THE STRESS RESPONSE Flashcards
1
Q
Which two nuclei release VSP? What
A
Magnocellular and parvocellular neurons in the Supraoptic and PVN. Parvocellular important for acute stress, magnocellular for water homeostasis.
2
Q
How does AVP travel?
A
Magnocellular: AVP then travels down the axon through the infundibulum in neurosecretory granules that are found in Herring bodies (localized swellings of the axons and nerve terminals). Stored in the posterior pituitary until release.
Parvocellular in the PVN: released into the median eminence, then through the hypophyseal portal circulation to the anterior pituitary
3
Q
What happens to VSP in anterior pituitary?
A
It works synergistically with the CRH to release ACTH from corticotrophin cells.
4
Q
What is stress?
A
A change in the internal or external environment which the organism perceives as a threat to homeostasis. It is physical or anticipatory
5
Q
What is the general adaptation syndrome?
A
Alarm, resistance - coping, exhaustion - illness
6
Q
What are consequences of stress?
A
- Adrenal hypertrophy of the cortex, producing more glucocorticoid
- Thymic atrophy
- Ulcers
- HT and cardiovascular dysfunction
- Behavioural issues
7
Q
Describe Cushings and an important note
A
- Immunosuppression
- Hypertension- can occur when the glucocorticoids act on the mineralocorticoid receptor in the kidney. This is normally prevented as cortisol is inactivated in the kidney to cortisone through 11bhsd2.
- Steroid diabetes
- Centropetal obesity but in other tissues you get lipolysis
- Muscle wasting. Cortisol is a catabolic hormone to provide a substrate for metabolism
- Impaired cognitive function
- Emotional lability
- Neurodegeneration
- Osteoporesis
- Impaired growth
- Impaired reproductive function
That people with stress related disorders do not have Cushing’s
8
Q
What are general functions of the glucocorticoids?
A
- Regulation of innate/acquired immunity
- Cell growth and differentiation
- Metabolism – energy mobilisation: increases blood glucose. Largely catabolic actions – hepatic glycogenolysis, gluconeogenesis/proteolysis, lipolysis (in some tissues, but centripetal obesity i.e. re-distribution of fat), inhibition of glucose uptake in peripheral tissues
- Cardiovascular function
- Bone turnover
- Cognitive function
9
Q
What 3 ways could you say glucocorticoids deal with stress?
A
Permissive, adaptive and protective
10
Q
What are examples of permissive?
A
At low physiological levels cortisol maintains or primes the defence mechanisms to enable the organism to respond appropriately to stress by for example augmenting expression of:
- Pro-inflammatory cytokine receptors
- β-adrenoceptors and phenylethanolamine methyl transferase
11
Q
What about adaptive?
A
- Energy mobilization
- Hepatic gluconeogenesis, raised blood sugar
- Mobilisation of fatty acids and amino acids
- Increased mental alertness
12
Q
What are protective functions?
A
- Inhibition of the synthesis and release of pro-inflammatory mediators, e.g. IL-1β, IL-6, prostaglandins, leukotrienes
- Impaired translocation of the glucose transporter, GLUT-4, to the cell membrane; hence reduced glucose uptake by cells.
- Suppression on non-vital functions, e.g. growth and reproduction.
13
Q
What factors influence circadian rhythm?
A
Through the suprachiasmatic nucleus
14
Q
What are factors affecting cortisol excretion?
A
- Neural - CRH and AVP neurones posses ionotropic glutamate and alpha – 1 adrenoceptors (stimulatory) and GABAa receptors (inhibitory)
- Humoral – cytokine, chemokine – circumventricular organ
- Local – prostaglandins (produced from pain
- Hormonal feedback
15
Q
How does stressed induced activation of the axis occur? Diagram
A
Through the limbic system (amygdala and hippocampus) to the PVN. The environmental and higher stressors go via the cortex to the limbic system. Finally homeostatic mechanisms go via the NTS to the PVN or limbic system. Cortisol is produced in relation to ACTH but there is a slight delay as it has to be synthesised (hence acth levels go down initially)
16
Q
What are experimental end points for measuring stress?
A
- Plasma ACTH and/or corticosterone – increasingly using repeated measures over time to examine hormone pulsatility. Also, following stress ACTH granules are released meaning levels rise very quicky and there is then a delay in cortisol release. One measurement at a specific time might therefore not be representative
- CRH and AVP in portal blood (very difficult).
