Behavioural Brain Research 275 (2014) 43–52

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Vocalizations associated with anxiety and fear in the common marmoset (Callithrix jacchus) Yoko Kato a,1,2 , Hayato Gokan a,b,1 , Arata Oh-Nishi a , Tetsuya Suhara a , Shigeru Watanabe b , Takafumi Minamimoto a,∗ a b

Molecular Neuroimaging Program, Molecular Imaging Center, National Institute of Radiological Sciences, 4-9-1 Anagawa, Inage-ku, Chiba 263-8555, Japan Graduate School of Human Relations, Keio University, 2-25-45 Mita, Minato-Ku, Tokyo 108-8345, Japan

h i g h l i g h t s • • • •

Tsik-egg call was identified as a multisyllabic call type rather than a sequence of tsik and egg call. Marmosets vocalized tsik-egg calls without tsik calls under anxiety-related situations. Marmosets vocalized tsik and tsik-egg calls when they were exposed to predatory stimuli. Tsik-egg with/without tsik calls appear to be vocal indices for fear/anxiety in marmosets.

a r t i c l e

i n f o

Article history: Received 1 May 2014 Received in revised form 19 August 2014 Accepted 23 August 2014 Available online 3 September 2014 Keywords: Affective behavior Negative emotion Separation Benzodiazepine Anxiety Marmoset

a b s t r a c t Vocalizations of common marmoset (Callithrix jacchus) were examined under experimental situations related to fear or anxiety. When marmosets were isolated in an unfamiliar environment, they frequently vocalized “tsik-egg” calls, which were the combination calls of ‘tsik’ followed by several ‘egg’. Tsik-egg calls were also observed after treatment with the anxiogenic drug FG-7142 (20 mg/kg, sc). In contrast, when marmosets were exposed to predatory stimuli as fear-evoking situations, they frequently vocalized tsik solo calls as well as tsik-egg calls. These results suggest that marmosets dissociate the vocalization of tsik-egg and tsik calls under conditions related to fear/anxiety; tsik-egg solo vocalizations were emitted under anxiety-related conditions (e.g., isolation and anxiogenic drug treatment), whereas a mixed vocalization of tsik-egg and tsik was emitted when confronted with fear-provoking stimuli (i.e., threatening predatory stimuli). Tsik-egg call with/without tsik can be used as a specific vocal index of fear/anxiety in marmosets, which allows us to understand the neural mechanism of negative emotions in primate. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Fear and anxiety are both emotional reactions to real or anticipated threats, but are dissociable; fear is generally held to be a reaction to an explicit threatening stimulus, whereas anxiety is usually considered a more general state of distress, more long-lasting, prompted by less explicit or more generalized cues [1,2]. Although these emotions are adaptive responses to negative situations, when

∗ Corresponding author. Tel.: +81 43 206 3249. E-mail addresses: [email protected] (Y. Kato), [email protected] (T. Minamimoto). 1 These authors contributed equally to this work. 2 Present address: Animal Physiology & Behavior Group, Department for Neuroscience, School of Medicine and Health Sciences, Carl von Ossietzky University Oldenburg, Carl von Ossietzky Str. 9-11, D-26129 Oldenburg, Germany. http://dx.doi.org/10.1016/j.bbr.2014.08.047 0166-4328/© 2014 Elsevier B.V. All rights reserved.

the negative emotions become excessive, they fall under a pathological state [3,4]. A large number of animal studies, mainly with rodents, have examined the neurobiological mechanisms of fear and anxiety, and their key brain structures (e.g., amygdala and bed nucleus of stria terminalis) and major target neurochemicals (e.g., benzodiazepine) were proposed [2,5]. For a better understanding of the pathological state and for developing treatments, non-human primate models of fear/anxiety have been developed, with their advantage of neurobiological proximity to humans, such as developed prefrontal cortex [6,7], as well as similar physiological and behavioral responses to situational contexts as humans [8–10]. In these models, specific and quantitative behavioral assessment of fear and anxiety are desired, although these two emotions are closely related and obviously overlap [1]. However, objective and dissociable measure of fear and anxiety in non-human primates has not yet been fully established.

