1. Principal Research Question: Why do mammals get old and die? How is this affected by: a. Reproduction b. Natural Stressors (competition, predators, etc.) c. Maternal and Cohort Effects

2. But physiological evidence obtained can be used to examine other areas simultaneously: 1. Genotypic and phenotypic variation. 2. Role of territory quality on variation in physiology. 3. Impact of reproductive effort on physiology. 4. Cohort and year effects.

3. Senescence the process of decline in physiological functioning that results in increasing mortality rates with increasing age after some point in the lifespan

4. Theory of Senescence 1. The age at which senescence is first expressed depends on how much mortality occurs independent of the effects of senescence. 2. Senescence should not be expressed until after the age at first reproduction.

5. Principal Research Approach: Target the key physiological system - the STRESS AXIS - that is crucial for survival, allows animals to cope with challenges, and integrates and reflects the past Examine at 2 levels: a. Changes in stress response. b. Changes in regulation of this response at the level of the Brain.

6. Stress Stress Response - the set of responses by birds and mammals by the stress axis to potentially harmful environmental challenges Stress Response - the set of responses by birds and mammals by the stress axis to potentially harmful environmental challenges Stressor - anything that upsets the Stressor - anything that upsets the homeostatic balance within an animal homeostatic balance within an animal Environmental Environmental Physical Physical Psychological Psychological

8. Response to Stressor is Crucial and Changes with Age, Condition, Experience, etc. Crucial components: 1. Response to the stressor - how rapid is it and how intense? 2. Negative Feedback - how rapidly is it terminated?

9. PITUITARY ADRENALSHIPPOCAMPUSBLOOD HYPOTHALAMUS ACTH CRF ACTH Cortisol STRESSNegativeFeedback

10. Hippocampus Hypothalamus Anterior Pituitary GR MR Adrenal Cortex Mobilization Of Energy Suppression Of Growth Immuno- suppression Suppression Of Digestion Reproductive Suppression ACTH Glucocorticoids AVP CRH PVN

11. Stress Response Good: if short term = Acute Response [Classic Flight or Fight Response] Bad: if long term = Chronic Response [short term effects are prolonged, with potential permanent consequences - Brain changes,etc.]

12. CATABOLIC ANABOLIC LIVER GLUCONEOGENESIS GLUCOSE GLYCOGEN MUSCLE LYMPHOID SKIN ADIPOSE ENERGY SUPPLY FATTY ACIDS GLYCEROL GLUCOSE CONNECTIVE AMINO ACIDS Cortisol has both these effects:

13. Stress Response is not Static 1. 1. May be modulated over annual cycle to optimize reproduction, survival, or both 2. 2. Modified during development: Programming of the Brain. 3. 3. Modified by experience.

14. Methods to study stress response in Natural Populations Before and After assessment (crude) Before and After assessment (crude) Measurement in feces and urine (rough and limited utility) Measurement in feces and urine (rough and limited utility) Challenge Protocol Challenge Protocol  Capture  Hormonal - Will Use this

15. ACTH Stimulation Test  Inject ACTH  Measure glucocorticoid levels  Moderate increase is normal  Excessive increase or reduced response (species dependent) indicative of chronic stress. response (species dependent) indicative of chronic stress.

16. PITUITARY ADRENALS HIPPOCAMPUS BLOOD HYPOTHALAMUS ACTH CRF ACTH Cortisol / Corticosterone ACTH

17. Hormonal Challenge Protocol 03060120 BASE Bleed ACTH Injection ACTH Bleeds ACTH Bleeds Units Time

18. Poor Condition Good Condition ACTH FreeCortisol Time

19. Hormonal Challenge Protocol 1. Capture squirrel 2. Bring to standard location 3. Bleed four times with suborbital sinus bleeds 4. Anesthetize each time with isofluorane and portable anesthetic device. 5. Bleeds : collect about 300 ul each time. Thus about 1.2 mls (note that a 250 g squirrel has about 25 ml blood; thus no problem here) 6. Bleeds: first = Baseline. Then inject intramuscular into Thigh approximately 100 ul ACTH. This Stimulates adrenals to mobilize cortisol 7. ACTH response: serial bleeds at 30, 60 and 120 min to observe ability to handle challenge

20. Blood Samples will be analyzed for 1. Cortisol and carrier protein - CBG. Will allow us to see rapidity and extent of response. 2.Testosterone (in males) 2. Measures of Energy mobilization: a. Glucose (principal fuel) b. Free fatty acids (secondary fuel) 3. Other measures of CONDITION a. hematocrit (packed cell volume - more is better) b. complete white blood cell profile: concentration and ratio of lymphocytes, neutrophils, and albumin etc. Will only do for BASE bleed. This must be done in Veterinary lab in Edmonton or Toronto

21. Assessment of Immune System and its ability to respond to a challenge is also be a good indication of physiological condition and status of the squirrel. Question - Does an animal’s ability to handle a immunocompetence challenge vary with: 1. Age 2. Condition of Territory (e.g.Fed vs Nonfed) 3. Investment in Reproduction 4. Cohort & Maternal Effects 5. Year Effects

22. Immunocompetence Challenge Protocol Measure: Cell Mediated Immunity Only 1. Day 1: capture animal, inject standardized antigen (sheep erythrocytes), release. 2. Day 7: recapture animal, collect blood sample, take sample to lab, do agglutination tests against antigen to assess animal’s ability to mount a immune response. 3. Simple, fast, and direct.

23. Possible Effects of Protocols on Red Squirrels 1. Have done Hormonal Challenge Protocol on about 50 Lloyd animals. All released alive, many called on release, and immediately went on with lives. 2. Is there a survival cost to hormonal challenge protocol? Don’t think so. But can test will Lloyd animals. In hares, animals challenged with a similar procedure in winter and released had similar survival of animals not challenge.

24. Sex to be examined Nonbreeding season: both sexes Nonbreeding season: both sexes Breeding Season: restrict tests to MALE red squirrels only to avoid any pregnancy or lactation effects on young. Breeding Season: restrict tests to MALE red squirrels only to avoid any pregnancy or lactation effects on young.

25. When? Early August at or before food gathering. Will allow examination of relationship to overwinter survival Early August at or before food gathering. Will allow examination of relationship to overwinter survival Late February or early March before breeding season. Will allow examination of relationship between reproductive fitness and physiological condition. Late February or early March before breeding season. Will allow examination of relationship between reproductive fitness and physiological condition.