![Total numbers of response calls emitted (A) for playback of 50-kHz as the first stimulus [95.25 ± 33.62 (mean ± SEM)] or as the second stimulus (152 ± 40.07) of WI rats. Latencies after stimulus onset (B): 50-kHz first: 16.04 ± 5.67; 50-kHz second: 24.67 ± 16.19.](https://www.researchgate.net/profile/Annuska-Berz/publication/357861457/figure/fig1/AS:11431281153690876@1682523624118/Total-numbers-of-response-calls-emitted-A-for-playback-of-50-kHz-as-the-first-stimulus_Q320.jpg)
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fnbeh-15-812142 January 10, 2022 Time: 14:26 # 1
ORIGINAL RESEARCH
published: 14 January 2022
doi: 10.3389/fnbeh.2021.812142
Edited by:
Susanna Pietropaolo,
UMR 5287 Institut de Neurosciences
Cognitives et Intégratives d’Aquitaine
(INCIA), France
Reviewed by:
Stefan Brudzynski,
Brock University, Canada
Sergio Marcello Pellis,
University of Lethbridge, Canada
*Correspondence:
Annuska C. Berz
Annuska.berz@staff.uni-marburg.de
†
†
†ORCID:
Annuska C. Berz
orcid.org/0000-0002-5522-0188
Markus Wöhr
orcid.org/0000-0001-6986-5684
Rainer K. W. Schwarting
orcid.org/0000-0002-4686-3974
‡These authors share last authorship
Specialty section:
This article was submitted to
Individual and Social Behaviors,
a section of the journal
Frontiers in Behavioral Neuroscience
Received: 09 November 2021
Accepted: 24 December 2021
Published: 14 January 2022
Citation:
Berz AC, Wöhr M and
Schwarting RKW (2022) Response
Calls Evoked by Playback of Natural
50-kHz Ultrasonic Vocalizations
in Rats.
Front. Behav. Neurosci. 15:812142.
doi: 10.3389/fnbeh.2021.812142
Response Calls Evoked by Playback
of Natural 50-kHz Ultrasonic
Vocalizations in Rats
Annuska C. Berz1,2*†, Markus Wöhr1,2,3,4†‡ and Rainer K. W. Schwarting1,2†‡
1Behavioral Neuroscience, Experimental and Biological Psychology, Faculty of Psychology, Philipps-University Marburg,
Marburg, Germany, 2Center for Mind, Brain and Behavior, Philipps-University Marburg, Marburg, Germany, 3Research Unit
Brain and Cognition, Laboratory of Biological Psychology, Social and Affective Neuroscience Research Group, Faculty
of Psychology and Educational Sciences, KU Leuven, Leuven, Belgium, 4Leuven Brain Institute, KU Leuven, Leuven,
Belgium
Rats are highly social animals known to communicate with ultrasonic vocalizations
(USV) of different frequencies. Calls around 50 kHz are thought to represent a positive
affective state, whereas calls around 22 kHz are believed to serve as alarm or distress
calls. During playback of natural 50-kHz USV, rats show a reliable and strong social
approach response toward the sound source. While this response has been studied
in great detail in numerous publications, little is known about the emission of USV
in response to natural 50-kHz USV playback. To close this gap, we capitalized on
three data sets previously obtained and analyzed USV evoked by natural 50-kHz USV
playback in male juvenile rats. We compared different rat stocks, namely Wistar (WI)
and Sprague-Dawley (SD) and investigated the pharmacological treatment with the
dopaminergic D2 receptor antagonist haloperidol. These response calls were found
to vary broadly inter-individually in numbers, mean peak frequencies, durations and
frequency modulations. Despite the large variability, the results showed no major
differences between experimental conditions regarding call likelihood or call parameters,
representing a robust phenomenon. However, most response calls had clearly lower
frequencies and were longer than typical 50-kHz calls, i.e., around 30 kHz and lasting
generally around 0.3 s. These calls resemble aversive 22-kHz USV of adult rats but
were of higher frequencies and shorter durations. Moreover, blockade of dopamine D2
receptors did not substantially affect the emission of response calls suggesting that
they are not dependent on the D2 receptor function. Taken together, this study provides
a detailed analysis of response calls toward playback of 50-kHz USV in juvenile WI
and SD rats. This includes calls representing 50-kHz USV, but mostly calls with lower
frequencies that are not clearly categorizable within the so far known two main groups of
USV in adult rats. We discuss the possible functions of these response calls addressing
their communicative functions like contact or appeasing calls, and whether they may
reflect a state of frustration. In future studies, response calls might also serve as a new
read-out in rat models for neuropsychiatric disorders, where acoustic communication is
impaired, such as autism spectrum disorder.
Keywords: ultrasonic vocalizations, animal communication, playback, stock, strain, haloperidol, Wistar, Sprague-
Dawley
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Berz et al. Response Calls Toward 50-kHz USV
INTRODUCTION
Acoustic communication among conspecifics is an important
aspect of the social life of many species and often essential
for maintaining stable social structures. A characteristic feature
of acoustic communication in several species is its reciprocal
nature where a signal emitted by the sender frequently
evokes the emission of a response signal in the receiver
(Seyfarth and Cheney, 2003).
Many rodent species communicate through so-called
ultrasonic vocalizations (USV), i.e., within frequencies not
audible for humans (Brudzynski, 2010). In juvenile and adult
rats, two main types of vocalizations are typically distinguished
(Brudzynski, 2013a;Wöhr and Schwarting, 2013). Vocalizations
with frequencies around 22 kHz are referred to as aversive or
distress calls, presumably representing a negative affective state
(Blanchard et al., 1991;Fendt et al., 2018). Vocalizations with
frequencies around 50 kHz are thought to represent a positive
affective state usually emitted during appetitive situations like
play or mating (Knutson et al., 1998;Panksepp, 2005). These
appetitive calls are typically characterized by frequencies between
35 and 80 kHz and durations in a range of 10–150 ms (Burgdorf
et al., 2008;Wöhr et al., 2008;Takahashi et al., 2010). Often,
such 50-kHz USV are categorized and the call categories flat,
step, trill, and mixed are commonly differentiated (Kisko et al.,
2018). Aversive 22-kHz USV, in contrast, have been defined
between frequencies of 18 and 32 kHz (Brudzynski, 2001)
and within this frequency range, short (<300 ms) and long
(>300 ms) calls were identified (Brudzynski et al., 1993). Long
22-kHz calls were found to be emitted during situations of
external danger, such as during the presence of a predator or
during predator odor exposure, and are usually associated with
freezing behavior (Blanchard et al., 1991;Fendt et al., 2018;
Simmons et al., 2018). Short 22-kHz USV, however, are much
more ambiguous and their function has not been identified
yet (Brudzynski, 2021). It was suggested that short 22-kHz
USV represent internal distress without external influence, like
frustration (Taylor et al., 2019). In addition, they were repeatedly
reported to occur during drug withdrawal (Ma et al., 2010;
Simmons et al., 2018).
