Sex Dependent Individual Differences in Rat Fear Responses During Fear Learning and

Extinction

By

Rebecca Bork

Submitted in Partial Fulfillment

of the Requirements for
Graduation with Honors from the
South Carolina Honors College

May, 2024

Approved:

______________________________________________________________________________

Dr. Marlene Wilson

Director of Thesis

______________________________________________________________________________

Dr. Jim Fadel

Second Reader

______________________________________________________________________________

Steve Lynn, Dean

For South Carolina Honors College

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Table of Contents

Acknowledgements………………………………………………………………………………..3

Abstract……………………………………………………………………………………………4

Introduction………………………………………………………………………………………..5

Post-Traumatic Stress Disorder: An Overview……………………………………………5

Pavlovian Fear Conditioning and Extinction……………………………………………...5

Fear Responses in Preclinical Rodent Models…………………………………………….6

Significance………………………………………………………………………………..8

Methods……………………………………………………………………………………………8

Subjects……………………………………………………………………………………8

Fear Conditioning and Extinction Procedures…………………………………………….9

Behavioral Analysis……………………………………………………………………...10

Statistical Analyses………………………………………………………………………11

Results……………………………………………………………………………………………12

Freezing Behavior in Males and Females………………………………………………..12

22 kHz Ultrasonic Vocalizations in Males and Females………………………………...15

50 kHz Ultrasonic Vocalizations in Males and Females………………………………...21

Discussion………………………………………………………………………………………..27

Conclusion……………………………………………………………………………………….30

References………………………………………………………………………………………..32
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Acknowledgements

I would like to give a special thanks to Dr. Marlene Wilson for all of her guidance and support

during my time in her lab and throughout the entirety of my thesis project. I would also like to

thank Dr. Jim Fadel, my second reader, as well as Dr. Sarah Hartvigsen, Dr. Christian O’Reilly,

Sabah Anis, and Kris F. Kaigler for their meaningful contribution to the project. Funding for this

research was supported by VA Merit Award I01BX001374 to MAW, ASPIRE I (USC Vice

President for Research) to SCH, and Magellan Scholar (USC Vice President for Research) and

SC Honors College Research awards (South Carolina Honors College) to RB. Some figures were

created using biorender.com.

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Abstract

Post-traumatic stress disorder (PTSD) is a condition in which individuals experience a

persistence in traumatic memories after undergoing a life-threatening event. Notable individual

variation and sex differences have been reported in the development of PTSD, with women

demonstrating higher susceptibility. However, little is understood as to why these individual and

sex differences exist, and how they may impact the efficacy of PTSD treatment. Hence, this

study aims to investigate variations in PTSD vulnerability using a preclinical rodent model of

Pavlovian fear learning and extinction. Individual and sex differences in fear responses,

including freezing behavior and 22 and 50 kHz ultrasonic vocalizations (USVs), were examined

in male and female Long-Evans rats during fear learning, contextual fear recall, and extinction

learning. Rats were divided into Extinction Competent (EC) and Extinction Resistant (ER)

phenotypes based on a median split of freezing behavior demonstrated during extinction

learning. Although as expected ER males and females displayed higher freezing during

extinction learning than EC groups, no ER-EC differences were seen during fear learning.

Variation was also present in 22 kHz distress calls, with males producing more vocalizations in

all trials than females, and ER males vocalizing more than EC males during fear extinction.

Analysis of acoustic parameters of 22 kHz USVs supported the increased distress vocalizations

in males than females, with ER males demonstrating a higher total duration of calls and number

of bouts than EC males during fear extinction. Females produced significantly more 50 kHz

positive USVs in all trials than males, especially during exposure to the novel context, and EC

females vocalized more than ER females during contextual fear recall. Analysis of 50 kHz USV

acoustic parameters also indicated sex differences in the total duration of calls during fear

extinction and in the number of bouts during fear learning and extinction. These results show
5

male and female rats demonstrate ER-EC differences in freezing behavior and USV production,

similar to individual risk for PTSD. The findings also suggest fear responses are sex-dependent,

with male and female rats having similar ER-EC differences in freezing but distinct fear behavior

in 22 and 50 kHz USVs.

