Playback of rat 22-kHz ultrasonic vocalizations as a translational assay of negative affective states: An analysis of evoked behavior and brain activity

The subjective nature of human emotions makes them uniquely challenging to investigate in preclinical models. While behavioral assays in rodents aim to evaluate affect (i.e., anxiety, hypervigilance), they often lack etho-logical validity. Playback of negatively valenced 22-kHz ultrasonic vocalizations (USVs) in rats shows promise as a translational tool to investigate affective processing. Much like how human facial expressions can communicate internal states, rats emit 22-kHz USVs that similarly convey negative affective states to conspecifics indicating possible threat. 22-kHz USV playback elicits avoidance and hypervigilant behaviors, and recruit brain regions comparable to those seen in human brains evoked by viewing fearful faces. Indeed, 22-kHz playback alters neural activity in brain regions associated with negative valence systems (i.e., amygdala, bed nucleus of the stria terminalis, periaqueductal gray) alongside increases in behaviors typically associated with anxiety. Here, we pre-sent evidence from the literature that supports leveraging 22-kHz USV playback in rat preclinical models to obtain clinically relevant and translational findings to identify the neural underpinnings of


Introduction
In rats, emission of ultrasonic vocalizations (USVs) is thought to communicate internal affective states (Burman et al., 2007;Knutson et al., 2002;Takahashi et al., 2010;Wöhr and Schwarting, 2013).USVs range from 20-kHz to 100-kHz in frequency and are largely outside of the human hearing range (Heffner and Heffner, 2007;MacDonald and Brudzynski, 2018).These vocalizations are emitted in a variety of behavioral and social situations, such as mother-infant interactions (Hofer, 1996;Kaidbey et al., 2019), play behavior (Kisko et al., 2015;Knutson et al., 1998), sexual behavior (Barfield et al., 1979;Börner et al., 2016), predator presence (Blanchard et al., 1991;Litvin et al., 2007), and defensive, aggressive, or social conflict situations (Burgdorf et al., 2008).There has been an increase in the utilization of USV recording as an indicator of affective state due to the frequency-and context-specific nature of these calls in rats (Burman et al., 2007;Portfors, 2007;Wöhr and Schwarting, 2013), and to assess overall communication in both rats and mice.However, while mice also vocalize in the ultrasonic range, mouse USVs as an index for affective state are not well characterized (Portfors, 2007;Portfors and Perkel, 2014), and may better reflect social interactions (Sangiamo et al., 2020) or motivation in sexual contexts (Karigo, 2022).Therefore, due to their more robust affective characterization, rat USVs are of particular interest in behavioral neuroscience to both infer (Barker et al., 2015;Burgdorf et al., 2020;Knutson et al., 2002) and induce (e.g., via emotional contagion; Saito et al., 2016), negatively valenced affective states.
Emotional contagion is the process by which emotional states are transferred from one organism to another and influenced by various social factors (Hatfield et al., 2014;Wróbel and Imbir, 2019).Emotional contagion in humans often extends beyond empathy, in which emotions are understood by conspecifics in proximity, and falls in the category of a genuine transfer of affect (Kleinke et al., 1998;Wild et al., 2001).This transfer of affect is also thought to be experienced in non-human animals and has been reported in ravens (Adriaense et al., 2019), pigs (Goumon and Špinka, 2016), parrots (Schwing et al., 2017), and rats (Saito et al., 2016).Emerging research points to the utility of rat USVs to alter emotional states, evidenced by behavioral changes, in rats via playback of affectively valenced calls.Indeed, rats exposed to differently valenced USV playback show bias toward ambiguous stimuli in line with USV-induced positive or negative affect (Saito et al., 2016).
Additionally, aversive USV playback shows increases in behavioral avoidance (Demaestri et al., 2019) and changes in indices of physiological arousal (Olszyński et al., 2020).In humans, the Fearful Face Task is a commonly used method to induce affective states in participants that extend beyond empathy (Kleinke et al., 1998;Wild et al., 2001).This task leverages social contagion and the innate, unspoken exchange of emotion conveyed via facial expressions from a sender to evoke similar emotions in a viewer/receiver (Wild et al., 2001).Upon presentation of human faces with fearful (or other) expressions, a congruent affective state can be induced in a typical viewer, though may lead to feelings of anxiety particularly in participants with an inability to regulate negative affect (Bishop et al., 2006;Fox et al., 2005).
Imaging studies utilizing the Fearful Face Task in the fMRI show changes in amygdala recruitment measured through blood oxygen level dependent (BOLD) response, finding increased activity that is left lateralized (Vuilleumier et al., 2001) and more strongly lateralized in men than women (Killgore and Yurgelun-Todd, 2001).In individuals with high trait anxiety, particularly women (Dickie and Armony, 2008), unattended fearful face presentation leads to a greater BOLD response in the amygdala compared to lower trait anxiety participants, with changes in activity also observed in the temporal sulcus for low perceptual load stimuli (Bishop et al., 2006).BOLD response in the Fearful Face Task can differ based on attention and perception of the face with unconsciously perceived fearful faces increasing responses in the basolateral amygdala for individuals with trait anxiety, with consciously encountered faces activating the dorsal amygdala (which includes central amygdala) (Etkin et al., 2004).More anatomically comprehensive studies have observed increased activity in the left temporal pole, left anterior cingulate gyrus, right fusiform gyrus, right lateral orbitofrontal cortex, and the superior colliculus upon presentation of fearful faces compared to those with neutral expressions (Vuilleumier et al., 2001).A summary of these studies indicating brain regions recruited by fearful face presentation can be seen in Fig. 2A.Despite the effectiveness of the Fearful Face Task to answer some questions about the neural underpinnings of human emotion, researchers are unable to investigate affective processing systematically or experimentally with the same degree of granularity afforded by non-human model systems.Therefore, it is necessary to identify stimuli for preclinical rodent assays that are analogous to those used in human studies to identify future areas of targeted research spanning molecular to therapeutic levels.

Building a translational assay to investigate affective [dys] function
In humans, anxiety can be a transient state which allows for adaptive heightened awareness and physiological arousal in contexts or situations that may be threatening or pose a possible risk to the individual.However, a prolonged or unprovoked state of anxiety beyond the passing of a possible threat -and/or attentional bias toward threat-related cues -is maladaptive and can be detrimental to a person's wellbeing, thereby constituting a disorder (Craske et al., 2009).Anxiety disorders are relatively common and have substantial long-term consequences and a high burden on society and those who suffer from them (Chisholm et al., 2016;McLean et al., 2011).Many people experience an anxiety disorder at some point in their lives and there is a high social and economic cost due to lost productivity of individuals with anxiety (Chisholm et al., 2016;Kessler et al., 2005;Murrough et al., 2015).However, a large gap in knowledge remains surrounding the causes and effective treatments of anxiety disorders, which is compounded by a lack of translational preclinical models (Honeycutt et al., 2022;Steimer, 2011;Tanaka et al., 2023).