- Hypothalamic CRH and AVP mRNA and peptide (note CRH and AVP protein levels are reduced in the median eminence after stress as stores have been released; delayed increases in CRH and AVP mRNA levels occur and serve to restore the depleted protein levels.
- Immediate early gene activation in the brain (usually cfos) mRNA or protein)
- Expression of CRH, AVP and glucocorticoid (GR and MR) receptors
- In vitro studies
17
Q
What is coping?
A
- Rapid mobilization of effector systems and effective feedback mechanisms.
18
Q
What happens in acute stress and what are the mediators?
A
Raised cortisol - Magnitude and duration of rise depend on the stress and the individual. Designed to mobilize and redirect energy stores, prime immune system, arousal, maintain blood pressure and inhibit HPG axis. Rapid activation (‘fight or flight response) closely followed by resolution/recovery phase = effective stress coping response. Principal effectors of the stress system: CRH, AVP, nor-adrenaline, adrenaline, glucocorticoids
19
Q
In chronic stress, what do adaptive responses depend on?
A
– Adaptive responses dependent on
• the stress and its predictability
• the individual and his/her ability to cope as well as their previous experiences
20
Q
What are the chronic responses to stress?
A
- Raised morning cortisol
- Blunted circadian rhythm
- Exaggerated or prolonged cortisol responses to novel stress. HYPER EXCITABILITY OF THE HPA AXIS
- Overall tissues experience a more continuous exposure of glucocorticoids
21
Q
What happens in repeat homotypic stress?
A
– Animals become habituated and the HPA response on each repeated stress exposure is attenuated
– Responses to novel stressors may be exacerbated.
22
Q
What happens in the onset of progressive disease?
A
– Development of HPA over-activity parallels the onset and severity of disease
– AVP becomes the primary driver of ACTH secretion. CRH Decreases significantly, neurons downregulated. AVP may be responsible for the hypersensitivity of the axis.
23
Q
What is chronic progressive disease characterised by?
A
- Downregulation of CRH mRNA in the parvocellular PVN
- Downregulation of CRH receptor binding in the anterior pituitary
- Sustained upregulation of AVP mRNA in the parvocellular PVN. No increase in the number of cells expressing AVP mRNA, but an increase in the amount of AVP mRNA per cell.
- Increase in V1b receptor mRNA and AVP receptor binding levels in the anterior pituitary
24
Q
What happens in chronic variable stress? What did the animal study show
A
Adaptive changes: increased CRH expression and impaired glucocorticoid feedback. Tissues may experience overall greater glucocorticoid exposure.
There is an increase in the number of cells expressing CRH as well as an increase in sympathetic inputs. This reflects the increase in stimulatory glutaminergic and noradrenergic inputs (measured through synaptophysin)
– Thymus weight unchanged (at very high levels of glucocorticoid would expect a decrease in weight)
– There is impaired negative feedback and a downregulation of glucocorticoid receptors in the hypothalamus and a downregulation of hippocampal MR and GR
– Rat experiment: Characterised by a modest but sustained hypersecretion of corticosterone, ACTH (the weight of the adrenal gland increases), prolactin (unknown the reason behind this, perhaps to modulate the immune system
25
What are the neuroplastic responses to stress?
Atrophy of regions with inhibitory influences on HPA & ANS responses to acute stress and hypertrophy of excitatory regions
26
What happens in the inhibitory neuroplastic responses?
• Hippocampus & mPFC
• Retraction of apical dendrites & reduced dendritic spine density (sites of excitatory glutamataergic input) in response to chronic restraint.
• Decreased GR expression
Net: chronic GC elevations (decreased GC feedback); impaired memory & reward
27
What happens in excitatory neuroplastic responses?
Hypertrophy/up-regulation in regions with excitatory influences on HPA & ANS responses to acute stress
• Basolateral amygdala (psychogenic stress/HPAA) - Increased dendritic branching & stress excitability
• Central amygdala (systemic stress/ANS activation) - Increased CRH expression and release
• Hypothalamic PVN: Increased CRH & AVP expression & release
• Decreased GR expression
Net: increased excitability to novel stress (GCs & emotionality) & decreased reward
28
What do all stresses ultimately do?
stress ultimately signal via the hypothalamic parvocellular PVN to restore homeostasis
29
What are the two main parts of the forebrain in terms of stress?
