Conspecific presence facilitates 22-kHz ultrasonic calls in male rats exposed to cat odor

Conspecific presence facilitates 22-kHz ultrasonic calls in male rats exposed to cat odor | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Conspecific presence facilitates 22-kHz ultrasonic calls in male rats exposed to cat odor Miguel Bedoya Perez, Mehek F. Rahman, Iain S. McGregor This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5028333/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted 10 You are reading this latest preprint version Abstract Rats ( Rattus norvegicus ) exhibit defensive behaviors such as hiding, freezing, and fleeing when confronted with predators. They often emit long-duration (~ 22-kHz) ultrasonic vocalizations (USVs) in response to predator detection, unlike shorter (~ 50-kHz) USVs associated with positive states. These vocalizations can also trigger defensive behaviors in other rats. We studied the impact of a predator-associated stimulus (cat fur) and the context in which it was experienced on ~ 22-kHz and ~ 50-kHz USVs in laboratory rats, along with how the presence of conspecifics (none, one, or two familiar rats) influenced these vocalizations. Group-housed rats were habituated to a testing arena with a hide box alone or in pairs or trios. The next day, they were exposed to cat fur (1 gram) in the arena, and defensive behaviors and USVs were recorded. Subsequently, the rats returned to the arena without cat fur to assess contextual fear. Cat fur prompted significant hiding and ~ 22-kHz USVs, especially in social groups, but not in the context alone. Cat fur also suppressed ~ 50-kHz USVs, with recovery faster in trios, indicating a social buffering effect. These findings suggest that predator cues induce ~ 22-kHz USVs influenced by social context, highlighting an altruistic communicative function. Biological sciences/Zoology/Animal behaviour Biological sciences/Neuroscience/Olfactory system olfactory predator rats vocalization 22-kHz 50-kHz Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Rats ( Rattus norvegicus ) exhibit a distinctive repertoire of defensive behaviors towards predators, including hiding, freezing, flight, and risk assessment. These behaviors are also elicited by proximate cues of predation risk, such as predator odors and contexts in which predators and their odors have been encountered. Laboratory rats also sometimes emit 22-kHz ultrasonic vocalizations (USVs) when a predator is detected, and these calls are hypothesized to serve as alarm calls alerting conspecifics to predator presence 1 . These calls can last from 100 ms to well over 3000 ms 2 , 3 and are also emitted when rats are confronted with other kinds of unavoidable negative situations, e.g., electric foot of tail shocks 4 – 6 , startle stimuli 7 , 8 , and drug withdrawal 9 , 10 . Therefore, these calls appear to reflect a negative emotional state 11 – 13 . Rats also emit higher frequency 50-kHz USVs that are thought to reflect a positive affective state 14 – 16 . Rats emit these calls in social contexts as contact calls 17 – 19 , during positive social interactions such as grooming 20 , mating 16 , 21 , 22 , and play 23 – 25 , while performing tasks that require cooperation with conspecifics 26 ; and after administration of euphorigenic drugs 16 , 27 , 28 . Further underlining their role in positive affective states, rats experiencing aversive stimuli, such as social defeat, frustrating, non-rewarding situations, and footshock, decrease their production of 50-kHz vocalizations 16 , 27 . Compared to the negative 22-kHz calls, these calls are brief, with an average duration of 30–40 ms and a frequency range of 35–72 kHz 29 , 30 . While the emission of 22-kHz calls by rats in the presence of a predator, i.e., a cat, is well established 1 , 2 , 31 , there is only very limited evidence that predator-related cues such as odors can have this effect 32 . However, it is generally assumed that proximate cues of predation, such as odor, do not represent a robust enough signal of predation risk to induce alarm calling 33 , 34 . In the current study, we first aimed to explore whether 22-kHz USVs could be induced in rats solely through exposure to a proximate olfactory predator cue. Cat odors, often presented as a cloth rubbed on a cat, or balls of cat fur, are a strong predation cue, capable of triggering overt fear behaviour in both laboratory and wild rats 35 – 37 . We, therefore, examined whether this cue would also be sufficient to elicit 22-kHz alarm calls in laboratory rats. Our second aim was to determine the degree to which conspecific presence influences these calls. Blanchard, et al. 1 first suggested that 22-kHz USVs are altruistic, alerting conspecifics of imminent danger. Brudzynski and Chiu 38 supported this hypothesis by showing that playback of these calls elicits a fear response in other rats, even in the absence of the predator. If 22-kHz calls are altruistic, we hypothesized that rats would emit more of these calls in groups of two or three compared to when they are alone. Our final aim was to determine if 50-kHz calls, linked to positive affective states, are suppressed by exposure to a proximate predator cue, i.e., cat fur. Results Hide Time.— The time during which at least one animal remained in the hide box (called “Hide Time“) was affected by day (i.e., Habituation, Exposure, or Conditioned Fear) (X 2 2 = 21.195, p < 0.0001), social grouping (i.e., single, pair or trio) (X 2 2 = 10.593, p < 0.0001), but not their interaction (X 2 4 = 5.670, p = 0.225). For all social groupings, hide times were longer on the Exposure day (singles and trios p < 0.0001 and pairs p = 0.001) and Conditioned Fear days (singles p = 0.020, pairs p = 0.002, and trios p = 0.001) compared to the Habituation day. This shows the effectiveness of the cat fur stimulus and the fur-associated context in eliciting defensive behavior. Hide times were longer on the Exposure day than the Conditioned Fear day for singles and pairs (p < 0.0001 and p = 0.002 respectively), but not trios (p = 0.190) (Fig. 1 A). On the Habituation and Conditioned Fear days, singles had lower hide times than trios (p = 0.018 and p < 0.0001, respectively), while pairs did not differ from singles or trios on any of these days (Habituation: p = 0.649 and p = 0.180; and Conditioned Fear: p = 0.079 and p = 0.219, respectively). Hide times did not differ between social groupings on the Exposure day (Singles vs. Pair: p = 0.832; Singles vs. Trios: p = 0.761; and Pairs vs. Trios: p = 0.996). Group Hide Time.— “Group Hide Time“ is defined as the time that all animals in the relevant social group spend in the hide box. In the case of single rats, this is equivalent to “Hide Time“. Group hide time was significantly affected by day (X 2 2 = 35.977, p < 0.0001) but not by social grouping (X 2 2 = 3.939, p = 0.140) or their interaction (X 2 4 = 3.392, p = 0.495). Group hide times were greater on the Exposure (p < 0.0001 for all comparisons) and Conditioned Fear days (singles p = 0.009, pairs p = 0.001, and trios p = 0.027) than on the Habituation day. They were also higher on the Exposure day than the Conditioned Fear day for singles (p = 0.002), pairs (p < 0.0001), and trios (p < 0.0001) (Fig. 1 B). Emission of ~ 22-kHz calls.— As shown in Fig. 3 A, ~ 22-kHz USVs were only evident on the Exposure day and not on the Habituation or Conditioned Fear days. They were also more prevalent in pairs and trios than in single rats. Accordingly, the total number of ~ 22-kHz calls emitted per rat was affected by day (X 2 2 = 6.575, p = 0.037) and the interaction of day and social grouping (X 2 4 = 33.219, p < 0.0001) but not by social grouping (X 2 2 = 3.166, p = 0.205). Only 14.29% of single rats produced ~ 22-kHz USVs on the Exposure day (Table 1 ) compared to 75.00% of pairs and 83.33% of trios. Pairs and trios, but not single rats, emitted more ~ 22-kHz USVs on the Exposure day than on the Habituation (Pairs: p < 0.0001; Trios: p < 0.0001; and Singles: p = 0.093) or Conditioned Fear days (Pairs: p < 0.0001; Trios: p < 0.0001; and Singles: p = 0.108). Regardless of day, pairs and trios did not differ in the number of calls (Habituation: p = 0.952; Exposure: p = 0.283; and Conditioned Fear: p = 0.920). Both pairs and trios emitted more ~ 22-kHz USVs calls than singles on Exposure days (p = 0.049 and p < 0.0001, respectively). However, they did not differ in the number of calls during Habituation (Singles vs. Pairs: p = 0.658; and Singles vs. Trios: p = 0.233) or Conditioned Fear (Singles vs. Pairs: p = 0.733; and Singles vs. Trios: p = 0.309) (Fig. 2 A). Table 1 Percentage of social units tested (singles, pairs, and trios) in which ~ 22-kHz or ~ 55-kHz calls were detected on the Habituation, Exposure, and Conditioned Fear days. Social grouping Habituation Exposure Conditioned Fear ~ 22-kHz calls Singles 0.00% 14.29% 0.00% Pairs 0.00% 75.00% 0.00% Trios 8.33% 83.33% 0.00% ~ 55-kHz calls Singles 28.60% 0.00% 0.00% Pairs 75.00% 0.00% 25.00% Trios 75.00% 16.67% 66.67% Examining the ~ 22-kHz USVs across 5-minute bins within the 20-minute sessions (Fig. 2 B) indicated that the number of ~ 22-kHz calls per rat was affected by day (X 2 2 = 27.224, p < 0.0001), day by social grouping (X 2 4 = 161.664, p < 0.0001) and bin by day (X 2 6 = 32.144, p < 0.0001), but not by bin (X 2 1 = 3.120, p = 0.373), social grouping (X 2 3 = 3.110, p = 0.211) or bin by social grouping (X 2 6 = 10.955, p = 0.090). On the Exposure day, the number of calls per rat was greatest during the first 5–10 minutes of testing and diminished over time regardless of social grouping (Fig. 2 B). As noted above, very few calls were recorded on the Habituation and Conditioned Fear days, and this did not change over time regardless of social grouping (p > 0.05 for all comparisons). Emission of ~ 50-kHz calls.— As shown in Fig. 3 A, ~ 50-kHz USVs were most evident on the Habituation day and in grouped rats and were much reduced on both the Exposure and Conditioned Fear days. Accordingly, the total number of ~ 50-kHz calls per rat was affected by day (X 2 2 = 6.626, p = 0.036) and social grouping (X 2 2 = 6.805, p = 0.033) but not by their interaction (X 2 4 = 3.090, p = 0.543). Only 28.60% of single rats produced ~ 50-kHz USVs on Habituation day (Table 1 ) compared to 75.00% of pairs and 75.00% of trios. On the Conditioned Fear day, none of the single rats produced ~ 50-kHz USVs, while 25.00% of pairs and 66.67% of trios did (Table 1 ). Pairs and trios emitted more ~ 50-kHz calls per rat on the Habituation day than on the Exposure day (p = 0.010 and p = 0.002, respectively)(Fig. 3 A). On the Exposure day, the number of ~ 50-kHz USVs emitted per rat was very low and did not differ between groupings (Singles vs. Pair: p = 0.992; Singles vs. Trios: p = 0.759; and Pairs vs. Trios: p = 0.681). Trios emitted more calls per rat than singles during the Habituation and Conditioned Fear days (p = 0.041 and p = 0.035, respectively) but not on Exposure day (p = 0.759). The total calls per rat emitted by pairs did not differ from singles or trios, regardless of the day (Habituation: Singles vs. Pair: p = 0.323 and Trios vs. Pairs: p = 0.791; and Conditioned Fear: Singles vs. Pair: p = 0.444 and Trios vs Pairs: p = 0.365). The total ~ 50-kHz calls by single rats were very few and did not differ across days (Habituation vs. Exposure: p = 0.077; Habituation vs. Conditioned Fear: p = 0.110; and Exposure vs. Conditioned Fear: p = 1.000). Examining the ~ 50-kHz USVs across 5-minute bins within the 20-minute sessions, showed an effect of social grouping (X 2 2 = 14.167, p = 0.001), day (X 2 2 = 6.044, p = 0.049), and the interaction between day and social grouping (X 2 4 = 155.453, p < 0.0001), but not of bin (X 2 3 = 6.173, p = 0.103), nor of interaction between bin and social grouping (X 2 6 = 7.612, p = 0.268) or bin by day (X 2 3 = 7.150, p = 0.307). Overall, this indicates that the number of ~ 50-kHz calls per rat did not tend to change within sessions regardless of social grouping or day (Fig. 3 B). Call latency.