Communication of Adult Rats by Ultrasonic Vocalization: Biological,
Sociobiological, and Neuroscience Approaches
Stefan M. Brudzynski
Abstract
Rats have developed antipredator defensive adaptations to
protect themselves from the large number of animals that
prey on them. One such adaptation is the ability to commu-
nicate by ultrasonic vocalization, which decreases the likeli-
hood of detection by a predator. Almost all rat vocalizations
are inaudible to the human ear as well, so laboratory studies
of ultrasonic vocalization require specialized electronic
equipment. The most popular of these is the “bat detector,”
which lowers ultrasonic frequency to a humanly audible fre-
quency. Adult rats emit two types of ultrasonic calls: alarm
(22 kHz) calls, in aversive and dangerous situations, and
appetitive (50 kHz) calls, in appetitive or “friendly” (i.e.,
nonaggressive) behavioral situations. These two types of
calls differ in all acoustic parameters, and their initiation de-
pends on activity in different ascending tegmental pathways
to the forebrain: 22 kHz calls require activity in the cholin-
ergic system, and 50 kHz calls in the mesolimbic dopamin-
ergic system. The release of acetylcholine in the diencephalon
and forebrain is associated with the emission of 22 kHz
vocalizations, and the release of dopamine in the nucleus
accumbens with 50 kHz calls. Thus the two calls are reliable
predictors of increased cholinergic or dopaminergic activity
in the brain. The calls serve as indices of the rat’s state, in-
cluding its affective state, induced by activity of one or the
other neurochemical system.
Key Words: alarm calls; cholinergic system; defensive be-
havior; dopaminergic system; ultrasonic vocalization; warn-
ing calls; 22 kHz calls; 50 kHz calls
Introduction
T
here is growing research interest in vocal communica-
tion in rats. Unlike many other rodent species, rats
are highly social animals (Barnett 1967; Blanchard
and Blanchard 1980; Lore and Flannelly 1977; Nyby and
Stefan M. Brudzynski, PhD, DSc, is Professor of Psychology and Neurosci-
ence in the Department of Psychology, Brock University, St. Catharines,
Ontario, Canada.
Address correspondence and reprint requests to Dr. Stefan M. Brudzyn-
ski, Department of Psychology, Brock University, 500 Glenridge Avenue,
St. Catharines, ON, L2S 3A1 Canada or email sbrudzyn@brocku.ca.
Volume 50, Number 1 2009
Whitney 1978) and vocal communication is a regular feature
of their social interactions. It is an intriguing phenomenon
that rats and other rodents have developed communication in
the ultrasonic range of sound frequencies. Although re-
searchers have known since the early 1960s, when bat detec-
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tors became commercially available, that rats could emit and
hear ultrasounds, studies of their ultrasonic communication
were hampered for at least a decade by technical difficulties
in recording and analyzing the ultrasonic signals.
Like other mammals, rats produce sound through the lar-
ynx. But unlike most other mammalian species, rodents can
use their larynx in two modes of functioning. The first, com-
mon to most other species, causes vibrations of vocal folds
and produces audible sound (squeals) of 2-4 kHz (Nitschke
1982). These calls express both physical pain or discomfort—
acute, approaching, or anticipated—and the affective dimen-
sion of pain or anticipated pain (Borszcz 2006). Rats use
similar audible vocalizations, sometimes not distinguishable
from pain calls, against predators. These signals not only in-
dicate a potentially painful encounter but also serve to intimi-
date or stop the advance of a predator, warning the approaching
animal that the rat is ready for an active defense (Litvin et al.
2007). The intensity of the call is dependent on the proximity
of the predator and independent of the proximity of other
conspecifics (Litvin et al. 2007). Rats may also direct these
vocalizations at humans in anticipation of, or when experi-
encing, physically unpleasant treatment or rough handling.
