camila demaestri – honors thesis · camila demaestri – honors thesis 1. introduction the...

Camila Demaestri – Honors Thesis

Anxiety-like responses in rats presented with playback of 22

kHz ultrasonic vocalizations

Camila Demaestri

Honors Thesis

Northeastern University Department of Psychology

College of Science 2017

Readers:

Heather C. Brenhouse, PhD Jennifer A. Honeycutt, PhD

Rebecca Shansky, PhD


Abstract: The basolateral amygdala (BLA) plays a crucial role in processing a variety of emotions,

including fear and anxiety-like states. When humans are presented with a fearful face (a strong

social cue) while undergoing functional magnetic resonance imaging (fMRI), amygdala activity

and corticolimbic circuitry associated with anxiety can be functionally studied. Previous work

supports the use of the fearful-face task to investigate aberrant corticolimbic circuitry in

populations who have experienced early life adversity. During this task, children with a history of

adversity show functional connectivity that is comparable to adolescents/young adults, which is

indicative of possible premature activation of this anxiety-related circuitry. Currently available

techniques and limitations in human studies make it difficult to systematically manipulate

neuronal and environmental variables to better understand the mechanistic underpinnings of

this precocial connectivity. Thus, we sought to model this phenomenon in rodents by

implementing novel methodology to investigate the mechanistic and neuronal changes

underlying aberrant connectivity. In order to create a rodent paradigm for use in fMRI research,

the present work was completed and provides key data to determine suitability of the novel task

to elicit anxiety-like responses. This was done in rats using an ethologically relevant analogue of

the fearful-face task via the presentation of pre-recorded fear-induced ultrasonic vocalizations

(USV; 22 kHz). The present study provides critical proof-of-concept, preliminary data

characterizing anxiety-like behavioral and physiological responses in rats presented with the

fear-induced USVs. Our findings show that rats exposed to 22 kHz USVs: 1) engage in anxiety-

like behaviors characterized by increased immobility and decreased exploration during

playback; 2) show increased heart rate variability; and 3) increased c-Fos activation within the

amygdala compared to rats exposed to a synthetic tone control stimulus in the 22 kHz range.

These results provide critical groundwork for developing a highly novel way of studying anxiety

circuitry in rodents and aid in improving our understanding of anxiety-related dysfunction and

psychiatric illnesses such as depression, anxiety, and schizophrenia.


1. Introduction

The corticolimbic system plays a major role in the regulation of emotionally

salient information. The brain structures involved in this circuit, such as the

hypothalamus, hippocampus, and amygdala, support critical functions including

motivation, long-term memory, and emotion. These regions work in synchrony with both

each other and the prefrontal cortex (PFC) in order to understand and process socially-

or emotionally-relevant stimuli. Specifically, strong reciprocal connections between the

PFC and the basolateral regions of the amygdala (BLA) are highly implicated in fear-

and anxiety-related reponses (Janak & Tye, 2015). The PFC has protracted

development and serves to regulate higher order cognitive functions (Alexander et al.,

1978). It is also a key region that receives the anxiogenic information emerging from the

BLA and modulates the BLA via inhibitory output (Adolphs et al., 1994; Phan et al.,

2002; Zald et al., 2003). In addition, the BLA itself selectively innervates the PFC (Janak

& Tye, 2015) and the connectivity between these regions plays a role in emotional

regulation and processing whereby meaning is attached to behaviorally relevant stimuli

and also drives behavioral output. These reciprocal connections allow the BLA to signal

the PFC regarding emotionally salient stimuli, which is subsequently modified by the

PFC, depending on the individual’s goals and visual and auditory confirmations of

possible threat stimuli (Shwartz et al., 2014).

The amygdala – including the BLA – has a well-established role in fear

processing, aversive conditioning, and social behavior (LeDoux, 2000; Amaral, 2002;

Schafe and LeDoux, 2004). Brain-imaging studies have shown evidence supporting that

amygdala activation via the presentation of emotionally salient pictures does not depend


on the subjects’ awareness of the stimuli (Whalen et al., 1998). Thus, the amygdala

responds early in the presentation of a stimulus and allows for quick regulation of

emotional responses. Although, BLA activation is seen in response to both negative and

positive stimuli, due to their direct importance to survival, negative stimuli will

preferentially activate the BLA (Costafreda et al., 2008).

BLA activation can be functionally studied by presenting humans with a fearful-

face in an fMRI. A fearful-face is a socially relevant cue in humans that has high

emotional significance. In addition, single neuron recordings have found that the

amygdala codes for the recognition of emotional facial expressions (Fried et al., 1997).

The fearful-face task in humans is useful in studying the functional connectivity of brain

regions implicated in anxiety. In fact, an altered pattern of PFC-BLA task-based

functional connectivity is seen in children who have experienced adverse rearing

conditions, which is comparable to the pattern seen in adults (Tottenham et al., 2012).