Introduction

Post-Traumatic Stress Disorder: An Overview

Post-traumatic stress disorder (PTSD) is a psychiatric condition which occurs after individuals

experience a life-threatening, traumatic event. It is characterized by the failure to terminate a

traumatic memory as well as the persistence of such memory leading to an increase in fear

responses1,10. While around 50 to 80% of the population sustains a life-threatening experience,

only 20% or less of those individuals have dysregulated fear extinction and subsequently present

with PTSD1,7. This deficit in normal extinction of the fear memory forms the basis of exposure

therapy, which acts as the primary treatment for most PTSD patients5. However, the efficacy of

exposure therapy varies greatly depending on the individual. Further variation in the

development of PTSD is seen between males and females, as women have a prevalence rate

which is twice as high as that of men1,9. Despite this increased vulnerability in women, it remains

unknown as to why these sex differences exist. Hence, it is imperative to gain a better

understanding of why males and females, as well as specific individuals, differ in fear learning

and extinction, so as to develop more appropriate treatments for patients with PTSD.

Pavlovian Fear Conditioning and Extinction

In order to investigate these differences in PTSD susceptibility, preclinical rodent models are

subjected to a Pavlovian fear conditioning and extinction paradigm, as they also exhibit variation

in fear learning and extinction, similar to humans2,7. During fear learning of Pavlovian

conditioning, an aversive unconditioned stimulus, such as a mild foot shock in rodents, is

6

repeatedly paired with a neutral conditioned stimulus (CS), such as a tone, until an association is

formed, at which point the CS can evoke fear behaviors. With further exposure to just the

conditioned tone during extinction learning and recall, a decrease in fear responses occurs 2,7. The

rate of this fear extinction varies among individuals, with rats presenting as either extinction

competent (EC) or extinction resistant (ER) depending on the persistence of their fear

behavior1,5,7. EC rats display increased extinction rates of fear behavior as compared to ER rats,

which maintain high levels of fear responses throughout extinction learning, reflective of patients

with PTSD.1,5,7 An example Pavlovian fear conditioning and extinction paradigm used in this

project, including fear learning, contextual fear recall, and extinction learning trials, is shown in

Figure 1.

Figure 1. Example protocol of a Pavlovian fear conditioning and extinction paradigm. Trials included are fear
learning, contextual fear recall, and extinction learning. Shocks are represented by lightning bolts, while tones are
represented by speaker notes. Created with biorender.com.

Fear Responses in Preclinical Rodent Models

In rodent models, fear responses are often measured through freezing behavior and emission of

ultrasonic vocalizations (USVs), Rodent USVs, in particular, are emitted at 2 different frequency

ranges, either 22 kHz or 50 kHz, to communicate emotional states,3,11. Typically, 22 kHz USVs

are considered to be distress calls, produced in response to threatening environments, including

7

presence of a predator, male on male social aggression, loud noises, or pain. Because of their

nature, an increase in 22 kHz distress calls is associated with tone-shock pairings during fear

learning and conditioned tones during extinction learning2,3,11. In contrast, 50 kHz vocalizations

are emitted during non-threatening activities such as sexual behavior and playing and are thought

to be positive in affect. Accordingly, 50 kHz USVs are not reported during aversive stimuli, but

rather are often correlated with initial periods within the testing environment 1,3,11. Figure 2 shows

example USVs produced in the 22 kHz (top) and 50 kHz (bottom) range.

Figure 2. Example 22 kHz USVs (top) and 50 kHz USVs (bottom) produced by a rat during fear learning (UltraVox
XT, Noldus Inc.). Time is shown in one second increments for 22 kHz USVs and 100 millisecond increments for 50
kHz USVs.

Previous research using rodent models suggests both individual and sex differences exist in

freezing behavior as well as USV emission1,2,4,7. More specifically, Laine et al. found that males

exhibit higher percent freezing during fear learning, and females display higher freezing

behavior during extinction learning, suggesting a strong influence of sex in the persistence of

fear responses2. In addition, it has been reported that males produce more 22 kHz distress calls
8

than females, while females produce more 50 kHz USVs than males, especially when exposed to

a novel context2,4. Although our lab has also indicated that both males and females display

variation in extinction phenotype, further research is needed to gain a full understanding of how

individual differences emerge based on sex1,7.

Significance
The goal of this project is to investigate how freezing behavior, as well as 22 kHz and 50 kHz

USVs vary by sex and extinction phenotype during fear and extinction learning. Although earlier

research has examined differential fear responses in males and females, the current study aims to

further explore whether individual variation is sex-dependent by comparing fear responses in

male and female ER and EC phenotypes2,4. Based on previous literature, it is hypothesized that

male rats will demonstrate higher rates of both freezing behavior and 22 kHz USV emission as

compared to female rats, with ER males demonstrating higher fear behaviors than EC males.

Further, females will produce a higher number of 50 kHz USVs than males, particularly when

introduced to a novel context.