The second mode of larynx function in the rat is in the
emission of ultrasounds. For these sounds, the larynx is sta-
bilized and used as a whistle with a very small orifice cre-
ated by the vocal cords (Sanders et al. 2001), which are tightly
constricted and cannot vibrate (Nyby 2001). Animals build
up considerable abdominal pressure and push air through
the orifice. It is possible sometimes to observe this so-called
“pressure breathing” (Fokkema et al. 1986) with deep inha-
lations and prolonged exhalations associated with the pro-
duction of long and loud alarm calls. The vibrating air
column produces the ultrasonic sound on the same principle
as a human whistle does, except that the small size of the
rat’s respiratory tract creates sound in the ultrasonic range
of frequencies. In this mode of vocalization, rats usually
emit pure sounds of a constant or variable frequency with
few or no overtones.
Rats use the ultrasonic call in a variety of social situa-
tions that include both aversive and appetitive encounters.
Ultrasonic acoustics and vocalizations are very natural for
43
rats and they emit these sounds from birth, although their
hearing develops at a slower rate (studies have reported the
onset of rat pup auditory function on postnatal day 12-14;
Freeman et al. 1999a,b; Geal-Dor et al. 1993). Rat pups emit
early ultrasonic “isolation calls” when separated from their
littermates or when they fall out and lose contact with the
nest. The isolation calls change only slightly in their acoustic
structure until the pups reach about 21 days of age, when a
more abrupt transformation occurs (Brudzynski 2006) and
the juvenile rats soon begin to emit calls similar to the 22
kHz and 50 kHz types of adult ultrasonic vocalization.
But why have rats, and rodents in general, developed vo-
calization in the ultrasonic range of frequencies?
Biological Causes of the Development of
Ultrasonic Communication
An impressively large number of species—mammals, birds,
and reptiles—feed on rodents, many of them almost exclu-
sively. In addition, other vertebrate species may eat rodents
occasionally or kill them during encounters without eating
them. This environmental pressure forced rats to develop, in
the process of natural selection, anatomical, physiological,
and behavioral adaptations enabling their individual and
species survival; such adaptations, which represent behav-
ioral adjustments to minimize bodily harm and/or death,
are termed defensive responses (McFarland 1987). Taking
into consideration the position of rats in the food chain,
researchers hypothesized that the rich repertoire of rat defen-
sive responses, including ultrasonic vocalization, developed
primarily as an antipredator behavior, and to a lesser degree
as a result of interactions between individuals within the spe-
cies (Blanchard and Blanchard 1988, 1989, 1990; Blanchard
et al. 1990).
Communication in the ultrasonic range of frequencies
(above 20 kHz) has a significant advantage over communi-
cation by sonic signals (below 20 kHz): many predators,
although not all, cannot hear ultrasounds (e.g., birds of
prey). Moreover, ultrasonic vocalizations are propagated
more directionally than audible sound, they are difficult to
detect and localize, and they dissipate easily in environ-
mental obstacles and weather conditions (e.g., wind, rain,
fog). Finally, predators on the ground or in the air cannot
hear these calls when they are emitted in underground
burrows.
Defensive behaviors may be primary or secondary in na-
ture (Edmunds 1974) but all are specific to the individual—
that is, only the animal demonstrating the behaviors benefits
from them. Primary defensive behaviors decrease the chance
of being noticed or found by a predator, whether the predator
is nearby or not. They include thigmotaxy, when an animal
avoids open spaces and remains or moves in contact with
objects, or anachoresis, when the animal remains for a pro-
longed period of time in burrows or other hiding spaces, usu-
ally during the daytime (Edmunds 1974; McFarland 1987;
Treit and Fundytus 1988). A secondary defensive behavior
44
appears once the animal has noticed the presence of a preda-
tor (or other localized source of danger) and acts to avoid or
eliminate the threat. This category includes many classic de-
fensive behaviors such as immobility (freezing), avoidance,
escape, threatening, or defensive attack (Edmunds 1974;
McFarland 1987).