The communicative functions of 22- and 50-kHz USV can be
studied by means of playback experiments (Seffer et al., 2014) and
it was shown that they elicit distinct behavioral responses pattern
in the receiver (Wöhr et al., 2016). Playback of natural 22-kHz
USV usually induces a defensive response, including avoidance
behavior and behavioral inhibition (Brudzynski and Chiu, 1995;
Fendt et al., 2018). Playback of natural 50-kHz USV, in contrast,
evokes social approach behavior toward the sound source (Wöhr
and Schwarting, 2007). At the physiological level, playback of 22-
and 50-kHz USV entail to distinct alterations. While playback
of 22-kHz leads to a decrease in heart rate during behavioral
inhibition, heart rate is increased during social approach behavior
in response to playback of 50-kHz USV (Olszy´
nski et al., 2020).
Likewise, distinct brain activation patterns are observed. Playback
of 22-kHz USV induces increased activity in the amygdala
(Sadananda et al., 2008;Parsana et al., 2012), whereas playback
of 50-kHz USV results in an activation of the nucleus accumbens
(Sadananda et al., 2008), where it causes a phasic release of
dopamine (Willuhn et al., 2014).
At the behavioral level, the social approach response toward
50-kHz USV playback can be accompanied by the emission of
response calls (Wöhr and Schwarting, 2007, 2009;Willadsen
et al., 2014;Willuhn et al., 2014;Engelhardt et al., 2017, 2018;
Berg et al., 2018, 2021;Kisko et al., 2020;Olszy´
nski et al.,
2020, 2021). Although echoing the reciprocal nature of acoustic
communication and repeatedly observed in studies applying the
50-kHz USV playback paradigm, still little is known about such
response calls. In previous studies, response calls toward 50-kHz
USV were observed in males and females (Berg et al., 2018, 2021),
albeit the emission of calls in response to 50-kHz USV playback
was found to be more prominent in males than females in one
study (Kisko et al., 2020). A developmental study further suggests
that age is another relevant factor, with juvenile rats emitting
more response calls than adult rats (Wöhr and Schwarting, 2009).
Finally, prior experiences (Olszy´
nski et al., 2021) and inter-
individual differences (Engelhardt et al., 2018) were also reported
to play a role. However, the function of response calls remains
elusive, which is why we wanted to shed light onto the meaning
and the importance of response calls in social situations like the
50-kHz USV playback.
To close this gap, we capitalized on a previously obtained
large data set and analyzed USV evoked by natural 50-kHz USV
playback in male juvenile rats (Berz et al., 2021). In our previous
study, we showed, amongst others, that the social approach
response toward 50-kHz calls is a stable phenomenon that occurs
in Wistar (WI) and Sprague-Dawley (SD) rats and that it can be
modulated by administration of the dopaminergic D2 receptor
antagonist haloperidol (Halo; Berz et al., 2021). Here, we present
three new data sets from these previous experiments. Data set
1 was comprised of WI rats exposed to 50-kHz USV playback.
We analyzed it in an initial attempt to better understand the
emission of response calls and to test whether response calls occur
specifically in reaction toward 50-kHz USV but not noise and
whether stimulus order of 50-kHz USV and noise plays a role.
Data set 2 consisted of WI and SD rats and their response calls
were compared to see whether there was a difference between the
stocks. In the final data set 3, rats received either Halo or saline
(Sal) to investigate whether Halo treatment not only affects social
approach behavior but also the emission of response calls toward
50-kHz USV playback. Our comprehensive analysis approach
included a detailed investigation of the temporal emission pattern
and an examination of acoustic features, focusing on numbers
of calls, latencies to start calling, mean peak frequencies, call
durations, and frequency modulations.
MATERIALS AND METHODS
Animals and Housing
In total, 108 experimentally naïve juvenile male rats around 5–
7 weeks of age (Charles River Laboratories, Sulzfeld, Germany)
were analyzed. The sample consisted of 90 Wistar (WI) wildtype
rats and 18 Sprague-Dawley (SD) wildtype rats. The animals were
kept in a vivarium with a 12-hour light/dark cycle with lights on
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Berz et al. Response Calls Toward 50-kHz USV
at 7 am and 32–50% humidity. They were housed in groups of
five to six rats in polycarbonate cages (macrolon type IV, size
380 ×200 ×590 mm with high steel covers) where food and
water were provided ad libitum. After arrival from the breeder,
the animals had seven days to acclimate to the vivarium, followed
by a standardized protocol of handling for three consecutive days,
each day for 5 min. The procedures had been approved by the
ethical committee of the local government (Regierungspräsidium
Gießen, Germany, TVA Nr. 6 35-2018).
Overview
Response calls were analyzed in three data sets. These sets were
obtained as part of a recently published study focusing on the
habituation of the social approach response to repeated playback
of 50-kHz USV (Berz et al., 2021). In this previous study,
rats were exposed twice to playback of 50-kHz USV and their
behavioral response was quantified, i.e., locomotor activity and
approach behavior. Here, we now analyzed response calls evoked
by playback of 50-kHz USV that were also recorded in this study.
We focused on the emission of response calls during the first
playback exposure because preliminary data indicate that call
emission decreases with repeated playback presentations similar
to social approach behavior (Berz et al., 2021). In the first data
set, we analyzed response calls in WI rats (N= 24) and tested
whether their emission occurs specifically during playback of 50-
kHz USV but not noise and whether their emission depends on
stimulus order. Rats were weighing 144.25 ±1.88 g (range 128.5–
164.5 g). In the second data set, we compared the production
of response calls between WI rats (N= 18) to that of SD rats
(N= 18). Rats were weighing 163.47 ±2.85 g (range 138.5–205 g).
In the third data set, we studied the role of the dopaminergic
system in regulating the emission of response calls and compared
response calls emitted by WI rats systemically treated with the
dopaminergic D2 receptor antagonist Halo (N= 24) and saline
treated controls (N= 24). Rats were weighing 189.57 ±2.95 g
(range 147.5–233 g).
Drug Treatment
In the third data set, rats received the dopaminergic D2
receptor antagonist Halo (0.5 mg/kg; Haldol, Janssen, Belgium)
or saline (Sal, 0.9% NaCl solution, Braun, Germany). The ip
injection took place 60 min before the start of the playback
experiment and during the time between the injection and the
playback experiment, rats were kept singly (in a small cage with
bedding and water ad libitum) in a dark room (according to
Tonelli et al., 2017).
50-kHz Ultrasonic Vocalizations
Playback: Setup
As experimental setups, an eight-arm radial maze (data sets 1
and 2) and a squared platform (data set 3), each elevated 52 cm
above the ground, were employed. On two opposite sides of
the given apparatus, an ultrasonic speaker (ScanSpeak, Avisoft
Bioacoustics, Berlin, Germany) and an ultrasonic condenser
microphone (CM16, Avisoft Bioacoustics) were placed 20 cm
away from the end of the arm or platform. Only one of the
speakers was active, whereas the other one served as a visual
control. Experiments were conducted under red light (∼10 lux).