Human studies provide useful insight on the aberrant connectivity patterns of this

circuitry. However, human studies do not allow for systematic manipulation of – and

control for – other variables that may contribute to such findings. Thus, a translational

rodent paradigm is needed in order to more effectively study the mechanistic

underpinnings of possible circuitry dysfunction. Here, we sought to model the fearful-

face task for humans by implementing a novel task for rodents in order to study this

anxiety-related circuitry in a task-based functional manner. The analogue to the

behaviorally-relevant, emotionally-stimulating and fear-inducing face task in humans are

aversive ultrasonic vocalizations (USVs) in rats. Because rats communicate via the use


of USVs, they are a socially-relevant stimulus that are behaviorally and meaningfully

akin to emotionally-charged facial expressions in humans.

USVs are the primary means of communication between rats. Specifically, USVs

in the 22 kHz range are emitted from juvenile and adult rats when in aversive situations

and when aroused by potential threats (Portfors, 2007). USVs in the 22-kHz range can

be recorded from rats who are exposed to predators (Blanchard et al., 1991; Brudzynski

et al., 1992), to pain (Borta et al., 2006), and during social defeat (Vivian and Miczek,

1993). Additionally, recent evidence suggests that there is an audience effect in vocal

behavior, such that rats will reliably vocalize when in proximity to another rat versus

being alone (Seagrave et al., 2016). Thus, aversive USVs are usually expressed by the

rat in a social context and are used to communicate an aversive state and, possibly, to

signal to conspecifics the possible presence of danger. This type of communication is

analogous to fearful facial expressions evoked in humans when exposed to a possible

or perceived threat because they are both socially relevant cues. In addition, studies in

rodents have shown that playbacks of aversive USVs induce behavioral inhibition, flight

or fight responses, and an increase in BLA activation (Neophytou et al., 2000; Endres et

al., 2007; Sadananda et al., 2008). Here, we propose the novel utilization of pre-

recorded aversive USVs to instigate a subtle anxiety-like state in rats as an analogue of

the fearful-face task in humans.

The proposed study determined the anxiogenic effects of aversive USV

presentation with the future goal of implementing this novel task to study the effects of

ELS on functional corticolimbic activity. In order to determine that the fear-induced

USVs are anxiety-provoking in rats, we played back either a pre-recorded aversive 22


kHz vocalizations or a synthetic tone control (imitating the kHz range of aversive USVs)

and examined the behavior, physiology and signs of neural activation within a subset of

rats. We hypothesized that the rats exposed to recorded USVs in the 22 kHz range

would 1) engage in anxiety-like behaviors; 2) show a change in heart rate variability;

and 3) show increased neural activation in the BLA compared to rats who were exposed

to a synthetic tone. Here, we provide compelling evidence for the use of aversive USVs

as socially relevant, anxiety provoking stimuli in the rat as an analogue to the human

fearful-face task, thereby allowing further analyses of corticolimbic circuitry and its role

in anxiety.

2. Methods

2.1 Animals

Twenty-four male postnatal day 35 (P35) Sprague-Dawley rats were received from

Charles River Laboratories (Wilmington, MA) and were housed under constant

temperature- and humidity-controlled conditions within a 12h light/dark cycle, with water

and food available ad libitum. Rats were pair housed and left undisturbed for 6 days to

acclimate to the colony room. They were then handled for 4 days for 10 minutes per day

in order to habituate them to experimenter contact and movement to and from the

experimental room. At P45 heart rate and behavior were monitored during the

presentation of either aversive USVs in the 22 kHz range or with a comparable synthetic

tone also in the 22 kHz range. At P85, a subset of 18 rats were presented with either a

playback of USVs, synthetic tone or no noise in order to evaluate c-Fos expression

within the BLA and auditory cortices. This was done in a counter-balanced manner with


rats placed in a stimulus group different to that of the previous experiment to avoid

possible confounds from stimulus-specific previous exposure.

2.2 Natural vocalizations and synthetic tone

The fear-induced aversive USVs in the 22 kHz range were recorded from an

adult male rat using Avisoft-RECORDER Bioacoustics Recording Software (Glienicke,

Germany). The rat was restrained for 15 min and placed in a cage inside a sound-

attenuating box infused with cat odor via a rag with cat urine and a bag of cat fur (both

collected 24 hours prior to the vocalization collection). In addition, and in order to

increase likelihood of vocalizations due to audience effect (e.g., Seagraves et al., 2016),

a rat matched for age and sex was anesthetized with 0.3 mL/kg of a ketamine/xylazine

cocktail and placed in the same cage in close proximity to the restrained rat.

Avisoft-SASLab Pro was used to analyze and refine the recordings for playback.

The average frequency (22.97 kHz), duration (2.51 sec), and intensity (-71 dB) of the

recorded USVs were analyzed and used to prepare the synthetic tone (Table 1). The

aversive USV recording was high-pass filtered with a cut-off frequency of 15 kHz to

reduce the presence of low frequency background noise for experimental playback.

The synthetic tone was prepared using Audition 3.0 software and was created to

imitate the frequency, length and intensity (dB) from the natural vocalization recordings

(Table 1B). The frequency spectrum of the stimulus was 22 kHz and consisted of a

series of segments lasting 3 seconds long and spaced by 2 seconds. Frequency

spectrograms for both the aversive USV and the synthetic tone can be seen in Figure 1.