• Hippocampus Contextual, episodic & spatial memory; down-regulation of stress response. Primarily activated by psychogenic stress: terminates HPAA response – regulates duration rather than magnitude [GR] decreases autonomic tone
• Amygdala Emotion, fear, anxiety, aggression, up-regulation of stress response. Central Amygdala activated by systemic > psychogenic stress; activates ANS > HPAA via projections to NTS
Prefrontal cortex decision making, working memory, controls impulsivity, down-regulation of stress response. It is primarily activated by psychogenic stress. Activation of prelimbic & dorsal regions terminates HPA response and reduces ANS response [GR]. Infralimbic regions initiate ANS & HPA responses via projections to NTS.
30
What does the pre frontal cortex do?
Prefrontal cortex decision making, working memory, controls impulsivity, down-regulation of stress response. It is primarily activated by psychogenic stress. Activation of prelimbic & dorsal regions terminates HPA response and reduces ANS response [GR]. Infralimbic regions initiate ANS & HPA responses via projections to NTS.
31
What neural inputs affect AVP and CRH release from the hypothalamus?
Neural - CRH and AVP neurones posses ionotropic glutamate and alpha – 1 adrenoceptors (n-ad), which are stimulatory, and GABAa receptors (inhibitory)
32
What simple experiment is there to assess corticosterone?
Rat. Measure corticosterone release after an injection of LPS versus a vehicle. Also induce hypo glycaemia, - Effects of hypoglycaemic stress on expression of CRH mRNA in the parvocellular PVN of the rat.
33
What evidence is there for chronic homotypic stress?
– Effect of repeated restraint stress for 1 h per day on expression of CRH and VP heteronuclear RNA levels in the medial parvocellular PVN of the rat. After repeated stress there is down-regulation of CRH mRNA. After repeated stress there is NO down-regulation of AVP mRNA
34
What is the experiment to look at chronic variable stress?
Experiment: Protocol:
– Random exposure to variety of mild stressors, e.g. novel environment, tilted cage, noise, wet bedding, strobe lighting
– Key features is the unpredictability of the nature and timing of the stress and the lack of ‘control’ the animal has of the situation.
– Compared to control, increased ACTH, Corticosterone, prolactin, increased adrenal gland weight and no change in thymus weight
Increase in number of cells expressing CRH mRNA. NB: these increases reflect increases in glutamatergic and noradrenergic inputs
Note that chronic variable stress does not increase the number of cells expressing AVP but does increase the amount of mRNA per cell
35
Where does the SNS response come from?
medullary preganglionic neurones of the intermediolateral cell column of the SC
36
Where does the PNS go through?
e.g. activation of NTS, then nucleus ambiguus & dorsal motor nucleus of the vagus nerve
37
What is the difference between the HPA and antonomic responses?
Slower onset (mins), more protracted than ANS response
38
What does the prefrontal cortex do?
Primarily activated by psychogenic stress
Activation of prelimbic & dorsal regions terminates HPAA response and reduces ANS response [GR]
infralimbic regions initiate ANS & HPA responses via projections to NTS
39
What does the amygdala do?
Central Amygdala activated by systemic > psychogenic stress; activates ANS > HPAA via projections to NTS
Medial & basolateral amygdala activated by psychogenic > systemic stress to up-regulate HPAA via interactions with PVN-projecting neurons
Think - involved in fear.
40
What does the hippocampus do?
Primarily activated by psychogenic stress
terminates HPAA response – regulates duration rather than magnitude [GR]
decreases autonomic tone
41
How do the brain stem and PVN communicate?
Through reciprocal connections. Brainstem NTS to PVN. and from dorsolateral parvocellular connection to brainstem.
42
Where do catecholaminergic inputs come from?
NTS
43
What is Hans Selye's general adaptation syndrome
Initially there is adrenal NA and GC, homeostasis restored (dips then goes up again). Then defence and adaptation sustained and optimal (curve plateaus at peak). Then adaptive responses ceases - illness and death. He thought exhaustion is a failure to cope but this model is now outdated. Now thought to be excessive exposure to stress mediators.