50-kHz Ultrasonic Vocalizations
Playback: Acoustic Stimuli
We presented two types of acoustic stimuli: (A) 50-kHz USV
recorded from an adult male WI rat (ca. 350 g) during exploration
of a cage containing scents from a recently removed cage
mate (for details see Wöhr et al., 2008). This recording was
composed of a sequence of 3.5 s with 13 different 50-kHz calls
(total calling time 0.9 s) presented in a loop (for details see
Wöhr and Schwarting, 2007). The peak amplitude was 70 dB
(measured from a distance of 40 cm), being in the typical range
of 50-kHz USV (Kisko et al., 2020). (B) Time- and amplitude-
matched noise was generated with SASLab Pro (Version 4.2,
Avisoft Bioacoustics) by replacing each 50-kHz call by noise with
matching duration and amplitude modulation. Accordingly, each
noise playback series had the same temporal pattern and all call
features were identical, except that the sound energy was not in
a certain frequency range as in the natural 50-kHz USV playback
(for details see Wöhr and Schwarting, 2012). The acoustic stimuli
were presented via an ultrasonic speaker (ScanSpeak, Avisoft
Biosacoustics) with a frequency range of 1–120 kHz and a flat
frequency response (±12 dB) between 15 and 80 kHz. Sounds
were played via a portable ultrasonic power amplifier with a
frequency range of 1–125 kHz (Avisoft Bioacoustics) and via an
external sound card with a sampling rate of 192 kHz (Fire Wire
Audio Capture FA-101, Edirol, London, United Kingdom).
50-kHz Ultrasonic Vocalizations
Playback: Paradigm
At the beginning of the playback experiment, rats were placed
individually in the center of the eight-arm radial maze (data sets
1 and 2) or the squared platform (data set 3). After an initial
habituation period of 15 min, the first playback presentation of
5 min duration commenced. The second playback presentation
of 5 min duration followed after an inter-stimulus interval of
10 min. Acoustic stimuli (i.e., 50-kHz USV, noise) were presented
in a counterbalanced manner. The trial ended with a post-
stimulus interval of 10 min. The whole paradigm lasted 45 min.
Recording and Analysis of Response
Calls
For recording response calls emitted by the given experimental
rat, two ultrasonic microphones were placed symmetrically on
two sides of the maze (data sets 1 and 2) or the platform
(data set 3) next to the speakers. They were connected via an
UltraSoundGate 416H USB audio device (Avisoft Bioacoustics) to
a computer, where acoustic data were recorded with a sampling
rate of 250 kHz (16-bit format; recording range 0–125 kHz)
using RECORDER USGH (Avisoft Bioacoustics). For acoustical
analysis, recordings were transferred to DeepSqueak (version
2.6.1, Windows standalone), a deep learning-based system for
detection and analysis of USV (Coffey et al., 2019). Recorded
files were converted into high-resolution spectrograms and were
analyzed using the pre-trained automated “short rat call network
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Berz et al. Response Calls Toward 50-kHz USV
V2.” The settings for call detection were “high recall,” with
an overlap of 0.001 s. This setting was chosen because it
minimizes the possibility that a call is missed, albeit at the cost
of false positives by including noise. Therefore, a custom trained
network for denoising was applied afterward. The detected
events were then transferred into the DeepSqueak Screener
(Fork on GitHub by L. Lara-Valderrábano and R. Ciszek:
10.5281/zenodo.3690137),1where the files were reviewed and
denoised again manually by an experienced observer accepting
(response calls) or rejecting (noise or playback calls) events.
All response calls, irrespective of frequencies and durations,
were counted. For later analysis, response calls during the
5 min before, during, and after the playback presentations
(50-kHz USV or noise) were taken into account (referred to
as stimulus phase). Outside this time window, calls occurred
rarely. Acoustic features, i.e., call duration, peak frequency, and
frequency modulation (difference between highest and lowest
frequency), were defined and analyzed as described previously
(Kisko et al., 2018). For classifying response calls, we applied
previously established frequency thresholds (Brudzynski, 2001).
Calls with frequencies higher than 32 kHz were classified as
50-kHz USV and calls below 32 kHz were defined as 22-kHz USV.
Recording and Analysis of Overt
Behavior
As pointed out above, the behavioral data (locomotion, approach)
were part of a recently published study focusing on the
habituation of the social approach response to playback of 50-
kHz USV (Berz et al., 2021). Here, we reconsidered these data
in the context of the new data on response calls in order to
address the question whether locomotor activity and approach
behavior evoked by playback of 50-kHz USV are associated
with the emission of response calls. Briefly, overt behavior was
recorded and analyzed using EthoVision XT (Version 13, Noldus,
The Netherlands). Locomotion was measured by the distance
traveled. For quantifying approach behavior on the maze (data
sets 1 and 2), the numbers of entries into the three arms proximal
and distal to the active speaker and the time spent thereon were
measured. For quantifying approach behavior on the platform
(data set 3), it was virtually divided into 25 equal quadrants, with
the six quadrants close to the active speaker serving as proximal
zone, while the six quadrants close to the inactive speaker were
defined as distal zones. Entries and time spent in these zones were
measured (for details see Berz et al., 2021).
Statistical Analysis
Analyses of variance (ANOVAs) for repeated measurements
were calculated with the between-subject factors playback
order (50-kHz USV first vs. second), stocks (WI vs. SD), or
drug treatment (Halo vs. Sal), and the within-subject factors
stimulus phase (5 min before, during, or after playback) and
playback stimulus (50-kHz USV or time- and amplitude-
matched noise). This was followed by two-tailed t-tests for
comparing individual experimental groups. The ratio between
calling and non-calling rats was evaluated by a χ2-test (calculated
1https://github.com/UEFepilepsyAIVI/DeepSqueak.git
using https://www.socscistatistics.com/tests/chisquare2/default2.
aspx). Approach behavior was quantified by subtracting the times
spent on proximal arms (or in proximal zones) before the 5 min
of 50-kHz USV playback from the time spent there during
the 5 min of playback. The same was done with the entries
into proximal arms or zones. Pearson correlation coefficients
(bivariate) were calculated for the correlation between numbers
of emitted calls and approach behavior. For testing a possible
correlation with locomotor behavior, locomotion (distance
traveled in cm) during the 5 min before playback were subtracted
from that during the 5 min during playback. This number
was then correlated with the numbers of response calls emitted
using the Pearson correlation coefficient. For general locomotor
activity correlations, the distance traveled during the initial 15-
min habituation period were taken into account. All t-tests,
ANOVAs, and correlations were calculated with IBM SPSS
Statistics (version 25). Graphs were made using GraphPad
Prism (version 8). Data are represented as means ±SEM
(standard error of mean). A p-value of <0.050 was considered
statistically significant.
RESULTS
Data Set 1: Response Calls
Call Numbers and Latencies
Playback of 50-kHz USV induced response calls in the majority
of WI rats. Among the 24 rats of data set 1, 23 of them
emitted response calls. The mean number of response calls was
123.5 ±26.21, ranging between 0 and 414 calls in total per rat
(Figure 1A). During the 5 min before 50-kHz USV playback,
no calls were emitted. The occurrence of response calls was not
dependent on whether 50-kHz USV were presented as the first or
the second stimulus (t22 = 0.82, p= 0.21). Importantly, high levels
of response calls were emitted specifically in reaction toward
playback of 50-kHz USV but not noise, irrespective of whether
50-kHz USV were presented as the first (t11 = 2.8, p= 0.017) or the
second stimulus (t11 = 4.013, p= 0.002; Figure 1A). The latency
to start calling after onset of 50-kHz USV was 20.17 ±88.17 s
(Figure 1B). Stimulus order did not affect call latency (t21 = 0.52,
p= 0.61). Therefore, we abstained from differentially considering
stimulus order further in all following analyses.