2.3 Auditory stimulus exposure

At P45, following habituation to the testing environment and apparatus, ECGs of

awake behaving rats were recorded non-invasively using an ECGenie apparatus

(Mouse Specifics, Inc.). The ECGenie apparatus was used to record cardiac electrical

signals at 2 kHz in the awake rat by placing the animal on a 6.5 cm x 7 cm recording

platform that acquires signal through footpad electrodes located on the floor of the

platform. Each rat was placed in the ECG recording chamber individually, and the

chamber was hooked up to the heart rate monitor and placed 24 cm away from the

playback speaker. The test consisted of 3-minutes of acclimation, followed by 3-minute

baseline recording and then a 3-minute stimulus exposure of either the synthetic tone or

aversive USV playback. The recording chamber was cleaned with 30% ethanol solution

between each animal.

Raw ECG signals were analyzed using eMOUSE software (Mouse Specifics).

Heart rate variability (HRV) was calculated at baseline and during stimulus exposure

time points. A one-way ANOVA compared HRV between groups, followed by post hoc

Bonferroni t-test analyses to determine differences. An independent sample t-test

compared change in HRV between synthetic tone and USV presentation when

compared to baseline. Rats with insufficient heart rate data due to technical difficulties

with the recording equipment were excluded from analysis.

Video recordings of each rat during ECG collection were collected and behavioral

measures were later analyzed by two blind investigators (the results were averaged

between the two scores). The amount of time immobile, grooming, and

sniffing/exploring was recorded and compared between baseline and stimulus exposure


time points. Amount of time immobile was characterized as the animal being completely

still. Amount of time sniffing/exploring was characterized as the animal moving around

the chamber and sniffing. A one-way ANOVA compared the groups and was followed

by post hoc Bonferroni t-test analyses to determine main effects and group differences.

2.4 c-Fos Immunohistochemistry

At P85, a subset of the HRV experimental rats were assigned to either synthetic

tone, an aversive USV, or no stimulus (counterbalanced and to be assigned to a

stimulus they had previously not been exposed to) for c-Fos immunohistochemistry

analysis. Rats were presented with their assigned stimulus for 45 minutes while single

housed and in a cage identical to their home cage. Fifteen minutes following the

completion of stimulus presentation, they were transcardially perfused with physiological

(0.9%) saline followed with a 4% paraformaldehyde fixative in 0.1 phosphate buffer (pH

7.4). Brains were postfixed in 4% paraformaldehyde for 4 days at 4°C and then moved

to 30% sucrose solution at 4°C until ready for sectioning. Serial sections (40µm thick)

were collected using a freezing microtome and stored in freezing solution at -20°C until

c-Fos staining.

C-fos immunohistochemistry was conducted via a two-day procedure. On day

one, free floating sections were washed 3 times for 5 minutes in 0.1% PBS-T and then

placed in blocking buffer (5% normal donkey serum in 0.1% PBS-T) at room

temperature for 60 minutes. Sections were then washed 3 times for 5 minutes each in

0.1% PBS-T and incubated overnight at 4°C with primary antibody (Millipore rabbit anti-

c-Fos 1:1,000 + 2.5% block). On day two, slices were washed 3 times for 5 minutes


each with 0.1% PBS-T and then placed in biotinylated anti-rabbit secondary solution

(AF488; 1:600 in 2.5% block) for 90 minutes. Immunohistochemistry was concluded

with 3 washes of 5 minutes each in 0.1% PBS-T, sections were mounted on glass slides

and coverslipped with DPX for stereological analysis.

C-Fos immunoreactivity was examined in the BLA, which was delineated

according to the anatomical atlas (Paxinos and Watson, 2010). Qualitative analysis was

completed, comparing a control rat, who received no noise to a rat who was presented

with the synthetic tone and to another rat who was presented with the USV.

3. Results

3.1 Behavior

A one-way ANOVA of immobility revealed a significant main effect between

immobility during baseline, synthetic tone and USV presentation F(2,40) = 3.94, p = .027.

There was a significant increase in immobility during the aversive USV presentation (M

= 53.06, SE = 7.15) in comparison to the baseline (M = 30.33, SE = 5.45) t(40) = 2.69, p

= 0.031. See Figure 2A.

A one-way ANOVA of sniffing and exploratory behavior revealed a significant

main effect between sniffing and exploration during baseline, synthetic tone and USV

presentation F (2,40) = 4.95, p = 0.012. As seen in Figure 2B, sniffing behaviors during the

aversive USV presentation were significantly lower (M = 79.44, SE = 7.30) than during

baseline (M = 11.7, SE = 9.23) (t(40) = 2.59, p = 0.04) and to synthetic tone sniffing (M =

121.40, SE = 11.81) t(40) = 2.69, p = 0.031. There was no significant effect of group on

grooming behavior (Figure 2C).