The evolution of social life in most species created new
forms of defensive behavior. Animals living in groups do not
need to be in a constant state of utmost vigilance and careful
observation for the possible appearance of predators, since
warning signals will likely arise from other group members
that spot the predator first (Edmunds 1974). Thus the social
group provides some security. Rats developed well-organized
colonies with 22 kHz vocalizations that serve as alarm calls
(Blanchard et al. 1991, 1992) and thus form a higher-order
defensive system (Brudzynski and Holland 2005). The
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system depends on the fact that ultrasonic calls emitted
by one individual alarm and activate the whole colony, thus
the entire social group benefits from the behavior of one
individual.
But communication in the ultrasonic range of sound
frequencies only increases the chances of survival, it does
not fully protect rats from predators. Indeed, the rat emit-
ting alarm calls (usually the alpha or other dominant male;
Blanchard et al. 1991) may do so even when the calls could
attract the predator’s attention to the vocalizing rat. There-
fore many other factors contribute to rodent survival,
such as the location of a particular rodent on its territory,
duration of residence in a colony, and age, as was recently
shown for prairie dogs (Hoogland et al. 2006). Also, the
animal’s involvement in a particular type of activity, or its
preoccupation with important biological functions such
as sexual behavior or fighting in males, or pregnancy in
females, may further influence its susceptibility to capture
by predators despite early warning signals (Hoogland et al.
2006).
Detection of Ultrasounds
Equipment
Because ultrasounds are (by definition) beyond the human
hearing threshold, they require computerized electronic
equipment to transform the frequency by lowering it to the
human hearing range. The transformation is done by bat de-
tectors, which were initially, as the term implies, designed
and used to detect the ultrasonic signals of bats (Pierce and
Griffin 1938). These instruments lower or divide the sound
frequency by a given factor (usually 10 or 16 times); thus,
for example, the bat detector would transform a frequency of
20,000 Hz to 2000 Hz or 1250 Hz, well within the range of
human audibility.
Acoustic analysis of detected ultrasonic vocalizations re-
quires their storage, for which several options are available:
the divided signal can be recorded on a classical tape
recorder, or digitized and stored on the computer drive as
ILAR Journal
a divided frequency record, or the original signal can be
digitized by the analyzing system. The most modern acoustic
recording and analyzing systems do not require bat detectors,
instead recording, digitizing, and saving the signals directly
on the hard drive of a computer in the original, undivided
form. This method of recording is more accurate, eliminating
the distortions introduced by bat detectors during frequency
division (Holland and Brudzynski 2004), but it requires more
expensive equipment.
Acoustic analysis also requires the use of a sonograph
or sonographic computer program, which uses digitized
records to show the sound as it is emitted, displaying it in a
sonogram, a graph showing the sound frequency on the y-
axis and time on the x-axis, thus illustrating changes of
sound over time. Sonographs can further generate spectro-
grams (also called power spectra), which show all individual

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sound frequencies (in kHz) and their relative power (in dB)
on a separate graph.
Parameters
The acoustic outcome from the bat detector is not how the
original call sounded because the instrument changes the
frequency, but the output faithfully conveys other parame-
ters that are useful to researchers’ efforts to characterize
the calls. These parameters usually include the duration of
ultrasonic signals (in ms), bandwidth (i.e., the difference
between the call’s minimal and the maximal sound frequen-
cies, in kHz), frequency modulation, and the overall sono-
graphic pattern of the call (e.g., sweep, trill, step). Use of
the spectrogram is necessary to calculate the original peak
frequency of an individual call (its strongest frequency
component, i.e., the one that had the highest amplitude and/
or was the most abundant) by, first, identifying the peak
frequency and then multiplying it by the frequency division
of the bat detector. The basic acoustic parameters are illus-
trated in Figure 1.