Methods
Subjects

Upon arrival, adult male and female Long Evans rats (125-150g; Envigo, Indianapolis, IN,

United States) were housed individually in a climate-controlled vivarium on a 12-h light/dark

cycle with ad libitum access to food and water. Animals were treated following the guidelines of

the National Institute of Health Guide for the Care and Use of Laboratory Animals (Department

of Health and Human Services) and all procedures were approved by the University of South

Carolina Institutional Animal Care and Use Committee. The rodents were handled daily for 2

weeks before behavioral testing commenced. Daily vaginal lavage was performed in female rats

for 2 weeks prior to behavioral testing, as well as after behavioral testing, avoiding additional
9

handling stress, to assess estrous cycle phase. Fresh cytology samples were examined under a

microscope to determine vaginal cytology. The slides were then fixed using 95% ethanol and

stained with hematoxylin and eosin for further verification (vaginal cytology results not

shown)1,4.

Fear Conditioning and Extinction Procedures

Behavioral procedures were adapted from previously performed fear conditioning and extinction

protocol1,5,7. The described procedures were used to generate amygdala brain slices for studying

electrophysiological responses (electrophysiological data not shown). On day 1, fear acquisition

began as the rats were individually placed into a shock box, labeled Context A, within a sound-

attenuated chamber comprised of a ventilation fan, speaker, and house light (CleverSys, Inc.,

Reston, VA, United States), as well as a microphone below the shock box to record USVs

(Noldus Information Technology, Leesburg, VA, United States). The shock box was cleaned

with mild (7%) ammonium hydroxide between animals. Once placed in the shock box, the

animal was given 3 minutes to explore freely, so as to assess unconditioned behavioral responses

including freezing and USVs. At 3 minutes, the rat was presented with 3 tone-shock pairings at

1-minute intervals, consisting of a 10 second tone (2 kHz, 80 dB) and a foot shock (1mA, 1s). On

day 2, animals were placed back into Context A for 8 minutes, with no tones or foot shocks, and

freezing behavior and USVs were recorded to assess contextual fear recall. On day 3, rats were

placed in a new environment (Context B) to assess cued fear recall and extinction learning.

Context B included a round bottom Plexiglas bowl, aspen bedding, lemon scent, and a

microphone placed on the upper side of the bowl, and was cleaned with ethanol (70%) between

each animal. Unconditioned behavior was recorded for the first 3 minutes, followed by the

presentation of 20 tones (2 kHz, 10s, 80 dB) at 1-minute intervals. Animals were immediately
10

returned to their home enclosures after completing extinction learning while awaiting

electrophysiological testing, although this data is not presented in the current report.

Behavioral Analysis

FreezeScan software (CleverSys, Inc., Reston, VA, United States) was used to analyze freezing

behavior in 1-minute bins, with automatic parameters set to detect freezing as the absence of

movement excluding respiration. Percent freezing was calculated as the percent of time an

animal displayed freezing behavior in each 1-minute bin. Using a median split of average

recorded freezing behavior during the last 10 minutes of extinction learning, the rats were

divided into Extinction Competent (EC) and Extinction Resistant (ER) groups.

Ultrasonic vocalizations (USVs) were recorded using an ultrasonic microphone (16-bit

resolution, 250 kHz sample rate, full-spectrum, USB port) placed below the shock box during

fear acquisition and context recall trials (Context A) and above the rats for extinction learning

and extinction recall trials (Context B). Both 22 and 50 kHz range USVs were manually labeled

using UltraVox XT (version 3.2.108, Noldus Information Technologies, Leesburg, VA), with

spectrographic settings including SFT length of 2048, Zero Padding of 1, and Overlap of 90%.

The UltraVox XT program supplied quantitative data such as call duration (ms), call start and

stop time, peak frequency (Hz), and mean amplitude for each call. These variables plus others

not provided by UltraVox XT, specifically latency to call, number of bouts, and calls per bout,

were calculated using a Python script developed and verified via collaborations with Dr.

Christian O’Reilly and Sabah Anis (not shown). This script also automated binning of variables

into 1-minute bins, as well as provided descriptive statistics for each variable.
11

Statistical Analyses

For each trial, percent freezing, 22 kHz USVs, and 50 kHz USVs over time were compared using

a three-way analysis of variance (ANOVA) to compare time, sex, and extinction phenotypes (ER

vs EC). Further, a two-way ANOVA was performed to compare average percent freezing, as

well as number of 22 kHz USVs and 50 kHz USVs for each trial, comparing sex and extinction

phenotypes (ER vs EC). For 22 kHz and 50 kHz calls, additional two-way ANOVAs were used

to compare acoustic parameters, including total duration, average duration of each call, average

peak frequency of each call, mean amplitude of each call, average number of bouts across all

bins, average number of calls per bout, and average new bout latency. Chi square analysis was

also performed to compare the number of rats in each group producing 22 kHz or 50 kHz USVs.