3.2 Heart rate variability (HRV)

A one-way ANOVA of HRV did not reveal a significant effect of HRV during the

baseline, synthetic tone, and USV presentation (F(2,21) = 2.538, p = 0.10). A two-tailed,

paired t-test revealed a significant increase in HRV in the USV group (M = 11.60, SE =

1.56) compared to baseline (M = 8.21, SE = 1.30) t(4) = 3.31, p = 0.029; Figure 3. A two-

tailed, unpaired t-test between synthetic tone and USV revealed no difference in HRV

after aversive USV presentation (M = 11.60, SE = 1.56) compared to synthetic tone (M

= 8.06, SE = 0.90) (Figure 3). In addition, a separate analysis was completed measuring

the change in HRV (BPM) from baseline HRV to synthetic tone and USV HRV

(determined via [USV/TONE Presentation HRV] – [Baseline HRV] = [change in HRV]). A

two-tailed, unpaired t-test revealed a trending increase in HRV when presented with the

aversive USVs (M = 5.24, SE = 2.21) compared to the synthetic tone (M = -2.55, SE =

2.21) t(12)= 2.16, p=0.517. See Figure 4).

3.3 C-Fos IHC

c-Fos immunoreactivity of the BLA, PFC, hippocampus and auditory cortex in

response to the synthetic tone, 22 kHz USV and no noise groups is currently being

analyzed. Figure 5 shows a representative image of c-Fos activity in the BLA for the

three groups.

4. Discussion This study provides evidence for the successful and novel use of aversive USV

playback to provoke a state of arousal and anxiety in a preliminary socially-relevant rat

analogue to the human fearful-face task. Rats showed an increase in immobility and an


increase in heart rate variability when exposed to a playback of aversive 22 kHz USV

but not when exposed to the comparable 22 kHz synthetic tone. Preliminary neural

activation results show an increase in c-Fos activation of the BLA only when presented

with the aversive USV stimulus.

4.1 Ultrasonic vocalizations

Ultrasonic vocalizations are crucial for the general communication between rats

and are particularly important when communicating about a potential threat in the

environment (Seagraves, 2016). In this case, the rat emitted USVs in the 22 kHz range,

which is an index of negative affect (Blanchard et al., 1991). The USVs were used

instead of predator odor in order to stimulate an anxiety- and emotionally-arousing

response in the rat rather than a fearful one. Predator odors are well understood to elicit

a fear and “fight or flight” response in the rat (Wallace and Rosen, 2000). Fear and

anxiety are distinct concepts that share neurobiological models but are caused by

different events. A fear response tends to occur in response to something specific (such

as cat odor), while an anxiety response tends to be elicited by something less specific

and sometimes harder to pinpoint (Perusini and Fanselow, 2015). Here, we propose

that the playback of aversive 22 kHz USVs elicits an anxiety-like response in the rat,

which then leads to the regulation of the BLA by the PFC.

4.2 Behavior

Rats showed an increase in immobility in response to the aversive USV but not

the synthetic tone. The increase in immobility seen in response to aversive USVs is

distinct to the freezing behavior seen during fear-conditioning. Rather than the


crouched position seen in fear-conditioning, we see immobility, which does not

resemble a fear-like state. This behavior has not been characterized in previous

research. However, we believe that this immobility is an index of attentiveness and

alertness. A decrease in locomotor activity and the number of approaches to the loud-

speaker is seen in rats exposed to a playback of 22 kHz calls by a defeated rat

(Brudzynski and Chiu, 1994). However, some studies find moderate to no behavioral

inhibition with 22 kHz playback, despite clear changes in brain areas related to anxiety

(Bang et al., 2008; Sadananda et al., 2008). The fact that neural activation can be

measured without robust behavioral changes indicates that playback of these USVs is

processed in the limbic areas of the brain without showing a clear defensive response.

A limitation was that behavior here was recorded inside the small apparatus that

was used to record HRV. Thus, we were only permitted to study a limited range of

behaviors. In future studies, additional behavior should be analyzed in order to further

investigate the anxiety-like responses in the rat in response to USV playback. The

open-field test can be used to study time spent in the center of the arena as an index of

anxiety while also continuing to measure immobility times. Also, light-dark box can be

used to study aversion to the playback of USVs, in addition to immobility.

4.3 Heart rate variability

Here, we provide evidence that the playback of aversive USVs, and not synthetic

tone, results in increased HRV in comparison to baseline. HRV is more than just an

index of a healthy heart; it also provides information on the degree to which the brain is

regulating the periphery. HRV is primarily an index of regulation, specifically in response


to an arousing stimulus. In fact, HRV is often increased during successful performance

in emotional regulation tasks, both in social situations and during regulation of unwanted

behavior (Butler et al., 2006; Ingjaldsson et al., 2003). This physiological response has

been linked to both emotional regulation and to mPFC activity (Wager et al., 2009;

Thayer et al., 2012). The PFC has an important role in the integration of information and

regulates both behavior and physiology. Thus, in addition to regulating behavior, the

mPFC also regulates peripheral physiology (Thayer et al., 2012). Through its

connectivity with the brainstem and the vagal nerve, the PFC can regulate heart rate

changes related to social threat (Wager et al., 2009). An increase in both HRV and

activation of the thalamus and mPFC is seen when humans are presented with a

‘disgust’ face and/or disgusting picture (Lane et al., 2009). In our study, the aversive

USVs, and not synthetic tone, were emotionally relevant and arousing, which lead to the

successful interpretation of the possible threat and emotional regulation, shown by the

increase in HRV seen.