44
What is allostasis?
Allostasis – ‘The process by which bodily functions change, in response to environmental challenges e.g. stressors:
‘The ability to achieve stability through change’
45
What does stress do to allostatic responses?
Stress – Activates allostatic responses. For stability to be achieved , allostatic responses must be switched off.
46
When does the allostatic load not switch off?
Failure of habituation such as public speaking (load constantly fluctuating), or inabillity to switch off such as blood pressure (remains high)
47
How is an event stressful?
Must be perceived as stressful. 1) Primary appraisal – the initial evaluation when the situation is irrelevant, relevant but not perceived as a threat or stressful.
2) Secondary appraisal - evaluation of the capabilities of the individual to deal with the situation.
Activates ANS, HPA and behaviour
48
Describe the fight and flight response.
- Is linked more to the male phenotype
- Involves learned behavior and experiential, eg the effect of culture (the murder rate is higher in the Southern states than the Northern)
- Innate programming in utero
- Brain is hard wired to react to certain stimuli
- But is it valid in 21st century? Suddenly examined, unlikely to fight or flee. Flight may now be drugs of abuse, social withdrawal, TV watching/ gaming
49
What is entrapment?
When there are no mechanisms to deal with fight or flight and leads to depression
50
What is a female equivalent of fight and flight?
Tend or befriend
- Tend : women protect and care for their children
- Befriend : women seek out and receive social support
- Exclusion study – increased cortisol and increased affliative behaviour
- Frightening maternal behaviour – females more likely to approach
- Sports competition – female bonding versus male focus
51
What is thought to drive the female response?
The female response is thought to be driven by OXYTOCIN, which tends to diminish the production of ACTH and thus the stress response. OESTROGEN has a positive effect compared to TESTOSTERONE’S negative.
52
How can you quantify allostatic load?
1) Commonly the HPA axis through parameters such as BP, obesity, HT, T2DM, CVS, hippocampal atrophy
2) Urinary cortisol – pulsatile release and huge variability in what’s normal, so not best measure
3) Cardiovascular. Risk Factor; Increased mortality, cardiovascular disease, cognitive functioning.
4) Metabolic. Risk Factor; Increased mortality, cardiovascular disease, cognitive functioning.
5) Inflammation. Risk Factor; Increased mortality, cardiovascular disease, cognitive functioning.
6) Behavioural. Self reported questionaires. Stress increases with all major diseases.
7) Brain markers
53
What is the biological cost of stress?
Biological cost of stress
This occurs when stress uses some of the resources that are normally allocated to other tissues.
Stress can divert energy normally required for other functions, for example stunting growth in children.
54
What can the failure to cope be due to?
- Excessive wear and tear in tissues activated by chronic stress such as the hippocampus
- Dysregulation due to diverted energy resources
- Direct inhibitory effect of stress hormones on certain systems.
55
What is the McArthur study of successful raging?
Cohort – High functioning 70-79 year olds
Baseline: Higher AL
Lower baseline functioning, poorer cognitive performance and weaker
physical performance
After 3 years: Higher AL
Greater declines in cognitive and physical functioning and
augmented risk of incident cardiovascular disease (CVD) indepen-
dently of socio-demographics and baseline health status.
(NB No individual biomarkers significantly explained these outcomes,
supporting the utility of a multi-systemic composite approach)
After 7 years: Higher AL
Threshold effect revealed
Greatest risk of cognitive and physical declines and risk of CVD.
Most importantly, higher AL was related to increased risk of all-cause mortality
56
What clinical conditions can you see raised ACTH? Low?
Low cortisol. Addisons and CAH. In cortisol secreting tumours, ACTH is reduced
57
What does inflammatory suppression involve?
Inflammatory suppression involves direct interactions of the glucocorticoid receptor with AP-1 and NF-κB
58
What is controlled by ACTH production?
Sex steroids and glucocorticoids however not aldosterone as much
59
What happens in congenital adrenal hyperplasia?
In Congenital adrenal hyperplasia you do not have 21-hydroxylase (*) meaning that you cannot produce cortisol or aldosterone. Can synthesise some forms but these are salt wasting. However, the pathways to produce androgens is still viable as well as increased precursors and overactive from the increased ACTH meaning that females can get secondary sexual characteristics. The increased ACTH from the lack of negative feedback from the absent cortisol.