— Latency to the first call to appear was affected by Class, i.e., 22-kHz or 50-kHz, (X 2 1 = 6.517, p = 0.011), the interactions between Class and Social grouping (X 2 4 = 14.482, p = 0.006), Class and Day (X 2 3 = 65.745, p < 0.0001), and the triple interaction between Class, Social grouping and Day (X 2 1 = 19.342, p < 0.0001)( Table SI 11). The 22-kHz calls showed a shorter latency to appear than 50-kHz calls (1.84 ± 0.23 minutes vs 3.83 ± 0.51 minutes, p = 0.009). The latency of 22-kHz calls during Exposure days did not differ across Social groupings (Singles: 2.00 ± 0.63 minutes vs. Pairs: 1.67 ± 0.31 minutes, p = 0.107; Singles vs. Trios: 1.20 ± 0.30 minutes, p = 0.358; and Pairs vs. Trios p = 0.674). Comparisons of 22-k-Hz calls between social groupings during Habituation and Conditioned Fear days were not possible due to the zero occurrence of these calls on these days. The latency of 50-kHz calls during Habituation days did not differ across Social groupings (Singles: 2.75 ± 1.69 minutes vs. Pairs 4.17 ± 2.38 minutes, p = 0.993; Singles vs. Trios: 3.11 ± 0.55 minutes, p = 0.982; and Pairs vs. Trios p = 1.000). During Conditioned Fear days, there was no difference in latency of 50-kHz calls between Pairs and Trios (8.00 ± 2.67 minutes vs 4.67 ± 1.53 minutes, p = 0.087), and comparisons to Singles were not possible due to the absence of these calls by singles on these days. Comparisons of latency of 50-k-Hz calls between social groupings during Exposure days were not possible due to the zero occurrence of these calls on these days. Discussion As predicted 39 , 40 , laboratory rats exhibited profound defensive behaviors towards a cat fur stimulus, despite never having experienced an actual cat. This included high levels of hiding from the fur and the context in which fur had been experienced (Conditioned Fear). In addition, cat fur strongly elicited long ~ 22-kHz USVs in rats, mainly when conspecifics were present. In contrast, cat fur strongly suppressed ~ 50-kHz USVs. The cat fur-associated context also suppressed ~ 50-kHz calls, although it did not elicit ~ 22-kHz USVs, even in the presence of conspecifics. This suggests that proximate cues of predation risk must reach a certain level of intensity, or imminence, to trigger alarm calls in rats. Our results extend the early findings of Blanchard, et al. 1 , where the sudden appearance of a cat elicited ~ 22-kHz USVs, termed “alarm cries“, in laboratory rats living in a social colony. These cat-elicited USVs were accompanied by defensive responses, including flight, concealment, freezing, risk assessment, and the suppression of non-defensive behaviors such as eating 1 . Our results also recall the findings of Fendt, et al. 32 , who reported ~ 22-kHz USVs in a small proportion of individually exposed rats (4 out of 19) exposed to the presumed predatory cue of lion urine, but not in any rats exposed to fox urine or TMT (an odorous component of fox feces). Similarly, we found that only a small proportion of our individually exposed rats (2 out of 14) emitted ~ 22-kHz USVs. Social groupings of pairs and trios of rats exhibited a greater number of ~ 22-kHz USVs (Fig. 2 A) and spent more time emitting these USVs (Table SI 5, Fig. SI 1A) than single rats, despite normalizing the data by the number of rats present. This suggests the importance of social context in motivating these calls. It has been suggested that 22-kHz calls have an altruistic “alarm“ function 1 , 13 , 32 . This hypothesis has been supported by studies demonstrating that playback of recordings of ~ 22-kHz USVs induces a reduction in activity in conspecifics 32 , 38 , 41 and even freezing 42 . However, some studies have found that freezing response upon playback of recorded ~ 22-kHz USVs only occurs in rats previously exposed to an aversive experience and who have produced ~ 22-kHz USVs in response to such an experience 42 . In our experiment, the number of ~ 22-kHz USVs tended to decrease over the 20 minutes of cat fur exposure, with more calls (Fig. 2 B) and more time spent (Table SI 6, Fig. SI 1B) emitting these calls in the first 5 to 10 minutes of the session. On the Exposure day, most rats only spent the first 1–2 minutes of testing outside the hide box (Fig. 1 , 1.624 ± 0.380 SEM minutes), a time interval corresponding to the latency of the first ~ 22-kHz calls recorded. It may be that the alarm calls serve as a trigger for hiding behavior in conspecifics, or that rats only tend to emit these calls from the relative safety of the hide box. Accordingly, Fendt, et al. 32 hypothesized that alarm calls might be more readily emitted when the immediate threat has dissipated and animals are in a place of relative safety, similar to findings in other rodent species. For example, yellow-bellied marmots ( Marmota flaviventris ) emit alarm calls when a predator is detected, but only from positions close to the safety of their burrows 43 . Additionally, it is plausible that the lower number of 22-kHz USVs in the last 10 minutes of the session indicates completion of the alarm function (i.e., alerting conspecifics) and habituation of fear following completion of risk assessment from within a safe location. The termination of alarm calling in other species is commonly attributed to fatigue 44 or group member behaviors 45 . For example, Thomas langur ( Presbytis thomasi) males only stop alarm calling after all other social group members have emitted at least one alarm call 45 . It is suggested that this behavior ensures that all group members are aware of the danger 45 . The ~ 22-kHz calls may serve additional functions: it has been suggested, for example, that high call frequency following the detection of a predator can serve to deter attacks by predators that depend on surprise 46 . By calling from the hide box, in relative safety, rats might be announcing to the “predator” (i.e., cat) that it has been detected and, therefore, its chances of capturing a rat are low. If this is the case, two conditions have been proposed for these calls to be evolutionary advantageous: (1) the prey has to be capable of assessing predation risk accurately, and (2) awareness of the predator's presence translates into a greater chance of escape by the prey 47 . Cat fur is, therefore, perceived as an accurate predator presence signal, and following the “risk dilution effect”, increasing group size correlated with a greater survival probability 48 . The emission of alarm calls is risky in terms of increasing detectability by the predator, with cats sensitive to sounds within the ultrasonic range 49 . A calling rat in the presence of two conspecifics has a lower chance of predation than a lone rat, or one within a pair, and this may explain the higher frequency of alarm calls in trios 48 , 50 . The suppression of ~ 50-kHz “social“ USVs in pairs and trios during cat fur exposure was a striking finding in the current study and recalls the inhibitory effects of other aversive stimuli (e.g., social defeat, non-reward, drug withdrawal, footshock) on ~ 50-kHz USVs 16 , 27 . Interestingly, when returned to the cat fur-associated context, the suppression of ~ 50-kHz USVs continued but appeared to recover more quickly, i.e., shorter latency, in trios than in pairs. Social grouping not only provides immediate survival advantages through dilution effects but also allows the enhanced performance of non-defensive behaviors during predatory threats 39 . This is sometimes referred to as social buffering, whereby social interaction with conspecifics during or after exposure to a stressor reduces the impact of that stressful situation 51 – 53 . Bowen, et al. 39 reported that rats exposed to cat fur in groups showed higher levels of grooming and locomotion than those exposed alone. Social buffering may also be directly related to increased predator detection capabilities, i.e., more eyes looking for predators, allowing individuals in the group to devote more time to critical non-defensive behaviors such as grooming and foraging 52 , 54 . The more rapid re-emergence of ~ 50-kHz USVs in trios may be another manifestation of this phenomenon. There is ample evidence that ~ 50-kHz USVs themselves can have social buffering effects 20 , 55 , reducing stress and anxiety through the release of oxytocin 56 , 57 . An alternative explanation of the reduced latency to restore ~ 50-kHz social calls by trios might be that with more rats present, there is a greater chance for one of them to commence ~ 50-kHz USVs, which, in turn, encourages more calls by the other individuals present. However, response calls evoked by playback of natural 50-kHz USVs appear to be short (around 0.3 s) and ~ 30-kHz 58 , not the type of calls we detected and reported in the current experiment. The elevated hiding behavior observed in a cat fur-associated context (Fig. 1 ) parallels previous findings from the literature and reflects conditioned contextual fear 40 . Despite this increased hiding, no ~ 22-kHz calls were elicited by the context (Fig. 2 A, Figure SI 1A, Table SI 3, and Table SI 5). Additionally, hiding responses tended to be somewhat reduced on Conditioned Fear day relative to Exposure day (see “Group Hide Times“). Rats appeared to assess lower risk in the cat fur-associated context than in the presence of cat fur itself. However, the level of fear was still sufficient to suppress ~ 50-kHz social calls. This is consistent with the Predatory Imminence Continuum (PIC) hypothesis 59 , which states that prey species will express a continuum of species-specific defensive reactions according to the level of risk perceived 59 . In our case, the cat fur-associated context represents a lower risk than the presence of cat fur itself; thus, hiding was reduced somewhat, ~ 22-kHz calls were absent, but the suppression of ~ 50-kHz calls continued, albeit to a lesser extent. Persistent avoidance responses are biologically ‘cheap’ if no significant survival advantage accrues from approaching the potentially threatening zone. Resistance towards the normal defensive behaviors towards proximate cues of predation can be seen in situations where this is manipulated. For example, Blanchard and Blanchard 60 placed rats where food and water were only available in a predator-associated location. Similarly, Bedoya-Pérez, et al. 37 showed wild-caught rats approaching feeding areas previously paired with cat odors, even with an alternative food source available. Clearly, there is a need for flexible sensitivity in response to predatory threats when other biological needs are present 40 . There