Bonferroni post hoc analysis was used to determine specific differences between groups. PRISM

GraphPad 8.0 (GraphPad Software, San Diego, CA, United States) was used to perform

statistical analysis and construct relevant figures. Significance level was set at P < 0.05 for all

statistical analyses.
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Results

Freezing Behavior in Males and Females

Male and female rats were categorized into Extinction Competent (EC) or Extinction Resistant

(ER) phenotypes based on a median split of percent freezing during the last 10 tones of

extinction learning. High-freezing rats were categorized as ER (N = 34 for males and N = 13 for

females) and low-freezing rats were categorized as EC (N = 35 for males and N = 14 for

females). Freezing behavior over time in each trial of the fear conditioning and extinction

protocol, including fear learning, contextual fear recall, and extinction learning is shown in

Figure 3. Both males and females showed a significant difference in freezing behavior between

ER and EC groups during extinction learning, but not fear acquisition. During fear learning,

there was a significant effect of time (F (5, 465) = 229.2, P<0.0001), as all four groups

demonstrated a similar increase in freezing at the presentation of the 3 tone-shock pairings. In

13

addition, there was an effect of sex (F (1, 93) = 10.92, P0.0014), with male rats freezing

significantly more than female rats, but no effect of phenotype (F (1, 93) = 1.212, P=0.2738).

During contextual fear recall, there was a main effect of time (F (7, 651) = 15.79, P<0.0001) and

ER rats froze significantly more than EC rats (main effect of phenotype, F (1, 93) = 13.87,

P=0.0003), although there was no significant difference between males and females (F (1, 93) =

3.137, P=0.0798). During extinction learning, because freezing increased upon the introduction

of the tones and diminished over time, there was a significant effect of time (F (22, 2046) =

109.6, P<0.0001). In addition, there was a significant effect of phenotype (F (1, 93) = 110.5,

P<0.0001), with ER rats freezing more than EC rats, as well as a significant effect of sex (F (1,

93) = 9.872, P=0.0023), with male rats freezing more than female rats. Further, for extinction

learning, there was a significant time × sex interaction (F (22, 2046) = 1.718, P=0.0201), time ×

phenotype interaction (F (22, 2046) = 14.14, P<0.0001), and sex × phenotype interaction (F (1,

93) = 4.345, P=0.0399).

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In Figure 4, the effects of sex and phenotype on average percent freezing during fear learning,

contextual fear recall, the first 5 tones of cued fear, and the last 10 tones of extinction learning

are shown. The results reiterate those seen in Figure 1, as males and females showed a significant

ER-EC difference during cued fear and extinction learning, but not during fear learning. There

was no significant difference in average percent freezing during the tone-shock pairings between

males and females (F (1, 93) = 0.06060, P=0.0704) or between ER and EC groups (F (1, 93) =

1.262, P=0.2642) in fear learning. However, similar to trends seen in freezing overtime, in

contextual fear recall there was a significant effect of phenotype (F (1, 93) = 13.86, P=0.0003),

as ER rats demonstrated higher freezing than their EC counterparts, although post-hoc analysis
15

demonstrated this effect was only significant between female ER-EC groups . During cued fear

(first 5 tones during extinction learning), female ER rats showed significantly higher freezing

than female EC rats, with a main effect of phenotype (F (1, 93) = 18.10, P<0.0001) as well as a

significant phenotype × sex interaction (F (1, 93) = 3.980, P=0.0490). For the last 10 tones of

extinction learning, similar to the effects seen on freezing over time, there was both a main effect

of phenotype (F (1, 93) = 137.4, P<0.0001) and sex (F (1, 93) = 8.207, P=0.0052), with ER rats

freezing more than EC rats, and males freezing more than females.

22 kHz Ultrasonic Vocalizations in Males and Females

The proportion of ER and EC males and females that were vocalizing or non-vocalizing in the 22

kHz range during fear learning, contextual fear recall, and extinction learning is shown in Figure

5. Neither ER or EC males or females showed a significant difference between vocalizing and

16

non-vocalizing during fear learning (2 = 0.4633, p = 0.2480 for males; 2 = 0.5163, p = 0.2362

for females) or contextual fear recall (2 = 1.167, p = 0.1400 for males), although all females

were non-vocalizing during contextual fear recall. However, during fear learning, male ER and

EC groups had a significantly higher proportion of vocalizing rats than non-vocalizing rats (2 =

4.503, p = 0.01692), while female ER and EC groups had a significantly higher proportion of

non-vocalizing rats than vocalizing rats (2 = 2.854, p = 0.04559).