60
How can steroid release be described?
* Rapid onset: Reduces the release of AVP/CRH and ACTH from preformed stores
* Slow onset: Reduces the synthesis of AVP/CRH and ACTH and thus the amount of hormone available for release
61
What is an example of stress axis feedback?
• At 0 mins, give either saline or cortisol to rats, then after 75 minutes give either saline or IL-1b. Much reduced response if give cortisol first.
This can also be seen by either doing a sham operation or removing the adrenal glands. Adx causes increased parvocellular AVPmRNA PVN and CRH
Talk about what happens when you give exogenous glucocorticoids to patients.
62
What are key features of steroid feedback in terms of mediating it?
Mediated principally by intracellular receptors, MR and GR
Some rapid onset effects may also involve membrane-bound receptors.
63
What is the glucocorticoid receptor like?
* Low affinity
* Glucocorticoid selective
* Widespread – found in most cells
64
What is the mineralocorticoid receptor like?
* High affinity
* Binds to glucocorticoids and mineralocorticoids equally
* Discrete locations: pituitary gland, hypothalamus distal nephrons, colon, sweat gland, hippocampus and vascular smooth muscle
65
What is the physiological role of mineralocorticoids? Evidence
High affinity receptors – therefore detect low (non-stress) levels of corticosterone in the blood.
Unless protected by 11βHSD2 are saturated at high physiological (stress) levels of GCs
Expressed in the pituitary gland, hypothalamus, hippocampus and probably also other brain areas. Immunohistochemical localisation of the mineralocorticoid receptor in the murine brain .
High affinity receptors – therefore detect low (non-stress) levels of corticosterone in the blood.
Unless protected by 11βHSD2 are saturated at high physiological (stress) levels of GCs
Expressed in the pituitary gland, hypothalamus, hippocampus and probably also other brain areas.
Hippocampal receptors are not ‘protected’ by 11βHSD2, therefore readily accessible to circulating GCs
Evidence suggests that MRs, particularly hippocampal MRs, have an important role in regulating CRH/AVP and ACTH secretion in basal, non-stress conditions at the nadir of the circadian rhythm.a
66
What role does the glucocorticoid receptor have?
Low affinity receptors
Do not detect low GC levels (nadir of the circadian rhythm)
Are sensitive to circadian and stress-induced elevations in GCs
Widely distributed, abundant in all areas associated with GC feedback
Sensitivity increased by 11βHSD1
Primary mediators of GC feedback
67
What increases the sensitivity of the glucocorticoid receptor?
11bHSD1
68
How is cortisol found in the blood?
In the bloodstream, cortisol is bound to CBG however only the free form enters the cell.
69
How do glucocorticoids work?
By transactivation (increase in the rate of gene transcription) or transrepression or NON-GENOMIC ACTIVATION OF KINASE CASCADE
70
How does transactivation work?
- GR are nuclear receptors
- Cortisol (F) accesses cell GR in cytoplasm
- Found as complex with heat shock proteins (chaperones)
- When F binds, chaperones dissociate
- Receptor dimerised translocated to nucleus
- In the nucleus, associates with co-activators or co-repressors
- Activated receptor acts as a transcription factor- directly binds to DNA
- Activated receptor targets the promoter of specific target genes
71
How is the glucocorticoid receptor found?
With heat shock protein
72
What are examples of transactivated genes
```
Transactivation
– β-adrenoceptors
– PNMT
– GLUT-4
– PEPCK
– Other metabolic genes
– Annexin 1 and other anti-inflammatory molecules, e.g. IқB
```
73
What about transrepressed genes?
```
Transrepression
– CRH
– AVP (parvocellular)
– POMC (pituitary)
– NFқB
– Pro-inflammatory cytokines
– COX-2
– iNOS
PEPCK is involved with regulating processes such as gluconeogenesis in the liver.
INOS is in inflammatory cells and produces ROS.
```
74
How does transrepression work?
- Receptor doesn’t dimerise
- Activated receptor binds and interacts with other TFs such as AP1 TF
- Receptor translocated to nucleus and binds to other TFs, not DNA itself. So changes activity of other TFs.
- Prevents the action of other TFs, and reduce proteins they are producing, so downregulates target gene expression.