are a few caveats to our experiment. One is the artificial nature of our experiment, in which laboratory rats were tested in a spatially constrained arena with a single predator-related stimulus 61 . Field studies are generally conducted at much larger spatial scales and involve animals exposed to multidimensional predator-related stimuli 61 . According to the “risk allocation“ hypothesis, antipredator responses are situation-specific, and optimal behavior will vary depending on the broader temporal context of a given scenario 33 . We must consider this within the context of our experimental protocol, where rats are exposed briefly to a ‘pulse' of high risk – such as sudden exposure to cat fur – preceded and followed by extended periods of low risk. This intensifies the perceived threat of the chosen stimulus and leads to heightened antipredator behavior that would not be typical in the wild 33 . Hence, the degree of antipredator behavior expected under realistic field conditions may be overestimated in laboratory experimental designs such as ours, which may increase the likelihood of triggering alarm calls. Another caveat is that our experiment did not include a non-predatory odor control. It can be argued that the production of ~ 22-kHz and suppression of ~ 50-kHz calls might be a natural response when exposed to any unusual odor cue. This seems unlikely, given that previous research with individual rats has shown that ~ 22-kHz calls do not occur with any novel odor cue. Fendt and Endres 62 demonstrated that only a minority of rats exposed to fox or lion urine emitted ~ 22-kHz calls, while none of the rats exposed to TMT (2,3,5-trimethyl-3-thiazoline) emitted such calls, even when the behavioral response to the different odors was similar. Although TMT is derived from fox feces, much debate has arisen regarding whether it is perceived as a predator cue or simply a pungent and noxious odor 35 , 62 – 64 . We therefore suggest that the production of ~ 22-kHz and suppression of ~ 50-kHz calls in our experiment are in response to the cue being a predator cue. Finally, our experimental design did not allow for the identification of the individuals calling. We do not know if all or only one of the individuals present in the social group called. Rats may either avoid “synchronizing” calls, and only one of the individuals present would call, or might purposely “synchronize” calls. Unfortunately, this is a limitation of our experimental set-up; however, we think it is unlikely rats avoid synchronous calls since it was apparent during data processing that calls from different animals overlapped in the spectrograms. In conclusion, the current study shows for the first time that cat fur is a sufficiently intense predator-related cue to elicit ~ 22-kHz USVs in laboratory rats. These calls appear much more likely to be emitted in the presence of conspecifics, confirming previous suggestions that these calls may serve as altruistic “alarm cries”. This proximate cue induced strong defensive behaviors and was enough to cause the development of contextual fear conditioning. Cat fur also strongly suppressed ~ 50-kHz social calls, which remained suppressed in the location where cat fur had been previously experienced. Methods Experimental Model and Subject Details.— The experiments used 90 experimentally naïve male Wistar rats (6–8 weeks) from the Animal Resource Centre (ARC, Perth, WA, Australia). They were housed in groups of 2 or 3 animals in standard Tecniplast 1500U IV cages (48 cm × 37.5 cm × 21 cm) with water and standard laboratory rodent chow available ad libitum and environmental enrichment in the form of a perplex box, a toilet roll, and small wooden block. The cages were housed in a temperature- and humidity-controlled vivarium (22 ± 2°C, 50–55%) with a reverse 12:12 h light/dark cycle (lights on at 9:30 pm). All behavioral tests were performed in the dark cycle between 10:00 am and 5:00 pm. All methods were performed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes and approved by The University of Sydney Animal Ethics Committee (ARA #2021/1900). All rats were handled for 2 min for at least five consecutive days before experimentation commenced. Rats were transported to and from the procedural rooms in their home cages. Experimental Apparatus.— The behavioral responses of rats were tested in a cat odor avoidance apparatus, which our group has routinely used to record antipredator behaviors in rats presented with odor cues (Fig. 4 ). This apparatus aims to replicate a predator odor encounter a rat would experience in the wild, whereby defensive behaviors are elicited, such as avoidance, risk assessment, and increased vigilance. The apparatus consists of a rectangular chamber (80 cm x 44 cm x 49 cm) with a smaller red Perspex “hide box” (23 cm x 15 cm x 49 cm) placed at one end. A glass Petri dish either containing the odor cue (cat fur) or empty (control) is fixed at the opposite end of the chamber using a Velcro sticker (Velcro brand, 2022 Velcro IP Holdings LLC). An ultrasonic microphone (UltraSoundGate CM16/CMPA, 2022 Avisoft Bioacoustics) and recording hardware (UltraSoundGate 416H, 2022 Avisoft Bioacoustics) were fitted to the top of the chamber on the opposite side of the hide box to record any vocalizations the animals emitted. This was accomplished using Avisoft RECORDER software (2022 Avisoft Bioacoustics). An infrared CCTV camera (Panasonic WV-CP300 Series 650 TVL Day/Night IR Dual Voltage Fixed Camera, fitted with a computer CS-Mount 2.9–8.2 mm Varifocal Lens) was located above the chamber for video recording. Cat fur sourcing and storage .— Cat fur was obtained from veterinary clinics and animal shelters across Sydney, Australia. Sampling was opportunistic, either as part of routine grooming or through shaving areas for medical procedures, and stored at -20 o C until use. Experimental procedure.— The experimental procedure occurred over three consecutive days, with rats placed in the testing apparatus for 20 minutes each day according to social grouping allocation (i.e., singles (n = 17 rats), in 14 pairs (n = 28 rats) or 15 trios (n = 45 rats)). Social grouping allocation was done with animals housed together, with pairs and trios housed in the same home cage; thus, all social groupings were composed of familiar individuals throughout the experiment. The experimental apparatus contained no cat fur on the first Habituation day. On the second Exposure day, 1 g of cat fur was placed in a petri dish, warmed to 36 o C, and then located opposite the hide box (Fig. 4 ). The fur was heated (36 o C) before Exposure to mimic the natural body temperature of a cat and increase the potential release of volatiles. Finally, on the third Conditioned Fear day, an empty heated petri dish was placed opposite the hide box. The testing apparatus was thoroughly cleaned with 70% ethanol at the end of each test. Data acquisition and analysis.— Video recordings of behavior were manually scored to determine the time at least one rat remained in the hide box and the time all rats present spent in the hide box. Avisoft SASLab Pro (2022 Avisoft Bioacoustics) software was used to record and analyze USVs. Spectrograms from each recording were obtained with the following parameters: Temporal resolution overlap of 50%, frequency resolution of 977 Hz, Bandwith 1699 Hz, frame size 100%, and Kaiser–Bessel window. Denoising was achieved by using a -80 dB threshold and a reduction by 90 dB. Spectrograms were matched for start and end times to the video recordings of behavior. The Automated Parameter measurements function, with Element separation set to “whistles”, allowed filtering of the data from the spectrogram into two frequency ranges: 18-32kHz frequency and longer than 100 ms in duration, i.e., ~ 22-kHz USVs 1 – 3 and 35-72-kHz frequency, i.e., ~ 50-kHz 29 , 30 . Examples of the spectrograms for each call can be found in Figure SI 3. Behavioral data were analyzed per 20-minute session, and USV data were analyzed per 20-minute session and per 5-minute bins within each session. Statistical analyses were accomplished using Generalized Linear Mixed Models (GLMM) using R v.4.2.1 65 , testing the effect of the Social Grouping, Day, i.e., Habituation, Exposure, and Conditioned Fear days, and their interaction on: “Time spent in the hide (at least one animal)”, “Time spent in the hide (all animals)”, “Time spent emitting 22-kHz calls”, “Number of 22-kHz calls”, “Time spent emitting 50kHz calls” and “Number of 50-kHz calls”. The rate of change over time (5-minute bins) was also measured for: “Time spent emitting 22-kHz calls”, “Number of 22-kHz calls”, “Time spent emitting 50-kHz calls” and “Number of 50-kHz calls”, according to Social Grouping, Day, and their interaction. The number of animals per test (one, two, or three) was used as an offset in all relevant models since more animals would be expected to lead to more calls. Additionally, latency until the first call was recorded and examined relative to “call type“ (i.e., ~ 22-kHz or ~ 50-kHz), Social Grouping, Day, the two-way interactions of “call type” by Social Grouping, and “call type” by Day, and the three-way interaction of “call type” by Social Grouping and Day. In all models, we included the experimental unit, i.e., the identity of the group of individuals tested, as the only random factor to account for repeated measures effects. Model construction and fitting used the functions lmer , glmer , or glmer.nb from the package “lme4” version 1.1–30 66 or the function glmmTMB from the package “glmmTMB” 67 . The data type (integers or continuous); residual plots; Shapiro–Wilk test of normality, using the function shapiro.test from the package “stats” 65 ; Levene's Test for Homogeneity of Variance (Homocedasticity) using the function leveneTest from the package “car” 68 ; and Pearson's dispersion test were used to identify the best distribution (i.e., Gaussian, Gamma, or Negative Binomial) and link (i.e., Identity, Inverse, Log or Square root) for each model 69 . P-values were generated by the type III Wald chi-square test using the function Anova from the package “car” 68 . Pairwise comparisons were performed using Dunn-Šidák corrections through the functions emmeans and emtrends from the package “emmeans” 70 . Statistical significance was defined by α = 0.05. The packages “ggplot2” 71 , “cowplot” 72 , “lemon” 73 , “scales” 74 , and “ggpubr” 75 were used to generate plots. Declarations Competing interests The authors declare no competing interests. Author Contribution M.B.P. and I.S.M. conceived and designed research and obtained funding. M.B.P. and M.F.R. collected, processed, and analyzed the data. M.B.P. and I.S.M. drafted the manuscript, which all authors reviewed and revised. All authors read and approved the final version of the manuscript. Acknowledgement This research was funded by The University of Sydney Animal Veterinary Bio-Sciences Honours program to M.F.R., under the supervision of M.B.P. and I.S.M., and conducted with permission from the University of Sydney Animal Research Authority 2021/1900. Data Availability All the data supporting this study's findings will be made available from Dryad:https://url.au.m.mimecastprotect.com/s/BZCcCnx1jnil17OPRt9fYCJKbmK?domain=datadryad.org References Blanchard, R. J., Blanchard, D. 