Figure 6 illustrates the number of 22 kHz USVs produced over time during each trial period.

Males produced significantly more 22 kHz USVs than females in all trials, with male ER rats

demonstrating particularly high levels during extinction learning. In fear learning, there was a

significant effect of time (F (5, 395) = 57.88, P<0.0001), with an increase in 22 kHz

vocalizations occurring at the introduction of the 3 tone-shock pairings. This increase was
17

significantly higher in males, indicating a main effect of sex (F (1, 79) = 18.75, P<0.0001),

although there was no significant difference between ER and EC rats (F (1, 79) = 0.07398,

P=0.7863). Similar trends were seen in contextual fear, as there was both a significant effect of

time (F (7, 644) = 3.535, P=0.0010) and sex (F (1, 92) = 11.80, P=0.0009), with males producing

a significantly higher number of 22 kHz calls than females, but no main effect of phenotype (F

(1, 92) = 0.7471, P<0.3896). However, in extinction learning there was a main effect of time (F

(22, 2002) = 7.501, P<0.0001), as well as a significant difference between males and females

(main effect of sex, F (1, 91) = 18.90, P<0.0001) and ER and EC rats (main effect of phenotype,

F (1, 91) = 11.97, P=0.0008). Further, for extinction learning, there was a significant time × sex

interaction (F (22, 2002) = 5.410, P<0.0001), time × phenotype interaction (F (22, 2002) =

3.716, P<0.0001), and sex × phenotype interaction (F (1, 91) = 4.372, P=0.0372). This

interaction was due to ER males showing more 22 USVs than EC males during extinction

learning.

The effects of sex and phenotype on the average number of 22 kHz USVs produced by both male

and female ER and EC rats during fear learning, contextual fear recall, and fear extinction are

shown in Figure 7. The results reflect those seen in Figure 3, with significant differences

between males and females seen in all 3 trials, as well as a significant difference between ER and

EC rats during fear extinction. More specifically, in fear learning and contextual fear recall, there

was a main effect of sex (F (1, 79) = 18.75, P<0.0001 for fear learning and F (1, 92) = 11.80,

P=0.0009 for contextual fear recall), with males producing a significantly higher number of 22

kHz calls compared to females. In addition, similar to trends seen in 22 kHz USVs produced

overtime, there was a main effect of sex (F (1, 91) = 18.93, P<0.0001) and phenotype (F (1, 91)
18

= 11.95, P=0.0008), as well as a significant sex × phenotype interaction (F (1, 91) = 4.486,

P=0.0369) during fear extinction, with males, particularly in the ER group, vocalizing

significantly more than females. This ER-EC difference during fear extinction was significant in

males (P<0.05).
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Figure 8 shows a comparison of the acoustic parameters of the 22 kHz USVs, including total

duration, average duration of each call, average peak frequency of each call, mean amplitude of

each call, average number of bouts across all bins, average number of calls per bout, and average

new bout latency, across different trials. Statistical comparison for most 22 kHz parameters was

excluded in contextual fear recall due to a lack of 22 kHz vocalizations during the trial in most

rats, especially in females. Significant sex differences in average peak frequency and mean

amplitude were shown during fear learning, while during fear extinction, there were significant

male ER-EC differences in total duration, average number of bouts, and average new bout

latency. For total duration of 22 kHz USVs, there was a significant effect of trial (F (2, 262) =

19.79, P<0.001), group (F (3, 262) = 25.24, P<0.001), and a significant trial × group interaction

(F (6, 262) = 13.62, P<0.0001), with post-hoc analysis revealing this difference to be in male

ER-EC groups during fear extinction. For average 22 kHz call duration, there were no significant

differences between trials (F (1, 117) = 2.243, P=0.1369), between groups (F (3, 117) = 1.042,

