Principal Research Question:

Why do mammals get old and die?How is this affected by:a. Reproductionb. Natural Stressors

(competition, predators, etc.)c. Maternal and Cohort Effects

But physiological evidenceobtained can be used to examineother 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.

Senescence

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

Theory of SenescenceTheory of Senescence

1. The age at which senescence is first expressed1. The age at which senescence is first expresseddepends on how much mortality occursdepends on how much mortality occursindependent of the effects of senescence.independent of the effects of senescence.

2. Senescence should not be expressed until2. Senescence should not be expressed untilafter the age at first reproduction.after the age at first reproduction.

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 pastExamine at 2 levels:a. Changes in stress response.b. Changes in regulation of this response at the level of the Brain.

Stress Stress Response - the set of Stress Response - the set of responses by birds and mammals by responses by birds and mammals by the stress axis to potentially harmful the stress axis to potentially harmful environmental challengesenvironmental challenges

Stressor - anything that upsets the Stressor - anything that upsets the

homeostatic balance within an animalhomeostatic balance within an animal EnvironmentalEnvironmental Physical Physical PsychologicalPsychological

Response to Stressor is Crucial and Changes with Age,

Condition, Experience, etc.

Crucial components:Crucial components:

1.1. Response to the stressor - Response to the stressor - how rapid is it and how how rapid is it and how intense?intense?

2.2. Negative Feedback - how Negative Feedback - how rapidly is it terminated?rapidly is it terminated?

PITUITARYPITUITARY

ADRENALSADRENALS

HIPPOCAMPUSHIPPOCAMPUS

BLOODBLOOD

HYPOTHALAMUSHYPOTHALAMUS

ACTH

CRFCRFCRFCRF

ACTHACTHACTHACTH

CortisolCortisolCortisolCortisol

STRESSSTRESS

NegativeNegativeFeedbackFeedback

Hippocampus

Hypothalamus

AnteriorPituitary

GR

MR

AdrenalCortex

MobilizationOf Energy

SuppressionOf Growth

Immuno-suppression

SuppressionOf DigestionReproductive

Suppression

ACTH

Glucocorticoids

AVPCRH

PVN

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.]

CATABOLICCATABOLIC

ANABOLICANABOLIC

LIVER

GLUCONEOGENESIS GLUCOSE

GLYCOGEN

MUSCLE

LYMPHOID

SKIN

ADIPOSE

ENERGYSUPPLY

FATTYACIDS

GLYCEROLGLUCOSE

CONNECTIVE

AMINOACIDS

Cortisol has both these effects:

Stress Response is not Static

1. May be modulated over annual cycle to optimize reproduction, survival, or both

2. Modified during development: Programming of the Brain.

3. Modified by experience.

Methods to study stress response in Natural

Populations Before and After assessment (crude) Before and After assessment (crude)

Measurement in feces and urine Measurement in feces and urine (rough and limited utility)(rough and limited utility)

Challenge ProtocolChallenge Protocol CaptureCapture HormonalHormonal - - Will Use thisWill Use this

ACTH Stimulation Test

Inject ACTHInject ACTH Measure glucocorticoid levelsMeasure glucocorticoid levels

Moderate increase is normalModerate increase is normal

Excessive increase or reduced Excessive increase or reduced

response (species response (species dependent) dependent) indicative of chronic indicative of chronic stress. stress.

PITUITARYPITUITARY

ADRENALSADRENALS

HIPPOCAMPUSHIPPOCAMPUS

BLOODBLOOD

HYPOTHALAMUSHYPOTHALAMUS

ACTH

CRFCRFCRFCRF

ACTHACTHACTHACTH

Cortisol / Cortisol / CorticosteroneCorticosterone

Cortisol / Cortisol / CorticosteroneCorticosterone

ACTHACTH

Hormonal Challenge Hormonal Challenge ProtocolProtocol

00 3030 6060 120120

BASE BleedBASE BleedACTH InjectionACTH Injection

ACTH ACTH BleedsBleeds

UnitsUnits

TimeTime

Poor ConditionPoor Condition

Good ConditionGood Condition

ACTHACTH

Free Free CortisolCortisol

Time

Hormonal Challenge ProtocolHormonal Challenge Protocol

1. Capture squirrel2. Bring to standard location3. Bleed four times with suborbital sinus bleeds4. 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 toobserve ability to handle challenge

Blood Samples will be analyzed forBlood 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: concentrationand ratio of lymphocytes, neutrophils, and albuminetc. Will only do for BASE bleed. This must be done in Veterinary lab in Edmonton orToronto

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 Question - Does an animal’s ability to handle

a immunocompetence challenge vary a immunocompetence challenge vary with:with:

1.1. AgeAge

2.2. Condition of Territory (e.g.Fed vs Condition of Territory (e.g.Fed vs Nonfed)Nonfed)

3.3. Investment in ReproductionInvestment in Reproduction

4.4. Cohort & Maternal EffectsCohort & Maternal Effects

5.5. Year EffectsYear Effects

Immunocompetence Challenge Protocol

Measure: Cell Mediated Immunity Measure: Cell Mediated Immunity OnlyOnly

1. Day 1: capture animal, inject 1. Day 1: capture animal, inject standardizedstandardized

antigen (sheep erythrocytes), release.antigen (sheep erythrocytes), release.

2. Day 7: recapture animal, collect blood 2. Day 7: recapture animal, collect blood sample, take sample to lab, do sample, take sample to lab, do agglutinationagglutination

tests against antigen to assess tests against antigen to assess animal’sanimal’s

ability to mount a immune response.ability to mount a immune response.

3. 3. Simple, fast, and direct.Simple, fast, and direct.

Possible Effects of Protocols on Red Squirrels1.1. Have done Hormonal Challenge Have done Hormonal Challenge

Protocol on about 50 Lloyd Protocol on about 50 Lloyd animals. All released alive, many animals. All released alive, many called on release, and immediately called on release, and immediately went on with lives.went on with lives.

2.2. Is there a survival cost to hormonal Is there a survival cost to hormonal challenge protocol?challenge protocol?Don’t think so. But can test will Don’t think so. But can test will Lloyd animals. In hares, animals Lloyd animals. In hares, animals challenged with a similar challenged with a similar procedure in winter and released procedure in winter and released had similar survival of animals not had similar survival of animals not challenge. challenge.

Sex to be examined

Nonbreeding season: both Nonbreeding season: both sexes sexes

Breeding Season: restrict tests Breeding Season: restrict tests to MALE red squirrels only to to MALE red squirrels only to avoid any pregnancy or avoid any pregnancy or lactation effects on young.lactation effects on young.

When?

Early August at or before food Early August at or before food gathering. Will allow examination gathering. Will allow examination of relationship to overwinter of relationship to overwinter survival survival

Late February or early March Late February or early March before breeding season. Will before breeding season. Will allow examination of relationship allow examination of relationship between reproductive fitness and between reproductive fitness and physiological condition.physiological condition.