In addition, the call rate (number of calls per time unit) is
a useful parameter to determine the magnitude of calling. A
series of individual calls is separated by breaks (intercall
intervals) of about 100 ms apiece (although they may be
much longer). The challenge is to determine how short these
breaks might be, a difficult measure as rats sometimes emit
fragmented calls. Fragmentation happens with long calls
when the animal emits the call with a low air pressure and
occasionally loses the sound, as a weak human whistle may
be interrupted by too little airflow. The ability to recognize
two sounds as either separate vocalizations or two fragments
of one call comes only with experience. Call fragmentation
also poses a significant problem in studies that depend on the
use of automated vocalization counting devices, which count
all sounds separated by a given time interval (including bro-
ken calls) as two or more separate calls, thus inflating the
number of emitted calls.
Ultrasonic calls, and particularly the 22 kHz alarm calls,
are usually emitted in series. Thus, another acoustic param-
Figure 1 Exemplary sonogram of a pup isolation call with explana-
tion of the main acoustic parameters. The difference between the
maximal and the minimal sound frequency provides bandwidth,
while the peak frequency was found from a spectrogram, which
showed that the peak frequency of 44.48 kHz was the most abun-
dant frequency in this particular call, which is neither a 22 kHz nor
50 kHz call. The duration of the call is shown below in ms. The
graph (modified) is from Brudzynski SM, Kehoe P, Callahan M.
1999. Sonographic structure of isolation-induced ultrasonic calls of
rat pups. Dev Psychobiol 34:195-204.
eter describes the number of calls per series or the number of
series and the interseries time interval.
Emission of 22 kHz Alarm Vocalizations
Circumstances
Rats emit the 22 kHz alarm call when facing danger, which
includes any circumstance that causes significant anxiety
in anticipation of an aversive, potentially harmful, or life-
threatening situation (e.g., an aggressive opponent, predator,
large mammal, loud noise). The animal may also emit such
calls when it expects a known unpleasant stimulus without
exact information about when it will happen (e.g., when an
aversive stimulus appears in an unpredictable way or startles
the animal). Classic examples are the presence of a predator
(e.g., a cat at a distance that allows the rat to escape), detec-
tion of a predator’s odor (Blanchard et al. 2005), confronta-
tion with a dominant and aggressive rat (Panksepp et al.
2004; Thomas et al. 1983), an encounter with an unfamiliar
human (Blanchard et al. 1986; Brudzynski and Ociepa
1992), or even a light but unpredictable and sudden air puff
(Brudzynski and Holland 2005; Knapp and Pohorecky 1995)
or startling acoustic stimulus (Kaltwasser 1990, 1991).
Other circumstances in which rats emit 22 kHz vocaliza-
tions are dyadic encounters such as fights or sexual contact
(Barfield and Geyer 1972; Barfield and Thomas 1986; Lore
et al. 1976; Thomas et al. 1983). In these situations, only one

Volume 50, Number 1 2009 45

rat in the pair—for example, the losing rat during a fight—
emits the calls (Panksepp et al. 2004; Thomas et al. 1983),
which not only signal an aversive and potentially dangerous
situation for the loser but may also serve to appease the ag-
gressor so that it reduces or stops the attacks, although stud-
ies have not confirmed the latter (Thomas et al. 1983). In
sexual contacts, males emit 22 kHz calls toward the end of
the series of copulations, probably signalling either a change
from an appetitive to an aversive affective state and expres-
sion of the absolute refractory period (Barfield and Geyer
1972) or cessation of contact with the female (Van der Poel
and Miczek 1991). Males may also emit such vocalizations
if they experience unsuccessful intromission while attempt-
ing to copulate (McIntosh et al. 1984) or if a female shows a
low level of lordosis and a high number of agonistic re-
sponses, making the sexual encounter difficult and aversive
(Brown 1979).
Social conditions and status may also influence the mag-
nitude (i.e., number and/or duration) of 22 kHz calls. Indi-
vidually reared rats emitted significantly fewer such calls in
response to a mild aversive stimulus than rats reared in pairs
(but tested individually) (Inagaki et al. 2005). Among rats
that remained in established pairs, the subordinate rats emit-
ted significantly more 22 kHz calls than the dominant ones
(Inagaki et al. 2005).