P=0.3767), or trial × group interaction (F (3, 117) = 1.638, P=0.1843). For average peak

frequency, there was a main effect of trial (F (1, 117) = 6.764), P=0.0105) and a main effect of

group (F (3, 117) = 3.960, P=0.0099), with post hoc Bonferroni comparisons demonstrating

significant sex differences during fear learning between male and female EC groups as well as

male and female ER groups. Similar trends were seen for mean amplitude, as there was a main

effect of trial (F (1, 117) = 4.776, P=0.0308) and group (F (3, 117) = 4.677, P=0.0040) due to

significant differences between male and female EC groups and male and female ER groups

during fear learning. For average number of bouts across all one-minute bins there was a main

effect of trial (F (2, 262) = 25.03, P<0.0001), group (F (3, 262) = 13.55, P<0.0001), and trial ×

group interaction (F (6, 262) = 2.428, P=0.0266), with significant differences between male and
21

female ER groups during contextual fear recall, as well as male ER-EC groups, male and female

EC groups, and male and female ER groups during fear extinction, as revealed by post hoc

analysis. For number of 22 kHz calls per bouts, there was no significant differences between

trials (F (1, 117) = 3.168, P=0.0777), between groups (F (3, 117) = 1.703, P=0.1702), or trial ×

group interaction (F (3, 117) = 1.522, P=0.2124). For average new bout latency, there was a

main effect of trial (F (1, 117) = 11.00, P=0.0012), group (F (3, 117) = 5.536, P=0.0014), and

trial × group interaction (F (3, 117) = 5.346, P=0.0017), with post hoc analysis demonstrating

specific significant differences during fear extinction between male ER-EC groups as well as

male and female ER groups.

50 kHz Ultrasonic Vocalizations in Males and Females

In Figure 9, the proportion of male and female ER and EC rats that were vocalizing or non-

vocalizing in the 50 kHz range during fear learning, contextual fear recall, and extinction

learning is shown. During fear learning, there were no significant differences between vocalizing

or non-vocalizing ER and EC groups in males or females (2 = 0.4633, p = 0.2480 for males),
22

although notably, all ER and EC females were vocalizing. During contextual fear recall, there

was no significant difference in the proportion of ER and EC groups vocalizing or not vocalizing

in both males and females (2 = 0.9694, p = 0.1624 for males; 2 = 1.854, p = 0.0866 for

females). Similarly, in fear learning, there was no significant difference seen in the proportion of

vocalizing or non-vocalizing male or female rats (2 = 0.592, p = 0.2208 for males), but all ER

and EC females were vocalizing.

In Figure 10, the number of 50 kHz USVs produced over time by male and female ER and EC

rats during fear learning, contextual fear, and fear extinction is shown. Females produced a

significantly higher number of 50 kHz USVs during all 3 trials, with the number of 50 kHz

USVs increasing during exposure to novel contexts and declining upon the introduction of the

tone-shock pairings or conditioned tones. For fear learning, there was a main effect of time (F (5,

395) = 28.90, P<0.0001) and sex (F (1, 79) = 27.81, P<0.0001), with female rats vocalizing

significantly more than males, although there was no significant difference between the ER and
23

EC phenotypes (F (1, 79) = 0.06160, P=0.8046). In contextual fear recall, there was a significant

effect of time (F (7, 644) = 9.130, P<0.0001), sex (F (1, 92) = 15.68, P=0.0001), and phenotype

(F (1, 92) = 4.000, P=0.0485), as well as a significant time × sex interaction (F (7, 644) =8.515,

P<0.0001). This was due to females, particularly in the ER group, producing significantly more

50 kHz USVs than males upon re-exposure to the conditioned context. During extinction

learning, there was a main effect of time (F (22, 2002) = 10.46, P<0.0001) and sex (F (1, 91) =

17.99, P<0.0001), as females continued to produce a significantly higher number of calls

especially upon exposure to the new context. In addition, while there was no main effect of

phenotype (F (1, 91) = 1.688, P=0.1971), there was a significant time × sex interaction (F (22,

2002) = 5.521, P<0.0001) and time × phenotype interaction (F (22, 2002) = 5.718, P<0.0001).
24

Figure 11 compares the effects of sex and phenotype on the average number of 50 kHz USVs

produced before fear learning, during the first tone-shock pair of fear learning, during contextual

fear recall, before cued fear, and during the 20 tone presentations of fear extinction. Similar to

the results seen in Figure 5, females produced a significantly higher number of 50 kHz USVs

throughout all trials, although there was also a notable difference between ER and EC groups in

unconditioned vocalizations during the 3 minutes prior to cued fear. In the 3 minutes of recorded

behavior prior to fear learning, there was a main effect of sex (F (1, 79) = 22.22, P<0.0001), with

females producing a significantly higher number of 50 kHz USVs than males. A similar trend is

seen during fear learning, as females vocalized significantly more, indicating a main effect of sex