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Vocalization is one of the main channels of emotional expression and affective communication in non-human primates. The marmoset, a New World primate, employs rich vocal repertoires and vocal communication [11,12]. Marmosets vocalize several specific call types under negative emotional situations. The ‘phee’ call is described as a contact call between conspecific subjects [11–13], and is vocalized under negative emotional situations such as social separations [14,15]. The ‘egg’ call is vocalized with vigilant behavior against a strange human approaching [11] and with mobbing a potential predator [12]. The ‘tsik’ call is vocalized with mobbing behavior against potential predators [11,12] and is also increased under experimental situations related to fear and anxiety [9,16]. In addition, ‘tsik-egg’ call is described as a combination or sequential call of tsik followed by egg, and is vocalized with mobbing to predators and vigilance behavior [11,12]. Tsik-egg and tsik were used to dissociate behavioral states during human intruder test [25,26]. However, the vocalization index in relation to fear and anxiety has not been specifically examined. Based on the definition, fear is characterized as a response to threatening objects. Accordingly, vocal analysis under the contexts with or without threatening objects would provide specific and quantitative vocal indices for fear and anxiety, respectively. In the present study, we examined vocal and behavioral responses under anxiety- or fear-provoking conditions to identify vocal indices reflecting each emotion. We first examined vocal and behavioral responses of marmosets under a novel isolated environment as an anxiety-provoking experimental condition. After the marmosets had become habituated to the environment, we examined whether the same vocal and behavioral responses could be pharmacologically induced by injection of an anxiogenic drug, FG7142, which is a partial inverse agonist of benzodiazepine receptor [5,17,18]. Finally, we performed a visual presentation test in which the isolated marmoset was exposed to predatory photo stimuli as a fear-provoking condition. By a comparison of the three experimental situations, we identified two types of vocal usage related to fear and anxiety.

be detected in the chamber. Room temperature was maintained at 27–31 ◦ C and relative humidity at 30–40%. The internal space of the sound-attenuated chamber was ventilated and illuminated with fluorescent lightning. A testing cage (same size as used in the colony room) was set in the sound-attenuated chamber. An infrared detector (Ohara Medical Industry, Tokyo, Japan) was attached to the ceiling of the cage in order to measure spontaneous movement. Vocalization sounds were recorded by linear PCM recorder (PCMD1, SONY, Tokyo, Japan) via a hypercardioid microphone (NTG2, RØDE Microphones, New South Wales, Australia) with 16 bits of digital sampling at a rate of 44.1-kHz. The animals’ behavior was monitored by digital video camcorder (HDR-XR500V, SONY, Tokyo, Japan), and recorded as a movie file by the direct-show capture on a PC. The microphone and camcorder were attached to the ceiling of the sound-attenuated chamber at a distance of 0.6 m from the top of the cage. 2.3. General experimental procedures All experiments were performed between 10:00 and 13:00. Before the experimental session, each subject was captured and transferred individually from the colony room to the experimental room in a small cage (165(h) × 140(w) × 255(d) mm), and placed in the testing cage in the sound-attenuated chamber. The experiments were performed once a day for each subject. Three experimental conditions were performed in the following order: isolation test, pharmacological stimulation test, and visual presentation test. Vocalization sounds, behaviors and spontaneous activity were recorded throughout the sessions. The subject was returned to the colony room after the end of each session. 2.4. Isolation test

2. Materials and methods

The animals were isolated in the testing cage in the soundattenuated chamber for a 30-min session each day. This isolation session was repeated daily for 5 days at ca. 24-h intervals. Since none of the 6 subjects had experienced this set-up before the test, the situation was similar to an open field test of anxiety in rodent studies.

2.1. Subjects

2.5. Pharmacological test

Six adult common marmosets (Callithrix jacchus, 3 males and 3 females, average age 2.6 years, range 2.0–2.9) were used in this study. All subjects were naïve to any live, stuffed models or pictures of predators. All the experimental procedures were carried out in accordance with the Guide for the care and use of laboratory animals (National Research Council 2011) and approved by the Animal Care and Use Committee of the National Institute of Radiological Sciences. All animals were born at the National Institute of Radiological Sciences. They weighed 250–450 g, and were housed in pairs, with one family member of the same sex, in cages (0.66 (h) × 0.33 (w) × 0.6 (d) m). Environmental lighting was provided from 8:00 a.m. to 8:00 p.m. in this colony room. Room temperature was maintained at 24–30 ◦ C and relative humidity at 40–60%. Balanced marmoset food pellets (CMS-1, CLEA Japan, Tokyo, Japan) were provided at sufficient amounts once a day, and water was available ad libitum in their home cages.

After the isolation session and a subsequent 2-day break, an additional isolation session was performed to verify their habituation to the environment. Subsequently, pharmacological testing was performed in 3 consecutive sessions with intervals of ca. 24 h. FG-7142 (N-methyl-b-carboline-3-carboxamide; SigmaAldrich, Saint Louis, MO, USA) was injected subcutaneously at a dose of either 5 or 20 mg/kg, in a volume of 200 ␮l dimethyl sulfoxide (DMSO) used as vehicle. The doses of FG-7142 were chosen from a previous study, in which 5 and 20 mg/kg were the minimal and maximum doses used, leading to anxiogenic effects in common marmosets [19]. Three treatments (two doses of FG-7142 and DMSO vehicle alone) were tested in a within-subject design. The order of the treatments was counterbalanced among subjects. The subjects received the treatment in their home cages. Within the following 3 min, the subjects were transferred to the testing cage and the isolation test was started.

