eD.G-P. conceptualized the study, conducted the investigation and data curation, performed the formal analysis and co-wrote the manuscript. J.H-K. conducted the visualization, organization, and formatting of data and results, co-wrote the manuscript, and contributed to the interpretation of findings. S.P. and B.A.H. conducted the investigation, data curation, and data analysis. J.P.B. conducted/supervised the investigation, conducted analysis, and co-wrote the manuscript. E.A. conceptualized the study design, supervised the study investigation, conducted analysis, supervised visualization, and co-wrote the final version of the manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll raw data has been included in supplemental files.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdditional information\u003c/strong\u003e (Supplementary information)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSupplementary figures and tables can be found in the supplemental file.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eShaw KA, et al. Prevalence and Early Identification of Autism Spectrum Disorder Among Children Aged 4 and 8 Years. 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Hum Brain Mapp. 2006; doi.org:10.1002/hbm.20213.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-autism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mola","sideBox":"Learn more about [Molecular Autism](http://molecularautism.biomedcentral.com/)","snPcode":"13229","submissionUrl":"https://submission.nature.com/new-submission/13229/3","title":"Molecular Autism","twitterHandle":"@MolecularAutism","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Social salience, lateral habenula, anterior cingulate cortex, prairie voles, social transmission of fear or threat, affective empathy, anterior insula, basolateral amygdala, autism, negative valence","lastPublishedDoi":"10.21203/rs.3.rs-7443708/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7443708/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eSocial learning is the process of acquiring social skills, new information, or associating negative or positive valence to a context through the observation of others and through direct social interaction with others. Neurodevelopmental disorders such as autism spectrum disorder or ASD show deficits in social salience and reciprocal affective responses. Social learning is known to implicate brain areas that relate to both aspects of social salience and affective empathy such as basolateral amygdala (BLA), anterior cingulate cortex (ACC), and anterior insula (AI). Lateral Habenula (LHb), a brain area renowned for its role in negative reinforcement learning and reward prediction error has not been yet extensively studied in the domain of social learning.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eWe developed an adapted version of fear conditioning by proxy paradigm called \u0026ldquo;Social Transmission of Negative Valence\u0026rdquo; or STNV and tested social rodent species prairie voles on the task. Observers experienced negative social conditioning through a proxy cage mate that served as the demonstrator during retrieval of a cued fear memory. Observers went through a social memory recall session 24 hours after observation. We measured observers\u0026rsquo; freezing time, self-grooming, rearing, and the range of frequency of ultrasonic vocalizations emitted as sign of distress. We also quantified immediate early gene translation as a proxy for neural activity using c-Fos immunochemistry 80 min after observing demonstrators going through memory recall.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSocially conditioned observers that were exposed to the fear-conditioned demonstrators displayed increased freezing time, self-grooming, and rearing during social recall sessions compared to control observers. They also displayed higher USVs frequency on average compared to controls. Socially conditioned observers showed increased c-Fos expression in the LHb and BLA, ACC and AI, in comparison to controls.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003ePrairie voles can be conditioned to threat through social transmission of negative valence. They activate brain areas known to be involved in affective processes and social salience. LHb can be another area of interest for neural correlates of social learning and may further be investigated as a part of a Social Affect Salience Network.\u003c/p\u003e","manuscriptTitle":"Increased c-Fos Expression of Lateral Habenula during Social Transmission of Negative Valence in Prairie Voles","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-25 18:21:32","doi":"10.21203/rs.3.rs-7443708/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-27T15:41:39+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-09T19:12:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-06T15:42:07+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-04T14:43:39+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"84950993808955547295059929635638136644","date":"2025-09-29T00:07:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"334083944566593274733769390027159635041","date":"2025-09-25T17:44:25+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"108920783105139495184150132942550413583","date":"2025-09-25T15:06:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"191589155667003472041339287204169107382","date":"2025-09-17T15:42:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-17T10:44:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-27T14:57:03+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-26T13:28:16+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Autism","date":"2025-08-24T02:13:24+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-autism","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mola","sideBox":"Learn more about [Molecular Autism](http://molecularautism.biomedcentral.com/)","snPcode":"13229","submissionUrl":"https://submission.nature.com/new-submission/13229/3","title":"Molecular Autism","twitterHandle":"@MolecularAutism","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8d32a1f7-8b20-4be8-b9cb-dfda0f7986a8","owner":[],"postedDate":"September 25th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-03-30T16:25:35+00:00","versionOfRecord":{"articleIdentity":"rs-7443708","link":"https://doi.org/10.1186/s13229-026-00712-5","journal":{"identity":"molecular-autism","isVorOnly":false,"title":"Molecular Autism"},"publishedOn":"2026-03-26 16:10:41","publishedOnDateReadable":"March 26th, 2026"},"versionCreatedAt":"2025-09-25 18:21:32","video":"","vorDoi":"10.1186/s13229-026-00712-5","vorDoiUrl":"https://doi.org/10.1186/s13229-026-00712-5","workflowStages":[]},"version":"v1","identity":"rs-7443708","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7443708","identity":"rs-7443708","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}