*Article* **Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization**

**Markus Fendt 1,2,\* , Claudia Paulina Gonzalez-Guerrero 1,3 and Evelyn Kahl <sup>1</sup>**


**\*** Correspondence: markus.fendt@med.ovgu.de

**Abstract:** Rats can acquire fear by observing conspecifics that express fear in the presence of conditioned fear stimuli. This process is called observational fear learning and is based on the social transmission of the demonstrator rat's emotion and the induction of an empathy-like or anxiety state in the observer. The aim of the present study was to investigate the role of trait anxiety and ultrasonic vocalization in observational fear learning. Two experiments with male Wistar rats were performed. In the first experiment, trait anxiety was assessed in a light–dark box test before the rats were submitted to the observational fear learning procedure. In the second experiment, ultrasonic vocalization was recorded throughout the whole observational fear learning procedure, and 22 kHz and 50 kHz calls were analyzed. The results of our study show that trait anxiety differently affects direct fear learning and observational fear learning. Direct fear learning was more pronounced with higher trait anxiety, while observational fear learning was the best with a medium-level of trait anxiety. There were no indications in the present study that ultrasonic vocalization, especially emission of 22 kHz calls, but also 50 kHz calls, are critical for observational fear learning.

**Keywords:** anxiety; observational fear learning; rat; ultrasonic vocalization

### **1. Introduction**

In potentially threatening situations, rats—as other animals and humans—express a variety of defensive behavior that ultimately helps to survive such situations [1]. The choice of the defensive behavior is dependent on several variables, such as the intensity and the proximity of the threatening stimulus but also on the local conditions, e.g., whether there is a way to escape or a place to hide [2]. A defensive attack may occur if the threat is too close, whereas freezing, a cessation of movements [3], is expressed if the threat is not too close, but escape is not possible. After the initial defensive response, e.g., hiding or freezing, many rats emit ultrasonic vocalizations [1,4,5]. These calls are characterized by a peak frequency around 22 kHz, a narrow bandwidth, and call durations around one second [6,7]. The function of these 22 kHz calls is not completely understood. It is well recognized that they reflect the negative affective state of the emitting rat [8]. Thereby, they serve as species-specific aversive communication signals, i.e., they are recognized by conspecifics and have the capability to change their emotional state [9] or even to warn them about a threat [4]. However, only modest effects are reported in experiments using playbacks of natural 22 kHz calls [10–13], eventually questioning the direct behavioral effects of 22 kHz calls.

Of note, threatening situations are also associated with very robust and rapid learning processes [14,15]. Both contextual and discrete stimuli that are present during the threatening situation can be associated with the threat, a process called threat or fearconditioning [16]. Laboratory protocols of fear-conditioning are widely used in neuroscience research to investigate the neural basis of fear/anxiety learning [17–20]. There

**Citation:** Fendt, M.; Gonzalez-Guerrero, C.P.; Kahl, E. Observational Fear Learning in Rats: Role of Trait Anxiety and Ultrasonic Vocalization. *Brain Sci.* **2021**, *11*, 423. https:// doi.org/10.3390/brainsci11040423

Academic Editor: Stefan Brudzynski

Received: 25 February 2021 Accepted: 23 March 2021 Published: 26 March 2021

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is a high translational relevance of this research field since fear-conditioning itself, its specificity, extinction, and inhibition are affected in patients with anxiety disorder [21–23]. Some of these changes are not only seen in clinical populations but also in human subjects with high but not yet pathological trait anxiety [24–26]. Of note, also rats with high trait anxiety showed exaggerated fear learning, a less specific fear memory, and impaired fear extinction [27,28].

Several studies have described observational fear learning, also called "social fear learning", "vicarious fear learning", or "fear-conditioning by proxy", in rats [29–32]. In these studies, rats learned fear by observation, i.e., they joined an already fear-conditioned conspecific (the "demonstrator") in a retention session on conditioned fear and could observe its fear responses to the conditioned context and/or discrete stimulus (that are neutral for the observer rat). To test whether fear was acquired by observation, a further retention test was performed one day later in which the observer rat alone was exposed to the conditioned context or stimulus [33]. Observational fear learning has been successful in male and female cage-mates [30,32], was more pronounced in related and submissive rats [31,32] and less pronounced in dominant rats [31]. Furthermore, the amount of social interaction and ultrasonic vocalization during the observational fear learning session was positively correlated with the observers' fear response on the following day [30–32]. Of note, only 22 kHz calls were analyzed in this experiment [31]. Of potential interest would also be the so-called "50 kHz calls" that are typically emitted during social contacts or other appetitive situations [34–36]. Since social interaction was positively correlated with the observers' fear learning [30–32] and 50 kHz calls are typically increased during social contacts, the amount of 50 kHz calls should also positively correlate with the observers' fear learning.

