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 (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 ) Non - Stress (12 females ) Stress (12 females ) Non - Stress (12 females ) 6 Females Stress on E3-E19, then sacrifice on E19 ( tailnick at 0’ 15’ 30’ and decap at 90’) giving birth, culling 6females/6males in one cage on PD2 Maternal Watch on PD3-PD14 ( video recording ), each cage has 12 pups Pups, 4Females/4MalesPups, 2Females/2Males On PD33, decap at 0’ 15’ 30’ and 90’ PD light/Dark testing PD EPM testing PD tailnick at 0’ 15’ 30’ and decap at 90’ Behavioral Testing Mom, on one week after weaning Tailnick at 0’ 15’ 30’ and decap at 90’ Brain Blood In Situ Hybridization Corticosterone ACTH 4 cages with Mom and Pups ( Handling ) (Pups are away from the foster mother and cage 15min/day) Gestation on E1-E19

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. dayGestationAM (between 9am-noon)PM (between 1pm-6pm) Stress DurationStressDuration 1E3 Swim( warm water )15 min 2E4Crowding(6-8 rats)2 h 3E5 Fox Odor+Food, drink deprivation 4h(11:00-3:00PM) 4E6Swim( warm water)15 min 5E7 Fox Odor+Food, drink deprivation 4h(11:00-3:00PM) 6E8Swim(Cold Water)5min 7E9 Crowding(6-8 rats)2 h 8E10Restraint40minSwim( warm water 22-25ºC)15 min 9E11 Fox Odor+Food, drink deprivation 4h(11:00-3:00PM)Restraint1h 10E12Crowding(6-8 rats)2 h 11E13Swim(Cold Water 10ºC)10 minRestraint2h 12E14Restraint2h Fox Odor+Food, drink deprivation 4h(11:00-3:00PM) 13E15Swim(Cold Water)5min 14E12Crowding(6-8 rats)2 h 15E17 Fox Odor+Food, drink deprivation 2h(11:00-3:00PM)Restraint2h 16E18Rest(Decap at PD33) 17E19Restraint30min 16E18Rest (Decap at E19) 17E19Restraint 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) Amygdala Stress Hippocampus Hypothalumus (PVN) Anterior Pituitary Adrenal Cortex (Arginine Vasopressin ) AVP CRH (Corticotropin Releasing Hormone) ACTH (Adrenocorticotropin Hormone) - - ( Negative-feedback regulation system ) Acute Stress: Adaptive and essential for the immediate survival Chronic Stress or Dysregulation: Cell Death, Mood and Affective Disorders (Paraventricular nucleaus) Cortisol (primates) Corticosterone (rat/mice) Glucocorticoids