2.2. Apparatus

2.6. Visual presentation test

All experiments were carried out in a sound-attenuated chamber (Muromachi Kikai, Tokyo, Japan; 1.6 (h) × 0.7 (w) × 1.6 (d) m) in an experimental room, which was away from the colony room. Any vocalization sounds from the colony room could not

The visual presentation test was performed in the testing cage with an LCD display (RDTI95LM, 376 mm × 301 mm, 90◦ angle rotation, Mitsubishi, Tokyo, Japan) attached and facing one side of the cage. In advance of the visual presentation test, the subjects

Y. Kato et al. / Behavioural Brain Research 275 (2014) 43–52

were habituated to the new environment for three 30-min sessions with intervals of ca. 24 h. White background stimuli were presented throughout the habituation sessions. The 3rd habituation session was treated as baseline. Following the habituations, two sessions of visual presentations were performed. In the 4th session, a non-picture image (gray rectangle image; 25% black by grayscale, 125 mm × 285 mm) was presented two times with a 10-min interval, serving as control for the following picture presentation. In the 5th session, four picture images (125 mm × 285 mm) were presented with 4-min intervals. The four images included predator A (wild cat), predator B (leopard), natural scene (mountains), and mosaic image of predator B. All picture images were obtained online. The mosaic image, spatially randomized as 20 × 30 pieces of the image of predator B, was created using Adobe Photoshop. The images were presented once in the session in the following order: natural scene, predator A, mosaic, and predator B. All stimuli of the 4th and 5th sessions were automatically presented using Microsoft PowerPoint in the following manner: the image was presented at the center of the screen (1 min), image expansion (expanded by 300% per 5 s, repetitively for 30 s), expanded image (steady: 300%, 30 s). Hence, the duration of each presentation was 2 min. White background was presented during an inter-stimulus interval (10 min and 4 min for non-picture and picture presentation sessions, respectively). 2.7. Data analysis 2.7.1. Acoustic analysis Vocalizations were detected and analyzed based on spectrograms (FFT length 512, Hamming window, bandwidth 112) using SAS-lab Pro 5.1 software (Avisoft Bioacoustics, Berlin, Germany). Recorded acoustic data were processed with a 1-kHz high pass filter and noise reduction (GoldWave audio editor version 5.58, GoldWave Inc., St. John’s, NL, Canada) to reduce background noise. As for the classification of call types, we defined two hierarchical components: element and unit. Element refers to distinctive sound figures on the spectrogram. Unit refers to sounds consisting of single or multiple elements. Units were separated by silent intervals longer than the minimum inter-unit-interval (IUImin ), which was determined as the antimode of a bimodal distribution of frequency of logarithmic silent intervals between all successive elements. For example, IUImin for subject CJ0044M in the isolation test was about 200 ms (Fig. 1B). Units were classified into call types based on spectrograms previously reported (Supplementary Fig. 1) [11]. For example, ‘tsik’ is characterized as an element of very sharp sweep (Fig. 1A, right) [11]. ‘Tsik-egg’ call is classified

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as having ‘tsik’ element followed by a few ‘egg’ elements with intervals shorter than IUImin (e.g., 90% agreement for training data set. 2.7.4. Statistical analysis Statistical analyses were performed on Kyplot ver 5.0 (KyensLab Inc., Tokyo, Japan) and R ver 2.13.2 (R Development Core Team, http://www.R-project.org). Comparisons of frequency of vocalizations or behaviors were performed by one-way repeated measures ANOVA followed by multiple comparisons using either Tukey’s HSD test and Dunnett’s test (for equal group size) or Games-Howell test (for unequal group size). In case unequal variances were detected by Bartlett’s test, Friedman one-way repeated measures of analysis of variance was performed followed by Steel-Dwass test for multiple comparisons. Level of significance was set at p < 0.05. 3. Results 3.1. Vocal classifications Under the three experimental contexts, we identified call types previously reported: phee, egg, tsik, twitter, tse, and cough calls. Besides these call types, we classified a multisyllabic call type,

Fig. 1. Definition of units and elements. (A) Example of vocal units. Tsik-egg call units consist of multiple elements of tsik and egg, whereas tsik consists of a single element. (B) Frequency of the logarithmic silent intervals between all successive elements in one subject. The histogram was fitted by a mixture of two normal distributions (the modes are shown as black curves). The distribution of the silent interval can be divided at the antimode (IUImin = ca. 200 ms, gray vertical line); intervals shorter and longer than this correspond to inter-element and inter-unit intervals, respectively.