Data Set 2: Stock Differences
Call Numbers and Latencies
Consistent with data set 1, response calls were seen in the majority
of rats in data set 2 focusing on possible stock differences between
WI and SD rats. From the two different stocks, 10 out of the 18 WI
rats emitted calls in response to 50-kHz USV playback and 12 out
of 18 SD rats did. The ratios between calling and non-calling rats
did not differ between stocks (x21,36 = 0.468, p= 0.49). Likewise,
the mean numbers of response calls (Figure 2A;t34 = 0.032,
p= 0.975; WI: 44.39 ±17.81; SD: 45.17 ±16.45) as well as the
latencies to start calling (Figure 2B;t20 = 0.547, p= 0.590; WI:
50.56 ±41.16 s; SD: 29.88 ±4.93 s) did not differ between WI and
SD. In both stocks, high levels of response calls were exclusively
evoked by playback of 50-kHz USV, while response calls rarely
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Berz et al. Response Calls Toward 50-kHz USV
FIGURE 1 | Total numbers of response calls emitted (A) for playback of
50-kHz as the first stimulus [95.25 ±33.62 (mean ±SEM)] or as the second
stimulus (152 ±40.07) of WI rats. Latencies after stimulus onset (B): 50-kHz
first: 16.04 ±5.67; 50-kHz second: 24.67 ±16.19.
occurred during noise playback (WI: t17 = 2.717, p= 0.015; SD:
t17 = 2.727, p= 0.014).
Data Set 1 and 2: Detailed Analyses
Temporal Emission Pattern
We next pooled the data sets 1 and 2 and performed more
detailed analyses. First, a detailed temporal analysis revealed
that the emission of response calls was strongly dependent
on stimulus (F1,58 = 21.260, p<0.001) and stimulus phase
(F2,116 = 21.120, p<0.001), with an interaction between
stimulus and stimulus phase (F2,116 = 21.002, p<0.001), while
stock had no major impact (stock: F1,58 = 2.311, p= 0.134;
stock ×stimulus: F1,58 = 2.253, p= 0.139; stock ×stimulus
phase: F2,116 = 2.308, p= 0.104; stock ×stimulus ×stimulus
phase: F1,116 = 2.290, p= 0.106; Figure 3). Specifically, playback
of 50-kHz USV but not noise led to a prominent increase in
response calls, which occurred during the 5 min of 50-kHz USV
playback and up to 5 min thereafter. The peak of vocalization
typically occurred in the second or third minute after 50-kHz
USV playback onset. With onset of the 50-kHz USV playback,
the numbers of emitted response calls increased significantly in
WI (F1,41 = 27.940, p<0.001) and SD rats (F1,17 = 7.436,
p= 0.014). After that, calling rate decreased to zero at the latest
5 min after the playback had ended. In both stocks, substantial
calling only occurred in response to 50-kHz USV playback and
not in response to noise, reflecting high specificity of response
call emission (WI: F1,41 = 25.387, p<0.001; SD: F1,17 = 7.538,
p= 0.014). Furthermore, the call emission sequence showed
that most animals started calling with higher frequencies around
50 kHz and quickly changed to emit calls of frequencies around
22 kHz (Supplementary Figure 1A).
Response Call Features
Secondly, detailed analyses of acoustic features revealed that the
calls in response to 50-kHz USV playback were heterogeneous
since they were characterized by a large variability in acoustic
features and shapes. Both, WI and SD rats emitted calls
below and above 32 kHz. These calls had rather different
FIGURE 2 | Total numbers of response calls (A) and latencies to call (B) in
Wistar (WI) and Sprague-Dawley (SD) rats.
durations and shapes, and the temporal spaces between them
varied substantially.
For a further quantification of the response calls, mean
peak frequencies, mean call durations, and mean frequency
modulations were quantified (Figure 4; for examples of
response calls, see Figure 5). Peak frequencies of WI rats
(32.48 ±1.46 kHz) and SD rats (37.82 ±3.2 kHz; Figure 4A) did
not differ significantly from each other (t15.82 = 1.52, p= 0.149).
Call durations of WI rats (0.34 ±0.03 s) tended to be longer than
those of SD rats (0.24 ±0.05 s; t43 = 1.859, p= 0.07). Frequency
modulations did not differ between stocks (t43 = 0.98, p= 0.33;
WI: 6.68 ±0.51 kHz; SD: 7.68 ±0.97 kHz).
To visualize the different call parameters and the distribution
of individual calls, scatter plots for either call durations
or frequency modulations were plotted vs. peak frequencies
(Figure 6). This analysis showed that most calls were below
32 kHz, with durations above and below 0.3 s. Frequency
modulations were mainly below 5 kHz. The main distribution of
the calls was around mean peak frequencies below 32 kHz in both
stocks, but in SD rats also another distribution peak occurred
around 50 kHz, with call durations typically shorter than 0.3 s
and frequency modulations below 5 kHz (Figures 6B,D).
Next, we quantified call numbers depending on acoustic call
features and divided response calls into those with mean peak
frequencies below or above 32 kHz, durations shorter or longer
than 0.3 s, and frequency modulations below or above 5 kHz
(Table 1). This analysis showed that in both stocks the majority
of response calls was below 32 kHz. Considering durations, most
calls were shorter than 0.3 s, particularly in SD rats. Frequency
modulations were mainly below 5 kHz. When comparing the
percentages of calls with mean peak frequencies below 32 kHz
among stocks, WI rats were found to have higher percentages
of calls below 32 kHz (t43 = 2.137, p= 0.038). Considering
percentages of calls with durations below 0.3 s, stocks did not
differ (t43 =−1.95, p= 0.058). The same was true for frequency
modulations. Similar percentages of calls were emitted with
modulations below 5 kHz in both stocks (t43 = 0.173, p= 0.864).
In addition, we asked whether response calls below or
above 32 kHz were related to each other in individual
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FIGURE 3 | Mean numbers (±SEM) of response calls emitted during each minute of WI (blue dots; A,B) and SD (magenta squares; C,D) rats. **p<0.01,
***p<0.001.
animals (Figure 7), but did not find significant correlations
between the two in WI (r= 0.08, p= 0.66) or SD rats
(r=−0.26, p= 0.44).
Relationships Between Response Calls and
Playback-Induced Approach
Thirdly, we asked whether the emission of the response calls was
correlated with social approach behavior evoked by playback of
50-kHz USV. As stated in the Introduction, the present response
call data sets were obtained in a study where social approach
behavior evoked by 50-kHz USV playback was examined (Berz
et al., 2021). In that study, approach behavior was quantified
by subtracting the time spent on the proximal arms (i.e., close
to the speaker) before playback from the time spent thereon
during the presentation of 50-kHz USV. The same was done
for the proximal entries (see detailed analysis in Berz et al.,
2021). These numbers were now correlated with the total amount
of response calls evoked by playback of 50-kHz USV to see
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FIGURE 4 | Bar graphs and individual data points of mean peak frequency (A), mean duration (B), and frequency modulation (C) of WI (blue dots) and SD rats
(magenta squares).
whether social approach behavior was related to the emission
of response calls across individual rats. In WI rats, this tended
to be the case. The more time the rats spent close to the active
speaker, the more calls in response to 50-kHz USV playback
they tended to emit (r= 0.314, p= 0.075). A more prominent
correlation was evident in SD rats, where social approach and
the emission of response calls were strongly associated (SD:
r= 0.662, p= 0.019). No such correlations were found with
respect to proximal arm entries (WI: r= 0.01, p= 0.952; SD:
r=−0.017, p= 0.948). To test whether these correlations
were only a byproduct of locomotor activity during playback,
the total numbers of response calls were correlated with the
degree of locomotor activation using the distance traveled during
playback in comparison to the distance traveled before playback.
