Anna M.

Hedges

Dr. Cassella

Intro Draft 1

November 9th, 2018

Pre-natal Stress Research

“Prenatal maternal stress refers to the stress that a mother experiences during her

pregnancy” (“What is…”). Prenatal stress is one major issue occurring in the world today. This

type of stress is actually more commonly happening than you would think. Given how common

prenatal stress is, it seems to be very improbable there would be effects on the development of

the child. Contrary to this belief, there have been multiple studies showing this to be the opposite

of true. Prenatal stress has been shown to be “associated with low birth weight, preterm birth,

preeclampsia, spontaneous abortion, growth-retardation (specifically reduced head

circumference), developmental delays, heightened emotionality, externalizing behaviors,

irritability, psychopathology, and deficits in attention, cognition, and neurodevelopment”

(LaPrairie, 2011). Specifically, this research is looking into how prenatal stress can lead to

neuropsychiatric diseases. According to other studies, prenatal stress can lead to many diseases

like anxiety (Vallee, 1997). (look more into this source and maybe add another paragraph below)

o Dutch Hunger Winter info here and cite

 (Shultz, 2010) article

o Excitatory vs. inhibitory imbalance (GABA & Glutamate)

 2 sources (1 general brain and one in the pre-fontal cortex)

o State 3 reasons why interested in the prefrontal cortex

 o Insert paper stating with linking psychiatric disease in the pre-fontal cortex

o Then talk about GAD 65

 What it is and what it does and how it fits in here with research

o Bring up hypothesis

 Expected there will be a lower conc. of GAD 65 in restricted males

Methods Section

Pregnant Female Sprague Dawley rats were used. These rats were received into the lab on day

E7 of pregnancy. Three days later on day E10, all of the rats were separated and housed individually

form one another. All of the pregnant rats, dams, were then separated into 3 different groups. Two of

the groups of dams were considered to be a part of the control group. This means they received 30

grams of regular chow food every 24 hours. This was found to be the normal amount of food in a 24-

hour period for dams. The other group was the considered to be the experimental group. This group is

the group that received the restricted by 50% food source by receiving 15 grams of food every 24 hours.

Once the pups were born, then food was given to the rats ad libitum, as needed.

24 hours after the pups were born, they were weighed and measured. The liters were then

reduced to just 8 pups a litter, four males and four females. These pups were then placed with a

different mother. Pups from the restricted food group were placed with the mom from control group 2.

The pups from control group 2 were placed with the mom from control group 1. Pups from control

group 1 were euthanized and not placed with the restricted food group mom to make sure the mom

being hungry did not change the offspring’s development. These mice were then raised by their “new
moms” and at the 30-day mark, one female and one male were then sacrificed. They were sacrificed

through perfusion. At the 60-day mark this process was repeated again in the same fashion with one

female and one male pup.

A total of 8 brains were used in this study, four control male pups and four restricted male pups.

In order to see GAD65, present in the prefrontal cortex, immunohistochemistry was used. To begin this

process, the perfused brains were first sectioned on a cryostat machine to 30 nm thick. Then the tissue

was washed in a 1X concentration of phosphate-buffered solution, PBS. After that, the tissue was then

washed in a 10% Donkey Serum solution for 30 minutes. Anti-GAD65 which was grown in a mouse was

then used at a 1:2000 dilution was used as the primary anti-body. The tissue was placed into the primary

anti-body solution for 24 hours in 4° C on the stir plate. The next day, the tissue was then washed again

with 1X PBS to rise all of the primary anti-body solution from the tissue. After washing, then the

secondary anti-body of anti-mouse FITC at a concentration of 1:500 was used. All tissue samples were in

the secondary anti-body solution for 60 minutes. They were then rinsed once again with the 1X PBS

solution and then mounted on positive slides. Poly-Mount was used to seal the cover slip onto the slide

and then slides were then stored in the fridge at 4° C.

The 8 slides were taken to Dr. Cooper’s lab in the Loras College Science hall to be imaged. An

Olympus electron microscope was used. This microscope was connected to a desktop computer and

images were then taken using cellSens Dimension software. All of the images were taken at 20x

magnification. Of each of the slides, 3 sections were imaged 3 times a piece for a total of 72 images.

After all data was collected from Dr. Cooper’s lab, the data was then analyzed using (insert info

on how data was analyzed and what tests ran to do so)

LaPrairie, J. L., & Brennan, P. A. (2011). Prenatal stress. Retrieved from

https://www.sciencedirect.com/topics/neuroscience/prenatal-stress

Schulz, L. C. (2010, September 28). The Dutch Hunger Winter and the developmental origins of

health and disease. Retrieved from http://www.pnas.org/content/107/39/16757

Vallee, M., Mayo, W., Dellu, F., Moal, M. L., Simon, H., & Maccari, S. (1997). Prenatal Stress

Induces High Anxiety and Postnatal Handling Induces Low Anxiety in Adult Offspring:

Correlation with StressInduced Corticosterone Secretion. The Journal of

Neuroscience,2626-2636. Retrieved November 8, 2018, from

http://www.jneurosci.org/content/jneuro/17/7/2626.full.pdf

“What is Prenatal Maternal Stress?” (2013, January 31). Retrieved from

https://www.mcgill.ca/spiral/spiral/prenatal-stress