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Table 1 Acoustic properties for each vocalization type identified in this study. Type

Duration (s)

Peak frequency (kHz) Start

Tsik-egg Tsik Egg Phee

0.33 0.22 0.10 1.53

± ± ± ±

0.05a 0.05a,b 0.03b 0.13

10.37 9.44 2.28 6.90

Entropy

End ± ± ± ±

1.72c 1.58c 0.58 0.36

2.83 5.58 2.70 7.49

Start ± ± ± ±

0.31d 0.95 0.24d 0.52

0.25 0.26 0.40 0.19

End ± ± ± ±

0.02e 0.03e 0.02 0.02

0.40 0.59 0.37 0.22

± ± ± ±

0.04f 0.03 0.04f 0.03

All analyses were performed on randomly resampled sonograms obtained from the isolation test and the visual exposure test (tsik-egg, egg, phee were obtained from 6 subjects, tsik was obtained from 5 subjects). Mean ± SD across subjects is shown. Non-significant differences are indicated by pairs of italicized letters (Tukey’s HSD test, p > 0.05).

“tsik-egg”, which is characterized as having the same element of tsik followed by 1∼3 elements similar to egg (Fig. 1, see Materials and methods) [11,12]. This sequence of elements mostly appeared without silent intervals (>99% of total tsik followed by egg(s)). Since twitter, tse and cough calls were observed very few times and from not all subjects, these calls were not reported in this study. We report four call types: phee, egg, tsik and tsik-egg (see Supplementary Fig. 1). Among them, all measured acoustical properties were different (Table 1; one-way repeated ANOVA; e.g., peak frequency at onset, F(3,12) = 88.36, p < 0.05). Tsik-egg and tsik calls were indistinguishable at their onset based on the peak frequency or entropy (post hoc Tukey’s HSD test, p > 0.05). Tsik-egg and egg calls were also indistinguishable at their offset based on the peak frequency or entropy (post hoc Tukey’s HSD test, p > 0.05).

General motor activity counts decreased as the session progressed (F(4,20) = 5.00, p < 0.05; Fig. 2). The frequency of other distinctive behaviors was indifferent across sessions (sway, F(4,20) = 2.77; gnaw, F(4,20) = 0.77; groom, F(4,20) = 0.66; p > 0.05; Fig. 2). Thus, two behavioral reactions, the number of tsik-egg calls and general motor activity, decreased as the subjects were repetitively exposed to the isolation environments. To compare the progress of these changes across the 5 isolation sessions, we plotted the cumulative percentage of the total number of tsik-egg calls against that of motor activity in each individual subject (Fig. 3). The curves were all convex, indicating that reduction of tsik-egg calls occurred in an earlier session during habituation than reduction of motor activity counts. Moreover, the curves were almost overlapping, suggesting that the relative speed of decrease in the two behavioral indices was consistent across subjects.

3.2. Vocalization and behavior in a novel isolated environment: initial and repetitive exposure

3.3. Vocalization and behavior under anxiogenic drug treatments

When the marmosets were isolated in a novel environment, tsik-egg calls were frequently observed in the 1st session. Repeated exposure to that environment reduced the frequency of tsik-egg calls (repeated measures ANOVA, F(4,20) = 4.99, p < 0.05; 1st vs. 25th, post hoc Tukey’s HSD test, p < 0.05, Fig. 2). Phee and egg were consistently observed across 5 sessions (phee, F(4,20) = 1.56, p = 0.41; egg, F(4,20) = 0.84, p = 0.26, Fig. 2). Tsik calls were less than 1% of the total vocalizations throughout the 5 sessions (Table 2).

In the pharmacological test, subjects were re-isolated with either high (20 mg/kg) or low dose (5 mg/kg) of FG-7142 treatment or vehicle injection. The frequency of tsik-egg calls was significantly affected by treatment conditions (repeated measures ANOVA, F(2,10) = 4.82, p < 0.05; vehicle vs 20 mg/kg, post hoc Dunnet’s test, p < 0.05, Fig. 4). By contrast, tsik calls were never induced by the treatments. The frequencies of phee and egg calls were not significantly affected (phee, F(2,10) = 0.38, p = 0.69; egg, F(2,10) = 1.25,

Fig. 2. Behavioral change during isolation test. Total number of vocalizations, distinctive behaviors and motor activity counts in each session (mean ± SEM) in the isolation test. Tsik-egg calls and motor activity counts significantly decreased across sessions (p < 0.05). Asterisk indicates significant difference in number from session 1 (p < 0.05; post hoc Tukey’s HSD test).