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Supplementary Files 20240903USVsforScientificReportsSI.docx 20240829 USVs for Scientific Reports.docx 22kHzCallsexamples.mp4 22-kHz Calls examples.mp4 50kHzCallsexamples.mp4 50-kHz Calls examples.mp4 Cite Share Download PDF Status: Published Journal Publication published 05 Mar, 2025 Read the published version in Scientific Reports → Version 1 posted Editorial decision: Revision requested 11 Nov, 2024 Reviews received at journal 08 Nov, 2024 Reviews received at journal 04 Nov, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers agreed at journal 23 Oct, 2024 Reviewers invited by journal 23 Oct, 2024 Editor assigned by journal 23 Oct, 2024 Editor invited by journal 13 Sep, 2024 Submission checks completed at journal 11 Sep, 2024 First submitted to journal 03 Sep, 2024 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-5028333","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":376626833,"identity":"a8f36bd0-220a-4f12-999b-81df25f6d94c","order_by":0,"name":"Miguel Bedoya Perez","email":"data:image/png;base64,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","orcid":"","institution":"The University of Sydney, Lambert Initiative for Cannabinoid Therapeutics","correspondingAuthor":true,"prefix":"","firstName":"Miguel","middleName":"Bedoya","lastName":"Perez","suffix":""},{"id":376626835,"identity":"cfb4ab80-f70d-4625-a83a-207ecd5f74bf","order_by":1,"name":"Mehek F. 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Results show time where (A) at least one rat was hiding, or (B) all rats were hiding (i.e., Group Hide Time) during each Day. Individual data points are shown as circles. Superscripts represent Dunn–Šidák corrected pairwise comparisons comparing Exposure and Conditioned Fear sessions to the Habituation session (baseline): p\u0026lt;0.001 '***'; p\u0026lt;0.01 '**'; p\u0026lt;0.05 '*'.\u003c/p\u003e","description":"","filename":"Fig.11.png","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/df1fd1e43097c393e63a865a.png"},{"id":71032003,"identity":"0e4ba635-5787-4ac6-a385-3d8bb4e7e71d","added_by":"auto","created_at":"2024-12-10 11:54:21","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":415064,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of ~22-kHz (18-32-kHz, \u0026gt; 100 ms long) USVs emitted per rat (Mean ± SE, log scale) on each Day according to Social Grouping: (A) USVs per rat present summed over the whole 20-minute trial; and (B) USVs per rat present for every 5 minutes of the test. Points represent the raw data. Superscripts represent Dunn–Šidák corrected pairwise comparisons to habituation (baseline): p\u0026lt;0.001 '***'; p\u0026lt;0.01 '**'; p\u0026lt;0.05 '*'.\u003c/p\u003e","description":"","filename":"Fig.12.png","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/752870c3ae790c6e7e7ed51e.png"},{"id":71031995,"identity":"b8d9849d-8169-411f-8cf2-3effcd32445e","added_by":"auto","created_at":"2024-12-10 11:54:19","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":404657,"visible":true,"origin":"","legend":"\u003cp\u003eThe number of ~50-kHz (35-72-kHz) USVs emitted per rat present (Mean ± SE, log scale) on each day according to social grouping: (A) USVs per rat present summed over the whole 20-minute trial; and (B) USVs per rat present for every 5 minutes of the test. Points represent the raw data. Superscripts represent Dunn–Šidák corrected pairwise comparisons for Exposure and Conditioned Fear days relative to Habituation day (baseline): p\u0026lt;0.001 '***'; p\u0026lt;0.01 '**'; p\u0026lt;0.05 '*'.\u003c/p\u003e","description":"","filename":"Fig.13.png","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/ac38ecfbf726f16f614dc063.png"},{"id":71032006,"identity":"625e6f06-07bd-4970-8fee-ad6154fa7a06","added_by":"auto","created_at":"2024-12-10 11:54:23","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":992091,"visible":true,"origin":"","legend":"\u003cp\u003eThe cat odor avoidance experimental apparatus (left) and a still image showing an aerial view of the apparatus (right) showing the position of the hide box (a), the cat fur stimulus (b), the ultrasonic microphone (c), and the infrared video camera (d). Created with BioRender.com.\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/841ba1829b042a9dea5a63ed.png"},{"id":78190608,"identity":"44ec7fa3-ec66-44a6-8827-62a56df7afe9","added_by":"auto","created_at":"2025-03-10 19:50:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2918747,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/19070c2c-cb35-4aaf-9bed-10b6c84e7738.pdf"},{"id":71031993,"identity":"cc42579f-a46d-45ea-ba0a-1646cf10f0e4","added_by":"auto","created_at":"2024-12-10 11:54:16","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":474856,"visible":true,"origin":"","legend":"\u003cp\u003e20240829 USVs for Scientific Reports.docx\u003c/p\u003e","description":"","filename":"20240903USVsforScientificReportsSI.docx","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/e17b399c98dc083e1ed5087a.docx"},{"id":71032097,"identity":"e1dc6c2e-5841-43a8-bcab-f95a342b6673","added_by":"auto","created_at":"2024-12-10 12:02:21","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":40987744,"visible":true,"origin":"","legend":"\u003cp\u003e22-kHz Calls examples.mp4\u003c/p\u003e","description":"","filename":"22kHzCallsexamples.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/3513f4e5b7cb55a80f55ca61.mp4"},{"id":71032004,"identity":"552705b2-7d78-4f69-85a9-3f455c4b26af","added_by":"auto","created_at":"2024-12-10 11:54:22","extension":"mp4","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":38868654,"visible":true,"origin":"","legend":"\u003cp\u003e50-kHz Calls examples.mp4\u003c/p\u003e","description":"","filename":"50kHzCallsexamples.mp4","url":"https://assets-eu.researchsquare.com/files/rs-5028333/v1/8a789c65a2c7bad8a6498e96.mp4"}],"financialInterests":"No competing interests reported.","formattedTitle":"Conspecific presence facilitates 22-kHz ultrasonic calls in male rats exposed to cat odor","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) exhibit a distinctive repertoire of defensive behaviors towards predators, including hiding, freezing, flight, and risk assessment. These behaviors are also elicited by proximate cues of predation risk, such as predator odors and contexts in which predators and their odors have been encountered. Laboratory rats also sometimes emit 22-kHz ultrasonic vocalizations (USVs) when a predator is detected, and these calls are hypothesized to serve as alarm calls alerting conspecifics to predator presence \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. These calls can last from 100 ms to well over 3000 ms \u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and are also emitted when rats are confronted with other kinds of unavoidable negative situations, e.g., electric foot of tail shocks \u003csup\u003e\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e, startle stimuli \u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e, and drug withdrawal \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Therefore, these calls appear to reflect a negative emotional state \u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eRats also emit higher frequency 50-kHz USVs that are thought to reflect a positive affective state \u003csup\u003e\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Rats emit these calls in social contexts as contact calls \u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e, during positive social interactions such as grooming \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e, mating \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e,\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e, and play \u003csup\u003e\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e, while performing tasks that require cooperation with conspecifics \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e; and after administration of euphorigenic drugs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e,\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Further underlining their role in positive affective states, rats experiencing aversive stimuli, such as social defeat, frustrating, non-rewarding situations, and footshock, decrease their production of 50-kHz vocalizations \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Compared to the negative 22-kHz calls, these calls are brief, with an average duration of 30\u0026ndash;40 ms and a frequency range of 35\u0026ndash;72 kHz \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eWhile the emission of 22-kHz calls by rats in the presence of a predator, i.e., a cat, is well established \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e, there is only very limited evidence that predator-related cues such as odors can have this effect \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, it is generally assumed that proximate cues of predation, such as odor, do not represent a robust enough signal of predation risk to induce alarm calling \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. In the current study, we first aimed to explore whether 22-kHz USVs could be induced in rats solely through exposure to a proximate olfactory predator cue. Cat odors, often presented as a cloth rubbed on a cat, or balls of cat fur, are a strong predation cue, capable of triggering overt fear behaviour in both laboratory and wild rats \u003csup\u003e\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. We, therefore, examined whether this cue would also be sufficient to elicit 22-kHz alarm calls in laboratory rats.\u003c/p\u003e \u003cp\u003eOur second aim was to determine the degree to which conspecific presence influences these calls. Blanchard, et al. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e first suggested that 22-kHz USVs are altruistic, alerting conspecifics of imminent danger. Brudzynski and Chiu \u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e supported this hypothesis by showing that playback of these calls elicits a fear response in other rats, even in the absence of the predator. If 22-kHz calls are altruistic, we hypothesized that rats would emit more of these calls in groups of two or three compared to when they are alone. Our final aim was to determine if 50-kHz calls, linked to positive affective states, are suppressed by exposure to a proximate predator cue, i.e., cat fur.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cem\u003eHide Time.