Analyzing Individual and Sex Differences in Rat Behavior during Fear Extinction

Rebecca Bork, Biology Major, Psychology Minor

Mentor: Dr. Marlene Wilson
University of South Carolina School of Medicine Department of Pharmacology, Physiology, & Neuroscience
Background
Post-traumatic stress disorder (PTSD) is a psychiatric disorder that occurs after individuals undergo a life-threatening
traumatic event. It is characterized by the failure to terminate a traumatic memory as well as an increase in fear responses
due to the persistence of such memory1,10. The development of PTSD differs between individuals, with only ~20% or less
of those experiencing a traumatic event subsequently presenting with PTSD. Further variation is seen between males and
females, as women have a prevalence rate which is twice as high as that of men 1,9. Deficits in normal extinction of the fear
memory are seen in PTSD patients and form the basis of exposure therapy 10. In order to investigate these differences in
PTSD vulnerability, preclinical rodent models use Pavlovian fear conditioning and extinction, as they also exhibit
variation in fear learning and extinction, similar to humans2,7. Rats present as either extinction competent (EC) or
extinction resistant (ER) depending on the persistence of their fear behavior 1,5,7.
During fear learning of Pavlovian conditioning, an aversive unconditioned stimulus (mild foot shock in rodents) is
repeatedly paired with a neutral conditioned stimulus (CS; tone) until an association is formed, at which point the CS can
evoke fear behaviors. With further exposure to just the tone during extinction learning and recall, a decrease in fear
responses occurs2,7. In rodent models, these fear responses are measured through freezing behavior and emission of
ultrasonic vocalizations (USVs). In particular, rodent USVs are emitted at 2 different frequency ranges, either 22 kHz or
50 kHz, to communicate emotional states. Typically, 22 kHz calls are produced in response to threatening environments,
including presence of a predator, male on male social aggression, loud noises, or pain. In contrast, 50 kHz vocalizations
are emitted during non-threatening activities such as sexual behavior and playing 3,11. Previous research using rodent
models suggests both individual and sex differences exist in freezing behavior as well as USV emission 1,2,4. Our studies
and others indicate such variation could be explained by the role of the cholinergic system in fear regulation 6. An example
of freezing differences in ER and EC males and females during fear learning and extinction learning are shown in Figure
1; I recently completed data analysis of freezing, 22 kHz, and 50 kHz USVs in another set of males that looks very
similar. Note the shaded areas represent periods of unconditioned freezing before any tones or shocks (Ns=13-18 per
group).
Due to its involvement in cognition, arousal, and memory, the cholinergic system is an important neurobiological
pathway for fear learning and extinction4,6. In the cholinergic system, acetylcholine signaling is transmitted through
muscarinic or nicotinic receptors throughout the fear extinction circuit, which includes the amygdala, hippocampus, and
prefrontal cortex. Extinction learning is thought to specifically involve the prefrontal-amygdalar circuits, with the
basolateral amygdala receiving much of its cholinergic innervation from the basal forebrain cholinergic system 5,6.
Scopolamine (SCOP) is a cholinergic muscarinic receptor antagonist, which inhibits cholinergic signaling, and thus has
the potential to decrease fear behavior during extinction. This project will examine the impact of SCOP on both freezing
and USVs during extinction learning and recall, to better understand the role of the cholinergic system in the development
of PTSD and other stress disorders.
Research Question
How does blocking the muscarinic cholinergic receptors influence
individual and sex variation in rat freezing and ultrasonic vocalization
behavior during fear extinction?
Project Goals and Objectives
The overarching Goal of this project is to understand the function of the
muscarinic cholinergic system in producing behavioral responses during fear
extinction and determine if pharmacological manipulation of this receptor
has therapeutic potential for treating PTSD. Through the administration of
SCOP before extinction learning, which blocks muscarinic cholinergic
receptors, the project Aims (Objectives) are to understand how inhibiting
cholinergic regulation can influence various fear responses, including
freezing behavior and production of 22 and 50 kHz USVs. It specifically
focuses on how these behaviors may differ between ER and EC individuals
as well as males and females throughout fear learning and extinction.
Project Impact
Although it is known that the development of PTSD or other stress disorders
Fig 1. Freezing behavior, 22 kHz USVs, and 50 kHz USVs in EC
vs ER males and females during fear learning and extinction
is variable, with females more likely to exhibit symptoms than males and
learning. * denotes p<0.05 individuals presenting as either at-risk or resilient, little is understood about
why such differences appear. Using preclinical rodent models, more can be discovered about the underlying
neurobiological pathways which may influence differences seen between sexes and individuals in the development of
PTSD symptoms. This improved understanding of the neurobiology of PTSD susceptibility can, in turn, help advance
treatment methods as well as minimize the likelihood of symptom development. Exposure therapy, for example, can be
restructured or combined with pharmacological treatments, such as SCOP (as studied in this project) or other muscarinic
agents, so as to be more effective for extinction resistant patients.
Project Design and Methodology Experimental Vehicle Scopolamine
In this study, male and female Long Evans rats will be used (see table). Groups
Fear conditioning and extinction protocols used by our lab and Male N=8 ER/ N=8 ER/
pharmacological manipulations will be conducted by other lab research (N=16/group) N=8 EC N=8 EC
staff. Briefly, rats will be fear conditioned with three tone-shock pairings Female N=8 ER/ N=8 ER/
and 24 hours contextual fear recall will be assessed. The next day rats will (N=16/group) N=8 EC N=8 EC
receive an injection of either SCOP (1mg/kg, i.p.) or vehicle (Veh, sterile saline) 30 minutes prior to extinction learning in
a new Context B, where they will experience 20 conditioned tones at 1-min intervals. Using a median split of freezing,
which is assessed via an automated system (Clever Sys Inc), during the last 10 minutes of extinction learning, the rats will
be divided into EC and ER groups.
Aim 1: My project will be analyzing the freezing and scoring USVs using Clever Sys Inc. Freezescan and Noldus