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Table 2 Total number of each type of vocalization in 5 isolation sessions. Subject

Tsik-egg

AA0011F CA0016M CJ0012F CJ0021M CJ0043F CJ0044M

864 87 86 212 923 743

Tsik (83%) (23%) (18%) (39%) (41%) (54%)

19 0 0 0 0 19

Egg (2%) (0%) (0%) (0%) (0%) (1%)

53 4 4 13 171 363

Phee (5%) (1%) (1%) (2%) (8%) (27%)

99 287 394 318 1148 243

Total (10%) (76%) (81%) (59%) (51%) (18%)

1035 378 484 543 2242 1368

Percentage indicates the frequency of call types per total number of vocalizations for each subject.

p = 0.33, Fig. 4). The frequencies of distinctive behaviors (sway F(2,10) = 0.70, gnaw F(2,10) = 0.32, groom F(2,10) = 1.13, p > 0.05) or motor activity counts (F(2 ,10) = 0.94, p > 0.05) were not affected (Fig. 4).

3.4. Vocalization and behavior during exposure to visual stimuli

Fig. 3. Reduction pattern of tsik-egg call and motor activity during isolation test. Cumulative percent of total number of tsik-egg calls (y axis) and motor activity (x axis) during the isolation test are plotted individually.

After the marmosets were habituated to the testing environment, they were exposed to non-picture or pictures (Fig. 5). When marmosets faced the stimulus on the display, all subjects reacted by staring with swaying, jumping backwards, piloerection and vocalizations. The predatory stimuli (e.g., Fig. 5A), but not non-picture or neutral stimuli (e.g., Fig. 5B), provoked intensive reactions, e.g., escape behaviors such as running inside the cage and clinging on the wall away from the display with frequent vocalizations. The repertory of behavioral expressions was similar to previous results using 3D objects [21,22]. Tsik-egg calls increased during the nonpicture presentation session, and became more prominent during the picture presentation session (Friedman test, df = 2, 2 = 9.65, p < 0.05; Post hoc Steel-Dwass test, baseline vs. picture stimuli, p < 0.05, Fig. 6). Tsik calls were rarely observed during baseline and non-picture presentation, but they were frequent during picture image presentations (Friedman test, df = 2, 2 = 10.38, p < 0.05; Post hoc Steel-Dwass test, picture vs. baseline and non-picture, p < 0.05, Fig. 6). To characterize vocalizations of the two call types further, we examined the timing of the vocalizations during the picture presentation session (Fig. 7A). Tsik-egg calls were consistently vocalized regardless of the presence or absence of the stimuli, as indicated

Fig. 4. Behavioral change in the pharmacological test. Total number of vocalizations, distinctive behaviors and motor activity counts in each treatment condition are shown (mean ± SEM). FG 5 and FG 20 indicate treatment with FG-7142 at 5 and 20 mg/kg, respectively. Tsik-egg calls are significantly increased across sessions (p < 0.05). Asterisk indicates significant difference from vehicle (p < 0.05; post hoc Dunnett’s test).

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Fig. 5. Images and presentation time course in the visual exposure test. (A) An example of predatory stimuli (wild cat). (B) An example of neutral stimuli (mountains). (C) Illustration for a sequence of stimulus presentations. The image was presented at the center of the screen for 1 min (left), expanded by 300% per 5 s repetitively for 30 s (middle), and then the expanded image was presented for 30 s (right). The presentation was followed by an inter-stimulus interval (10 min and 4 min for non-picture and picture presentation session, respectively).

by the linearly accumulated number of calls during the session (thin curve in Fig. 7A). In contrast, tsik calls were regularly observed within picture presentations but occasionally during the intervals (thick curve in Fig. 7A). Especially, predatory stimuli increased the number of tsik calls (repeated measures ANOVA F(2,12) = 4.47, p < 0.05, post hoc test by Games-Howell method p < 0.05; Fig. 7B). Tsik-egg calls were consistently observed during the pre-stimulus period, as well as during predatory and non-predatory picture presentations (F(2,12) = 2.51, p > 0.05; Fig. 7B). Other vocalizations, phee or egg calls, were not significantly different across sessions (one-way repeated ANOVA, phee, F(2,10) = 2.26; egg, F(2,10) = 4.10; p > 0.05, Fig. 6). Among distinctive behaviors, sway was increased in the picture presentation session (Friedman test, df = 2, 2 = 10.33, p < 0.05; Post hoc Steel-Dwass test, picture stimuli vs. baseline, p < 0.05, Fig. 6). There were no significant differences in motor activities, groom or gnaw across sessions (motor activity, F(2,10) = 3.51; groom, F(2,10) = 3.48; gnaw, F(2,10) = 0.45; p > 0.05, Fig. 7). In the picture presentation session, the subjects tended to be away from the display while the stimulus was presented (repeated measures ANOVA, F(2,12) = 27.91, p < 0.05; Post hoc Games-Howell, pre-stimulus vs. neutral, pre-stimulus vs. predator, p < 0.05; neutral vs. predator, p > 0.05, Fig. 7C).