A limitation with HRV measurement in our study was the difficulty in receiving a

clean signal from several of the rats. The apparatus was made in a way that requires

the rat’s four paws to be on the floor at the same time and in a specific location, apart

from each other. Thus, HRV recordings were not analyzed when the rat was grooming

or turning around.

4.4 Neural activation

Preliminary neural activation evaluation provided evidence of BLA activation, via

c-Fos expression, in response to the aversive USV and not the synthetic tone. The BLA


is reliably activated in response to emotionally salient information, with preference

towards negative stimuli, and projects onto the PFC, which in turn is able to regulate

responses and anxiety by inhibiting BLA (Adolphs et al., 194; Phan et al., 2002; Zald et

al., 2003; Costafreda et al., 2008). An increase in c-Fos immunoreactivity has been

reported in the BLA, and hippocampus in rats exposed to both live and recorded 22 kHz

vocalizations (Ouda, Jilek, & Syka, 2016). Furthermore, Parsana and colleagues (2012)

uncovered a tonic increase in the firing rates of the BLA in response to 22 kHz playback

and decreases in firing rates in response to 50 kHz playback. Neural activity in the BLA

as well as other regions (PFC, hippocampus, and auditory cortex) are currently being

analyzed.

4.5 Conclusion and future directions

We can now use this novel methodology in order to further investigate functional

and mechanistic changes underlying aberrant corticolimbic connectivity in rats that have

experienced early-life stress. As mentioned, the PFC has protracted development and is

involved in the integration and regulation of stimuli from subcortical brain regions (such

as the BLA). The amygdala, contrary to the PFC, undergoes early structural

development and has an early disposition to respond to stressors (Callaghan et al.,

2011; Gee et al., 2013). In addition, PFC inhibitory projections onto the BLA exhibit a

delayed emergence, which suggest underdeveloped inhibitory control in response to

BLA excitation earlier in life (Arruda-Carvalho et al., 2017). Precocially mature

corticolimbic functional connectivity is seen in children who have experienced early life

adversity (Gee et al., 2009) and similar behavioral and neural phenotypes are observed


in rodent models of early life adversity via maternal separation during the postnatal

period (Brenhouse et al., 2011; Cabungcal et al., 2006). Maternal deprivation in rodents

alters the bidirectional circuits between the PFC and the BLA that, when fully

developed, serve to regulate responses to anxiety-provoking stimuli (Janak & Tye,

2015). Perhaps the accelerated development of this circuitry in response to stress

earlier in life has implications for the aberrant behavioral outputs seen in humans and in

rodents and explains the risk factor for depression, anxiety, and schizophrenia later in

life (Ritchie et al., 2009; Miller and Cole, 2010; Carr et al., 2013; Holland et al, 2014).

However, the underlying neurobiological substrates of this early environmental and

neural circuitry maturation are not known. Thus, this study has provided evidence for a

novel methodology to further investigate the mechanistic changes underlying aberrant

frontoamygdala connectivity.

Adult rats do not exclusively emit ultrasonic vocalizations in the low-frequency 22

kHz range. Future direction involves studying the neural, behavioral, and physiological

responses to USVs with a playback of USVs in the 50 kHz range. These high-frequency

USVs are emitted when rats are mating (Barfield et al., 1979), eating food high in

sucrose (Browning et al., 2011), during cocaine self administration (Burgdorf et al.,

2001), with nucleus accumbens amphetamine microinjections (Browning et al., 2011)

and when tickled by an experimenter (Panksepp and Burgdorf, 2000). In addition, rats

show an increase in 50 kHz USVs when receiving electrical stimulation of the ventral

tegmental area (Burgdorf et al., 2000) and show increased neural activity of the nucleus

accumbens with 50 kHz playback (Sadananda et al., 2008). Thus, these high-frequency

USVs are emitted from rats who are experiencing pleasure and are involved with brain


regions associated with reward. However, others have found these 50 kHz calls to

occur in situations that are neither pleasurable nor aversive. They are also emitted

when put into used cage which previously housed a conspecific (Brudzynski and Pniak,

2002), when separated from a cage mate (Wöhr et al., 2007), and when approaching a

new rat (White and Barfield, 1987). In addition, playback of 50 kHz USVs induces

approach behavior and increases distance traveled in the open field (Wöhr and

Schwarting, 2007). Thus, these findings suggest that the 50 kHz calls are not only

involved in reward and pleasure but also serve for general communication during non-

aversive situations. We would expect to see differing effects of 50 kHz and 22 kHz USV

playback, which can help us understand the effects of early-life stress on both anxiety

and reward circuitries of the brain.

