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 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 8 Females X 8 Males 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 57-60 light/Dark testing On PD33, decap at 0’ 15’ 30’ and 90’ Behavioral Testing Brain PD 65-68 EPM testing Blood Corticosterone ACTH PD 77-81 tailnick at 0’ 15’ 30’ and decap at 90’ 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.

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 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.  

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