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.
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 )
(Pups are away from the foster mother and cage 15min/day)
4 cages with Mom and Pups (Non - Handling)
Maternal Watch on PD3 - PD14 (Manually)
3X 15min in the AM, 3X15min in the PM
Licking,
Nursing/Licking
Nursing
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 )
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
Stress on E3-E19, then sacrifice
on E19 ( tailnick at 0’ 15’ 30’
and decap at 90’)
Maternal Watch on PD3-PD14 ( video recording ), each cage has 12 pups
Mom, on one week
after weaning
Tailnick at 0’ 15’ 30’
and decap at 90’
Brain
In Situ Hybridization
Non - Stress (12 females )
Blood
Corticosterone
ACTH
Pups, 4Females/4Males
On PD33, decap at
0’ 15’ 30’ and 90’
Pups, 2Females/2Males
PD 57-60 light/Dark testing
PD 65-68 EPM testing
PD 77-81 tailnick at 0’ 15’
30’ and decap at 90’
Behavioral
Testing
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 - 5-8 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)
Stress
PM (between 1pm-6pm)
Duration
Stress
Duration
Swim( warm water )
15 min
Fox Odor+Food, drink deprivation
4h(11:00-3:00PM)
Fox Odor+Food, drink deprivation
4h(11:00-3:00PM)
Crowding(6-8 rats)
2h
1
E3
2
E4
3
E5
4
E6
5
E7
6
E8
7
E9
8
E10
Restraint
40min
Swim( warm water 22-25ºC)
15 min
9
E11
Fox Odor+Food, drink deprivation
4h(11:00-3:00PM)
Restraint
1h
10
E12
Crowding(6-8 rats)
2h
11
E13
Swim(Cold Water 10ºC)
10 min
Restraint
2h
12
E14
Restraint
2h
Fox Odor+Food, drink deprivation
4h(11:00-3:00PM)
13
E15
Swim(Cold Water)
5min
14
E12
Crowding(6-8 rats)
2h
15
E17
Fox Odor+Food, drink deprivation
2h(11:00-3:00PM)
Restraint
2h
16
E18
17
E19
16
E18
17
E19
Crowding(6-8 rats)
Swim( warm water)
Swim(Cold Water)
2h
15 min
5min
Rest(Decap at PD33)
Restraint
30min
Rest (Decap at E19)
Restraint 30min-----Tail nick at 0', 30', 60' and decap at 90'
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.
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.
Endocrine Stress Response
Stress: various physiologic alternations including: homeostatic
imbalances and activation of the Hypothalamic-Pituitary-Adrental
Axis (HPA)
Stress
Amygdala
-
Hippocampus
Hypothalumus
(PVN)
(Arginine Vasopressin )
(Negative-feedback
regulation system)
AVP
CRH
(Paraventricular nucleaus)
(Corticotropin Releasing Hormone)
Anterior Pituitary
ACTH (Adrenocorticotropin Hormone)
Adrenal Cortex
Glucocorticoids
Cortisol (primates)
Corticosterone (rat/mice)
Acute Stress: Adaptive and essential for the immediate survival
Chronic Stress or Dysregulation: Cell Death, Mood and Affective Disorders