4. Discussion In this study, we examined vocal and behavioral responses under anxiety- or fear-provoking conditions to find vocal indices reflecting each emotion in common marmosets. We identified a multisyllabic call, tsik-egg, in the common marmoset. We consider

tsik-egg as a distinctive vocalization pattern for the following two reasons: (1) This sequence of vocalization was frequent in all subjects (Table 2). (2) Silent intervals between the two types of elements in tsik-egg were absent or short enough to allow the call to be considered a single vocal unit (Fig. 1B). Tsik-egg has been reported previously as a sequential call emitted in several contexts (e.g., foraging, vigilance and watching a conspecific group) [11,12,24]. In these reports, however, tsik-egg appeared to be recognized as one variation of tsik calls rather than a distinct vocal pattern. The exception was a recent study that distinguished tsikegg from tsik to dissociate behavioral states during human intruder test [25,26]. Based on our clear definition of the multisyllabic tsikegg call, we found that marmosets emitted tsik and tsik-egg calls differentially depending on fear- and anxiety-related experimental conditions.

4.1. Vocalizations under anxiety-provoking conditions Social separation has often been used as one of the experimental conditions that provoke anxiety in non-human primates, where the absence of social members and exposure to unfamiliar environments may lead to anticipated threats [27,28]. The anticipated threat level was assumed to be highest in the first session because the subjects did not know whether they could return to their home cage safely until the session ended. It was then likely to become lower as the subjects were repetitively exposed to the same situation. We found that tsik-egg calls were frequently vocalized, especially in the first session, and were less frequent as the sessions progressed (Fig. 2). This suggests that vocalization of tsik-egg

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Fig. 6. Vocalization and behavior in the visual exposure test. Total number of vocalizations, distinctive behaviors and motor activity counts (mean ± SEM) in the 3rd isolation session (baseline), non-picture presentation (Non-pic) and picture presentation sessions (Picture) are shown. Asterisks indicate significant differences (p < 0.05; post hoc Steel-Dwass test,).

calls can be a behavioral/emotional response to anxiety-provoking feature in our isolated environment. In contrast, other vocalizations, phee and egg calls, did not differ across the sessions (Fig. 2). Since phee calls are considered a contact call [11–13], it would be vocalized to find separated members. This is consistent with previous studies in which frequent phee calls were observed under separation from social members in marmosets [14,15]. Motor activity was also highest in the first session of isolation, then becoming lower in the following sessions (Fig. 2). Decreasing motor activity of marmosets during habituation to an isolated environment was also reported previously [29]. In our study, reduction of both motor activity and tsik-egg calls reflect habituation to an unfamiliar isolated environment. The relative reduction patterns of tsik-egg calls and motor activity were consistent across subjects, and demonstrated faster reduction of tsik-egg calls over the habituation (Fig. 3). This suggests that sensitivity of tsik-egg calls to anxiety-provoking features (i.e., unfamiliarity) was higher than that of motor activity in our isolation test. The possibility that tsik-egg calls reflect anxiety was further tested in the pharmacological test, in which the subjects were

injected with the anxiogenic drug FG-7142 and were isolated in the environment to which they had already become habituated. FG-7142 is a partial inverse agonist at the benzodiazepine allosteric site of the GABAA receptor and changes neural signaling in multiple functional circuits related to anxiety [18,30]. Injection of FG-7142 induces anxiety-related physiological responses (e.g., heart rate and blood pressure increases or plasma cortisol level elevation) [18,31] as well as anxiety-related behavioral changes (e.g., elevated plus maze test of rat [18], specific vocalizations in social separation of rat pups [32] and squirrel monkeys [27]). We found that tsik-egg calls were specifically increased by the treatments whereas other call types and behavioral patterns were not different among treatments (Fig. 4). A previous study in the marmoset demonstrated that administration of FG-7142 at a dose of 5- to 20-mg/kg increased anxious behavior as indicated by the reduction of time spent in the front part of the cage under unspecified threatening condition [19]. These observations are consistent with the interpretation that increasing tsik-egg calls reflect anxiety provoked by FG-7142. Moreover, pharmacological-induced tsik-egg calls were also dosedependent; low-dose FG-7142 (5 mg/kg) did not increase tsik-egg