Individuals with anxiety disorders typically exhibit exaggerated fear, worry, and apprehension in scenarios where there is no direct danger and these feelings can be prolonged well beyond the occurrence of the initial anxiety-producing event (Davis et al., 2010;Gelenberg, 2000).While feelings of fear can be entwined with the experience of anxiety, it is important to differentiate between these two behavioral states.Indeed, the experience of fear can be distinguished by the acute and proximal nature of a threat, whereas anxiety is prompted by distal, ambiguous, or anticipatory threat and can be displayed as avoidance behaviors (Perusini and Fanselow, 2015).Pathological anxiety, on the other hand, is characterized as an enhanced, maladaptive, and persistent state of fear, often in the absence of a direct threat (Rosen and Schulkin, 1998).Affective dysfunction resulting from an anxiety disorder can have severe consequences on a person's health and quality of life.Despite the prevalence and severity of anxiety disorders, pharmacotherapies can lack effectiveness in certain populations and can be accompanied by a variety of unpleasant side effects (Bandelow et al., 2017).Thus, there is a need to further understand the physiological and neural basis for such disorders to better target treatments for maximum efficacy.
The most significant challenges in the etiological understanding and treatment of anxiety disorders involve the complex and overlapping circuits and mechanisms implicated in disorders characterized by affective dysregulation.Individual variation and differences across patients complicate our understanding of anxiety disorders further.There are documented individual differences in anxiety onset (Lewinsohn et al., 1998;Nelemans et al., 2018), etiology (Zinbarg et al., 2022), and comorbidities (Eisenberg et al., 1998) which add complexity to diagnosis and treatment.Due to the similarity of fear and anxiety, the amygdala, which is known for threat and fear processing (Adolphs, 2008), is commonly involved in descriptions of anxiety circuitry (Kim et al., 2011;Ouda et al., 2016).However, other regions, such as the bed nucleus of the stria terminalis and nucleus accumbens, have also been implicated in anxiety and affective dysregulation (Kalin et al., 2005;Walker et al., 2003).Many anxiety-related regions overlap with learning and memory systems including the hippocampus, insula, and dorsal anterior cingulate cortex, which may be responsible for acquisition and retention of fear and anxiety responses (Hartley and Phelps, 2012).The activity, connectivity, and discrete functioning of these numerous regions remains to be further characterized in the context of anxiety to better model anxiety disorders at a preclinical level.
There are clear sex differences in rates of anxiety disorders as well as experiences with affective dysfunction (Bangasser and Cuarenta, 2021;Maeng and Milad, 2015).In humans, women are up to twice as likely as men to develop an anxiety disorder in their lifetime and the neural underpinnings of this difference are just beginning to be explored in preclinical models (e.g., Bangasser and Cuarenta, 2021;Honeycutt et al., 2020;Maeng and Milad, 2015;Tolin and Foa, 2008).Additionally, the age of onset of anxiety disorders often occurs much earlier in females; in humans by age six, girls are twice as likely to have an anxiety disorder compared to boys (Lewinsohn et al., 1998).Similarly, the illness burden of anxiety and frequency of comorbid mood disorders are greater in women, meaning that both prevalence and toll of anxiety disorders are different across sex (McLean et al., 2011;Vesga-López et al., 2008).The presentation of anxiety symptoms can also differ as a function of sex, with women reporting more symptoms overall, including fatigue, irritability, muscle tension, and gastrointestinal, cardiovascular, autonomic, and respiratory issues (Vesga-López et al., 2008).Considering these differences, further research is needed to develop individualized anxiety treatments that would benefit all patients, though we must first leverage preclinical models to identify sex-specific and/or developmental opportunities for intervention.
The development and characterization of more valid and translational animal models and assays of anxiety is critical to furthering our understanding of anxiety disorders, their underlying neural mechanisms, targeted treatments, and relevant sex differences.In rodent models, a negative affective state can be inferred through various behavioral manifestations consistent with features of anxiety (e.g., increased behavioral avoidance) in assays such as the elevated plus maze, elevated zero maze, open field test, light-dark box, hole board assay, Vogel conflict task, and successive alley task, among others (La-Vu et al., 2020;Lezak et al., 2017).Though there is substantial literature validating the use of these behavioral measurements to infer a negative affective state, such interpretation-dependent assays leave room for inconsistency, issues with replication, and misinterpretation.However, there is emerging evidence that playback of rat USVs may serve as a promising translational model/assay to reliably induce and/or evaluate an acute and reversible negative affective state in rats.Importantly, this method may serve as a translational analogue to the human Fearful Face Task, thereby providing researchers a valid model that can bridge the human and animal literature for an enhanced ability to screen for or induce a negative affective state in a more nuanced and ethologically relevant way.This review highlights the use of aversive USV playback as an In rats, yellow highlighted regions indicate changes to regional neural activity via exposure to 22-kHz USV playback (locations given relative to Bregma) as measured through cFos immunoreactivity following playback.Regions that are responsive to 22-kHz playback compared to silence and/or other USV comparisons include: (B1) inferior colliculus, periaqueductal gray (including rostral dorso-medial, dorsal raphe nuclei); (B2) perirhinal cortex, ectorhinal cortex, periaqueductal gray; (B3) perirhinal cortex, ectorhinal cortex, primary auditory cortex, hippocampus; (B4) perirhinal cortex, ectorhinal cortex, primary auditory cortex, hippocampus; (B5) perirhinal cortex, ectorhinal cortex, periventricular nucleus of thalamus, hippocampus, central amygdala, basolateral amygdala; (B6) bed nucleus of the stria terminalis (oval and antero-dorsal nuclei); and (B7) prefrontal cortex.Notably, there is cross-species overlap in activity changes in homologous regions (i.e., prefrontal cortex and amygdala) that are important for threat processing and response, and activity in these regions is thought to be altered in both patients and animal models of affective disorders.Human data presented here summarized from fMRI work using fearful face presentation (Vuilleumier et al., 2001;Killgore and Yurgelun-Todd, 2001;Bishop et al., 2007).Figure adapted from (Mai et al., 2016;Paxinos and Watson, 2007) with data summarized from research assessing rat cFos in response to 22-kHz playback (see Table 2).
ethological, translational model for inducing negative affect in rats with the goal of furthering our understanding of the neural correlates of affective processing.First, we provide an overview of USVs, with specific attention to aversive 22-kHz USV alarm calls.Next, we explore the utility of playback of pre-recorded, ethologically relevant 22-kHz USVs as a reproducible method to induce a negative affective state through a comprehensive analysis of the literature.A consideration of sex differences in affective processing and sex-specific dysregulation will be presented.We highlight the potential for 22-kHz playback as a method of inducing a negative affective state in rats as a translational animal model of anxiety which would allow for the exploration of the putative neural underpinnings of anxiety and other disorders characterized by affective dysregulation.