Although rats generally emit 22 kHz alarm calls in aver-
sive situations, the calls do not express direct pain. In fact, a
vocalizing rat subjected to acute pain ceases to make ultra-
sonic calls. One study reported the emission of similar au-
dible calls as a result of direct painful stimuli, and noted that
the acoustic structure of the calls was dependent on the in-
tensity of the nociceptive stimulus (Jourdan et al. 1995). The
same group more recently demonstrated that the 22 kHz
calls do not represent affective pain (Jourdan et al. 2002): the
calls signal anxiety caused by potential danger, not an actual
painful or nociceptive event. For example, highly aversive
foot shock associated with more intensive anxiety (unavoid-
able shock) caused more 22 kHz calls than the same foot
shock associated with less anxiety (avoidable shock; Kikusui
et al. 2003). Thus, the magnitude of the anxiety and not the
physical nature of the aversive stimulus is the driving force
for expression of alarm calls.
The biological role of these calls may be further illustrated
by the example of a rat threatened by a cat. If the intensity of
the danger is high enough (e.g., the cat is within striking dis-
tance), the rat will either become silent or emit audible warn-
ing squeals (Blanchard et al. 1991; Litvin et al. 2007). If the
rat can escape, it will be silent until it reaches a safe place
(e.g., in or close to its burrow); then, when the immediate life-
threatening danger is over, it will emit the alarm calls. The 22
kHz vocalizations signal a proximate danger to the calling rat,
and the rats who hear the alarm call receive a message that
the individual emitting it is only in potential danger and still
has a chance to escape. The rats in the colony may therefore
not respond to the distant 22 kHz alarm call—unless they hear
an abrupt stop to an ongoing call, when they respond with
freezing and alert attention (Brudzynski and Chiu 1995).
46
Characteristics and Mechanisms
Rats’ alarm calls are long—a single call may last from 100
ms to well over 3000 ms (Figure 2A)—and the peak fre-
quency remains mostly within a narrow band of 20-23 kHz,
although some calls can reach 26 kHz or even 30 kHz (the
alarm calls of females may be shorter and of a slightly higher
sound frequency than those of males; Blanchard et al. 1992;
Haney and Miczek 1993). The vocalizing rats try to maintain
a constant sound frequency, so frequency modulation is al-
most absent and 22 kHz calls appear on a sonogram as
straight flat lines with a narrow bandwidth of 1-4 kHz. These
calls are emitted usually in short series of 2 to 7 calls. There
are also some short 22 kHz calls (less than 100 ms in dura-
tion), emitted almost exclusively after the long calls, but
their communicative role is unknown (Brudzynski et al.
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1993; Brudzynski and Holland 2005).
All adult rats of different strains and ages know how to
reach the “alarming” frequency. How? There are undoubt-
edly some inborn mechanisms that enable them to do so.
Rats do not always begin their call at the right frequency,
so they “tune” the call down to the narrow band of 20-23
kHz. Thus the very first alarm call, or the first in a series of
calls, often has a mild downward frequency modulation (i.e.,
decreasing from a slightly higher to lower sound frequency;
the tuning is never upward). Once the call reaches the low
band, the frequency remains constant. This observation sug-
gests that rats attempt to emit their alarm calls at as low an
ultrasonic frequency as possible, reaching a common frequency
Figure 2 Sonograms of typical 22 kHz (A) and 50 kHz (B) calls.
During 2 s of recording, a rat emitted one 22 kHz call (A) and seven
50 kHz calls (B). The duration of the 22 kHz call is 1125 ms, while
the 50 kHz calls do not exceed 50 ms in duration. Average peak
frequency for the 22 kHz call is 23 kHz and peak frequencies of the
50 kHz calls are between 54.5 and 62.5 kHz. The sonogram in (B)
(modified) is from Brudzynski SM, Pniak A. 2002. Social contacts
and production of 50 kHz short ultrasonic calls in adult rats. J
Comp Psychol 116:73-82.