(F (1, 79) = 31.25, P<0.0001). As also seen in the results of 50 kHz USVs produced over time,

there was both an effect of sex (F (1, 92) = 15.68, P=0.0001) and phenotype (F (1, 92) = 4.000,

P=0.0485) for contextual fear recall, with significant differences seen between males and females

as well as the ER and EC phenotypes. In the 3 minutes before cued fear, females produced a

significantly higher number of 50 kHz USVs with a main effect of sex (F (1, 91) = 18.94,

P<0.0001), but no effect of phenotype (F (1, 91) = 1.042, P=0.3100). Similarly, during the 20

tones of extinction learning, there was a significant effect of sex (F (1, 91) = 14.48, P=0.0003),

with females again producing a higher number of 50 kHz calls than males, but the ER-EC

difference in females was not statistically different.

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26

A comparison of the acoustic parameters of the 50 kHz USVs, including total duration, average

duration of each call, average peak frequency of each call, mean amplitude of each call, average

number of bouts across all bins, average number of calls per bout, and average new bout latency,

across different trials is depicted in Figure 12. There were significant sex differences in the

average number of bouts in fear learning, as well as significant sex and female ER-EC

differences in total duration and average number of bouts during fear extinction. For total 50 kHz

call duration, there was a main effect of trial (F (4, 436) = 6.957, P<0.0001), group (F (3, 436) =

11.08, P<0.0001, and a trial × group interaction (F (12, 436) = 3.801, P<0.0001), although post

hoc analysis revealed these significant differences to be between male and female EC groups and

female ER-EC groups during fear extinction. For average 50 kHz call duration, there were no

significant differences between trials (F (2, 181) = 2.231, P=0.1104), between groups (F (3, 181)

= 1.478, P=0.2220), or trial × group interaction (F (6, 181) = 0.5190, P=0.7934). Similarly, for

average peak frequency, there was no main effect of trial (F (2, 181) = 0.7309, P=0.4829), group

(F (3, 181) = 0.8709, P=0.4573), or a significant trial × group interaction (F (6, 181) = 0.4819,

P=0.8213). For mean amplitude, although there was a main effect of trial (F (2, 181) = 9.309,

P=0.0001), post hoc Bonferroni comparisons revealed no significant differences between groups

across all trials. Similar trends were also seen in the average number of calls per bout and

average new bout latency, as there was a main effect of trial (F (2, 181) = 6.294, P=0.0023 for

number of calls per bout and F (2, 181) = 20.77, P<0.0001 for new bout latency), but post hoc

analysis revealed no significant differences between groups across all trials. For average number

of bouts across all bins, there was a main effect of trial (F (2, 262) = 36.78, P<0.0001) and group

(F (3, 262) = 24.59, P<0.0001), with post hoc analysis revealing specific differences between

male and female EC and ER groups during fear learning and fear extinction.
27

Discussion

This study examined the freezing behavior and USV production of male and female Long-Evans

rats in response to a Pavlovian cued fear learning and extinction paradigm. Previous studies have

demonstrated sex differences in fear behaviors, as well as individual variation in males and

females, with both sexes exhibiting multiple levels of fear extinction1,2,4,7,12. The current results

aim to further contribute to this expanding literature, comparing male and female extinction

phenotypes to show that individual differences in behavioral responses during fear learning and

extinction are sex dependent. Contrary to previous research in our lab, which has shown females

display higher freezing than males during extinction learning, as well as other labs, which

indicate no sex differences in freezing during extinction learning, the current results found males

freeze significantly more than females during both fear and extinction learning2,4. These findings

add to the mixed reports on sex differences in freezing behavior observed during fear learning,

contextual fear recall, and extinction learning, suggesting experimental conditions and behavioral

protocols may influence this variation between males and females2,4,12. More aligned with past

studies, the results show males and females display individual differences in freezing behavior

during extinction learning, with ER rats freezing more than their EC counterparts 1,5,7. However,

similar to previous findings, these individual differences in freezing did not appear during fear

learning and are therefore not because of variation in sensitivity to aversive stimuli between

phenotypic groups1,7. Instead, the current results, as well as similar, past findings could suggest

that ER-EC differences emerge due to neural mechanisms associated with fear memory

consolidation and extinction learning1,5,7.