Ultrasonic vocalizations (USVs) in rats: a means of conveying social and affective information
USVs can be categorized by distinct acoustic characteristics and include three main frequency bands which are associated with certain behavioral and affective states (Portfors, 2007).Vocalizations around 40-kHz frequency are the first to develop and are emitted by rat pups to prompt retrieval by the dam (Kaidbey et al., 2019).USVs around 50-55-kHz frequencies (Fig. 2B) are thought to generally reflect a positive affective state and are typically produced in appetitive environments (Schwarting, 2023) and may communicate pleasure to conspecifics (Burgdorf et al., 2011;Kisko et al., 2015).Finally, vocalizations around 22-kHz (Fig. 2C) are considered alarm calls and are emitted under a variety of conditions, most of which can be categorized as aversive and suggest that these USVs may indicate a negative affective state of the emitter (Blanchard et al., 1991;Knutson et al., 2002).Rats emit 22-kHz USVs as an alarm call to conspecifics when an individual is in direct danger, approaching danger, distress, discomfort, fear, or anxiety (Blanchard et al., 1991;Brudzynski, 2019;Wöhr and Schwarting, 2013).USVs are produced by laryngeal vibrations (Johnson et al., 2010) as well as a whistle mechanism (Riede et al., 2017), similar to a bird call (Simola and Brudzynski, 2018).Rats also vocalize in the 2-10-kHz frequency range which is audible to humans and is produced with laryngeal activity without whistle tones (Roberts, 1975).These calls within the audible range of humans have mainly been observed in situations of pain and discomfort (Han et al., 2005).Due to these distinct physical mechanisms and situations in which vocalization takes place, it is clear that USVs are of evolutionary importance with a clear purpose of conspecific communication (Brudzynski, 2013(Brudzynski, , 2014;;Brudzynski and Burgdorf, 2021;Simola and Brudzynski, 2018).
Vocalizations of the 50-kHz category can range from 32 to 96-kHz in frequency yet are generally centralized around 50-55-kHz and are distinctly short in duration with high frequency modulation (Kelm--Nelson et al., 2018;Portfors, 2007).In comparison, 22-kHz calls contain less frequency modulation, but are longer in duration with calls lasting up to 3.5 s presented in sequence with short gaps between (Brudzynski et al., 1993;Brudzynski, 2019).Within the category of 22-kHz calls, there are short calls (20-300 ms) and long calls (greater than 300 ms) (Brudzynski et al., 1993).Long calls typically serve as an alarm call, and while the function of short 22-kHz calls is lesser known, evidence suggests that they also communicate an aversive affective state (Barker et al., 2010;Blanchard et al., 1991).
Playback of positively valenced 50 kHz-USVs has been shown to elicit changes in behavior, such as increased approach behavior, indicating its value as an appetitive social cue (Wöhr and Schwarting, 2007), and showing promise as a means by which to study nuanced positive affect in rat models (Burgdorf, 2011;Schwarting, 2023).Such USVs can also be characterized as pro-social, social contact, or play signals which are positively valenced (Seffer et al., 2014;Wöhr, 2018).Due to the communicative role of USVs and distinct meaning and reactions to 50-kHz USV playback, it is likely that playback of 22-kHz USV recordings have equally distinct effects on behavior indicating the transfer of a negative affective state.Underlying neural activity upon exposure to 22-kHz USV playback may also point to valence-specific neural correlates of these affective states.Indeed, recent studies comparing playback of the two have pointed to both neural and behavioral differences based on the type of USV presented (Demaestri et al., 2019).
Due to the social and communicative purpose of USVs, 22-kHz USVs are not emitted as frequently when an animal is knowingly alone (Inagaki et al., 2005).Interestingly, 22-kHz USVs have been recorded from male rats post-ejaculation and these calls are thought to maintain social contact while prompting separation of the mating pair (Barfield et al., 1979).Rats also emit 22-kHz USVs in laboratory settings due to handling stress, fear conditioning, and in anticipation of pain (Borta et al., 2006;Brudzynski, 2019).Since USVs are emitted during distinct affective states and often during what could be considered emotionally salient situations, characterizing, quantifying, and analyzing USVs can be used to better assess affective states in rats (Brudzynski, 2021).Playback of USVs may thereby impart distinctly valenced affective states depending on frequency, which opens the door for USV playback studies to enhance translational emotion research to better understand the neural underpinnings of affective dysfunction.

Behavioral changes supporting the aversive valence of 22-kHz USV playback
Many interpretations of the affective influence of 22-kHz USVs center around changes in locomotion -such as increased freezing and thigmotaxis -as anxiety-like indices in response to playback.Notably, the term 'anxiety-like' has often been used to categorize a variety of behaviors in preclinical rodent research, including: behavioral avoidance, startle response, and freezing, among others.In the interest of clarity, and in recognition that we cannot directly infer a subjective emotional state from a rodent, the findings described here from playback studies will be discussed largely in terms of their dependent measures.Numerous studies have confirmed alterations in these behaviors during playback; however, the method of playback, behavioral assays used, and overall results can vary greatly.One of the first studies focusing on behavior during 22-kHz USV playback used a 5-minute male recording and saw altered locomotion and speaker approach during playback compared to background tape noise or a 38-kHz tone played for the same duration (Sales, 1991).In this two-chamber preference test, both playback conditions deterred rats from the speaker chamber and reduced locomotion during and 5 min after the recording ended, yet only the 22-kHz USV playback decreased sniffing of the speaker (Sales, 1991).Subsequent studies have further examined approach and avoidance as measures of the effects of 22-kHz playback (e.g., Sadananda et al., 2008;Schönfeld et al., 2020;Shukla and Chattarji, 2021;Wöhr and Schwarting, 2007).In a study with 30 min of continuous 22-kHz playback, animals in the 22-kHz USV condition had decreased locomotor activity measured as decreased entries into a speaker compartment compared to a 50-kHz USV condition or background noise (Sadananda et al., 2008).Similarly, playback at one end of a linear track for two 3-minute episodes also led to avoidance behavior unique to the 22-kHz USV condition compared to 50-kHz USVs or white noise (Shukla and Chattarji, 2021).Self-administration of USVs through nose-poke triggered playback also shows avoidance through decreased nose pokes into a previously inactive hole when the hole was paired with 22-kHz USV playback (Burgdorf et al., 2008).Avoidance is thus a common measure indicating the aversiveness of 22-kHz USV playback and confirms the conveyance of a negative affective state on a listener through increased avoidance of the USVs.