The role of trait anxiety in observational fear learning has not been investigated in rats so far. There are hints for a possible role of trait anxiety from a human study. In this study, participants had to observe mock panic attacks while their fear responses were measured [37]. "Anxiety sensitivity" was correlated with self-reported fear levels but not physiological arousal during this observational fear challenge procedure. However, potential learning processes during the challenge were not investigated in this study.

In the present study, the role of trait anxiety and ultrasonic vocalization on observational fear learning in male Wistar rats was investigated. Two experiments using a published protocol [33] were performed: In the first experiment, trait anxiety was assessed in the light–dark box test before the rats were submitted to the observational fear learning procedure. In the second experiment, ultrasonic vocalization was recorded throughout the whole observational fear learning procedure and 22 kHz and 50 kHz calls were analyzed. Our working hypotheses were that (1) rats with higher trait anxiety show more pronounced observational fear learning and that (2) higher emission of both 22 kHz and 50 kHz calls in the observational fear learning session leads to enhanced fear memory in the observer rats.

#### **2. Materials and Methods**

#### *2.1. Animals and Housing*

Testing was carried out using 102 experimentally naive male Wistar rats aged 8–12 weeks. They were bred and reared at the local animal facility (original breeding stock: Taconic, Silkeborg, Denmark). They were housed in groups of six animals in standard Macrolon Type IV cages (58 cm × 33 cm × 20 cm) with water and standard lab chow (ssniff, Soest, Germany) available ad libitum. Cages were kept in temperature- and humidity-controlled rooms (22 ± 2 ◦C, 55 ± 10%) with a 12:12 h light/dark cycle (lights on at 6:00 am). All behavioral tests were conducted during the light phase between 8:00 am and 3:00 pm. Our study was carried out in accordance with international guidelines for the use of animals in experiments (2010/63/EU) and was approved by the local ethical committee (Landesverwaltungsamt Sachsen-Anhalt, Az. 42502-2-1587 UniMD).

#### *2.2. Apparatus*

The light–dark box system consisted of four identical boxes (49.5 cm × 49.5 × 41.5 cm; TSE Systems, Bad Homburg, Germany) divided into two compartments of the same size. The light compartment (135–310 lux) had transparent acryl glass walls, while the dark compartment (0.2–1.5 lux) had black walls. The two compartments were connected by an 8 cm × 6 cm opening. Position and movements of the rats were detected by animal detection infrared sensor frames (detector distance: 14 mm) and analyzed by TSE PhenoMaster software (version 4.9.4).

The fear-conditioning system consisted of four identical transparent acryl glass box (46 cm × 46 cm × 32 cm) located in sound-attenuating chambers (70 cm × 80 cm × 70 cm; TSE Systems, Bad Homburg, Germany). The chambers were equipped with loudspeakers for the acoustic stimuli (background noise of 55 dB SPL and the tone stimuli for fearconditioning), light sources (continuous illumination of ca. 10 lux), ventilation fans, and video cameras (for monitoring and videotaping) mounted in the ceiling of the chambers. The floor of the boxes consisted of removable stainless steel grids (bars: 4 mm diameter, distance: 9 mm), which were connected to a shock unit and able to deliver foot shocks. Delivery of the different stimuli was controlled by TSE fear-conditioning software (version 09.10). This software also analyzed the position and movements of the rats, recorded by infrared animal detection sensor frames (detector distance: 14 mm). Freezing behavior was defined as no infrared beam crosses for more than 1 s. This automated measurement of freezing was previously validated by demonstrating a high correlation with observer scoring of freezing [13,38].

Recording and analyses of ultrasonic vocalization were performed with the Ultra-SoundGate system (Avisoft Bioacoustics, Berlin, Germany). For recording, an ultrasound condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies of 15–180 kHz (flat frequency response between 25 and 140 kHz; ±6 dB) mounted on one corner of the box was used, which was connected to a laptop via a USB audio device (UltraSoundGate 116H). Acoustic data were recorded by Avisoft Recorder USGH software (version 5.2) using a sampling rate of 250,000 Hz in 16-bit format and a recording range of 0–125 kHz.