Neither in WI nor SD rats a correlation was found (r= 0.065,
p= 0.681; r= 0.151, p= 0.551, respectively). Also, the numbers
of response calls were not correlated with locomotor activity
during the first 15 min on the maze as a measure of general
locomotor activity (WI: r= 0.031, p= 0.864; SD: r= 0.187,
p= 0.540).
Data Set 3: Effects of Drug Treatment
Call Numbers and Latencies
In the third data set, rats were treated either with the
dopaminergic D2 receptor antagonist Halo or saline as a control.
The pharmacological treatment had no prominent effect on the
emission of response calls and the proportion of vocalizing rats
(saline: 15 out of 24, Halo: 20 out of 24) did not differ between Sal
and Halo (x21,48 = 2.64, p= 0.104). Moreover, treatment did not
affect response call numbers (t46 = 0.465, p= 0.644; Figure 8A;
Sal: 66.5 ±31.18; Halo: 86 ±31.53) and latencies to start calling
(t33 = 0.578, p= 0.567; Figure 8B; Sal: 19.41 ±4.18 s; Halo:
26.33 ±9.86 s).
Temporal Emission Pattern
Similar to the previous data sets 1 and 2, the emission of response
calls was strongly dependent on stimulus (F1,46 = 11.771,
p= 0.001) and stimulus phase (F2,92 = 14.443, p<0.001),
with an interaction between stimulus and stimulus phase (F2,
92 = 14.373, p<0.001), while treatment had no major impact
(treatment: F1,46 = 0.194, p= 0.662; treatment ×stimulus: F1,
46 = 0.232, p= 0.632; treatment ×stimulus phase: F2,92 = 0.842,
p= 0.434; treatment ×stimulus ×stimulus phase: F1,92 = 0.797,
p= 0.454; Figure 9). Specifically, as in the previous data sets 1
and 2, playback of 50-kHz USV but not noise led to a prominent
increase in response calls, which occurred during the 5 min of
50-kHz USV playback and up to 5 min thereafter. The peak was
again typically seen during the second or third minute after 50-
kHz USV playback onset. With onset of 50-kHz USV playback,
the numbers of emitted response calls increased significantly in
rats treated with Sal (F1,23 = 6.443, p= 0.018) but also in rats
treated with Halo (F1,23 = 8.068, p= 0.009). After that, calling
rate decreased to zero at the latest 5 min after playback had ended.
Substantial calling only occurred in response to 50-kHz USV and
not in response to noise and was therefore specific to the 50-
kHz USV playback in both treatment groups (Sal: F1,23 = 4.687,
p= 0.041; Halo: F1,18 = 7.613, p= 0.013). Furthermore, the call
emission sequence showed that most animals started calling with
higher frequencies around 50 kHz and quickly changed to emit
calls of frequencies around 22 kHz (Supplementary Figure 1B).
Response Call Features
For a further characterization of response calls in the third
data set, their mean peak frequencies, durations, and frequency
modulations were analyzed. Sal-treated animals had peak
frequencies around 33.76 ±2.8 kHz, which was not significantly
different from Halo-treated animals (30.89 ±2.49 kHz;
t33 = 0.898, p= 0.376; Figure 10A). Call durations in controls
were 0.282 ±0.036 s, which was significantly shorter than those
of Halo-treated rats (0.395 ±0.039 s; t33 = 2.048, p= 0.049,
Figure 10B). Frequency modulation did not differ between
treatment groups and Sal-treated rats called with a frequency
modulation of 5.33 ±0.56 kHz compared to 6.16 ±0.66 kHz in
HALO-treated rats (t33 = 0.919, p= 0.365; Figure 10C).
The response calls were various in shape and differed in
call parameters (for examples of response calls, see Figure 11).
For better visualization of the different call parameters and
the distribution of the individual calls, scatter plots for either
call durations or frequency modulations were plotted vs. peak
frequencies (Figure 12). The accompanying histograms show
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FIGURE 5 | Exemplary response calls during 50-kHz USV playback. The first picture is always the 50-kHz USV playback sequence and the following pictures show
response calls in addition to the 50-kHz USV playback sequence (red arrows) <32 kHz (A) or >32 kHz (B) of WI and SD rats. Exemplary high-resolution
spectrograms (frequency resolution 488 Hz; time resolution 0.512 ms) were generated with SASLab Pro software 5.2.09 (Avisoft Bioacoustics) via fast Fourier
transformation (512 FFT length, 100% frame, Hamming window, and 75% time-window overlap).
the main distribution at mean peak frequencies around 25 kHz
in both treatment groups; meaning that the majority of calls
were below 32 kHz. Especially in Halo-treated rats, few calls
were above 32 kHz. Call durations were as well above as below
0.3 s in Sal- and Halo-treated rats. Frequency modulation was
mainly below 5 kHz.
Next, we again quantified call numbers depending on acoustic
call features and divided response calls into those with mean
peak frequencies below or above 32 kHz, durations shorter or
longer than 0.3 s, and frequency modulations below or above
5 kHz (Table 2). When comparing the percentages of calls
with mean peak frequencies below 32 kHz among treatment
groups, no significant difference was detected (t33 =−0.978,
p= 0.335). Considering durations below 0.3 s, there was likewise
no difference (t33 = 1.996, p= 0.054). The same was true for
frequency modulations, since similar percentages of calls were
emitted with modulations smaller than 5 kHz in both groups
(t33 = 0.979, p= 0.335).
In addition, we again asked whether response calls below or
above 32 kHz were related in individual animals (Figure 13), but
found no significant correlations in Sal- (r=−0.161, p= 0.566)
or Halo-treated rats (r= 0.123, p= 0.606).
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FIGURE 6 | Wistar (WI) (A,C) and Sprague-Dawley (SD) rats (B,D) scatter plots with histograms (blue for WI and magenta for SD rats) of duration or frequency
modulation vs. mean peak frequency. Duration is divided into <> 0.3 s (A,B: horizontal gray dashed lines), frequency modulation is divided into <> 5 kHz (C,D:
horizontal gray dashed lines) and mean peak frequencies are divided into <> 32 kHz (vertical gray dashed line). For each section, an exemplary call with the
respective parameters is shown (red arrows).
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TABLE 1 | Scatter plot distributions for Wistar (WI) and Sprague-Dawley (SD) rats.