\u0026mdash;\u003c/em\u003e The time during which at least one animal remained in the hide box (called \u0026ldquo;Hide Time\u0026ldquo;) was affected by day (i.e., Habituation, Exposure, or Conditioned Fear) (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21.195, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), social grouping (i.e., single, pair or trio) (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.593, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not their interaction (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.670, p\u0026thinsp;=\u0026thinsp;0.225). For all social groupings, hide times were longer on the Exposure day (singles and trios p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and pairs p\u0026thinsp;=\u0026thinsp;0.001) and Conditioned Fear days (singles p\u0026thinsp;=\u0026thinsp;0.020, pairs p\u0026thinsp;=\u0026thinsp;0.002, and trios p\u0026thinsp;=\u0026thinsp;0.001) compared to the Habituation day. This shows the effectiveness of the cat fur stimulus and the fur-associated context in eliciting defensive behavior. Hide times were longer on the Exposure day than the Conditioned Fear day for singles and pairs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and p\u0026thinsp;=\u0026thinsp;0.002 respectively), but not trios (p\u0026thinsp;=\u0026thinsp;0.190) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). On the Habituation and Conditioned Fear days, singles had lower hide times than trios (p\u0026thinsp;=\u0026thinsp;0.018 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively), while pairs did not differ from singles or trios on any of these days (Habituation: p\u0026thinsp;=\u0026thinsp;0.649 and p\u0026thinsp;=\u0026thinsp;0.180; and Conditioned Fear: p\u0026thinsp;=\u0026thinsp;0.079 and p\u0026thinsp;=\u0026thinsp;0.219, respectively). Hide times did not differ between social groupings on the Exposure day (Singles vs. Pair: p\u0026thinsp;=\u0026thinsp;0.832; Singles vs. Trios: p\u0026thinsp;=\u0026thinsp;0.761; and Pairs vs. Trios: p\u0026thinsp;=\u0026thinsp;0.996).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eGroup Hide Time.\u0026mdash;\u003c/em\u003e \u0026ldquo;Group Hide Time\u0026ldquo; is defined as the time that \u003cem\u003eall\u003c/em\u003e animals in the relevant social group spend in the hide box. In the case of single rats, this is equivalent to \u0026ldquo;Hide Time\u0026ldquo;. Group hide time was significantly affected by day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;35.977, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but not by social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.939, p\u0026thinsp;=\u0026thinsp;0.140) or their interaction (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.392, p\u0026thinsp;=\u0026thinsp;0.495). Group hide times were greater on the Exposure (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 for all comparisons) and Conditioned Fear days (singles p\u0026thinsp;=\u0026thinsp;0.009, pairs p\u0026thinsp;=\u0026thinsp;0.001, and trios p\u0026thinsp;=\u0026thinsp;0.027) than on the Habituation day. They were also higher on the Exposure day than the Conditioned Fear day for singles (p\u0026thinsp;=\u0026thinsp;0.002), pairs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and trios (p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e\u003cem\u003eEmission of ~\u0026thinsp;22-kHz calls.\u0026mdash;\u003c/em\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, ~\u0026thinsp;22-kHz USVs were only evident on the Exposure day and not on the Habituation or Conditioned Fear days. They were also more prevalent in pairs and trios than in single rats. Accordingly, the total number of ~\u0026thinsp;22-kHz calls emitted per rat was affected by day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.575, p\u0026thinsp;=\u0026thinsp;0.037) and the interaction of day and social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;33.219, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) but not by social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.166, p\u0026thinsp;=\u0026thinsp;0.205). Only 14.29% of single rats produced\u0026thinsp;~\u0026thinsp;22-kHz USVs on the Exposure day (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) compared to 75.00% of pairs and 83.33% of trios. Pairs and trios, but not single rats, emitted more\u0026thinsp;~\u0026thinsp;22-kHz USVs on the Exposure day than on the Habituation (Pairs: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Trios: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; and Singles: p\u0026thinsp;=\u0026thinsp;0.093) or Conditioned Fear days (Pairs: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; Trios: p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001; and Singles: p\u0026thinsp;=\u0026thinsp;0.108). Regardless of day, pairs and trios did not differ in the number of calls (Habituation: p\u0026thinsp;=\u0026thinsp;0.952; Exposure: p\u0026thinsp;=\u0026thinsp;0.283; and Conditioned Fear: p\u0026thinsp;=\u0026thinsp;0.920). Both pairs and trios emitted more\u0026thinsp;~\u0026thinsp;22-kHz USVs calls than singles on Exposure days (p\u0026thinsp;=\u0026thinsp;0.049 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001, respectively). However, they did not differ in the number of calls during Habituation (Singles vs. Pairs: p\u0026thinsp;=\u0026thinsp;0.658; and Singles vs. Trios: p\u0026thinsp;=\u0026thinsp;0.233) or Conditioned Fear (Singles vs. Pairs: p\u0026thinsp;=\u0026thinsp;0.733; and Singles vs. Trios: p\u0026thinsp;=\u0026thinsp;0.309) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePercentage of social units tested (singles, pairs, and trios) in which\u0026thinsp;~\u0026thinsp;22-kHz or ~\u0026thinsp;55-kHz calls were detected on the Habituation, Exposure, and Conditioned Fear days.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSocial grouping\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eHabituation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eExposure\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eConditioned Fear\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e~\u0026thinsp;22-kHz calls\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSingles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e14.29%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePairs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e75.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrios\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e8.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e83.33%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e~\u0026thinsp;55-kHz calls\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSingles\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e28.60%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePairs\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e0.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e25.00%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTrios\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e75.00%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e16.67%\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e66.67%\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eExamining the ~\u0026thinsp;22-kHz USVs across 5-minute bins within the 20-minute sessions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) indicated that the number of ~\u0026thinsp;22-kHz calls per rat was affected by day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;27.224, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), day by social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;161.664, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and bin by day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e6\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;32.144, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not by bin (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.120, p\u0026thinsp;=\u0026thinsp;0.373), social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.110, p\u0026thinsp;=\u0026thinsp;0.211) or bin by social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e6\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;10.955, p\u0026thinsp;=\u0026thinsp;0.090). On the Exposure day, the number of calls per rat was greatest during the first 5\u0026ndash;10 minutes of testing and diminished over time regardless of social grouping (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). As noted above, very few calls were recorded on the Habituation and Conditioned Fear days, and this did not change over time regardless of social grouping (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05 for all comparisons).\u003c/p\u003e \u003cp\u003e\u003cem\u003eEmission of ~\u0026thinsp;50-kHz calls.\u0026mdash;\u003c/em\u003e As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, ~\u0026thinsp;50-kHz USVs were most evident on the Habituation day and in grouped rats and were much reduced on both the Exposure and Conditioned Fear days. Accordingly, the total number of ~\u0026thinsp;50-kHz calls per rat was affected by day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.626, p\u0026thinsp;=\u0026thinsp;0.036) and social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.805, p\u0026thinsp;=\u0026thinsp;0.033) but not by their interaction (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.090, p\u0026thinsp;=\u0026thinsp;0.543). Only 28.60% of single rats produced\u0026thinsp;~\u0026thinsp;50-kHz USVs on Habituation day (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) compared to 75.00% of pairs and 75.00% of trios. On the Conditioned Fear day, none of the single rats produced\u0026thinsp;~\u0026thinsp;50-kHz USVs, while 25.00% of pairs and 66.67% of trios did (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Pairs and trios emitted more\u0026thinsp;~\u0026thinsp;50-kHz calls per rat on the Habituation day than on the Exposure day (p\u0026thinsp;=\u0026thinsp;0.010 and p\u0026thinsp;=\u0026thinsp;0.002, respectively)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). On the Exposure day, the number of ~\u0026thinsp;50-kHz USVs emitted per rat was very low and did not differ between groupings (Singles vs. Pair: p\u0026thinsp;=\u0026thinsp;0.992; Singles vs. Trios: p\u0026thinsp;=\u0026thinsp;0.759; and Pairs vs. Trios: p\u0026thinsp;=\u0026thinsp;0.681). Trios emitted more calls per rat than singles during the Habituation and Conditioned Fear days (p\u0026thinsp;=\u0026thinsp;0.041 and p\u0026thinsp;=\u0026thinsp;0.035, respectively) but not on Exposure day (p\u0026thinsp;=\u0026thinsp;0.759). The total calls per rat emitted by pairs did not differ from singles or trios, regardless of the day (Habituation: Singles vs. Pair: p\u0026thinsp;=\u0026thinsp;0.323 and Trios vs. Pairs: p\u0026thinsp;=\u0026thinsp;0.791; and Conditioned Fear: Singles vs. Pair: p\u0026thinsp;=\u0026thinsp;0.444 and Trios vs Pairs: p\u0026thinsp;=\u0026thinsp;0.365). The total\u0026thinsp;~\u0026thinsp;50-kHz calls by single rats were very few and did not differ across days (Habituation vs. Exposure: p\u0026thinsp;=\u0026thinsp;0.077; Habituation vs. Conditioned Fear: p\u0026thinsp;=\u0026thinsp;0.110; and Exposure vs. Conditioned Fear: p\u0026thinsp;=\u0026thinsp;1.000).\u003c/p\u003e \u003cp\u003eExamining the ~\u0026thinsp;50-kHz USVs across 5-minute bins within the 20-minute sessions, showed an effect of social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.167, p\u0026thinsp;=\u0026thinsp;0.001), day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.044, p\u0026thinsp;=\u0026thinsp;0.049), and the interaction between day and social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;155.453, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), but not of bin (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.173, p\u0026thinsp;=\u0026thinsp;0.103), nor of interaction between bin and social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e6\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.612, p\u0026thinsp;=\u0026thinsp;0.268) or bin by day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.150, p\u0026thinsp;=\u0026thinsp;0.307). Overall, this indicates that the number of ~\u0026thinsp;50-kHz calls per rat did not tend to change within sessions regardless of social grouping or day (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003cem\u003eCall latency.