1. Effects Of Glucocorticoid Exposure During First Week of Life
The objectives of this project is to identify brain mechanisms that may be involved in the generation and persistence of these problems. We use the rat as a model and concentrate in the Corticotropin Releasing Hormone Stress System and Growth Hormone axis.
Methodology: 1) In situ hybridization
2) Protein identification by immunostaining or binding methods
3) Radioimmunoassay
4) Animal procedures
5) Video watching and scoring
Student Tasks: Student will be involved in animal protocols, processing of tissue, and quantification. The student will help with the molecular biology and biochemical methods (slide subbing, preparation of solutions, sectioning, pipetting ). With the exception of quantification, all of these tasks require team work. Students will be paired with other students or technicians to accomplish these tasks. Students will be analyzing previously collected data.
Minimum Qualifications: General biology courses, general chemistry courses, computer skills (Excel, Word, Endnote). Willingness to work with small animals (rats).
The person can help with animal study, insitu hybridization, sectioning etc.

2. Maternal Care Study --- Project Flow Chart
8 Females X 8 Males
Giving birth (PD1, culling 6Females/6Males in one cage on PD2)
4 cages with Mom and Pups ( Handling )
4 cages with Mom and Pups (Non - Handling)
(Pups are away from the foster mother and cage 15min/day)
Licking, Nursing/LickingNursing
Maternal Watch on PD3 - PD14 (Manually)
3X 15min in the AM, 3X15min in the PM
PD22 Weaning ( separate with the foster mother )
PD57 – PD65 (Adult, only need female rats)
Females X other Males
Handling (4 cages x 6 females /cage = 24 females)
Non - Handling (4 cages x 6 females / cage= 24 females)
Stress (12 females )
Non - Stress (12 females )
Stress (12 females )
Non - Stress (12 females )
6 Females
6 Females
giving birth, culling 6females/6males in one cage on PD2
Gestation on E1-E19
Maternal Watch on PD3-PD14 ( video recording ), each cage has 12 pups
Stress on E3-E19, then sacrifice on E19 ( tailnick at 0’ 15’ 30’ and decap at 90’)
Mom, on one week after weaning
Pups, 4Females/4Males
Pups, 2Females/2Males
Tailnick at 0’ 15’ 30’ and decap at 90’
PD light/Dark testing
On PD33, decap at 0’ 15’ 30’ and 90’
Behavioral Testing
Brain
PD EPM testing
Blood
Corticosterone
ACTH
PD tailnick at 0’ 15’ 30’ and decap at 90’
In Situ Hybridization

3. Take Home Message
Parenting style can influence child’s health as an adult…
Will there be repercussions on stress response in the offspring of mothers subjected to stress during pregnancy?
Can maternal care mitigate the effect of prenatal stress?

4. Handling…
Touching the pups for 15 min daily results in the same biology as that given by high licking moms…

5. Design: First Phase Purpose: Create High Lickers
Daily Handling
3x AM
3x PM
Maternal Behavior Scored
HAN= 15 min/daily
HAN
HAN
HAN
Birth: litters culled to 12 pups (6M:6F)
HAN LR
N-HAN= not handled
Weaned 21 days
N-HAN
N-HAN
As adults, females are mated
N-HAN
N-HAN
12/31/2018

6. Design: Second Phase Purpose: Rise Stress Hormone Levels in utero
Pregnant- E1-E Birth Lactation Adulthood
HAN
Stressed
HAN
Unpredictable Stressors
swim
predator odors
crowding, etc....
HAN
Control
(not-stressed)
HAN LR
N-HAN
Stressed
N-HAN
Control
(not-stressed)
N-HAN
N-HAN
12/31/2018