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Fig. 7. Tsik-egg and tsik vocalizations and time spent away from the display during picture presentation. (A) Cumulative distributions of tsik-egg (thin curve) and tsik (thick curve) against time from the onset of the first non-predatory stimulus are shown. Gray indicates the periods during presentation of neutral (N) or predatory stimuli (P). (B) The number of tsik-egg and tsik calls (mean ± SEM) in pre-stimulus period (Pre; preceding. 2-min period of the first presentation) and during presentation of neural or predatory stimuli (average of two presentations). (C) The duration spent away from the display in pre-stimulus and during presentations. Asterisks indicate significant difference (p < 0.05; post hoc Games-Howell test).

significantly (Fig. 4). Again, this is consistent with the previous observation in marmosets that dose-dependent change of anxietyrelated behavior was induced by FG-7142 treatment [19]. A limitation of our study was the potential influence of the order across three examinations. For example, we could not exclude the possibility that the behavioral effect of FG-7142 would reflect subjects’ previous experience in the testing environments. If this were the case, the results would be different when the visual presentation test was performed in advance of the pharmacological test. Taken together, tsik-egg calls were specifically induced under isolation in unfamiliar situations and with anxiogenic drug

treatment, both of which are anxiety-provoking conditions. The relative frequency of tsik-egg calls may reflect the intensity of anxiety within the subject. 4.2. Fear-provoking context with presentation of predatory stimuli According to the clinical dissociation of fear and anxiety, confronting a specific threat is considered as a fear-provoking situation. When marmosets were exposed to picture images, they vocalized tsik-egg calls, and also tsik calls that were rarely emitted

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in anxiety-related situations (Fig. 6). Especially, predatory photo stimuli significantly increased tsik calls (Fig. 7). These observations are consistent with the previous studies reporting that tsik calls (i.e., tsik-tsik calls) were vocalized when marmosets were exposed to a taxidermized predator (e.g., hawk, snake or wild cat) [11,33], but not to a non-predatory toy [21,22]. Tsik calls are regarded as mobbing and alarm calls, uttered toward conspecifics and potential predators [12,34]. Tsik calls against human confrontation, however, have been inconsistent and controversial [11,25,35]. Thus, tsik calls could be related to predatory fear but not to fear in general. In addition to tsik calls, the duration of staying away from the display was significantly increased during presentation of visual stimuli. This was also comparable with marmosets’ behavior confronting 3D predatory objects [21]. In a previous study, a 2D photograph of snake evoked marmosets’ fearful responses including tsik calls only when the same object had been previously presented [36]. In our presentation, 2D photo images were expanded on the display, which increased visual saliency with motion in depth (i.e., as if the image approached the subjects). This additional feature on visual presentation may facilitate marmosets’ perception as a fearful object. Without tsik, tsik-egg calls were emitted during non-picture image presentation and pre-picture images (Figs. 6 and 7B). In addition, the frequency of tsik-egg calls did not differ during the test sessions regardless of stimulus category, or whether a stimulus was present or absent (Figs. 6 and 7). The time-course plot shows that tsik calls were vocalized mainly during stimulus presentation, whereas the tsik-egg calls were continuously vocalized before and after the stimuli presentations (cf. Fig. 7A). These results support our idea that tsik-egg calls represent a specifically emitted anxiety-provoking condition, in this case, an unfamiliar context where something is suddenly presented in the display. In the visual presentation test, we found two vocal expressions related to negative emotional response; one is tsik-egg solo vocalization (i.e., tsik-egg without tsik) that reflects the non-predatory or anticipatory nature of presentation that potentiates anxiety. The other is mixed vocalization of tsik and tsik-egg calls that reflects a specific threatening feature of stimuli that may relate to fear (e.g., confronting predatory stimuli) rather than anxiety. Besides, our result suggests that the intensity of fear may be reflected by the frequency of tsik calls during the mixed vocalizations of tsik and tsik-egg calls. 4.3. Tsik-egg with/without tsik call as a specific index of fear and anxiety In this study we found that marmosets dissociate to vocalize tsik/tsik-egg calls under conditions related to fear/anxiety; tsik-egg solo vocalizations were emitted under anxiety-related conditions (e.g., isolation and anxiogenic drug treatment), whereas mixed vocalization of tsik-egg and tsik was emitted when the subject confronted with fear-provoking stimuli (i.e., predatory stimuli). Conversely, these vocalizations allow us to infer negative emotion in marmosets; tsik-egg solo vocalizations indicate anxiety whereas mixed vocalizations indicate fear. Although fear and anxiety are closely related and occasionally untangled [1], our finding suggests that anxiety can be segregated from fear when tsik-egg vocalization is solely emitted without tsik. Our results dissociate that additional tsik call utterance with tsik-egg call could be a borderline of negative emotions of fear/anxiety. In addition to fear/anxiety dissociation, our results also suggest that the intensity of fear/anxiety may be inferred by the relative number of tsik/tsikegg calls within the subject. Analyzing tsik-egg and tsik calls may allow us to dissociate fear/anxiety of marmosets and to understand the underlying biological mechanisms. Indeed, a recent study used tsik/tsik-egg calls as one of the behavioral markers to dissociate