Response to USV playback can also be measured in an 8-arm radial maze where a speaker is located at the end of one arm (a silent-dummy speaker can also be positioned on the opposite arm) with decreased time spent proximal to the active speaker interpreted as behavioral avoidance (Schönfeld et al., 2020;Seffer et al., 2014Seffer et al., , 2015;;Wöhr and Schwarting, 2007).As little as 1 min of 22-kHz USV playback in the 8-arm radial maze increased avoidance of the area proximal to the speaker (Schönfeld et al., 2020).During this relatively short 22-kHz USV playback length, animals spent significantly less time in the arms proximal to the active speaker and more time in the arms distal to the speaker compared to 50-kHz USV playback (Schönfeld et al., 2020).Decreased locomotion or increased freezing in the 8-arm radial maze is also used as a behavioral measure of negative affect during playback and multiple studies with only 1 min of 22-kHz USV playback support this assertion finding that playback decreases average velocity and distance traveled (Fendt et al., 2018;Schönfeld et al., 2020;Wöhr and Schwarting, 2007).Inhibited locomotion with 22-kHz USV playback is also seen in a study with juvenile animals in an 8-arm radial maze; however, significant avoidance was not observed, leading the authors to conclude that this playback has a relatively weak effect (Wöhr and Schwarting, 2007).One recent study using the 8-arm radial maze observed greater approach behavior to 22-kHz USV playback over a 1-minute period compared to background noise that was unique to males and not seen in females (Bigelow et al., 2022).Regardless, the locomotion and avoidance measurements provided by the 8-arm radial maze when animals are exposed to playback makes it a useful assay with results that often confirm the anxiogenic effects of 22-kHz USV playback.
Locomotion and freezing have also been measured in cage environments and other assays such as an open field test or elevated zero maze.One of the first studies in an open cage environment found no significant change in locomotion of adult male rats during 22-kHz USV playback but did observe decreased locomotion following the termination of playback (Brudzynski and Chiu, 1995).A more recent study reported decreases in locomotion during playback in conjunction with decreased heart rate which persisted after playback ended (Olszyński et al., 2020).Interestingly, these results were more prominent in singly-housed animals as opposed to pair-housed, reinforcing assertions regarding the social importance of USVs and an increased communicative value for contact-deprived rats (Olszyński et al., 2020).Playback in the elevated zero maze appears to produce results consistent with 22-kHz USVs as aversive stimuli.In a study with 10 min of playback (5-minute recording looped twice) in the elevated zero maze, male rats spent less time in the open areas during playback of male 22-kHz USVs compared to a silent condition (Demaestri et al., 2019).However, in the same study, a 20 min open field test with the same recording looped 4 times produced results (though non-significant) indicating increased time in center and frequency to center during 22-kHz USV playback.Notably, average velocity and distance traveled were nearly equivalent to 50-kHz USV playback and silence conditions, suggesting that overall locomotion was not impacted.This study also reported decreased orienting behavior towards the speaker, suggestive of avoidance during 22-kHz playback (Demaestri et al., 2019).Of note, there was an initial increase in open field center-oriented behaviors at the onset of playback, though in the last 5 min of playback male rats showed a marked decrease in center duration in the 22-kHz condition, perhaps indicating a temporal component to USV responses.In this study heart rate increased in response to 3 min of 22-kHz playback which, combined with the open field findings, may be suggestive of an initial heightened arousal and a more active escape-like response (Demaestri et al., 2019).Another study with male F344 Fischer rats using a modified open field test found that 15 min of 22-kHz USV playback from a male of the same strain led to decreased time in the center of the open field (Inagaki and Ushida, 2021).When a hiding box was introduced, animals spent more time concealed in the box and less time with the head out of the box during playback as compared to a 1000-Hz long sine-wave tone (Inagaki and Ushida, 2021).In another emergence test with only 3 min of playback during which a rat was able to leave a small, enclosed box, 22-kHz USV playback increased latency to emerge from the box and decreased time spent outside of the box compared to 50-kHz USV playback or background noise (Burman et al., 2007).Together, these results indicate a general aversion to 22-kHz USV playback when given the opportunity for concealment, but varying results when additional behaviors are evaluated.
A few other methods have been used to assess preference or aversion to 22-kHz USVs.The acoustic startle response can be quantified and used as a measure of anxiety (Davis et al., 1997).Acoustic startle response has been found to be enhanced with prior playback of 22-kHz USVs in typical males (Granata et al., 2022) and with playback of 22-kHz USVs compared to a 25-kHz tone; however, it is important to note that when the 25-kHz tone was shortened in length, the acoustic startle response is increased (Inagaki and Ushida, 2017).These results confirm the importance of 22-kHz USVs as an alarm call and support the idea that playback of these USVs is affectively anxiogenic, but they also suggest further nuances in how the auditory characteristics of USVs may differentially impact behavior.
Researchers have also assessed behavioral response to 22-kHz USV playback with other measures related to pathology.A recent study measured cocaine intake during 7.5 s of 22-kHz USV playback at 5-minute intervals and found that cocaine intake increased during 22-kHz playback but decreased during appetitive 50-kHz playback (Montanari et al., 2020).In contrast, animals did not consume significantly different amounts of sucrose during each playback condition, indicating that 22-kHz USV playback did induce an aversive state that was behaviorally attenuated with drug administration (Goeders and Guerin, 1994;Montanari et al., 2020).Another study examined the effects of differently valenced USV playback on cataplexy time and found that 22-kHz playback did not reduce cataplexy time while 50-kHz playback did (Tonelli et al., 2018).A recent study found that spontaneously hypertensive rats (SHR; adult males) were slightly behaviorally inhibited by 10 s of 22-kHz USV playback compared to 50-kHz USV playback with less time near the playback speaker and less distance traveled (Olszyński et al., 2023).Interestingly, SHR rats had a lower heart rate during and following 22-kHz USV playback compared to 22-kHz-exposed Wistar and 50-kHz-exposed SHR rats (Olszyński et al., 2023).22-kHz playback also prompts hesitation in the receiver when used as a cue for reward conditioning (Kagawa et al., 2017), further highlighting its unique salience and meaning to conspecifics, and is also an especially effective, specific, and long-lasting stimulus for fear conditioning (Bang et al., 2008;Endres et al., 2007;Lindquist et al., 2004).