Ultravox XT software, respectively. I have been previously trained on how to score both 22 kHz and 50 kHz USVs and
will receive additional training on all the parameters related to USVs (Frequency, duration, amplitude, etc.). PRISM
GraphPad will be used to perform statistical analysis and construct figures of the data. The male behavioral analysis is
complete so that data will be available to score and analyze immediately, and female studies are planned for initiation in
the near future. We anticipate that SCOP will diminish freezing during extinction learning and 22 kHz USVS
differentially in ER and EC male and female groups. Brain tissue will be collected, and I will also assist in the molecular
analysis of acetylcholinesterase (ACHE) splice variants in brain sections using RNAScope (Advanced Cell Diagnostics,
Inc). ACHE is the enzyme that terminates cholinergic transmission and differs between ER and EC groups 5.
Aim 2: Control experiments (same experimental plan) will assess the impact of SCOP on freezing in an unconditioned
paradigm, namely predator odor as well as locomotor activity in an open field 8. I will be responsible for analyzing
locomotor activity (Noldus Ethovision software), freezing to predator odor (Freezescan, Clever Sys Inc.) and USVs
(Noldus Ultravox XT software).
Project Timeline
 Throughout project: Continue literature review and background research, participate in lab meeting and journal clubs
 March-Nov 2023: Complete data collection and scoring of freezing behavior and USVs, Aims 1 and 2
 Sept-Jan 2024: Start performing data analysis and graph results using PRSIM software
 Jan-Apr 2024: Finalize data analysis and graphs, Prepare for Discover USC and prepare final report write-up
 Apr 2024: Present poster at Discover USC
Anticipated Results and Dissemination
Based on previous research, when comparing sex differences in extinction learning and recall, it is expected that ER
male rats will demonstrate higher percent freezing and emit more 22 kHz USVs than EC males and emit more 22 kHz
USVs than their female counterparts. In contrast, females are expected to produce a higher number of 50 kHz USVs than
males, with EC females showing more 50 kHz USVs than ER females (see Figure). With the administration of SCOP, it is
anticipated that both male and female rats will experience a reduction in freezing behavior and emission of 22 kHz USVs,
with little to no change in the number of 50 kHz USVs produced. Further, it is expected that SCOP will be more effective
in EC rats as opposed to ER rats throughout extinction learning and recall, as seen through a reduction in freezing
behavior and USV emission, due to a lower endogenous level of cholinergic neurotransmission.
Upon completion of the study, the findings will be presented at Discover USC in April 2024. In addition, I hope to
further the research detailed in this proposal through the completion of my Honors Thesis. Finally, these studies will be
part of publications from the lab which I will contribute to and be an author.
Personal Statement
Throughout my previous work in Dr. Wilson’s lab, I have developed valuable skills in data collection and analysis, as
well as gained extensive knowledge in behavioral neuroscience and pharmacology. With such knowledge, my interest in
the function of various neurochemical pathways, especially in fear behavior, has only grown. Thus, receiving the
Magellan Grant would not only continue to cultivate my passion for neuroscience as a whole, but also allow me to
demonstrate my commitment to expanding the scientific and medical community. As someone planning to attend medical
school, I hope this opportunity to further advance my research will prepare me for a future career as a physician and give
me a unique perspective when caring for patients. With the Magellan Grant, I will be able to focus more of my time and
effort on the research project, giving me a greater chance to successfully reach my goals.
 References
1. Tryon, S. C., Sakamoto, I. M., Kellis, D. M., Kaigler, K. F., & Wilson, M. A. (2021). Individual Differences in
Conditioned Fear and Extinction in Female Rats. Frontiers in behavioral neuroscience, 15, 740313.
https://doi.org/10.3389/fnbeh.2021.740313
2. Laine, M. A., Mitchell, J. R., Rhyner, J., Clark, R., Kannan, A., Keith, J., Pikus, M., Bergeron, E., Ravaglia, I.,
Ulgenturk, E., Shinde, A., & Shansky, R. M. (2022). Sounding the Alarm: Sex Differences in Rat Ultrasonic
Vocalizations during Pavlovian Fear Conditioning and Extinction. eNeuro, 9(6), ENEURO.0382-22.2022.
https://doi.org/10.1523/ENEURO.0382-22.2022
3. Portfors C. V. (2007). Types and functions of ultrasonic vocalizations in laboratory rats and mice. Journal of the
American Association for Laboratory Animal Science : JAALAS, 46(1), 28–34.
4. Tryon, S. C., Sakamoto, I. M., Kaigler, K. F., Gee, G., Turner, J., Bartley, K., Fadel, J. R., & Wilson, M. A.
(2023). ChAT::Cre transgenic rats show sex-dependent altered fear behaviors, ultrasonic vocalizations and
cholinergic marker expression. Genes, brain, and behavior, 22(1), e12837. https://doi.org/10.1111/gbb.12837
5. Kellis, D. M., Kaigler, K. F., Witherspoon, E., Fadel, J. R., & Wilson, M. A. (2020). Cholinergic neurotransmission in the
basolateral amygdala during cued fear extinction. Neurobiology of stress, 13, 100279.
https://doi.org/10.1016/j.ynstr.2020.100279
6. Wilson, M. A., & Fadel, J. R. (2017). Cholinergic regulation of fear learning and extinction. Journal of
neuroscience research, 95(3), 836–852. https://doi.org/10.1002/jnr.23840
7. Sharko, A. C., Fadel, J. R., Kaigler, K. F., & Wilson, M. A. (2017). Activation of orexin/hypocretin neurons is
associated with individual differences in cued fear extinction. Physiology & Behavior, 178, 93-102.
doi:10.1016/j.physbeh.2016.10.008
8. Deslauriers, J., Toth, M., Der-Avakian, A., & Risbrough, V. B. (2018). Current Status of Animal Models of
Posttraumatic Stress Disorder: Behavioral and Biological Phenotypes, and Future Challenges in Improving
Translation. Biological psychiatry, 83(10), 895–907. https://doi.org/10.1016/j.biopsych.2017.11.019
9. Ramikie, T. S., & Ressler, K. J. (2018). Mechanisms of Sex Differences in Fear and Posttraumatic Stress
Disorder. Biological psychiatry, 83(10), 876–885. https://doi.org/10.1016/j.biopsych.2017.11.016
10. Ross, D. A., Arbuckle, M. R., Travis, M. J., Dwyer, J. B., van Schalkwyk, G. I., & Ressler, K. J. (2017). An
Integrated Neuroscience Perspective on Formulation and Treatment Planning for Posttraumatic Stress Disorder:
An Educational Review. JAMA psychiatry, 74(4), 407–415. https://doi.org/10.1001/jamapsychiatry.2016.3325
11. Brudzynski S. M. (2021). Biological Functions of Rat Ultrasonic Vocalizations, Arousal Mechanisms, and Call
Initiation. Brain sciences, 11(5), 605. https://doi.org/10.3390/brainsci11050605
Instructions and tips on completing this form, can be found here.