Rat ultrasonic vocalizations are ethologically essential social signals. Rats emit

22 kHz USVs during negative affect circumstances and use them to communicate

potential threat or danger to another rat. The playback of 22 kHz USVs induces an

anxiety-like state in the rat in the form of immobility (an index of arousal), increased

heart rate variability (an index of emotional regulation), and increased BLA activation

(an index of anxiety). This study provides evidence for the successful and novel use of

the playback of aversive USVs to provoke a state of arousal and anxiety in the rat.


References: Adolphs, R., Tranel, D., Damasio, H., Damasio, A. (1994). Impaired recognition of emotion in

facial expressions following bilateral damage to the human amygdala. Nature, 372, pp. 669–672

Alexander, GE., Goldman, PS. (1978). Functional development of the dorsolateral prefrontal

cortex: an analysis utilizing reversible cryogenic depression. Brain Res. 143; 233–249. Amaral, D.G. (2002). The primate amygdala and the neurobiology of social behavior:

implications for understanding social anxiety. Biol. Psychiatry 51, 11–17.

Arruda-Carvalho, MA., Wu, WC., Cummings, KA., Clem, RL. (2015) Optogenetic examination of prefrontal-amygdala synaptic development. Journal of Neuroscience. 37:2976-2985.

Bang, SJ., Allen, TA., Jones, LK., Boguszewski, P., Brown, TH. (2008). Asymmetrical stimulus

generalization following differential fear conditioning. Neurobiology Learning and memory. 90:200–216.

Barfield, RJ., Auerbach, P., Geyer, LA. (1979). Ultrasonic vocalisations in rat sexual behavior.

American Zoologist. 19: 469 – 480. Blanchard, RJ., Blanchard, DC., Agulla, R., Weiss, SM. (1991). Twenty-two kHz alarm cries to

presentation of a predator, by laboratory rats living in visible burrow systems. Physiology and behavior. 50:967–972.

Borta, A., Wöhr, M., Schwarting, RKS. (2006). Rat ultrasonic vocalization in aversively

motivated situations and the role of individual differences in anxiety-related behavior. Behavioural brain research. 166:271–280.

Brenhouse, HC., Anderson, SL. (2011). Nonsteroidal anti-inflammatory treatment prevents

delayed effects of early life stress in rats. Biol. Psychiatry 70;434–440. Browning, JR., Browning, DA., Maxwell, AO., Dong, Y., J, HT., Jansen, HT., Panksepp, J., Sorg.

BA. ( 2011). Positive affective vocalizations during cocaine and sucrose self-administration: A model for spontaneous drug desire in rats. Neuroparmacology. 61: 268-275.

Brudzynski, SM., Chiu EMC. (1994) Behavioral responses of laboratory rats in playback of 22

kHz ultrasonic calls. Physiology and behavior. Vol 57, 1039-1044. Brudzynski, SM., Ocepia, D. (1992). Ultrasonic vocalization of laboratory rats in response to

handling and touch. Physiology and behavior. 52:655–660. Brudzynski, SM., Pniak, A. (2002). Social contacts and production of 50-kHz short ultrasonic

calls in adult rats. Journal of comparative psychology. 116: 73-82. Butler, EA., Wilhelm, FH., Gross, JJ. (2006). Respiratory sinus arrhythmia, emotion, and

emotion regulation during social interaction. Psychophysiology. V. 43, I. 6. 612-622.


Burgdorf, J., Jnutson, B., Panskepp, J., Ikemoto, S. (2001) Nucleus accumbens amphetamine microinjections unconditionally elicit 50-kHz ultrasonic vocalizations in rats. Behavioral Neuroscience. 115: 949-4.

Cabungcal, JH., Nicolas, D., Kraftsik, R., Cuenod, M., Do, K., Hornung, JP. (2006) Glu- tathione

deficit during development induces anomalies in the rat anterior cingulate GABAergic neurons: relevance to schizophrenia, Neurobiology. Dis. 22 624–637.

Callaghan, BL., Richardson, R. (2011) Maternal separation results in early emergence of adult-

like fear and extinction learning in infant rats. Behavioral Neuroscience. 125(1):20–28. Carlini, VP., Monzon, ME., Varas, MM., Cragnolini, AB., Schioth, HB., Scimonelli, TN., Barioglio,

SR. (2002). Ghrelin increases anxiety-like behavior and memory retention in rats. Biochemical and biophysical research communications. Volume 299, Issue 5. 739 – 743.

Carr, CP., Martins, CM., Stingel, AM., Lemgruber, VB., Juruena, MF. (2013). The role of early

life stress in adult psychiatric disorders: a systematic review according to childhood trauma subtypes. Journal of Nervous and Mental Disease. V. 201, N 12.

Costafreda, S., Brammer, M., David, A., Fu, C. (2008) Predictors of amygdala activation during

the processing of emotional stimuli: A meta-analysis of 385 PET and fMRI studies. Brain research reviews. 58, 57-70.