While the majority of 22-kHz USV playback studies report alteration in behavior due to playback, some research in the field argues that behavioral responses to USVs are not innate and are instead acquired through experience with adverse events (Endres et al., 2007;Kim et al., 2010;Parsana et al., 2012b).Parsana and colleagues introduced the autoconditioning hypothesis in which they postulate that a negative experience, such as a foot shock prompting emission of 22-kHz USVs, self-conditions the animal to the negative valence of 22-kHz USVs (Parsana et al., 2012b).This conclusion was reached through a study using a novel chamber environment that found that animals who had experienced foot shocks prior to USV playback had higher levels of freezing than naïve animals while both groups were unresponsive to 50-kHz playback (Parsana et al., 2012b).Additionally, the same group found that playback of 7.92 s of 22-kHz USVs presented 20 times randomly mixed with other stimuli produced no significant increase in freezing during 22-kHz USV playback or during trials with a 22-kHz tone (Parsana et al., 2012a).A more recent study has contradicted the autoconditioning hypothesis with results that show significantly increased freezing during 22-kHz USV playback for devocalized animals previously exposed to foot shocks as well as sham animals who vocalized during foot shocks (Calub et al., 2018).Another study also found no effect of exposure to live 22-kHz USV emitted by a previously foot shocked animal on a naïve conspecific (Kim et al., 2010).This study confirmed that visible behavior or odor of the emitting rat did not lead to freezing in the receiver, indicating that behavioral effects of 22-kHz USV playback are primarily due to auditory cues (Kim et al., 2010).A study using haloperidol-induced catalepsy time to assess salience of playback found no difference in response to 22-kHz USV playback between animals that had been through autoconditioning or naïve individuals (Tonelli et al., 2018).Therefore, while there is some level of disagreement, overwhelming evidence supports the ability for aversive USVs to elicit changes in behavior associated with an induction of negative affect.Thus, it is likely that 22-kHz USVs have innately negative affective meaning that can be leveraged to translationally study the neural and behavioral correlates of emotion.

Brain activity during 22-kHz USV playback
Pinpointing the underlying neural mechanisms associated with 22-kHz USV processing may lead to a better understanding of the affective circuitry associated with anxiety disorders and concomitant behavior.Studies using cFos quantification as an activity index have reported changes in cFos density in brain regions thought to be involved in affective processing, suggesting differential activation by 22-kHz USV playback compared to control stimuli.When exposed to 30 min of 22-kHz USV playback in a two-chamber avoidance environment, increased cFos density was observed in the perirhinal cortex, the amygdala, and the periaqueductal gray (Sadananda et al., 2008).This pattern of activity corresponded with increased avoidance of the chamber containing the active speaker and decreased locomotion compared to a 50-kHz playback condition, indicating distinctly different processing of positively and negatively valenced stimuli (Sadananda et al., 2008).Another study in Long Evans rats with 20 min of USV playback in the open field showed increased cFos cell density in the auditory cortex for both 22-kHz and 55-kHz USV playback (indicating comparable processing of auditory input), with increased cFos density in the basolateral amygdala and bed nucleus of the stria terminalis that was unique to 22-kHz USV playback (Demaestri et al., 2019).This study also showed upregulation of cFos density in discrete nuclei of the bed nucleus of the stria terminalis that was dependent on playback type, with 22-kHz USVs eliciting significantly higher cell recruitment in the anterodorsal nucleus of the bed nucleus of the stria terminalis compared to 55-kHz or silence.This increased activity in the basolateral amygdala and bed nucleus of the stria terminalis likely indicate heightened neural response to negatively valenced social stimuli to regulate response to possible threat and mediate behavioral output.
There do appear to be regional activation patterns that are specific to the ethological quality and natural emission pattern of 22-kHz playback.In female Long Evans rats, playback of natural 22-kHz USVs, a temporally matched 22-kHz tone, and live emission of 22-kHz USVs by a conspecific prompted greater cFos density in the inferior colliculus compared to the silent control (Ouda et al., 2016).In contrast, the auditory cortex in these animals only had increased cFos density following natural USV playback and live USVs compared to artificial 22-kHz stimuli, even though all 22-kHz stimuli prompted greater activation than control (Ouda et al., 2016).In the same study, the basolateral amygdala, hippocampus, and periaqueductal gray had significantly higher levels of cFos upon exposure to natural 22-kHz USV playback and live USVs compared to artificial stimuli and silence (Ouda et al., 2016).In the periaqueductal gray, both the natural and artificial 22-kHz playback conditions showed significantly greater cFos expression than controls (Ouda et al., 2016).In male Sprague-Dawley rats, the perirhinal cortex is another region that is sensitive to ethological aspects of playback (Allen et al., 2007).While both 22-kHz USV playback and 22-kHz frequency-and temporally-matched tones have been found to alter single unit firing rates in the perirhinal cortex, continuous frequency-matched tones do not produce the same effect (Allen et al., 2007).Distinct presence of cFos in regions specific for emotional processing and regulation is a reliable method to visualize brain activity during playback, but the time course to induce cFos activation does not suit every experimental paradigm.
Given the amygdala's role in emotional processing, particularly fear and anxiety, a large portion of research on neural processes during 22-kHz USV playback center on amygdala activity during exposure to USVs.Single unit recording from the amygdala found neurons that fire in response to multiple kinds of negative stimuli including 22-kHz USV playback and an aversively valenced lever press (Kagawa et al., 2017).This study also found no such activity for positively valenced stimuli (including 50-kHz USV playback and a positively valenced lever press), supporting the assertion that this region may provide a generalized response to negative stimuli (Kagawa et al., 2017).Further research on the basolateral amygdala specifically points to its role in providing an avoidance response to aversive USVs (Shukla and Chattarji, 2021).Inactivation of the basolateral amygdala prevented the typical avoidance to 22-kHz USV playback on a linear track while the basolateral amygdala of non-lesioned animals displayed greater theta activity during playback and higher cFos levels after playback in addition to observed speaker avoidance (Shukla and Chattarji, 2021).Similarly, lesions in the basolateral amygdala alter behavior during playback in an 8-arm radial maze by creating an immobilization response where neither avoidance of 22-kHz USV playback nor uninhibited exploration occurs in the maze (Schönfeld et al., 2020).The authors of this study propose this diminished responsiveness to USVs in lesioned animals as a loss of affective value of the calls and thus a loss of interest (Schönfeld et al., 2020).Single unit recordings in the amygdala are also sensitive to USV playback and frequency (Parsana et al., 2012a).In a locomotion and freezing study which found no significant behavioral influence of 22-kHz USV playback, 22-kHz stimuli (including USV playback and a tone) increased overall firing rates relative to baseline and to a 50-kHz tone (Parsana et al., 2012a).Thus, amygdala activity certainly plays into the processing of aversive 22-kHz USVs and, in combination with other factors, likely contributes to behavioral output in response to playback.