75
What is the importance of pulsatile secretion of glucocorticoids?
For gene expression
76
How do the non genomic effects occur?
When cortisol binds to its receptor and receptor phosphorylation occurs that influences protein transcription. The non genomic activation of membrane bound receptors is rapid which induces a complex kinase signaling cascade that eventually leads to:
• K+ extrusion
• Blockade of Ca2+ entry
This is for the rapid effects
77
General GR effects
* GCs exert rapid effects on CRH/AVP and ACTH secretion which inhibit the release of hormone from pre-formed stores
* They also exert effects which are slow in onset which reduce the synthesis of CRH/AVP and POMC.
78
What are factors affecting glucocorticoid access to a cell?
11bHSD, MDRP, Neutrophil elastase that works on the proportion of free and bound cortisol
79
What does neutrophil elastase do?
Neutrophil elastase was identified in neutrophils (sites of infection and inflammation). Breaks down the binding protein that cortisol binds to. Get recruitment of neutrophil to area to activate stress response, helps also to increase the cortisol at a LOCAL level through neutrophil elastase. CBG has an exposed loop that is cleaved and decreases affinity of binding globulin for cortisol, meaning more cortisol • GC availability is thus increased in inflamed tissues, facilitating an anti-inflammatory effect to maintain homeostasis.
80
What is worth noting with neutrophil elastase?
• Probably not important in steroid feedback in normal physiological situations as there should be no local inflammation in the HPA axis. • However, may be important in conditions of brain/pituitary injury where there is a local inflammatory response – the consequent increase in local GC levels would enhance steroid feedback
81
What is 11bHSD1 like? What are its affinities like
```
– NADPH
– Widespread, liver, CNS, vascular SM, adipose
– Km (nM)
• cortisol - 17,000 (low affinity)
• cortisone - 200
• dexamethasone –negligible
```
82
What about 11bHSD2?
cortisol - 50 (high affinity)
cortisone - negligible
dexamethasone 150
83
What is a confusing result concerning 11bHSD1?
KO 11BHSD1: you actually INCREASE the levels of corticosterone. Reducing negative feedback as there is less cortisol produced through 11bhsd1 so more ACTH stimulating. KO also increases cortisol during stress. ESPECIALLY DURING RESTRAINT STRESS
84
What is the difference between 11bhsd1 and 2 in feedback?
Type 1 augments delivery to receptor and state ko results whereas type 2 protects MR so less likely to play a role and in protecting foetus
85
What are multi drug resistant proteins? What is a powerful substrate
Important role in the blood barrier, limiting drug access by promoting exportation of the drug from the cell.
Also found in many other cell types in the body, including brain neurones and pituitary cells
Dexamethasone is a powerful substrate
Cortisol and corticosterone are weaker substrates
86
How can the importance of ABC be assessed?
Forced swim Stress on ABC KO mice: have reduced ACTH and no difference cortisol. This is because more cortisol can access the brain and thus the negative feedback is greater. There is impaired rise in circadian rhythm due to impaired negative feedback→ plays a role in circadian rhythm. There is reduced CRH and POMC in the hypothalamus/pituitary
87
What is the role of ABC1B knock out in terms of dexamethasone?
• There are Dose-dependent effects of dexamethasone on decreasing plasma ACTH and corticosterone ABC-B1 (i.e. mrdp) knockout mice compared to wild type. Summary: increases the sensitivity of the axis to dexamethasone
88
What are examples of early life exposures influencing glucocorticoid sensitivity? What does this mean in terms of the brain?
Neonatal endotoxin (ENDO) treatment reduces GR expression in in the rat brain in adulthood. Early stressors in life change subsequent responses to stress. Maniuplation in early life has significantly reduced gr expression in three key brain areas. This means that there is less negative feedback→ Neonatal endotoxin treatment augments CRH and AVP expression in the pPVN in the adult rat. There is also an increased response under stress. There is a decreased inhibitory effect of dexamethasone.
89
What evidence is there for non genomic feedback? At the anterior pituitary
1. Corticosterone inhibits ACTH release from anterior pituitary within ten mins, and treatment with cyclohexamide does not stop this inhibition (stops translation)
2. Stress-induced ACTH release in adrenalectomised rats does not return rapidly to baseline (as it does in sham operated animals, but it is suppressed by GC administration.
