Effects Of Glucocorticoid Exposure During First Week of Life

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 (Non - Handling)
4 cages with Mom and Pups ( 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
PD 57-60 light/Dark testing
Tailnick at 0 15 30 and decap at 90
On PD33, decap at 0 15 30 and 90
Behavioral Testing
PD 65-68 EPM testing
Brain
Blood
PD 77-81 tailnick at 0 15 30 and decap at 90
Corticosterone ACTH
In Situ Hybridization
3
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) AM (between 9am-noon) PM (between 1pm-6pm) PM (between 1pm-6pm) PM (between 1pm-6pm) PM (between 1pm-6pm)
    Stress Duration Stress Stress Stress Duration
1 E3     Swim( warm water ) Swim( warm water ) Swim( warm water ) 15 min
2 E4 Crowding(6-8 rats) 2 h        
3 E5     Fox OdorFood, drink deprivation Fox OdorFood, drink deprivation Fox OdorFood, drink deprivation 4h(1100-300PM)
4 E6 Swim( warm water) 15 min        
5 E7     Fox OdorFood, drink deprivation Fox OdorFood, drink deprivation Fox OdorFood, drink deprivation 4h(1100-300PM)
6 E8 Swim(Cold Water) 5min        
7 E9     Crowding(6-8 rats) Crowding(6-8 rats) Crowding(6-8 rats) 2 h
8 E10 Restraint 40min Swim( warm water 22-25ºC) Swim( warm water 22-25ºC) Swim( warm water 22-25ºC) 15 min
9 E11 Fox OdorFood, drink deprivation 4h(1100-300PM) Restraint Restraint Restraint 1h
10 E12 Crowding(6-8 rats) 2 h        
11 E13 Swim(Cold Water 10ºC) 10 min Restraint Restraint Restraint 2h
12 E14 Restraint 2h Fox OdorFood, drink deprivation Fox OdorFood, drink deprivation Fox OdorFood, drink deprivation 4h(1100-300PM)
13 E15 Swim(Cold Water) 5min        
14 E12 Crowding(6-8 rats) 2 h        
15 E17 Fox OdorFood, drink deprivation 2h(1100-300PM) Restraint Restraint Restraint 2h
16 E18 Rest(Decap at PD33) Rest(Decap at PD33) Rest(Decap at PD33) Rest(Decap at PD33) Rest(Decap at PD33) Rest(Decap at PD33)
17 E19 Restraint 30min        
16 E18 Rest (Decap at E19) Rest (Decap at E19) Rest (Decap at E19) Rest (Decap at E19) Rest (Decap at E19) Rest (Decap at E19)
17 E19 Restraint 30min-----Tail nick at 0', 30', 60' and decap at 90' Restraint 30min-----Tail nick at 0', 30', 60' and decap at 90' Restraint 30min-----Tail nick at 0', 30', 60' and decap at 90'      
4
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 animals
  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.  

5
Behavioral Testing (Cont.)

• Elevated Plus MazeThe 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.

6
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