WI
N= 33/42
Mean peak frequency
=32 kHz >32 kHz Total calls
Total numbers (percentages) means ±SEM 3,328 (88.44%) 69.34 ±5.61 435 (11.56%) 30.66 ±5.61 3,763 (100%)
Duration <0.3 s 1,936 (51.44%) 48.69 ±4.5 1,644 (43.7%) 292 (7.8%)
>0.3 s 1,827 (48.56%) 51.34 ±4.5 1,684 (44.8%) 143 (3.8%)
Modulation <5 kHz 2,272 (60.38%) 51.62 ±4.18 2,104 (55.9%) 168 (4.5%)
>5 kHz 1,491 (39.62%) 48.38 ±4.18 1,224 (32.5%) 267 (7.1%)
SD
N= 12/18
Mean peak frequency
=32 kHz >32 kHz Total calls
599 (73.7%) 44.29 ±11.95 214 (26.3%) 55.71 ±11.95 813 (100%)
Duration <0.3 s 479 (58.9%) 66.79 ±9.2 273 (33.6%) 206 (25.3%)
>0.3 s 334 (41.1%) 33.21 ±9.2 326 (40.1%) 8 (1%)
Modulation <5 kHz 464 (57.1%) 50.21 ±7.62 324 (39.9%) 140 (17.2%)
>5 kHz 349 (42.9%) 49.79 ±7.62 275 (33.8%) 74 (9.1%)
Mean peak frequencies <or >32 kHz, Durations =or >0.3 s, frequency modulations =or >5 kHz.
FIGURE 7 | Correlation between calls <32 kHz and >32 kHz for Wistar (WI) (A) and Sprague-Dawley (SD) (B) rats. Each data point represents response calls below
and above 32 kHz of one animal.
Relationships Between Response Calls and
Playback-Induced Approach
To see whether social approach was associated with the emission
of response calls, these two parameters were again correlated.
The results were the same in both treatment conditions. In
Sal-treated rats, there were no significant correlations, neither
between the time spent in the proximal arms close to the
active speaker nor between the entries into those with the
amount of response calls (Sal time: r=−0.0195, p= 0.487;
Sal entries: r= 0.059, p= 0.783). In Halo-treated animals,
likewise no significant correlations between proximal time or
entries and number of emitted calls were detected (Halo time:
r= 0.143, p= 0.547; Halo entries: r=−0.112, p= 0.602).
Moreover, locomotor activity during 50-kHz USV playback in
comparison to the distance traveled before playback was not
correlated with the total numbers of response calls, irrespective
of treatment condition (Sal: r=−0.101, p= 0.639; Halo:
r=−0.113, p= 0.598). In addition, locomotor activity during
the first 15 min on the platform was not correlated with the
number of response calls (Sal: r=−0.224, p= 0.421; Halo:
r= 0.238, p= 0.312).
DISCUSSION
In this study, we characterized response calls emitted by rats
exposed to playback of appetitive 50-kHz USV, previously
shown to function as social contact calls (Wöhr, 2018). The
phenomenon that rats respond to playback of species-specific 50-
kHz calls by emitting response calls has been repeatedly reported
before, but has not been described in detail yet (Wöhr and
Schwarting, 2007, 2009;Willadsen et al., 2014;Willuhn et al.,
2014;Engelhardt et al., 2017, 2018;Berg et al., 2018, 2021;
Kisko et al., 2020;Olszy´
nski et al., 2020, 2021; for an overview
see Supplementary Table 1). First, we described the emission
of response calls in reaction toward 50-kHz USV playback in
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FIGURE 8 | Total numbers of response calls (A) and latencies to call (B) in
Sal- and Halo-treated rats. Data are presented as individual results and as
means ±SEM.
WI rats. Secondly, we compared these to SD rats. Thirdly, we
analyzed the effect of blocking DA receptors on response calls
using Halo, as compared to vehicle-injected WI subjects.
Through these means, we could demonstrate that most rats
emitted response calls. Importantly, the emission of response
calls was clearly linked to 50-kHz USV playback. In fact, response
calls were seen specifically in response to 50-kHz USV but not
in response to time- and amplitude-matched noise, replicating
previous results (Willadsen et al., 2014;Willuhn et al., 2014;
Engelhardt et al., 2017, 2018;Berg et al., 2018, 2021;Kisko et al.,
2020;Olszy´
nski et al., 2020, 2021). When exposed to 50-kHz USV,
receiver rats often started emitting response calls within the first
minute of playback and emission rates were typically peaking
after around 2–3 min, often outlasting playback for up to 5 min.
This certainly supports naming these calls “response calls.”
Most response calls were characterized by peak frequencies
below 32 kHz, the threshold typically applied to differentiate
between 22- and 50-kHz USV (Brudzynski, 2001). Although peak
frequencies were highly variable and ranged roughly between
20 and 80 kHz, the vast majority of response calls occurred in
a frequency range of 20–32 kHz. Similarly, call durations were
characterized by large variability, ranging from a few milliseconds
to up to 1.5 s. Call durations of about 0.3 s occurred at a
particularly high rate. Frequency modulations were typically
below 5 kHz. When comparing these values to the parameters of
typical 22- and 50-kHz USV, our values correspond more to 22-
kHz USV; more precisely the short 22-kHz USV type since the
durations were rarely longer than 0.3 s (Brudzynski, 2021).
The emission of response calls was seen in WI and SD rats,
suggesting that this is a robust phenomenon not dependent
on stocks. Specifically, we found that there were no substantial
differences between WI and SD rats, concerning numbers of
emitted calls, latencies to start calling, and call likelihood.
In both stocks there was a large variability among response
calls. However, their mean peak frequencies, call durations,
and frequency modulations did not differ significantly between
experimental conditions. SD rats only differed in one aspect by
clearly showing calls around frequencies of 50 kHz, which was
not that prominent in WI rats. This is somehow in line with
other studies that also showed higher emission of 50-kHz USV
and elevated rough-and-tumble play behavior in SD compared
to WI rats (Manduca et al., 2014). Other studies, however, found
that WI rats emitted more 50-kHz USV compared to SD rats
(Schwarting, 2018a,b), indicating that WI rats are more prone to
emit USV in general, which is also not represented by our data.
If at all, on a descriptive level, WI rats emit slightly less response
calls compared to SD rats. Regarding call parameters, previous
studies showed marginal differences between stocks, i.e., shorter
call durations in SD rats compared to WI rats (Schwarting,
2018b). On a descriptive level again, this aligns with our results,
albeit this difference in call duration did not yield significance.
Apart from stock differences, various other factors like breeding
or experience have to be taken into account. Moreover, inter-
individual differences should not be neglected, as our results also
suggest (Schwarting, 2018a,b).
In our study, the pharmacological treatment with the
D2 antagonist Halo did not affect call likelihood, call rates,
latencies, temporal distribution, peak frequency, and frequency
modulation. In Sal-treated WI rats, the majority of calls was
again below 32 kHz, however, in Halo-treated rats this was
even more prominent and Halo treatment also led to longer
call durations. Previous studies showed that exposure to 50-
kHz USV playback under the influence of systemically applied
amphetamine, a catecholaminergic agonist, resulted in response
calls with frequencies around 50 kHz at the expense of 22 kHz
(Engelhardt et al., 2017). Specifically, calls of lower frequencies
decreased drastically under the influence of amphetamine. In
contrast, response calls in the 50 kHz range increased dose-
dependently following amphetamine administration. This is in
line with a large number of studies showing that the emission
of 22- and 50-kHz USV are associated with the activation of
distinct neurotransmitter systems (for review: Brudzynski, 2021).