\u0026mdash;\u003c/em\u003e Latency to the first call to appear was affected by Class, i.e., 22-kHz or 50-kHz, (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;6.517, p\u0026thinsp;=\u0026thinsp;0.011), the interactions between Class and Social grouping (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e4\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.482, p\u0026thinsp;=\u0026thinsp;0.006), Class and Day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e3\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;65.745, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001), and the triple interaction between Class, Social grouping and Day (X\u003csup\u003e2\u003c/sup\u003e\u003csub\u003e1\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;19.342, p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001)( Table SI 11). The 22-kHz calls showed a shorter latency to appear than 50-kHz calls (1.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.23 minutes vs 3.83\u0026thinsp;\u0026plusmn;\u0026thinsp;0.51 minutes, p\u0026thinsp;=\u0026thinsp;0.009). The latency of 22-kHz calls during Exposure days did not differ across Social groupings (Singles: 2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.63 minutes vs. Pairs: 1.67\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31 minutes, p\u0026thinsp;=\u0026thinsp;0.107; Singles vs. Trios: 1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30 minutes, p\u0026thinsp;=\u0026thinsp;0.358; and Pairs vs. Trios p\u0026thinsp;=\u0026thinsp;0.674). Comparisons of 22-k-Hz calls between social groupings during Habituation and Conditioned Fear days were not possible due to the zero occurrence of these calls on these days. The latency of 50-kHz calls during Habituation days did not differ across Social groupings (Singles: 2.75\u0026thinsp;\u0026plusmn;\u0026thinsp;1.69 minutes vs. Pairs 4.17\u0026thinsp;\u0026plusmn;\u0026thinsp;2.38 minutes, p\u0026thinsp;=\u0026thinsp;0.993; Singles vs. Trios: 3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55 minutes, p\u0026thinsp;=\u0026thinsp;0.982; and Pairs vs. Trios p\u0026thinsp;=\u0026thinsp;1.000). During Conditioned Fear days, there was no difference in latency of 50-kHz calls between Pairs and Trios (8.00\u0026thinsp;\u0026plusmn;\u0026thinsp;2.67 minutes vs 4.67\u0026thinsp;\u0026plusmn;\u0026thinsp;1.53 minutes, p\u0026thinsp;=\u0026thinsp;0.087), and comparisons to Singles were not possible due to the absence of these calls by singles on these days. Comparisons of latency of 50-k-Hz calls between social groupings during Exposure days were not possible due to the zero occurrence of these calls on these days.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs predicted \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e,\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e, laboratory rats exhibited profound defensive behaviors towards a cat fur stimulus, despite never having experienced an actual cat. This included high levels of hiding from the fur and the context in which fur had been experienced (Conditioned Fear). In addition, cat fur strongly elicited long\u0026thinsp;~\u0026thinsp;22-kHz USVs in rats, mainly when conspecifics were present. In contrast, cat fur strongly suppressed\u0026thinsp;~\u0026thinsp;50-kHz USVs. The cat fur-associated context also suppressed\u0026thinsp;~\u0026thinsp;50-kHz calls, although it did not elicit\u0026thinsp;~\u0026thinsp;22-kHz USVs, even in the presence of conspecifics. This suggests that proximate cues of predation risk must reach a certain level of intensity, or imminence, to trigger alarm calls in rats.\u003c/p\u003e \u003cp\u003eOur results extend the early findings of Blanchard, et al. \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e, where the sudden appearance of a cat elicited\u0026thinsp;~\u0026thinsp;22-kHz USVs, termed \u0026ldquo;alarm cries\u0026ldquo;, in laboratory rats living in a social colony. These cat-elicited USVs were accompanied by defensive responses, including flight, concealment, freezing, risk assessment, and the suppression of non-defensive behaviors such as eating \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. Our results also recall the findings of Fendt, et al. \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, who reported\u0026thinsp;~\u0026thinsp;22-kHz USVs in a small proportion of individually exposed rats (4 out of 19) exposed to the presumed predatory cue of lion urine, but not in any rats exposed to fox urine or TMT (an odorous component of fox feces). Similarly, we found that only a small proportion of our individually exposed rats (2 out of 14) emitted\u0026thinsp;~\u0026thinsp;22-kHz USVs.\u003c/p\u003e \u003cp\u003eSocial groupings of pairs and trios of rats exhibited a greater number of ~\u0026thinsp;22-kHz USVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and spent more time emitting these USVs (Table SI 5, Fig. SI 1A) than single rats, despite normalizing the data by the number of rats present. This suggests the importance of social context in motivating these calls. It has been suggested that 22-kHz calls have an altruistic \u0026ldquo;alarm\u0026ldquo; function \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. This hypothesis has been supported by studies demonstrating that playback of recordings of ~\u0026thinsp;22-kHz USVs induces a reduction in activity in conspecifics \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e,\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e and even freezing \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. However, some studies have found that freezing response upon playback of recorded\u0026thinsp;~\u0026thinsp;22-kHz USVs only occurs in rats previously exposed to an aversive experience and who have produced\u0026thinsp;~\u0026thinsp;22-kHz USVs in response to such an experience \u003csup\u003e\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn our experiment, the number of ~\u0026thinsp;22-kHz USVs tended to decrease over the 20 minutes of cat fur exposure, with more calls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) and more time spent (Table SI 6, Fig. SI 1B) emitting these calls in the first 5 to 10 minutes of the session. On the Exposure day, most rats only spent the first 1\u0026ndash;2 minutes of testing outside the hide box (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, 1.624\u0026thinsp;\u0026plusmn;\u0026thinsp;0.380 SEM minutes), a time interval corresponding to the latency of the first\u0026thinsp;~\u0026thinsp;22-kHz calls recorded. It may be that the alarm calls serve as a trigger for hiding behavior in conspecifics, or that rats only tend to emit these calls from the relative safety of the hide box. Accordingly, Fendt, et al. \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e hypothesized that alarm calls might be more readily emitted when the immediate threat has dissipated and animals are in a place of relative safety, similar to findings in other rodent species. For example, yellow-bellied marmots (\u003cem\u003eMarmota flaviventris\u003c/em\u003e) emit alarm calls when a predator is detected, but only from positions close to the safety of their burrows \u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAdditionally, it is plausible that the lower number of 22-kHz USVs in the last 10 minutes of the session indicates completion of the alarm function (i.e., alerting conspecifics) and habituation of fear following completion of risk assessment from within a safe location. The termination of alarm calling in other species is commonly attributed to fatigue \u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e or group member behaviors \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. For example, Thomas langur (\u003cem\u003ePresbytis thomasi)\u003c/em\u003e males only stop alarm calling after all other social group members have emitted at least one alarm call \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. It is suggested that this behavior ensures that all group members are aware of the danger \u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe ~\u0026thinsp;22-kHz calls may serve additional functions: it has been suggested, for example, that high call frequency following the detection of a predator can serve to deter attacks by predators that depend on surprise \u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. By calling from the hide box, in relative safety, rats might be announcing to the \u0026ldquo;predator\u0026rdquo; (i.e., cat) that it has been detected and, therefore, its chances of capturing a rat are low. If this is the case, two conditions have been proposed for these calls to be evolutionary advantageous: (1) the prey has to be capable of assessing predation risk accurately, and (2) awareness of the predator's presence translates into a greater chance of escape by the prey \u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. Cat fur is, therefore, perceived as an accurate predator presence signal, and following the \u0026ldquo;risk dilution effect\u0026rdquo;, increasing group size correlated with a greater survival probability \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. The emission of alarm calls is risky in terms of increasing detectability by the predator, with cats sensitive to sounds within the ultrasonic range \u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. A calling rat in the presence of two conspecifics has a lower chance of predation than a lone rat, or one within a pair, and this may explain the higher frequency of alarm calls in trios \u003csup\u003e\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e,\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThe suppression of ~\u0026thinsp;50-kHz \u0026ldquo;social\u0026ldquo; USVs in pairs and trios during cat fur exposure was a striking finding in the current study and recalls the inhibitory effects of other aversive stimuli (e.g., social defeat, non-reward, drug withdrawal, footshock) on ~\u0026thinsp;50-kHz USVs \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Interestingly, when returned to the cat fur-associated context, the suppression of ~\u0026thinsp;50-kHz USVs continued but appeared to recover more quickly, i.e., shorter latency, in trios than in pairs. Social grouping not only provides immediate survival advantages through dilution effects but also allows the enhanced performance of non-defensive behaviors during predatory threats \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. This is sometimes referred to as social buffering, whereby social interaction with conspecifics during or after exposure to a stressor reduces the impact of that stressful situation \u003csup\u003e\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e. Bowen, et al. \u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e reported that rats exposed to cat fur in groups showed higher levels of grooming and locomotion than those exposed alone. Social buffering may also be directly related to increased predator detection capabilities, i.e., more eyes looking for predators, allowing individuals in the group to devote more time to critical non-defensive behaviors such as grooming and foraging \u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e. The more rapid re-emergence of ~\u0026thinsp;50-kHz USVs in trios may be another manifestation of this phenomenon. There is ample evidence that ~\u0026thinsp;50-kHz USVs themselves can have social buffering effects \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e,\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u003c/sup\u003e, reducing stress and anxiety through the release of oxytocin \u003csup\u003e\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e,\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eAn alternative explanation of the reduced latency to restore\u0026thinsp;~\u0026thinsp;50-kHz social calls by trios might be that with more rats present, there is a greater chance for one of them to commence\u0026thinsp;~\u0026thinsp;50-kHz USVs, which, in turn, encourages more calls by the other individuals present. However, response calls evoked by playback of natural 50-kHz USVs appear to be short (around 0.3 s) and ~\u0026thinsp;30-kHz \u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e, not the type of calls we detected and reported in the current experiment.