7. Chronic Unpredictable Stress Paradigm for Maternal Care Study
Stressor Used
Warm Swim - up to 18 rats are placed in a sink filled with warm water(22-25ºC) for 15 min.
Crowding rats are all placed in one cage for 2 h. Be sure to leave food & water and lid ajar to allow for enough air circulation.
Fox Odor - fill shake-away powder inside 50ml tube (making a several holes on the side) and tape it on the corner of each cage, leave cap pen. During this stress, there are no any food and water provided.
Cold Swim - up to10-12 rats are placed in a sink filled with cold water(10ºC) for 5 min.
day
Gestation
AM (between 9am-noon)
PM (between 1pm-6pm)
Stress
Duration 1 E3
Swim( warm water )
15 min 2 E4
Crowding(6-8 rats)
2 h 3 E5
Fox Odor+Food, drink deprivation
4h(11:00-3:00PM) 4 E6
Swim( warm water) 5 E7 6 E8
Swim(Cold Water)
5min 7 E9 8
E10
Restraint
40min
Swim( warm water 22-25ºC) 9
E11 1h 10
E12 11
E13
Swim(Cold Water 10ºC)
10 min 2h 12
E14 13
E15 14 15
E17
2h(11:00-3:00PM) 16
E18
Rest(Decap at PD33) 17
E19
30min
Rest (Decap at E19)
Restraint 30min-----Tail nick at 0', 30', 60' and decap at 90'

8. Design: Third Phase Purpose: What effect does CUS have in pregnant rat stress response and brain elements?
Pregnant- E1-E22
HAN
Experiment 1
Obtain stress profile at E19
Sacrifice at the end of stress
Obtain brain for in situ studies
Stressed
HAN
HAN
Control
(not-stressed)
HAN LR
N-HAN
Stressed
N-HAN
Control
(not-stressed)
N-HAN
N-HAN
12/31/2018

9. Design: Fourth Phase Purpose: What effect does stress during pregnancy have on…
Pregnant- E1-E Birth Lactation Adulthood
HAN
Experiment 2
Score Maternal Behavior
Obtain stress profile at adolescence and adulthood
Test for anxiety behavior
Obtain brains for circuit studies
What effect on:
maternal behavior?
offspring
stress reactivity and shut down?
behavioral reactivity?
Stressed
HAN
HAN
Control
(not-stressed)
HAN LR
N-HAN
Stressed
N-HAN
Control
(not-stressed)
N-HAN
N-HAN
12/31/2018

10. Place Preference (Light-Dark Box), Open Field, Elevated Plus Maze
Impact of Postnatal Dexamethasone Therapy on Development and Function of the LHPA Axis
Place Preference (Light-Dark Box), Open Field, Elevated Plus Maze

11. Behavioral Testing Light/Dark Preference
In the light/dark preference test, each animal was placed in the dark compartment first, and the amount of time elapsed before the animal entered the lit side ( the latency to leave dark) was recorded. Locomotor's activity as well as time spent in each compartment were monitored. Total testing time was 5 min. The animal’s avoidance of the lighted portion of the apparatus suggests elevated anxiety, while showing little or no preference for the dark area of the enclosure suggests a lack of anxiety.  

12. Behavioral Testing (Cont.)
Elevated Plus Maze The elevated plus maze is widely used as an anxiety paradigm and is based on unconditioned responses of rat/mice to a potentially dangerous environment. A combination of maze height, luminosity and open space is assumed to induce fear or anxiety, the degree of which is assessed by measuring the amount of time rat/mice spend in various areas of the maze.

13. Endocrine Stress Response
Stress: various physiologic alternations including: homeostatic imbalances and activation of the Hypothalamic-Pituitary-Adrental Axis (HPA)
Stress
Amygdala
Hippocampus
Hypothalumus
(PVN) -
(Paraventricular nucleaus)
(Arginine Vasopressin ) AVP
CRH (Corticotropin Releasing Hormone)
Anterior Pituitary -
ACTH (Adrenocorticotropin Hormone)
(Negative-feedback regulation system)
Adrenal Cortex
Cortisol (primates)
Glucocorticoids
Corticosterone (rat/mice)
Acute Stress: Adaptive and essential for the immediate survival
Chronic Stress or Dysregulation: Cell Death, Mood and Affective Disorders