While 22-kHz USV are associated with the cholinergic system
(Brudzynski, 2001;Kroes et al., 2007;Willadsen et al., 2018), the
dopaminergic system plays an important role in the regulation
of 50-kHz USV (Wöhr, 2021). For instance, electrolytic or 6-
hydroxydopamine lesions of the ventral tegmental area reduce
the emission of 50-kHz USV (Burgdorf et al., 2007). Conversely,
emission of 50-kHz USV can be evoked by electrical stimulation
of the ventral tegmental area or the nucleus accumbens (Burgdorf
et al., 2000, 2007). Moreover, psychostimulants, most notably
amphetamine, lead to a robust increase in 50-kHz USV emission
(Rippberger et al., 2015). Additionally, playback of 50-kHz USV
was shown to induce enhanced levels of activity in the nucleus
accumbens (Sadananda et al., 2008), where it elicits a rapid
phasic release of dopamine (Willuhn et al., 2014). Based on these
findings, one could have assumed that the dopaminergic receptor
blockade with Halo should decrease response call numbers,
especially those above 32 kHz, which was apparently not the case.
Possibly, these calls are not critically dependent on dopamine
D2 receptor function, and might be dependent on endogenous
opiates, as indicated by an earlier playback study with the opiate
receptor antagonist naloxone (Wöhr and Schwarting, 2009).
Together, the present findings indicate that the emission of
response calls is a robust phenomenon that is seen specifically
in response to playback of 50-kHz USV independent of
stock and despite blocking dopamine neurotransmission. These
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FIGURE 9 | Mean number of response calls emitted during each minute of Sal- (blue; A,B) and Halo-treated (orange; C,D) rats. Most calls were emitted during
50-kHz USV stimulus and almost no calls were emitted during noise. **p<0.01, ***p<0.001.
observations are in line with the idea that the emission of
response calls reflects changes in affect that are caused by
playback of 50-kHz USV. For example, one might expect the
induction of a positive affective state in response to appetitive
50-kHz USV. On the other hand, it was suggested that response
calls reflect frustration induced by the inability to reach the
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FIGURE 10 | Call parameters of Sal- (blue) and Halo-treated (orange) rats for mean peak frequency (A), mean duration (B), and frequency modulation (C).
conspecific emitting 50-kHz USV. Alternatively, response calls
might serve communicative functions as social contact calls
or as appeasement signals. While the present findings do not
allow drawing strong conclusions about causes and functional
significance of response calls, they provide first insights into
potential mechanisms underlying their emission.
In support of the idea that response calls might reflect an
affective state we hypothesize that the rats are not solely in
one affective state, but rather in an ambivalent state. There is
convincing evidence in support of the notion that USV emission
reflects prominent affective states (Brudzynski, 2021) and that
different call types are associated with distinct states (Brudzynski,
2013b). Because USV below 32 kHz are typically believed to
function as alarm or distress calls reflecting a negative affective
state, this would suggest that playback of 50-kHz USV induced
a negative state in the receiver rats. However, the strong level of
social approach behavior and the emission of 50-kHz response
calls, at least in SD rats, evoked by playback of 50-kHz USV
speaks against the induction of a solely negative affective state
through 50-kHz USV playback (Wöhr, 2018). Furthermore, the
positive and negative emotional states in rats were proposed
to be mutually exclusive and acting in an antagonistic manner
(Brudzynski, 2021). It is possible, however, that the two states
quickly alternate which leads to the hypothesis of an ambivalent
state, with negative and positive phases present in an oscillating
manner. This is also reminiscent of an approach/avoidance
conflict, i.e., a situation characterized by choices leading to either
reward or punishment (Aupperle et al., 2015). Interestingly, it
was shown that rats emit 22-kHz as well as 50-kHz USV during
neutral situations and not only aversive ones (Robakiewicz et al.,
2019). The study by Robakiewicz et al. (2019) also showed that
both call types and hence presumably both emotional states can
be present during an emotional neutral task of performing nose
pokes in order to change the light of the experimental apparatus.
Both call types were also found in a cocaine self-administration
task (Barker et al., 2010), where animals received either high or
low doses of cocaine. Low dose rats predominantly emitted short
22-kHz calls and high dose rats emitted mostly 50-kHz calls.
Nevertheless, both groups showed calls of both emotional states
and this supports the hypothesis of the ambivalent state. In the
present study, however, only SD rats emitted 50-kHz USV to a
higher extent and all other experimental groups mainly emitted
calls with frequencies below 32 kHz. Additionally, the emissions
of response calls below and above 32 kHz were not correlated
across individual rats, suggesting that there was no general
tendency for emitting response calls in both frequency ranges,
which speaks against the hypothesis of an ambivalent state.
With respect to the emission of 22-kHz calls, this phenomenon
might be explained by the hypothesis of a frustrated state in
the receiver rat, possibly induced by the violated expectation of
another rat being present. Other studies suggested that short
22-kHz calls (<0.3 s) represent a dysphoric state or displeasure
without any external threat (Simmons et al., 2018), which is
in line with the mean peak frequencies, durations, and low
frequency modulations of the response calls found in our study.
This might also be an indication that calls with low frequencies
in response toward 50-kHz USV playback are an expression of
internal distress, i.e., frustration, as suggested before (Wöhr and
Schwarting, 2009). Frustration is defined as a result of behavior
after an expected but not received reward (Scull et al., 1970;
Burokas et al., 2012). In our playback paradigm, the rat probably
realized that there was no rat physically present for interaction
after hearing the 50-kHz USV playback, and this could have led
to a state of frustration in the approaching rat. This might also
explain why the majority of response calls was emitted within 2
or 3 min after the onset of the 50-kHz USV playback. At first, the
animals heard and recognized the stimulus, exhibited a strong
social approach immediately afterward and as soon as the rats
realized that there was no conspecific present, the emission of
response calls increased as an expression of a frustrated state. In
line with the frustrated state hypothesis is our finding that the
first calls of most animals of data set 2 and 3 were of higher
frequencies, i.e., around 50 kHz and quickly changed to calls with
frequencies in the 22-kHz USV range (Supplementary Figure 1).
On the other hand, the positive correlation of response
calls and approach behavior might serve the hypothesis that
the response calls could also be characterized as social contact
calls. 50-kHz USV have been postulated to fulfill an affiliative
communication function to, for example, maintain a playful
state during rough-and-tumble play or as social contact calls
to reestablish social proximity after separation of conspecifics
(Wöhr et al., 2016). An indication that the response calls in
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FIGURE 11 | Exemplary response calls during 50-kHz USV playback. The first picture is always the 50-kHz USV playback sequence and the following pictures show
response calls in addition to the 50-kHz USV playback sequence (red arrows) <32 kHz (A) or >32 kHz (B) of Sal- and Halo-treated rats. Note that the calls depicted
for Sal- or Halo-treated rats are not specific to the treatment groups and calls were descriptively similar in all groups.
our study serve as social contact calls is that they are emitted
during social approach behavior. Further, such calls are emitted
frequently during the approach behavior like 50-kHz USV during
rough-and-tumble play (Knutson et al., 1998). In our study we
found a moderate positive correlation between response calls and
approach behavior, i.e., the time spent close to the active speaker,
in SD and, at least to some extent, in WI rats. Apparently, the
more the animals tried to reach a possible conspecific signaled
by the 50-kHz USV playback, the more calls they emitted,
supporting the hypothesis of response calls being contact calls.