Endres, T., Wildmann, K., Fendt, K. (2007). Are rats predisposed to learn 22 kHz calls as

danger-predicting signals? Behavioral Brain Research V. 185, Issue 2, 69 – 75. Fan, T., Herrera-Melendez, AL., Pestke, K., Feeser, M., Aust, S., Otte, C., Pruessner, JC.,

Boker, H., Bajbouj, M., Grimm, S. (2014). Early life stress modulates amygdala-prefrontal functional connectivity: Implications for oxytocin effects. (2014) Human Brain Mapping. Volume 35. Issue 10. 5328-5339.

Felix-Ortiz, AC., Burgos Robles, A., Bhagat, ND., Leppla, CA., Tye, KM. (2016). Bidirectional

modulation of anxiety-related and social behaviors by amygdala projections to the medial prefrontal cortex. Neuroscience 321, 197 – 209.

Fried, I., MacDonalds, KA., Wilson, CL. (1997). Single neuron activity in human hippocampus

and amygdala during recognition of faces and objects. Science Direct. V18, I5; 753-765. Gee, D., Gabard-Durnam, L., Flannery, J., Goff, B., Humphreys, K., Telzer, E., Hare, T.,

Bookheimer, S., Tottenham, N. (2013). Early developmental emergence of human amygdala-prefrontal connectivity after maternal deprivation. Psychological and cognitive sciences. Vol. 110, No 39.

Holland, FH., Ganguly, P., Potter, DN., Chartoff, EH., Brenhouse, HC. (2014). Early life stress

disrupts social behavior and prefrontal cortex parvalbumin interneurons at an earlier time-point in females than in males. Neuroscience Letters. 566 131 – 136.

Hoover, WB., Vertes, RP. (2007). Anatomical analysis of afferent projections to the medial

prefrontal cortex in the rat. Brain Structure and Function. 212:149-179. Janak, PH., Tye, KM. (2015). From circuits to behavior in the amygdala. Nature. Vol 517.


LeDoux, J.E. (2000). Emotion circuits in the brain. Annual review of neuroscience. 23, 155–184.

McEwen, BS. (2008) Understanding the potency of stressful early life experiences on brain and body function. Metabolism 57(2):S11–S15. 5.

McGarry, LM., Adam, CG. (2016) Inhibitory gating of basolateral amygdala inputs to the

prefrontal cortex. The Journal of Neuroscience, 36 (36) 9391-9046. Miller, GE., Cole, SW. (2012). Clustering of depression and inflammation in adolescents

previously exposed to childhood adversity, Biol. Psychiatry 72. 34–40. Neophytou, SI., Graham, M., Williams, J., Aspley, S., Marsden, CA., Beckett, SR. (2000) Strain

difference to the effects of aversive frequency ultrasound on behavior and brain topography of c-Fos expression in the rat. Brain research. 31;854(102):158-64.

Ouda, L., Jilek, M., Syka, J. (2016). Expression of c-Fos in rat auditory and limbic systems following 22 kHz calls. Behavioral brain research. V 308, 196-204. Panskepp, J., Burgdorf, J. (2000). 50-kHz chirping (laughter?) in response to conditioned and

unconditioned tickle-induced reward in rats: effects of social housing and genetic variables. Behavioral brain research. 115: 25-38.

Parsana, AJ., Li, N., Brown, TH. (2012). Positive and negative ultrasonic social signals elicit

opposing firing patterns in the rat amygdala. Behavioral Brain Research. 226(1):77-86. Paxinos, G. (2010). The rat brain in stereotaxic coordinates (6th ed.). Amsterdam:

Elsevier/Academic. Perusini, JN., Fanselow, MS. (2015). Neurobehavioral perspectives on the distinction between

fear and anxiety. Cold spring harbor laboratory press. 22:417-425. Phan, KL., Wager, T., Taylor, SF., Liberzon, I. (2002). Functional neuroanatomy of emotion: a

meta-analysis of emotion activation studies in PET and fMRI. Neuroimage, 16, pp. 331–348

Portfors, CV. (2007) Types and functions of ultrasonic vocalizations in laboratory rats and mice.

Journal of the American Association for Laboratory Animal Science. Vol 46: 28-34. Ritchie, K., Jaussent, I., Stweard, R., Dupuy, AM., Courtet, P., Ancelin, ML. (2009). Child

maltreatment and allostatic load: consequences for physical and mental health in children from low-income families. Developmental Psychopathology. 23, p 1107-1124.

Sadananda, M., Wöhr, M., Schwarting, RK. (2008). Playback of 22 kHz and 50 kHz ultrasonic

vocalizations induces differential c-Fos expression in rat brain. Neuroscience Letters V. 435, Issue 1, 17-23.

Schafe, G.E., LeDoux, E.J. (2004). The neural basis of fear. In: Gazzaniga, M.S. (Ed.), The

Cognitive Neurosciences III. The MIT Press. pp. 987–1004.


Schwarting, RKW., Wöhr, M. (2012). On the relationship between ultrasonic calling and anxiety-related behaviors in rats. Brazilian journal of medical and biological research. 45(5): 337-348.