ILAR Journal
band based on the physical features of their respiratory sys-
tem (for details, Brudzynski and Holland 2005).
Rats also have innate mechanisms associated with their
response to the alarm calls. In addition to the direct alarming
properties of the calls, data suggest that an innate mecha-
nism facilitates defensive responses to 22 kHz calls. Rats
readily associate aversive and dangerous situations with 22
kHz alarm calls and retain this association in their memory
for a significantly longer time than they do for events associ-
ated with other types of calls or ultrasonic stimuli (Endres
et al. 2007).
Emission of 50 kHz Appetitive
Vocalizations
Circumstances
Rats emit 50 kHz calls, sometimes termed social calls, in
many situations that appear neutral to the human observer
but that seem to be associated with a positive social function.
The circumstances for such calls may be appetitive or neu-
tral (i.e., nonaggressive, nonaversive, or “friendly”) behav-
ioral situations such as sexual contacts and the expectation
of such contact, other rewarding situations (e.g., anticipation
of play), encounters with an anesthetized (immobile and
therefore easily approachable) conspecific, meeting rats af-
ter a period of separation, or in the initial meeting of a resi-
dent-intruder pair (to signal nonaggression) (Bialy et al.
2000; Blanchard et al. 1993; Brudzynski and Pniak 2002;
McGinnis and Vakulenko 2003; Takahashi et al. 1983). Juve-
nile rats emit the same type of call during homospecific
(rough-and-tumble) play or heterospecific play with a human
hand (“tickling”) (Burgdorf and Panksepp 2001; Knutson
et al. 1998; Panksepp and Burgdorf 2000).1
Rats also emit large numbers of 50 kHz calls in anticipa-
tion of other rewarding situations—for example, conditioned
place preference, a feeding session, rewarding brain stimula-
tion, or receipt of a drug with rewarding properties (Burgdorf
et al. 2000; Knutson et al. 1999). They also emit many such
calls when they enter places frequently visited by other rats
(even in the absence of those animals), demonstrating that the
emission of these calls is associated with expecting and/or
seeking social contacts (Brudzynski and Pniak 2002). More
specifically, the number of calls is directly proportional to the
number of rats that had previously visited the cage (Brudzynski
and Pniak 2002). However, calling associated with direct so-
cial contact may decrease in the constant presence of familiar
conspecifics; and social housing, as opposed to isolation, de-
creased the number of 50 kHz calls that juvenile rats emitted
1It is worth mentioning that most juvenile rats readily learn to play with
humans as they play with their siblings—after just 2-3 repetitions most
juvenile rats will follow a human hand and emit many 50 kHz calls (Burgdorf
and Panksepp 2001; Burgdorf et al. 2005). Many rats in adulthood may still
emit such calls in contact with a familiar human (Schwarting et al. 2007),
although these contacts do not have the features of juvenile play.
Volume 50, Number 1 2009
in response to heterospecific play (“tickling”; Burgdorf and
Panksepp 2001). This result is the opposite of the effects of
social housing on the emission of 22 kHz alarm calls.
Characteristics
Appetitive 50 kHz calls are very brief, approximately a hun-
dredth of the duration of an alarm call, and their sound fre-
quency is subjected to more modulation than alarm calls
(Figure 2B). The average duration of a 50 kHz call is 30-40
ms, with a peak frequency of 45-55 kHz, although it may
reach 70 kHz. The bandwidth of these calls is 5-7 kHz,
broader than that of 22 kHz calls.
Researchers recently distinguished two major types of
50 kHz calls: constant frequency (flat) calls and frequency-
modulated calls (step calls with trills; Burgdorf et al. 2007;
Downloaded from http://ilarjournal.oxfordjournals.org/ at Istanbul University on April 22, 2014
Wöhr et al. 2008). Further experimental data indicate that rats
emit one or the other type of call in dissimilar situations. Calls
with the flat sound frequency seem to play a social coordinat-
ing function (Wöhr et al. 2008), to be associated with aggres-
sive situations, or to indicate social ambivalence (Burgdorf
et al. 2008), whereas the frequency-modulated calls (step calls
or combined step-trill calls) are characteristic of reward situa-
tions and the expression of positive affect (Burgdorf et al.