A handful of other regions have also been examined for their role in affective processing of 22-kHz USVs.The thalamo-amygdala pathway has been proposed to be influenced by 22-kHz USV playback, and lesions of the medial geniculate nucleus of the thalamus have been shown to block freezing responses in non-naïve rats (Kim et al., 2010;Parsana et al., 2012a).Additionally, lesions of the subthalamic nucleus removed anxiety-generated increases in cocaine taking behavior during aversive USV playback (Montanari et al., 2020).Together this regional data suggests unique and nuanced involvement of multiple brain regions and circuits involved in affective processing of 22-kHz USVs (see Table 2).
A summary of cFos changes in response to 22-kHz USV playback can be seen in Fig. 2B and highlights neural recruitment during aversive playback that has compelling overlap with homologous regions in humans that are responsive to fearful face presentation in the Fearful Face Task.It should be mentioned, however, that changes in cFos density in response to USV playback can be difficult to interpret.Indeed, cFos -in the absence of colocalized neural markers or other measurescan only reveal a short temporal window of neural activity and may be influenced by processes happening in parallel to the stimulus.Further, cFos is unable to indicate directionality of information flow (i.e., input or output processing) and can only provide information about gross regional activity and may not be upregulated in all cell types (Kovács, 2008).It is clear that substantial characterization is still needed to fully appreciate how the brain is processing and responding to these cues.However, these initial studies linking brain and behavior suggest promise for leveraging 22-kHz USV playback as a translational social stimulus indicating potential threat.

Sex differences in USV emission and playback outcomes
Like humans, there also appear to be sex differences in rat affective states (Inagaki, 2018).An examination of three common assays for depression and anxiety, the elevated plus maze, the open field test, and a social interaction paradigm, found sex differences in behavior with females spending more time in open areas and investigating novel objects as well as having higher locomotion regardless of estrous cycle phase (Scholl et al., 2019).Anxiogenic social isolation in adolescence also leads to sex differences in adrenal response later in life, thereby influencing behavioral responses (Weintraub et al., 2010).Female rats have higher basal corticosterone and norepinephrine levels and higher plasma levels of epinephrine and corticosterone after novel environment exposure and foot shock compared to males (Weinstock et al., 1998).There are sex differences in USV emission in the presence of a predator where female rats emit longer, higher pitched, and more frequent 22-kHz USVs compared to males in a visible burrow system (Blanchard et al., 1992).Neonatally isolated male and female rats have been shown to emit aversive USVs at different rates as adults with males emitting 22-kHz USVs with a longer duration during fear conditioning (Kosten et al., 2005).Similarly, behavioral responses induced by air puffs to the neck produced results showing that male rats emitted 22-kHz USV calls for a longer period after the stimulus and more frequently than females (Inagaki, 2018).In line with this, female Wistar rats emit fewer 22-kHz USVs than makes during fear conditioning, though time spent immobile was comparable regardless of sex (Willadsen et al., 2021).A recent review also considered 22-kHz USV emission across the estrous cycle in females, concluding that sex differences were present, but that cycle-specific studies of USV emission are scarce (Lovick and Zangrossi, 2021).This evidence supports the assertion that male and female rats exhibit different affective reactions to specific stressors.Despite these well-documented differences in behavioral avoidance and physiological response to stressors, research continues to be sparse regarding sex differences in response to playback of affectively and socially valenced USVs.
The methods utilized in USV playback experiments vary markedly from study to study with little consistency in playback parameters such as volume, duration, method of eliciting the call, and behavioral assay and interpretation.Seemingly one of the only consistencies throughout playback studies is the use of only male subjects and male-emitted 22-kHz USV recordings (see Table 1; Demaestri et al., 2019;Fendt et al., 2018;Inagaki and Ushida, 2017;Kagawa et al., 2017;Montanari et al., 2020;Sadananda et al., 2008;Schönfeld et al., 2020).To the best of our knowledge, only two studies have examined sex differences in response to 22-kHz USV playback.The first study conducted by Wöhr et al. in 2020 looked in part at sex differences in response to aversive USV playback for animals with or without the CACNA1C gene which is a cross-disorder risk gene for bipolar disorder, schizophrenia, and major depressive disorder (Green et al., 2010;Wöhr et al., 2020).Through analysis in an 8-arm radial maze with 22-kHz USV playback from a male conspecific, Wöhr and colleagues found that CACNA1C haploinsufficiency led to less behavioral avoidance only in male animals (Wöhr et al., 2020).Though the main experimental question of this study was not directly related to USV playback, the use of this methodology and the sex-specific, and genetically modulated effects of behavior in response to this social stimulus indicate that underlying behaviors in response to 22-kHz USV playback are indeed sex-specific in some way (Wöhr et al., 2020).A more recent study by Bigelow and colleagues explored how a single kainic acid-induced early-life seizure may alter behavioral responses to USV playback in an 8-arm radial maze later in life (Bigelow et al., 2022).This study found that seizure caused deficits in auditory communication in only adult males which led to changes in behavior in response to 50-kHz USV playback but did not alter male's increased approach behavior observed during 22-kHz USV playback (Bigelow et al., 2022).Both experiments using male and female subjects point to potential sex differences in how animals respond to USVs and other experimental variables; however, little analysis has been done to explain these differences in the context of affective processing of USVs.
In addition to differences in male and female responses to USV playback, there is also a lack of understanding of how these responses may also differ based on the sex of the recorded emitter.A recent study (without the use of USVs) also found prominent sex differences in multiple rat behavioral assays, further underscoring the need to consider how the field approaches the interpretation of behavioral findings within the context of sex as a biological variable (Börchers et al., 2022;Shansky, 2019).Therefore, we strongly urge that sex should be included as a biological variable to be considered in future playback studies.Additionally, to our knowledge, no published studies exist examining the possibility of sex differences in neural activity when exposed to 22-kHz USV playback.Given known sex differences in both humans and rats regarding affective processing, stress responses, avoidance behavior, and USV emission -coupled with a lack of mechanistic understanding of processing 22-kHz USV processing -the field must expand to include male and female experimental subjects to further understand these differences.