3. GC treatment inhibits CRH-induced ACTH secretion in vivo, even in animals treated with actinomycin-D (inhibits transcription, and so DNA synthesis)
90
What evidence is there for non genomic feedback at the hypothalamus?
GCs rapidly inhibit CRH release from hypothalamic slices and synaptosomes
Administration of GCs within 2 minutes of adx prevents the fall in hypothalamic CRH content
Dex treatment decreases electrical excitability in PVN CRH neurones
Dex infusion into PVN 5 mins before restraint stress blocks ACTH and cortisol responses.
Evidence for membrane bound receptors
91
How does the non-genomic action at the level of the hypothalamus occur?
* GCs can rapidly inhibit CRH release from PVN neurons by acting on membrane-associated receptors on the CRH neurones
* Receptor activation leads to increased retrograde endocannabinoid (eCB) signalling at CB1 receptors at presynaptic terminal which suppresses excitation of presynaptic glutamatergic neurons
92
What evidence is there for the non genomic effects at CRH nerve terminals? Think KO models
• GR is localized to post-synaptic neuronal membranes
in PVN (electron microscoopy, immunocytochemistry)
• Glucocorticoid suppression of excitation of CRH neurones is absent in PVN GR KOs
• Administration of GC conjugated to BSA into PVN suppresses restraint-induced activation of HPA axis. (Localising it and forcing
it to stay outside cell, and as cannot get into cell, any effect it has is on membrane-bound receptors)
• CB1 receptors are ubiquitous in pre-synaptic terminals (not CRH neurones themselves)
• eCBs (synthesized from arachidonic acid metabolites) are rapidly produced in PVN dendrites following GC exposure
• CB1 antagonism or KO increases PVN CRH mRNA and plasma ACTH and corticosterone
93
Evidence for suppression of brain structures with direct stimulatory innervation of the PVN?
* Specifically the NTS: Within 30 mins stress (acute & chronic) → GCs suppress preproglucagon (GLP-1; and HPA-stimulatory peptide) mRNA and peptide levels in NTS by destabilizing mRNA pools, without affecting gene transcription. GC inhibit the production of GLP-1 in the NTS, as these GLP-1 producing neurones also stimulate CRH neurones
* Influences in limbic brain regions
* Annexin 1-mediated actions
94
What is something you would never think of that stimulates CRH neutrons?
GLP1
95
What are the 3 interacting domains of GC feedback?
GCs provide rapid, non-genomic inhibition of glutamate release from
presynaptic terminals
GC inhibit the production of GLP-1 in the NTS, as these GLP-1 producing neurones also stimulate CRH neurones.
GCs enhances glutamate input and so activates GABA pathways, which have inhibitory input to CRH neurons
96
What are the rapid feedforward actions of the GC?
Depending on physiological demand and anticipatory signals from the forebrain, GCs may provide feedforward excitation of the HPA axis.
Largely mediated through the amygdala, where cortisol has a positive effect on CRH production, or glutamate production which feeds on to the hypothalamus to increase CRH neurone stimulation
GCs can upregulate CRH signaling in the amygdala and BST, potentially prolonging GC secretion.
GCs may also act on glutamatergic neurons in the ilPFC and BLA to excite PVN
CRH neurons via synaptic relays in the hypothalamus
97
Evidence for glucocorticoids rapid effects on behaviour?
Rapid facilitation of hypothalamic aggression by corticosterone in adrenalectomized rats may explain why stress is a major factor in stimulating aggressive behaviour in humans and rodents
Hypothalamic aggression, in turn, was rapidly facilitated by a corticosterone injection in rats in which the natural adrenocortical stress response was prevented by adrenalectomy. Effects not blocked by cyclohexamide
The rapidity of both effects points to a fast, mutual, positive feedback of the controlling mechanisms within the time frame of a single conflict.
Such a mutual facilitation may contribute to the precipitation and escalation of violent behavior under stressful conditions.
98
What is the function of Annexin 1?
```
Glucocorticoid-regulated
Anti-inflammatory
Anti-proliferative, pro-apoptotic
Neuroprotective
Modulator of neuroendocrine function
```
99
What levels can annexing 1 feedback?
The pituitary and hypothalamus
100
How are glucocorticoids anti-inflammatory?
