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 ) 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 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

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?

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

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

Design: Second Phase Purpose: Rise Stress Hormone Levels in utero Pregnant- E1-E22 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

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) 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'

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

Design: Fourth Phase Purpose: What effect does stress during pregnancy have on… Pregnant- E1-E22 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

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

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) - (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