Seagraves, KM, Arther, BJ., Egnor, SE. (2016). Evidence for an audience effect in mice: male

social partners alter the male vocal response to female cues. Journal of Experimental Biology. 219, 1437-1448.

Thayer, JF., Ahs, F., Fredrikson, M., Sollers, JJ., Wager, TD. (2012). A meta-analysis of heart

rate variability and neuroimaging studies: implications for heart rate variability as a marker of stress and health. Neuroscience and biobehavioral reviews. 36, 747-756.

Tottenham, N,. Hare, TA., Millner, A., Gilhooly, T., Zevin, J., Casey, BJ. (2012). Elevated

amygdala responses to faces following early deprivation. Dev Sci. 14(2) 190-204. Tottenham, N., Hare, T.A., Quinn, B.T., McCarry, T., Nurse, M., Gilhooly, T., et

al. (2000). Prolonged institutional rearing is associated with atypically large amygdala volume and emotion regulation difficulties. Developmental Science, 13 (1), 46–61.

Vivian, JA., Miczek, KA. (1993). Morphine attenuates ultrasonic vocalization during agonistic

encounters with male rats. Psycho- pharmacology 111:367–375. Wager, TD., Waugh, CE., Lindquist, M., Noll, DC., Fredrickson, BL., Taylor, SF. (2009). Brain

mediator of cardiovascular responses to social threat, Part 1: Reciprocal dorsal and ventral sub-regions of the medial prefrontal cortex and heart-rate reactivity. Neuroimage. 47, 821 – 835.

Wallace, KJ., Rosen, JB. (2000). Predator odor as an unconditioned fear stimulus in rats:

elicitation of freezing by trimethylthiazoline, a component of fox feces. Behavioral Neuroscience. 114(5):912-22.

Whalen, PJ. (1998). Fear, vigilance, and ambiguity: initial neuroimaging studies of the human

amygdala. Current direction in psychological science. Sci. 7, 177-188. White, NR., Barfield, RJ. (1987). Role of the ultrasonic vocalization of the female rat in sexual

behavior. Journal of Comparative Psychology. 101: 73-81. Wöhr, M., Schwarting, RK., (2007) . Ultrasonic communication in rats: can playback of 50-kHz

calls induce approach behavior? PLOS One. 2:e1365. Wöhr, M., Houx, B., Schwarting, RKW, Spruijt, B. (2007). Effects of experience and context on

50-kHz vocalizations in rats. Physiological behavior. V93: 766-776. Zald, DH. (2003). The human amygdala and the emotional evaluation of sensory stimuli. Brain

Res. Rev. 41; pp. 88–123


A) Aversive USV recording analysis

B) Synthetic tone analysis

Table 1: Analysis of played back recordings: aversive USVs and synthetic tone. (A) Mean+/-SEM kHz level of aversive USVs emitted by a restrained adult male rat. (B) shows the comparable parameters of the synthetic tone prepared using Audition 3.0 software.


Figure 1: Avisoft-RECORDER bioacoustics image showing aversive USV recording and synthetic tone recording. (A) 5 recorded aversive USVs emitted by a restrained adult male rat in a sound attenuated box infused with cat odor (22.97 kHz, 2.51 sec and -71 dB). (B) An example of the synthetic tone created using Audition 3.0 software imitating the frequency (22 kHz), length (3 sec), and intensity (-70dB) of the aversive USV recording.

kHz

sec


Figure 2: USV playback, and not synthetic tone playback, induces anxiety-like behavior in the form of increased immobility and decreased sniffing. Rats were placed in the chamber and habituated for three minutes. Baseline was recorded for 3 min (n=20) and then either the synthetic tone (n=7) or the aversive USVs (n=16) were played back. Data shown as means+SEM. *p<0.05 compared to immobility baseline (A), sniffing/exploration (B), and grooming (C).


Figure 3: USV playback, and not synthetic tone playback, induces anxiety-like physiological responses in the form of increased heart rate variability. Baseline HRV (3 min; n=12) was measured and compared to synthetic tone HRV (3 min; n=7) or USV HRV (3 min; n=9) measured as beats-per-minute (BPM) P45. Data shown as mean+SEM. *p<0.05 compared to baseline.


Figure 4: Positive change in HRV (BPM) when presented with an aversive USV when compared to synthetic tone. Increased change in HRV (determined via [USV/Tone Presentation HRV] - [Baseline HRV] = [change in HRV]. Synthetic tone HRV (n=7) was compared to baseline HRV (n=7) and USV HRV (n=7) was compared to baseline HRV (n=7). Data shown as means+SEM.


Figure 5: Representative picture showing increased c-Fos immunoreactivity in the basolateral amygdala when presented with an aversive 22 kHz USV. Rats were presented with either no noise, a synthetic tone in the 22 kHz range and the aversive 22 kHz USV for 45 minutes.

BLA BLA BLA

CONTROL SYNTHETIC TONE

USV

camila demaestri – honors thesis · camila demaestri – honors thesis 1. introduction the...

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