Annexin I belongs to the annexin family of Ca2+-dependent phospholipid-binding proteins.
Annexin A1 both suppresses phospholipase A2 and COX level, thereby BLOCKING EICASANOID PRODUCTION, and inhibits various leukocyte inflammatory events
101
What happens in resting human cells concerning annexin1?Then when activated
In resting conditions, human and mouse immune cells such as neutrophils and macrophages contain high levels of annexin A1 in their cytoplasm.
Annexin A1 is promptly mobilized to the cell surface and secreted. Annexin A1 promotes neutrophil detachment and apoptosis, and phagocytosis of apoptotic neutrophils by macrophages
102
What does annexin1 do to the innate immune system?
In vitro and in vivo analyses show that exogenous and endogenous annexin A1 counter-regulate the activities of innate immune cells, in particular extravasation and the generation of proinflammatory mediators, and this ensures that a sufficient level of activation is reached but not exceeded.
103
What experimental evidence is there concerning annexing 1?
Immunoneutralisation of ANXA1 reverses the inhibitory effects of corticosterone on IL-1B induced ACTH secretion
Animals treated with saline and IL-1B have increased ACTH production
When corticosterone added, it suppresses IL-1B induced ACTH secretion
When ANXA1 antibody added in vivo, ACTH increases, and so corticosterone is ineffective in suppressing IL-1B induced ACTH secretion
Therefore annexin mediates GC feedback in these rats
104
How are The acute regulatory actions of glucocorticoids on ACTH and CRH secretion in vitro by pituitary and hypothalamic tissue respectively controlled?
mimicked by ANXA1
inhibited by
anti-ANXA1 antisera
ANXA1 antisense ODNs
105
What do glucocorticoids do to annexin?
De novo annexin 1 synthesis:
A) Dexamethasone increases the expression of ANXA1 in the rat anterior pituitary gland, measured by western blot. Detected at cell surface, close to endocrine cells
b)Translocation of annexin 1 from the cytoplasm to the cell surface where it is retained by a Ca2+-dependent manner.
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Where is annexin 1 localised to?
Annexin 1 is localized to folliculostellate cells in the anterior pituitary gland
(Not present in the corticotrophs, but collects where folliculostellate cells come into contact with endocrine cells)
Increased detection of cell surface ANXA1 in co-cultures of pituitary cell line, AtT20 and pituitary FS-like cell line, TtT/GF cells by immunofluorescence
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What has flow cytometry shown?
If you increase the
concentration of annexin, increase the binding. If you give enough annexin, can saturate the binding sites.
Cell sorting identified annexin 1 binding sites on approximately 80% of the pituitary endocrine cells, including
Corticotrophs
Somatotrophs
Lactotrophs
Gonadotrophs
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What is one piece of evidence for how annexin works?
Reversal of the inhibitory effects of Annexin and dexamethasone on forskolin-stimulated ACTH release in vitro by PKC blockade (nhibitor of PKC caused ACTH release to increase, even when ANXA and Dex administered)
Dexamethasone administration causes translocation of annexin-1 to cell surface.
If you give PKC inhibitor, lose the dexamethasone-induced appearance of annexin 1 on the cell surface of rat anterior pituitary cells (Western blot)→ need protein kinase c
Dexamethasone causes increased translocation of GFP-tagged annexin to cell surface: Mutation of serine 27 of annexin 1, no translocation of GFP-tagged annexin 1 to the cell surface in FS cells.
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Overall what is the annexin1 pathway?
GC binds to GR
Upregulates annexin 1, serine phosphorylation, externalization. ABCA1 is important in facilitating this
Serine phosphorylated annexin 1 sits outside, in close position to corticotroph cells (FS cells touch corticotrophs)
Deliver ANXA1
Subsequent receptor is prpbably FPR that influences exocytosis, therefore ANXA interferes with exocytosis
Summary
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What are pathological changes from stress due to?3
Stress related pathology Can be due to; Excessive wear and tear in tissues activated by chronic stress Dys-regulation due to diverted energy resources Direct inhibitory effect of stress hormones on specific systems
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What are examples of pathological stress pathology?
Reduced hippocampal density in PTSD Vietnam veterans, reduced growth in a abused child, worked cancer survival in depressed patients, worsening of atherosclerosis in stressed