\u003c/p\u003e \u003cp\u003eThe elevated hiding behavior observed in a cat fur-associated context (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) parallels previous findings from the literature and reflects conditioned contextual fear \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Despite this increased hiding, no\u0026thinsp;~\u0026thinsp;22-kHz calls were elicited by the context (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA, Figure SI 1A, Table SI 3, and Table SI 5). Additionally, hiding responses tended to be somewhat reduced on Conditioned Fear day relative to Exposure day (see \u0026ldquo;Group Hide Times\u0026ldquo;). Rats appeared to assess lower risk in the cat fur-associated context than in the presence of cat fur itself. However, the level of fear was still sufficient to suppress\u0026thinsp;~\u0026thinsp;50-kHz social calls. This is consistent with the Predatory Imminence Continuum (PIC) hypothesis \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e, which states that prey species will express a continuum of species-specific defensive reactions according to the level of risk perceived \u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e. In our case, the cat fur-associated context represents a lower risk than the presence of cat fur itself; thus, hiding was reduced somewhat, ~\u0026thinsp;22-kHz calls were absent, but the suppression of ~\u0026thinsp;50-kHz calls continued, albeit to a lesser extent.\u003c/p\u003e \u003cp\u003ePersistent avoidance responses are biologically \u0026lsquo;cheap\u0026rsquo; if no significant survival advantage accrues from approaching the potentially threatening zone. Resistance towards the normal defensive behaviors towards proximate cues of predation can be seen in situations where this is manipulated. For example, Blanchard and Blanchard \u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e placed rats where food and water were only available in a predator-associated location. Similarly, Bedoya-P\u0026eacute;rez, et al. \u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e showed wild-caught rats approaching feeding areas previously paired with cat odors, even with an alternative food source available. Clearly, there is a need for flexible sensitivity in response to predatory threats when other biological needs are present \u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eThere are a few caveats to our experiment. One is the artificial nature of our experiment, in which laboratory rats were tested in a spatially constrained arena with a single predator-related stimulus \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. Field studies are generally conducted at much larger spatial scales and involve animals exposed to multidimensional predator-related stimuli \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e. According to the \u0026ldquo;risk allocation\u0026ldquo; hypothesis, antipredator responses are situation-specific, and optimal behavior will vary depending on the broader temporal context of a given scenario \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. We must consider this within the context of our experimental protocol, where rats are exposed briefly to a \u0026lsquo;pulse' of high risk \u0026ndash; such as sudden exposure to cat fur \u0026ndash; preceded and followed by extended periods of low risk. This intensifies the perceived threat of the chosen stimulus and leads to heightened antipredator behavior that would not be typical in the wild \u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Hence, the degree of antipredator behavior expected under realistic field conditions may be overestimated in laboratory experimental designs such as ours, which may increase the likelihood of triggering alarm calls.\u003c/p\u003e \u003cp\u003eAnother caveat is that our experiment did not include a non-predatory odor control. It can be argued that the production of ~\u0026thinsp;22-kHz and suppression of ~\u0026thinsp;50-kHz calls might be a natural response when exposed to any unusual odor cue. This seems unlikely, given that previous research with individual rats has shown that ~\u0026thinsp;22-kHz calls do not occur with any novel odor cue. Fendt and Endres \u003csup\u003e\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u003c/sup\u003e demonstrated that only a minority of rats exposed to fox or lion urine emitted\u0026thinsp;~\u0026thinsp;22-kHz calls, while none of the rats exposed to TMT (2,3,5-trimethyl-3-thiazoline) emitted such calls, even when the behavioral response to the different odors was similar. Although TMT is derived from fox feces, much debate has arisen regarding whether it is perceived as a predator cue or simply a pungent and noxious odor \u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan additionalcitationids=\"CR63\" citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e\u003c/sup\u003e. We therefore suggest that the production of ~\u0026thinsp;22-kHz and suppression of ~\u0026thinsp;50-kHz calls in our experiment are in response to the cue being a predator cue.\u003c/p\u003e \u003cp\u003eFinally, our experimental design did not allow for the identification of the individuals calling. We do not know if all or only one of the individuals present in the social group called. Rats may either avoid \u0026ldquo;synchronizing\u0026rdquo; calls, and only one of the individuals present would call, or might purposely \u0026ldquo;synchronize\u0026rdquo; calls. Unfortunately, this is a limitation of our experimental set-up; however, we think it is unlikely rats avoid synchronous calls since it was apparent during data processing that calls from different animals overlapped in the spectrograms.\u003c/p\u003e \u003cp\u003eIn conclusion, the current study shows for the first time that cat fur is a sufficiently intense predator-related cue to elicit\u0026thinsp;~\u0026thinsp;22-kHz USVs in laboratory rats. These calls appear much more likely to be emitted in the presence of conspecifics, confirming previous suggestions that these calls may serve as altruistic \u0026ldquo;alarm cries\u0026rdquo;. This proximate cue induced strong defensive behaviors and was enough to cause the development of contextual fear conditioning. Cat fur also strongly suppressed\u0026thinsp;~\u0026thinsp;50-kHz social calls, which remained suppressed in the location where cat fur had been previously experienced.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cem\u003eExperimental Model and Subject Details.\u0026mdash;\u003c/em\u003eThe experiments used 90 experimentally na\u0026iuml;ve male Wistar rats (6\u0026ndash;8 weeks) from the Animal Resource Centre (ARC, Perth, WA, Australia). They were housed in groups of 2 or 3 animals in standard Tecniplast 1500U IV cages (48 cm \u0026times; 37.5 cm \u0026times; 21 cm) with water and standard laboratory rodent chow available \u003cem\u003ead libitum\u003c/em\u003e and environmental enrichment in the form of a perplex box, a toilet roll, and small wooden block. The cages were housed in a temperature- and humidity-controlled vivarium (22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, 50\u0026ndash;55%) with a reverse 12:12 h light/dark cycle (lights on at 9:30 pm). All behavioral tests were performed in the dark cycle between 10:00 am and 5:00 pm. All methods were performed in accordance with the \u003cem\u003eAustralian Code of Practice for the Care and Use of Animals for Scientific Purposes\u003c/em\u003e and approved by The University of Sydney Animal Ethics Committee (ARA #2021/1900). All rats were handled for 2 min for at least five consecutive days before experimentation commenced. Rats were transported to and from the procedural rooms in their home cages.\u003c/p\u003e \u003cp\u003e \u003cem\u003eExperimental Apparatus.\u0026mdash;\u003c/em\u003e The behavioral responses of rats were tested in a cat odor avoidance apparatus, which our group has routinely used to record antipredator behaviors in rats presented with odor cues (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). This apparatus aims to replicate a predator odor encounter a rat would experience in the wild, whereby defensive behaviors are elicited, such as avoidance, risk assessment, and increased vigilance. The apparatus consists of a rectangular chamber (80 cm x 44 cm x 49 cm) with a smaller red Perspex \u0026ldquo;hide box\u0026rdquo; (23 cm x 15 cm x 49 cm) placed at one end. A glass Petri dish either containing the odor cue (cat fur) or empty (control) is fixed at the opposite end of the chamber using a Velcro sticker (Velcro brand, 2022 Velcro IP Holdings LLC). An ultrasonic microphone (UltraSoundGate CM16/CMPA, 2022 Avisoft Bioacoustics) and recording hardware (UltraSoundGate 416H, 2022 Avisoft Bioacoustics) were fitted to the top of the chamber on the opposite side of the hide box to record any vocalizations the animals emitted. This was accomplished using Avisoft RECORDER software (2022 Avisoft Bioacoustics). An infrared CCTV camera (Panasonic WV-CP300 Series 650 TVL Day/Night IR Dual Voltage Fixed Camera, fitted with a computer CS-Mount 2.9\u0026ndash;8.2 mm Varifocal Lens) was located above the chamber for video recording.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cem\u003eCat fur sourcing and storage .\u0026mdash;\u003c/em\u003e Cat fur was obtained from veterinary clinics and animal shelters across Sydney, Australia. Sampling was opportunistic, either as part of routine grooming or through shaving areas for medical procedures, and stored at -20\u003csup\u003eo\u003c/sup\u003eC until use.\u003c/p\u003e \u003cp\u003e \u003cem\u003eExperimental procedure.