2008). These calls may represent a vocal homologue or a pre-
served-in-evolution counterpart of human laughter (Panksepp
and Burgdorf 2000)—the calls themselves are not laughter in
the human sense, but rather are used by rats in a way that is
somewhat analogous to human laughter.
Neurochemical Substrates for Emission of
22 kHz and 50 kHz Calls
Rats emit either 22 kHz or 50 kHz calls depending on the
situation, and both the circumstances and pattern of the
emission of these calls seem to be mutually exclusive
(Brudzynski 2007). While 22 kHz calls are associated with
aversive situations, behavioral inhibition, and breaking con-
tacts among rats, 50 kHz calls are associated with appetitive
situations, reward, behavioral activation, and approach
(Brudzynski and Chiu 1995; Wöhr and Schwarting 2007).
There are extremely rare, behaviorally ambiguous situations
with brief bouts of emissions of both types of call, but rats
cannot acoustically mix the 22 kHz and 50 kHz calls.
Furthermore, the acoustic parameters of the two calls
are not overlapping and are often separated by a significant
safety space that ensures other rats’ unambiguous recogni-
tion of the calls (Brudzynski 2007). It is likely that each call
type signals a particular behavioral state and that the recipi-
ents of the calls recognize and understand them as conveying
information about the state of the caller. Such understanding
is crucial for the receiving rat to determine its own appropri-
ate behavioral response (Brudzynski 2007).
But how does the rat brain initiate these two different and
mutually exclusive types of ultrasonic vocalizations? Initia-
tion is regulated by two ascending systems that originate in
47
the brain stem tegmentum: the cholinergic system and the me-
solimbic dopaminergic system (Figure 3). In a broad sense,
these two systems are part of the ascending brainstem reticular
system, which evolved as an ancient generalized arousal sys-
tem (Pribram 1971) and regulates the entire state of the organ-
ism (for further discussion of the state of the organism, see
McCarley 1980 and Brudzynski 2007).
Activation of the rat’s ascending cholinergic system,
which originates from the laterodorsal tegmental nucleus
and travels to the basal forebrain and limbic areas (Figure 3,
filled arrows), induces defensive behavior and intensive 22
kHz vocalization (Brudzynski and Barnabi 1996; Brudzyn-
ski 2001). Pharmacological activation of this system by
cholinomimetics also induces 22 kHz alarm calls, while an-
tagonism of the system by anticholinergic agents blocks or
decreases the number of such calls (for details, see Brudzyn-
behavioral activation—increased locomotor activity, explora-
tion, and an increase in the number of 50 kHz vocalizations
(Brudzynski 2007; Burgdorf et al. 2001; Thompson et al.
2006). Systemic or intra-accumbens application of amphet-
amine significantly increases the number of 50 kHz calls.
Activity of the ascending cholinergic system induces a
general negative state, defined as a response to “external or
internal stimuli and/or situations (complex stimuli) that pres-
ent threat or danger to the organism, cause or can cause physi-
cal damage or impairment, biological or sociobiological
destabilization, and disruption of physiological functions and
balances” (Brudzynski 2007, 262-263). The negative state of
the organism includes not only adaptive changes in the so-
matic, autonomic, and endocrine systems but also a negative
affective state (for a full discussion, see Brudzynski 2007).
Activity of the ascending dopaminergic system induces a

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ski 2001).