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marmosets’ fear/anxiety state, and demonstrated the contribution of the ventral prefrontal cortex to controlling negative emotions [25,26]. Neurochemical analysis on marmosets confronted by taxidermized predator revealed that production of tsik-tsik call negatively correlated with the level of acetylcholine in the left hypothalamus [37]. Thus, tsik-egg/tsik vocalization analysis under contextual- or pharmacological-induced negative emotion, combined with neurophysiological and neurochemical investigation, will provide a promising avenue for addressing many intriguing questions regarding the neural mechanism of fear/anxiety in primates, including the search for novel anxiolytic drugs. Conflict of interest National Institute of Radiological Sciences holds a patent in Japan (5058312) on the use of vocalization described in this paper. H.G., A.O., T.S. and T.M. are listed as co-inventors of this patent. Other authors have no conflicts of interest to declare. Acknowledgements We thank Hajime Ishii, Yuji Nagai, Yoko Eguchi, Yuko Toyoda and Masayo Kurokawa for their technical assistance, and Yumiko Yamazaki for discussion and comments on an earlier version of the manuscript. This study was partly supported by Grant-in-Aid for Young Scientists B (25730103) to YK and Grant-in-Aid for Scientific Research on Innovative Areas (25135736) to TM (Japan Society for the Promotion of Science), and by the “Integrated Research on Neuropsychiatric Disorders” study carried out under the Strategic Research Program for Brain Sciences to TS (The Ministry of Education, Culture, Sports, Science and Technology of Japan). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bbr.2014.08.047. References [1] Öhman A. Fear and anxiety: overlaps and dissociations. In: Lewis MH-J, Barrett JM, Feldman L, editors. Handbook of emotions. New York: The Guilford Press; 2008. p. 709–29. [2] Lang PJ, Davis M, Öhman A. Fear and anxiety: animal models and human cognitive psychophysiology. J Affect Disord 2000;61:137–59. [3] McNaughton N, Corr PJ. A two-dimensional neuropsychology of defense: fear/anxiety and defensive distance. Neurosci Biobehav Rev 2004;28:285–305. [4] Öhman A, Mineka S. Fears, phobias, and preparedness: toward an evolved module of fear and fear learning. Psychol Rev 2001;108:483–522. [5] Treit D, Degroot A, Shah A. Animal models of anxiety and anxiolytic drug action. In: Kasper S, den Boer JA, Ad Sitsen JM, editors. Handbook of depression and anxiety: a biological approach. New York: Marcel Dekker, Inc; 2003. p. 681–702. [6] Passingham RE, Wise SP. The neurobiology of the prefrontal cortex: anatomy, evolutio, and the origin of insight. Oxford: Oxford University Press; 2012. [7] Roberts AC, Tomic DL, Parkinson CH, Roeling TA, Cutter DJ, Robbins TW, et al. Forebrain connectivity of the prefrontal cortex in the marmoset monkey (Callithrix jacchus): an anterograde and retrograde tract-tracing study. J Comp Neurol 2007;502:86–112. [8] Palit G, Kumar R, Chowdhury SR, Gupta MB, Saxena RC, Srimal RC, et al. A primate model of anxiety. Eur Neuropsychopharmacol 1998;8:195–201. [9] Barros M, Tomaz C. Non-human primate models for investigating fear and anxiety. Neurosci Biobehav Rev 2002;26:187–201. [10] Kalin NH, Sheltona SE. Nonhuman primate models to study anxiety, emotion regulation, and psychopathology. Ann N Y Acad Sci 2003;1008:189–200. [11] Bezerra BM, Souto A. Structure and usage of the vocal repertoire of Callithrix jacchus. Int J Primatol 2008;29:671–701. [12] Epple G. Comparative studies on vocalization in marmoset monkeys (Hapalidae). Folia Primatol (Basel) 1968;8:1–40. [13] Miller CT, Mandel K, Wang X. The communicative content of the common marmoset phee call during antiphonal calling. Am J Primatol 2010;72:974–80. [14] Yamaguchi C, Izumi A, Nakamura K. Time course of vocal modulation during isolation in common marmosets (Callithrix jacchus). Am J Primatol 2010;72:681–8. [15] Norcross JL, Newman JD. Effects of separation and novelty on distress vocalizations and cortisol in the common marmoset (Callithrix jacchus). Am J Primatol 1999;47:209–22.

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