\u0026mdash;\u003c/em\u003e The experimental procedure occurred over three consecutive days, with rats placed in the testing apparatus for 20 minutes each day according to social grouping allocation (i.e., singles (n\u0026thinsp;=\u0026thinsp;17 rats), in 14 pairs (n\u0026thinsp;=\u0026thinsp;28 rats) or 15 trios (n\u0026thinsp;=\u0026thinsp;45 rats)). Social grouping allocation was done with animals housed together, with pairs and trios housed in the same home cage; thus, all social groupings were composed of familiar individuals throughout the experiment. The experimental apparatus contained no cat fur on the first Habituation day. On the second Exposure day, 1 g of cat fur was placed in a petri dish, warmed to 36 \u003csup\u003eo\u003c/sup\u003eC, and then located opposite the hide box (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The fur was heated (36 \u003csup\u003eo\u003c/sup\u003eC) before Exposure to mimic the natural body temperature of a cat and increase the potential release of volatiles. Finally, on the third Conditioned Fear day, an empty heated petri dish was placed opposite the hide box. The testing apparatus was thoroughly cleaned with 70% ethanol at the end of each test.\u003c/p\u003e \u003cp\u003e \u003cem\u003eData acquisition and analysis.\u0026mdash;\u003c/em\u003e Video recordings of behavior were manually scored to determine the time at least one rat remained in the hide box and the time all rats present spent in the hide box. Avisoft SASLab Pro (2022 Avisoft Bioacoustics) software was used to record and analyze USVs. Spectrograms from each recording were obtained with the following parameters: Temporal resolution overlap of 50%, frequency resolution of 977 Hz, Bandwith 1699 Hz, frame size 100%, and Kaiser\u0026ndash;Bessel window. Denoising was achieved by using a -80 dB threshold and a reduction by 90 dB. Spectrograms were matched for start and end times to the video recordings of behavior. The Automated Parameter measurements function, with Element separation set to \u0026ldquo;whistles\u0026rdquo;, allowed filtering of the data from the spectrogram into two frequency ranges: 18-32kHz frequency and longer than 100 ms in duration, i.e., ~\u0026thinsp;22-kHz USVs \u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e and 35-72-kHz frequency, i.e., ~\u0026thinsp;50-kHz \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e. Examples of the spectrograms for each call can be found in Figure SI 3.\u003c/p\u003e \u003cp\u003eBehavioral data were analyzed per 20-minute session, and USV data were analyzed per 20-minute session and per 5-minute bins within each session. Statistical analyses were accomplished using Generalized Linear Mixed Models (GLMM) using R v.4.2.1 \u003csup\u003e65\u003c/sup\u003e, testing the effect of the Social Grouping, Day, i.e., Habituation, Exposure, and Conditioned Fear days, and their interaction on: \u0026ldquo;Time spent in the hide (at least one animal)\u0026rdquo;, \u0026ldquo;Time spent in the hide (all animals)\u0026rdquo;, \u0026ldquo;Time spent emitting 22-kHz calls\u0026rdquo;, \u0026ldquo;Number of 22-kHz calls\u0026rdquo;, \u0026ldquo;Time spent emitting 50kHz calls\u0026rdquo; and \u0026ldquo;Number of 50-kHz calls\u0026rdquo;. The rate of change over time (5-minute bins) was also measured for: \u0026ldquo;Time spent emitting 22-kHz calls\u0026rdquo;, \u0026ldquo;Number of 22-kHz calls\u0026rdquo;, \u0026ldquo;Time spent emitting 50-kHz calls\u0026rdquo; and \u0026ldquo;Number of 50-kHz calls\u0026rdquo;, according to Social Grouping, Day, and their interaction. The number of animals per test (one, two, or three) was used as an offset in all relevant models since more animals would be expected to lead to more calls. Additionally, latency until the first call was recorded and examined relative to \u0026ldquo;call type\u0026ldquo; (i.e., ~\u0026thinsp;22-kHz or ~\u0026thinsp;50-kHz), Social Grouping, Day, the two-way interactions of \u0026ldquo;call type\u0026rdquo; by Social Grouping, and \u0026ldquo;call type\u0026rdquo; by Day, and the three-way interaction of \u0026ldquo;call type\u0026rdquo; by Social Grouping and Day. In all models, we included the experimental unit, i.e., the identity of the group of individuals tested, as the only random factor to account for repeated measures effects.\u003c/p\u003e \u003cp\u003eModel construction and fitting used the functions \u003cem\u003elmer\u003c/em\u003e, \u003cem\u003eglmer\u003c/em\u003e, or \u003cem\u003eglmer.nb\u003c/em\u003e from the package \u0026ldquo;lme4\u0026rdquo; version 1.1\u0026ndash;30 \u003csup\u003e66\u003c/sup\u003e or the function \u003cem\u003eglmmTMB\u003c/em\u003e from the package \u0026ldquo;glmmTMB\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e. The data type (integers or continuous); residual plots; Shapiro\u0026ndash;Wilk test of normality, using the function \u003cem\u003eshapiro.test\u003c/em\u003e from the package \u0026ldquo;stats\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e; Levene's Test for Homogeneity of Variance (Homocedasticity) using the function \u003cem\u003eleveneTest\u003c/em\u003e from the package \u0026ldquo;car\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e; and Pearson's dispersion test were used to identify the best distribution (i.e., Gaussian, Gamma, or Negative Binomial) and link (i.e., Identity, Inverse, Log or Square root) for each model \u003csup\u003e\u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e\u003c/sup\u003e. P-values were generated by the type III Wald chi-square test using the function \u003cem\u003eAnova\u003c/em\u003e from the package \u0026ldquo;car\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u003c/sup\u003e. Pairwise comparisons were performed using Dunn-Šid\u0026aacute;k corrections through the functions \u003cem\u003eemmeans\u003c/em\u003e and \u003cem\u003eemtrends\u003c/em\u003e from the package \u0026ldquo;emmeans\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e\u003c/sup\u003e. Statistical significance was defined by α\u0026thinsp;=\u0026thinsp;0.05. The packages \u0026ldquo;ggplot2\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e\u003c/sup\u003e, \u0026ldquo;cowplot\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e\u003c/sup\u003e, \u0026ldquo;lemon\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e\u003c/sup\u003e, \u0026ldquo;scales\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e\u003c/sup\u003e, and \u0026ldquo;ggpubr\u0026rdquo; \u003csup\u003e\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e\u003c/sup\u003e were used to generate plots.\u003c/p\u003e"},{"header":"Declarations","content":" \u003ch2\u003e \u003cspan type=\"SmallCaps\" class=\"SmallCaps\" name=\"Emphasis\"\u003eCompeting interests\u003c/span\u003e \u003c/h2\u003e \u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e \u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eM.B.P. and I.S.M. conceived and designed research and obtained funding. M.B.P. and M.F.R. collected, processed, and analyzed the data. M.B.P. and I.S.M. drafted the manuscript, which all authors reviewed and revised. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThis research was funded by The University of Sydney Animal Veterinary Bio-Sciences Honours program to M.F.R., under the supervision of M.B.P. and I.S.M., and conducted with permission from the University of Sydney Animal Research Authority 2021/1900.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll the data supporting this study's findings will be made available from Dryad:https://url.au.m.mimecastprotect.com/s/BZCcCnx1jnil17OPRt9fYCJKbmK?domain=datadryad.org\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBlanchard, R. J., Blanchard, D. C., Agullana, R. \u0026amp; Weiss, S. M. 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(2020).\u003c/span\u003e\u003c/li\u003e\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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"olfactory, predator, rats, vocalization, 22-kHz, 50-kHz","lastPublishedDoi":"10.21203/rs.3.rs-5028333/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5028333/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRats (\u003cem\u003eRattus norvegicus\u003c/em\u003e) exhibit defensive behaviors such as hiding, freezing, and fleeing when confronted with predators. They often emit long-duration (~\u0026thinsp;22-kHz) ultrasonic vocalizations (USVs) in response to predator detection, unlike shorter (~\u0026thinsp;50-kHz) USVs associated with positive states. These vocalizations can also trigger defensive behaviors in other rats. We studied the impact of a predator-associated stimulus (cat fur) and the context in which it was experienced on ~\u0026thinsp;22-kHz and ~\u0026thinsp;50-kHz USVs in laboratory rats, along with how the presence of conspecifics (none, one, or two familiar rats) influenced these vocalizations. Group-housed rats were habituated to a testing arena with a hide box alone or in pairs or trios. The next day, they were exposed to cat fur (1 gram) in the arena, and defensive behaviors and USVs were recorded. Subsequently, the rats returned to the arena without cat fur to assess contextual fear. Cat fur prompted significant hiding and ~\u0026thinsp;22-kHz USVs, especially in social groups, but not in the context alone. Cat fur also suppressed\u0026thinsp;~\u0026thinsp;50-kHz USVs, with recovery faster in trios, indicating a social buffering effect. These findings suggest that predator cues induce\u0026thinsp;~\u0026thinsp;22-kHz USVs influenced by social context, highlighting an altruistic communicative function.\u003c/p\u003e","manuscriptTitle":"Conspecific presence facilitates 22-kHz ultrasonic calls in male rats exposed to cat odor","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-10 11:53:50","doi":"10.21203/rs.3.rs-5028333/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-11-11T11:49:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-08T09:34:33+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-11-04T07:47:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"144139362566981215851121493075508127108","date":"2024-10-23T14:01:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"255686338349505642516107737758320426721","date":"2024-10-23T11:34:45+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-10-23T05:05:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-10-23T04:51:59+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-09-13T21:03:15+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-09-11T13:25:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2024-09-04T03:37:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ada041ff-93cd-4fe5-8374-47d8588527db","owner":[],"postedDate":"December 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40089835,"name":"Biological sciences/Zoology/Animal behaviour"},{"id":40089836,"name":"Biological sciences/Neuroscience/Olfactory system"}],"tags":[],"updatedAt":"2025-03-10T19:48:08+00:00","versionOfRecord":{"articleIdentity":"rs-5028333","link":"https://doi.org/10.1038/s41598-025-90272-0","journal":{"identity":"scientific-reports","isVorOnly":false,"title":"Scientific Reports"},"publishedOn":"2025-03-05 15:58:21","publishedOnDateReadable":"March 5th, 2025"},"versionCreatedAt":"2024-12-10 11:53:50","video":"","vorDoi":"10.1038/s41598-025-90272-0","vorDoiUrl":"https://doi.org/10.1038/s41598-025-90272-0","workflowStages":[]},"version":"v1","identity":"rs-5028333","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5028333","identity":"rs-5028333","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}