On the other hand, activation of the rat’s ascending do-
paminergic system, which originates from the ventral tegmen-
tal area and terminates in the nucleus accumbens and other
basal forebrain structures (Figure 3, blank arrows), induces
general positive state, defined as a response to “stimuli and/
or situations that minimize threat or danger to the organism,
enhance its security, preserve physical and social integrity,
improve, or can improve biological or sociobiological
stability, maintain and stabilize physiological functions and

Figure 3 Two ascending tegmental systems responsible for the initiation of vocalization. The system originating from the laterodorsal teg-
mental nucleus (LDT) is cholinergic (filled arrows). Release of acetylcholine in the medial cholinoceptive vocalization strip (stippled area)
induces 22 kHz vocalization. Main targets of this system are anterior hypothalamus (AH), preoptic area (PO), bed nucleus of stria terminalis
(BS), and lateral septum (LS). The system originating in the ventral tegmental area (VTA) is dopaminergic (blank arrows). Release of do-
pamine in the nucleus accumbens (NA) and neighboring areas (TU, olfactory tubercle) induces 50 kHz vocalization. The diagram (modified)
is from Brudzynski SM. 2007. Ultrasonic calls of rats as indicator variables of negative or positive states: Acetylcholine-dopamine interaction
and acoustic coding. Behav Brain Res 182:261-273.

48 ILAR Journal
balances” (Brudzynski 2007, 263). The positive state of the
organism includes the relevant changes in the somatic, auto-
nomic, and endocrine systems, as well as a positive affective
state (for a full discussion, see Brudzynski 2007).
These observations and numerous experimental results
led to the postulation that 22 kHz alarm calls and 50 kHz
appetitive calls can predict the state of an organism and thus
serve as reliable indices of the general (including the affec-
tive) state of the rat organism2 (Brudzynski 2007; Knutson
et al. 2002 had already postulated the usefulness of the ultra-
sonic vocalization in assessing an animal’s affective state).
Based on the evidence presented in this review, one may fur-
ther postulate that rats’ 22 kHz and the 50 kHz calls are also
reliable predictors of increased cholinergic or dopaminergic
activity in the brain.
Conclusions
Rats are highly social animals that can emit audible sounds
but communicate among themselves predominantly in the
ultrasonic range of sound frequencies. Their ultrasonic vo-
calization evolved as a defensive behavior and an antipreda-
tor adaptation in particular.
Rats emit two basic types of ultrasonic calls: 22 kHz
(alarm) calls and 50 kHz (appetitive or social) calls. Behav-
ioral observations consistently indicate that a rat’s 22 kHz
vocalizations signal aversive situations and a negative state,
and 50 kHz calls signal appetitive situations and a positive
state. The calls differ dramatically in their acoustic param-
eters and are easily distinguishable: the alarm calls (22
kHz) are long (from 100 to 3000 or more ms) and have a
low sound frequency, whereas the appetitive calls (50 kHz)
are short (30 to 40 ms) and have a twofold higher sound
frequency.
The two call types are driven from the core limbic mid-
brain regions by two ascending projections: the activity of
the ascending cholinergic system initiates alarm calls and
the ascending dopaminergic system initiates appetitive calls.
These two systems work in a mutually exclusive way, thus
animals normally emit only one or the other type of call.
The underlying neurochemical mechanisms and consis-
tency of acoustic parameters of these two different calls
make them good indices of the rat’s general and affective
state. The 22 kHz alarm calls inform about the rat’s anxiety
and negative state, and the 50 kHz calls about its appetitive
and positive state. Although the calls may be useful in as-
sessments of a rat’s affective state, ultrasonic vocalization
does not provide information about a rat’s physical or affec-
tive (the psychological dimension) pain.
2The general state of an organism is a “sum of measurable values obtained
from a (practically infinitive) number of variables describing functions of
the whole organism” (Brudzynski 2007, 262, based on Rosen 1970). Thus
the state of the organism includes both the psychological (affective)
dimension as well as the physiological correlates from subcellular to
cognitive parameters.
Volume 50, Number 1 2009
Acknowledgments
Studies originating from our laboratory were supported by
the Natural Sciences and Engineering Research Council of
Canada.
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