Considerations for the experimental use of USV playback
Though most literature points to the social contagion properties of 22-kHz USV playback, there are inconsistencies and caveats which must be considered to fully appreciate the anxiogenic effects of playback (Wöhr and Schwarting, 2013).These inconsistencies mainly arise from the use of different protocols for USV playback that are specific to a given research group (see Tables 1, 2).Notably, 22-kHz USVs are emitted under a variety of situations with unique acoustic characteristics that may influence behavior differently upon playback (Knutson et al., 2002).22-kHz USVs induced by tactile stimulation and carbachol injection were found to have similar effects on behavior in a recording cage (Brudzynski and Chiu, 1995).22-kHz studies have produced similar behavioral results with playback of USVs elicited by foot shock (Shukla and Chattarji, 2021), defeat in a fight (Sales, 1991), restraint stress (Demaestri et al., 2019), and predator urine (Fendt et al., 2018).However, the age of the animal producing USVs affects acoustic startle response (Inagaki and Ushida, 2017), and unique effects of playback have been seen based on the individual animal that USVs were recorded from (Burman et al., 2007).It is also possible that the sex of the animal emitting USVs might lead to different behavior as has been shown with increased exploration of male rats during female 50-kHz USV playback (Inagaki and Ushida, 2021), but this question has not been examined thoroughly with 22-kHz USVs.
Playback parameters also differ greatly between research groups.Playback length ranges from 30 min (Sadananda et al., 2008) or 45 min (Ouda et al., 2016) to 10 seconds or less (Kagawa et al., 2017;Montanari et al., 2020;Olszyński et al., 2020) and this time may be occupied by a continuously recorded, 5-minute string of relatively ethologically accurate calls (Demaestri et al., 2019) or a single call repeated numerous times (Brudzynski and Chiu, 1995).Volume of playback is also inconsistent with reported sound levels centering around 60-70 dB but ranging from 30 dB (Brudzynski and Chiu, 1995) to 85 dB (Burgdorf et al., 2008) measured a variety of distances from the speaker.An examination of "high intensity" (70-80 dB) versus "low intensity" (40-50 dB) USVs played 20 cm from listening animals in an 8-arm radial maze demonstrated that a decrease in locomotion due to aversive playback was more specific in low intensity conditions (Fendt et al., 2018).In natural conditions, a 22-kHz call is usually emitted from a rat at 60-80 dB when measured from 20 to 30 cm away (Ouda et al., 2016;Wöhr et al., 2005;Wöhr and Schwarting, 2008), thus many playback studies use these parameters yet produce varying results or fail to report other details about their playback setup.These variables must be considered in analysis of behavior in response to these social signals and the field may benefit from standardizing some parameters of playback.
The accuracy of playback is also a consideration worth noting, as this can be influenced by a number of factors, including (but not limited to) the type of speaker used, filtering parameters, and acoustics within the testing environment.The distance of the speaker from the subject is also important, as the sound intensity of high frequency signals like USVs decay rapidly over distance (Fletcher, 2010), which might compromise the accuracy of the playback if not accounted for.The hardware used for playback often differs between research groups, which may interact  The included studies are those that assess behavioral and/or neural outcomes in response to 22-kHz USV playback.They do not include studies that solely examine effects of conditioning to 22-kHz USV playback nor effects of artificially generated 22-kHz tones; however, tones were used in addition to natural USVs as comparison groups in some of these studies.This table summarizes the methodological parameters for USV collection and playback and includes information on the strain and sex of the subjects in each study (M, male; F, female), the strategy used to elicit 22-kHz USV emission, the total length of playback (min (minutes); s (seconds)), the volume and distance from animals at which playback occurred (dB (decibels); cm (centimeters)), the general measured outcomes during playback, and the behavioral assay in which behavioral reactions to playback were measured.For the neural correlates of playback included in some studies, see Table 2 as indicated in the measured outcomes column.If information was not specified, this is indicated with "ns".with other non-standardized playback factors, including distance from subjects, sex and/or strain of recorded emitter, type of playback (ethological or looped), etc.While specialized ultrasonic recording and playback setups exist and are used in more recent studies which are thought to maintain acoustic fidelity, earlier studies used USVs that had been tape recorded (e.g., Sales et al., 1991).In some cases, frequency band pass filters are applied to USV recordings prior to playback to isolate the USVs and eliminate background noise or stimuli outside of the range of interest (e.g., Ouda et al., 2016;Demaestri et al., 2019).However, these approaches for collecting USV stimuli, as well as the equipment and parameters used for playback, are not standardized across research groups (see Table 1).Further, inclusion and/or details about external validation of frequency or volume of playback is rarely included in manuscripts.Indeed, the accuracy of the USV stimuli during playback is a critical feature to consider when we intend to present an ethological stimulus to elicit naturalistic behavior in response to a conspecific social cue indicating possible threat in the environment.
It is important to note that this review has focused on behavior and neural activity in response to playback of ethological, recorded 22-kHz USVs.In studies using tones, results sometimes match those of 22-kHz USV playback depending on the experimental questions and parameters of playback (Allen et al., 2007;Inagaki and Ushida, 2017;Ouda et al., 2016;Parsana et al., 2012a).Studies exclusively using tones have found strain differences in behavior during playback, reporting that Lister Hooded and Wistar rats had distinct changes in locomotor and escape behavior in response to tones with the same frequency and sound characteristics (Commissaris et al., 2000;Neophytou et al., 2000).As outlined in the present review, despite these differences in response to tones, weak yet relatively consistent avoidance behavior in response to ethological 22-kHz USV playback is reliably observed (Wöhr and Schwarting, 2013).
Even though 22-kHz USV playback is increasingly being used as an anxiogenic tool to study mood disorders in an animal model, this area of study has not sufficiently expanded to include analysis of sex differences.There is a need for the systematic exploration of sex differences in almost every aspect of human affect and affective disorders, as well as in preclinical models, to enhance translatability (Shansky, 2019).Overall, playback of 22-kHz USVs is a promising approach for modeling and understanding affective dysfunction in humans.By translating affective communication and the triggering of anxiety responses to social cues through paradigms including playback of 22-kHz USVs, affective processing and anxiety can be studied in a relatively accurate animal model.Further, using 22-kHz USV playback to expose and leverage underlying neural mechanisms of negative affective processing has the potential to further our understanding of anxiety circuitry and compensatory systems in the brain.