Magellan Scholar BUDGET FORM

Student’s Name: Rebecca Bork

Double-click on table to enter data

Student salary

Research h o
sem esters w h e
class
Research h o
sem esters w h en
Budget Justification/Description class
NOTE: Magellan Scholar awards are processed through “E” funds. All expenditures MUST remain compliant with E fund procurement
requirements. All budgets must be reviewed by department business managers prior to submission.

Student Salary: Indicate estimated number of student research hours per week and hourly rate separated by semesters when student is enrolled in
classes or not enrolled in classes (generally fall or spring vs summer semesters). Time during breaks (Fall, Winter or Spring break) are still hours
during semesters of enrolled classes.
While Taking Classes: ~7 to 7.5 hours per week for 27 weeks at $14.00 per hour

Materials/Supplies*: Indicate items, quantity, and estimated price. Be sure to include taxes on all purchases.
*Review guidebook for allowable/unallowable expenses.
 Are you requesting funds for participant incentives? You must attach an approval memo from business manager – see guidebook.
 ALL non-expendable items MUST be fully explained/justified, if not described in methods
 See guidebook regarding machine, electronic, and equipment purchases (typically NOT allowed)
 Publication costs are not permitted

Year subscription to GraphPad Prism for data analysis: $142

External Hard Drive for data storage: $100
Discover USC poster: $57.90

Travel: Indicate location, purpose of travel, provide itemized costs (list out each cost separately: transportation, lodging, registration, etc). For
conferences, provide name of conference, dates, and explain why this conference is most appropriate. No more than $1000 is permitted for
conference travel.

N/A
Additional Funding: List additional funding sources and dollar amounts, including both those that you have applied for and those that you
previously received, that will fund aspects of this project. Explicitly state how the funds requested in the Magellan Scholar proposal will be used
differently from the funds requested/awarded through other grants.
N/A