For Sal- and Halo-treated WI rats, however, this was not the case.
In Halo-treated rats, the absence of a positive correlation between
approach behavior and response call emission was probably due
to the drug-induced immobility (Berz et al., 2021). Since Sal-
treated rats also received an i.p. injection 60 min prior to testing,
this might have influenced their approach response, as well as
their calling behavior; even though Sal-treated rats significantly
approached the sound source (Berz et al., 2021) and emitted
similar numbers of response calls as WI rats. No correlation was
found, however, between overall activity and call numbers in
any group. Also or alternatively, the positive correlation between
approach behavior and response calls especially observed in SD
rats might not be in order to establish contact, but rather due to
hypervigilance. Olszy´
nski et al. (2021) showed that in response
to 50-kHz USV playback, heart rate and locomotor activity
increased as well as the emission of USV. The USV in response
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FIGURE 12 | Sal- (A,C) and Halo-treated rats (B,D) scatter plots with histograms (blue for Sal- and orange for Halo-treated rats) of duration or frequency modulation
vs. mean peak frequency. Duration is divided into <> 0.3 s (A,B: horizontal gray dashed lines), frequency modulation is divided into <> 5 kHz (C,D: horizontal gray
dashed lines) and mean peak frequencies are divided into <> 32 kHz (vertical gray dashed line). For each section, an exemplary call with the regarding parameters is
shown (red arrows).
to 50-kHz USV playback in that study were mainly 50-kHz calls,
possibly representing contact calls, in contrast to our study here,
where the animals mostly emitted calls of lower frequencies. Also,
the peak of call emission occurred shortly after the recipient of
the playback was in proximity to the sound source and ceased
after playback has stopped, which suggests that these calls could
function to establish social contact or in search of it. However, the
response calls linked to the 50-kHz USV playback do not classify
as 50-kHz calls because their mean peak frequencies are much
lower, the duration is longer, and there is hardly any frequency
modulation compared to 50-kHz calls.
Alternatively, response calls could serve appeasing purposes.
The age difference between the rat of the recorded playback
and the test subject might be of interest, because in our
study, a juvenile rat heard 50-kHz USV playback recorded
from an adult rat and accordingly, it seems plausible for the
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TABLE 2 | Scatter plot distributions for Sal- and Halo-treated rats.
Sal
N= 15/24
Mean peak frequency
=32 kHz >32 kHz Total calls
Total numbers (percentages) means ±SEM 1,490 (93.4%) 72.77 ±8.78 106 (6.6%) 27.23 ±8.78 1,596 (100%)
Duration <0.3 s 1,044 (65.4%) 59.39 ±5.91 960 (60.2%) 84 (5.3%)
>0.3 s 552 (34.6%) 40.61 ±5.91 530 (33.2%) 22 (1.4%)
Modulation <5 kHz 1,147 (71.9%) 60.27 ±6.73 1,098 (68.8%) 49 (3.1%)
>5 kHz 449 (28.1%) 39.73 ±6.73 392 (24.6%) 57 (3.6%)
Halo
N= 20/24
Mean peak frequency
=32 kHz >32 kHz Total calls
2,021 (97.9%) 83.44 ±6.78 43 (2.1%) 16.56 ±6.78 2,064 (100%)
Duration <0.3 s 919 (44.5%) 40.48 ±6.89 893 (43.3%) 26 (1.3%)
>0.3 s 1,145 (55.5%) 59.52 ±6.89 1,128 (54.7%) 17 (0.8%)
Modulation <5 kHz 1,633 (79.1%) 50.76 ±6.73 1,624 (78.7%) 9 (0.4%)
>5 kHz 431 (20.9%) 49.24 ±6.73 397 (19.2%) 34 (1.6%)
Mean peak frequencies <or >32 kHz, Durations = or >0.3 s, frequency modulations = or >5 kHz.
FIGURE 13 | Correlation between calls <32 kHz and >32 kHz for Sal- (A) and Halo-treated (B) rats. Each data point represents response calls below and above
32 kHz of one animal.
subject rat to cautiously approach the potential conspecific.
Supporting this hypothesis, is the fact that in adult male
rats, USV calls of lower frequencies were found during play
fighting (Burke et al., 2017, 2020). In social situations that
were at risk to escalate into aggression, the play partners
lowered their calls gradually from 50 kHz to around 30 kHz
with increasing durations (Burke et al., 2017). The authors
hypothesized that this group of calls might be a transition
from 50-kHz flats to 22-kHz flats or a unique new type of
calls. The function of these calls is probably the induction of
appeasement, i.e., to de-escalate a situation at risk to turn into
aggression (see also Sales, 1972;Lore et al., 1976). Our results
seem to support this hypothesis since we tested juvenile rats
subjected to calls from an older adult rat and the response
calls were in similar frequencies. Moreover, the response calls
had also similar frequency modulations, like the calls in the
study by Burke et al. (2017) and were not exclusively flat as the
common 22-kHz USV. So far, however, it is not known whether
receiver rats can gain information about the age of the sender
based on their USV.
Importantly, the response call phenomenon studied here in
detail appears sufficiently robust to be used as a measure for
the reciprocal nature of acoustic communication and can easily
be applied in rat model systems for neuropsychiatric disorders,
where acoustic communication is impaired, such as autism
spectrum disorder (Lai and Baron-Cohen, 2015). In preclinical
studies examining USV with the aim to reveal communication
deficits in rodent model systems, most laboratories have focused
exclusively on the sender. Although there is now an increasing
number of preclinical studies including playback paradigms to
learn about the responses evoked in the receiver as well (Berg
et al., 2018, 2020a,b;Kisko et al., 2018, 2020;Wöhr et al., 2020),
Frontiers in Behavioral Neuroscience | www.frontiersin.org 16 January 2022 | Volume 15 | Article 812142
fnbeh-15-812142 January 10, 2022 Time: 14:26 # 17
Berz et al. Response Calls Toward 50-kHz USV
an important aspect of acoustic communication that is often
still neglected is its reciprocal nature and the fact that a
signal emitted by the sender frequently evokes the emission
of a response signal in the receiver (Seyfarth and Cheney,
2003). Measuring response calls offers a unique opportunity to
overcome this limitation. It offers a new approach to studying
the reciprocal nature of communication in rodent models for
neuropsychiatric disorders.
DATA AVAILABILITY STATEMENT
The raw data supporting the conclusions of this article will be
made available by the authors, without undue reservation.
ETHICS STATEMENT
The animal study was reviewed and approved by
Tierschutzbehoerde, Regierungspraesidium Giessen, Germany,
TVA No. 35-2018.
AUTHOR CONTRIBUTIONS
RS and MW designed the study, acquired resources and funding,
and oversaw the project. AB performed the experiments. AB with
substantial help from MW analyzed the data. AB, RS, and MW
wrote the manuscript. All authors contributed to the article and
approved the submitted version.
FUNDING
This work was supported by grant SCHW 559/15-1 from the
Deutsche Forschungsgemeinschaft (DFG) and the DFG funded
Research Training Group “Breaking Expectation” (GRK 2271).
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnbeh.
2021.812142/full#supplementary-material
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Conflict of Interest: The authors declare that the research was conducted in the
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The handling editor declared a past co-authorship with one of the authors MW.
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