28

In addition to the observed individual and sex differences in freezing behavior, the results

showed significant individual and sex differences in both 22 kHz and 50 kHz USVs during fear

learning, contextual fear recall, and extinction learning. As expected, the number of 22 kHz

distress calls increased at the presentation of the shock-tone pairs in fear learning and the

conditioned tones during extinction learning2,4. Like findings in previous literature, males emitted

more 22 kHz USVs than females during all trials, with a significantly higher proportion of males

vocalizing than non-vocalizing during extinction learning2,4. Similar to other reports, females

produced little to no 22 kHz USVs, particularly during contextual fear recall and extinction

learning1,2,4. These pronounced sex differences in 22 kHz distress calls could be related to

hormonal variation between males and females, although more research is needed to fully

understand the influence of hormones on USV emission4. Because males emit more 22 kHz

USVs than females, it is reasonable that ER-EC differences in distress calls were only noted in

male rats, with ER males vocalizing more during extinction learning than EC males. This

difference in extinction phenotype observed only in males suggests individual variation in 22

kHz USV emission is sex dependent. Accordingly, the acoustic parameters of the 22 kHz

vocalizations further support individual differences observed exclusively in males during

extinction learning, with the total duration of calls and number of bouts also indicating higher

results in ER males than EC males. It was also shown that the average amplitude of calls was

greater in males than females and the average peak frequency was higher in females than males

during fear learning, findings aligned with previously observed results from our lab4. However,

in contrast to other reports, which showed males had a higher average duration of each call

compared to females, the current results found no significant differences in average call duration
29

between sexes in any trial4. More research on 22 kHz call parameters is needed to appreciate the

significance of these sex differences and why they might exist.

Contrasting with 22 kHz USVs, the number of 50 kHz USVs showed a marked increase during

the initial exposure to novel contexts and decrease upon the presentation of the tone-shock pairs

during extinction learning and the conditioned tones during fear extinction. This trend aligns

with those observed in past studies, with a higher number of 50 kHz USVs noted during

exploratory periods in a new, safe environment and less calls produced in response to aversive

stimuli1,2,4. Hence, it is reasonable to assume that this decrease in the positive valence 50 kHz

vocalizations is an expression of increased fear behavior1. Sex differences were also noted in 50

kHz vocalizations, with females emitting more 50 kHz USVs than males in all trials. In fact,

males emitted a limited number of 50 kHz USVs, a result also reported in previous

literature2,4.This notable variation between males and females is similar to previous studies,

which have also found females to vocalize more in the 50 kHz range than males, particularly

during the exploration periods of trials, indicating 50 kHz USVs are a sex dependent behavior2,4.

The pronounced sex differences in 22 kHz and 50 kHz vocalizations may suggest the

involvement of distinct neural mechanisms between males and females in the production of

USVs, but more research is needed to investigate this differential regulation. Although past

research in our lab has shown female ER-EC differences in 50 kHz USV production during fear

learning and extinction, no significant differences were seen between phenotypic groups in the

current results of those trials1. Instead, EC females emitted a higher number of 50 kHz USVs

than ER females during contextual fear recall. Disparities in findings of individual variation in

50 kHz USV production may be due to hormone fluctuations based on differing stages in estrous

cycles, as there is evidence that estrous cycle may impact USV emission in females 1,13. However,
30

the current study was underpowered to fully assess estrous cycle associations with fear

behaviors, and more research is needed to understand how it may be impacting female individual

variation in 50 kHz USVs. Nevertheless, the distinct sex differences observed in the number of

50 kHz USVs is further supported by differences in call parameters between males and females.

More specifically, females had a higher total duration of 50 kHz calls than males during

extinction learning, as well as a higher number of bouts than males during fear and extinction

learning, thus reiterating the significant sex differences observed in the number of 50 kHz USVs.

While no differences in average amplitude or peak frequency were noted between males and

females, past studies have found these parameters to be higher in females than males 4. Similar to

22 kHz USVs, a better understanding of 50 kHz call parameters is needed to grasp the

significance of these sex differences, as they may be reflective of individual affective state.

Conclusion

Overall, this study demonstrates that both males and females display individual variation in

freezing behavior, with ER rats freezing significantly more than EC rats during extinction

learning but not fear acquisition. Additional sex dependent individual variation was shown in 22

kHz USVs, as males produced more distress calls than females in all trials, and ER males

produced a significantly higher number of 22 kHz vocalizations than EC males during fear

extinction. Female rats were also shown to vocalize more in the 50 kHz range during all trials

than males, especially when exposed to a novel context. Acoustic parameters of both 22 kHz and

50 kHz USVs, including total duration of calls and number of bouts, support the individual and

sex differences in the number of 22 and 50 kHz USVs. This sex dependent individual variation

in freezing behavior and USV production suggests distinct neural processes are responsible for
31

fear responses in males and females, an important implication in understanding both individual

and sex differences in the vulnerability to PTSD and other stress-related disorders.
32

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