Conclusions
Playback of aversive rat 22-kHz USVs is a useful translational method to examine affective processing of social signals in a variety of experimental setups with relevance to anxiety disorders.This is underscored by findings that report increased behaviors that are consistent with features of anxiety (i.e., behavioral avoidance) in response to 22-kHz playback (e.g., Demaestri et al., 2019;Olszyński et al., 2023;Sadananda et al., 2008;Sales, 1991;Schönfeld et al., 2020;Shukla and Chattarji, 2021;among others), as well some reports of sex differences in behavioral response to playback (e.g., Bigelow et al., 2022) suggesting utility for understanding affective etiology.Importantly, when reviewing the literature characterizing areas of the rat brain that are recruited by 22-kHz USVs, several regions associated with processing affective information show changes in cFos activity, including regions of the amygdala (e.g., basolateral), periaqueductal gray, hippocampus, and prefrontal cortex.This is in line with homologous brain regions in humans known for their role in affective processing (for review see Hiser and Koenigs, 2018;Kim et al., 2011).Furthermore, when directly The included studies are all those that assess neural effects in response to 22-kHz USV playback following a lesion or measurement (i.e., immunohistochemistry, lesion, recording) at the neural level.Here, we present the region(s) of interest examined in each study, the method(s) used to assess playback effects and/or neural manipulation, and a brief summary of findings as it relates to the neural correlates of 22-kHz USV playback.Results are communicated for each study based on regional activity increase (↑) or decrease (↓).When applicable, results are also specified for the right (R) or left (L) hemisphere regional neural recruitment of negatively valenced affective stimuli in rats (via 22-kHz USV playback) and humans (via fearful face presentation), both the prefrontal cortex and amygdala show significant changes in activity (for review of regional findings, see Fig. 2) and these regions are implicated in attentional bias to threat in humans with anxiety (Cisler and Koster, 2010).The fact that both stimuli recruit discrete regions associated with anxiety and/or processing of emotion lends support for the use of 22-kHz USV playback as a means through which we can evoke similar neural and behavioral outcomes to those seen in humans, with a stimulus that holds comparable ethological relevance to a fearful face.
Based on this evidence, we propose the use of aversive, ethological rat USV playback as a translational assay comparable to the human fearful face task that can be leveraged in a variety of experimental contexts.Though there is some disconnect in the current literature over the ideal parameters and assessments for playback exposure, this stimulus holds promise to evaluate how an organism processes, and responds to, distal possible threat in the environment.While many of the experiments that have been conducted with playback of aversive USVs are done in typical animals, it is important to note that the most robust responses to possible threat in the environment would emerge in neuropathological models associated with affective dysfunction (i.e., those which induce states hypervigilance to possible threat), particularly since we see enhanced bias for negative stimuli in patients with anxiety disorders (e.g., Bradley et al., 1999;Waters et al., 2004).Despite this, we still observe significant effects of anxiogenesis evoked by 22-kHz playback in naive, non-model system rats, suggesting that playback could be used as both a model of acute anxiety induction, as well as a future means by which we can assess hypervigilance and susceptibility to aversive cues in disease models to probe underlying neural etiology.Of course, further research on this model of social contagion and affective processing is needed to fully elucidate its utility in the translational field.Leveraging 22-kHz USV playback in rat models of affective dysfunction (via behavioral, environmental, genetic, and/or other paradigms and preparations) will broaden our understanding of how the processing of -and response to -threat is altered in affective disorders.Combining model system approaches with 22-kHz playback to probe for subtle behavioral changes will also provide a more nuanced understanding of how experiences and risk factors converge to impact and regulate affective [dys]function.

Fig. 1 .
Fig. 1.Representative spectrograms of different types of rat ultrasonic vocalizations. A. 40-kHz vocalizations (retrieval call) emitted by a two-day old female pup separated from the dam.B. 50-kHz vocalizations emitted by an adult female upon receipt of a food reward.C. 22-kHz vocalizations emitted by an adult male under restraint stress in the presence of cat urine and an anesthetized conspecific.

Fig. 2 .
Fig.2.Neural activity changes due to social threat cue exposure: 22-kHz playback (rats) or fearful face presentation (humans).Figure depicts summary findings of regional changes in neural activity as measured through a social cue indicating possible threat through presentation of a fearful face in the fMRI in humans (A), or through playback of 22-kHz ultrasonic vocalization (USV) alarm calls in rats (B).In humans, blue highlighted regions indicate regional BOLD activity change in response to fearful face presentation (measurements in mm indicate distance from center anterior commissure).Regions that are responsive to fearful faces compared to baseline include: (A1) fusiform gyrus, anterior cingulate gyrus; (A2) fusiform gyrus, superior colliculus, anterior cingulate gyrus; (A3) fusiform gyrus, amygdala, basolateral amygdala; (B4) fusiform gyrus, amygdala, basolateral amygdala; (B5) fusiform gyrus, amygdala, basolateral amygdala; (B6) orbitofrontal cortex, prefrontal cortex; and (B7) orbitofrontal cortex, prefrontal cortex.In rats, yellow highlighted regions indicate changes to regional neural activity via exposure to 22-kHz USV playback (locations given relative to Bregma) as measured through cFos immunoreactivity following playback.Regions that are responsive to 22-kHz playback compared to silence and/or other USV comparisons include: (B1) inferior colliculus, periaqueductal gray (including rostral dorso-medial, dorsal raphe nuclei); (B2) perirhinal cortex, ectorhinal cortex, periaqueductal gray; (B3) perirhinal cortex, ectorhinal cortex, primary auditory cortex, hippocampus; (B4) perirhinal cortex, ectorhinal cortex, primary auditory cortex, hippocampus; (B5) perirhinal cortex, ectorhinal cortex, periventricular nucleus of thalamus, hippocampus, central amygdala, basolateral amygdala; (B6) bed nucleus of the stria terminalis (oval and antero-dorsal nuclei); and (B7) prefrontal cortex.Notably, there is cross-species overlap in activity changes in homologous regions (i.e., prefrontal cortex and amygdala) that are important for threat processing and response, and activity in these regions is thought to be altered in both patients and animal models of affective disorders.Human data presented here summarized from fMRI work using fearful face presentation(Vuilleumier et al., 2001;Killgore and Yurgelun-Todd, 2001; Bishop et al., 2007).Figure adapted from(Mai et al., 2016;Paxinos and Watson, 2007) with data summarized from research assessing rat cFos in response to 22-kHz playback (see Table2).

Table 1
Comprehensive list of studies using 22-kHz ultrasonic vocalization playback with analysis of behavior and/or brain activity.

Table 2
Summary of results from studies examining neural outcomes in response to 22-kHz USV playback.