Lectures 4-6 Flashcards
(132 cards)
1
Q
Define Chronobiology
A
A field of biology that examines timing processes, including periodic (cyclic) phenomena in living organisms, such as their adaptation to solar and lunar-related rhythms.
These cycles are known as biological rhythms.
2
Q
What are the 4 main selective day/night pressures that drive universal biological timers?
A
Sunlight
Temperature
Predation
Food availability
3
Q
What two things does have a biological timer allow?
A
The adaptation to and prediction of changes in our environment.
4
Q
Name 4 physiological parameters that are under rhythmic patterns/cycles.
A
Body Temperature.
Melatonin Secretion
Cortisol Secretion (high during day and low at night).
Urine Production. (High during day and low at night).
5
Q
What is interesting about the clock mechanism and the differences in it throughout the body?
A
The core clock mechanism is similar across the body.
However, the genes that control these rhythms are very different; they are tissue specific clock genes.
6
Q
Where is the SCN in the circadian hierarchy?
A
At the top.
7
Q
List 9 things that a disrupted clock increases the chances of.
A
Sleep Disorders
Metabolic Syndromes
Obesity
Diabetes
Cardiovascular Risks
Stroke
Arthritis
Hypertension
Cancer
8
Q
What does circadian mean?
A
About a day.
9
Q
What was the seminal Jean-Jacques de Mairan (1729) study?
A
He observed that plants show circadian rhythms and wanted to test if they were solely driven by light or were internally driven.
So he left one plant in a window to receive light information and another in the darkness to observe the effects.
He found that in both conditions there were circadian rhythms; they were internally driven.
10
Q
What studies into circadian rhythms were done by Nathaniel Kleitman?
A
Cave Studies - he is known as the father of modern clock research.
He took a student to a cave in Kentucky and they stayed there for more than a month.
They withdrew ALL time cues; no clocks, no light, no temperature cues etc.
They also imposed a 28 hour clock on themselves.
After a month, they revealed that there was about a 24.3 hour rhythm that was maintained in the absence of light.
11
Q
Who was Jurgen Aschoff and what influential study did he do?
A
He was one of the 3 founders of chronobiology.
In his study he:
Changed ex-WW2 bunkers into sleep study ‘labs’ as he could measure and control a lot of variables within these.
He tested the following:
- Ion concentration in Urine (CA and K)
- Volume of urine
- Body Temp
- Sleep/wake cycle
The results showed for the first time that there was a ~24 rhythm in humans.
12
Q
Who were the 3 scientists who won the noble prize in chronobiology and why?
A
Jeffrey C. Hall
Michael Roshbash
Michael W. Young
They discovered the period gene in drosophila that was in charge of it’s circadian rhythm.
This discovery (and noble prize) solidified Chronobiology as an important field.
13
Q
What has genome analysis shown us about the basic building blocks of the molecular clock?
A
They are conserved between all animals.
14
Q
What did the 1971 study by Konopka and Benzer discover?
A
The first ever mutant fruit fly with a different periodicity was discovered; called it the period mutant as they couldn’t clone genes at this point (no PCR technology).
*This gene was cloned for the later nobel prize discovery.
15
Q
What famous discovery happens in 1984 and was done by Hall, Roshbash and Young?
A
They cloned the Period gene from Drosophila.
16
Q
What was the finding of the 1988 study by Ralph & Menaker?
A
They identified the Tau hamster that had a 20 hour period instead of 24 (gene wasn’t cloned yet).
This was the first found mammalian mutation that had a 20 hour period.
17
Q
What was the discovery of the 1990 study by Ralph & Menaker?
A
They identified that the SCN was the central pacemaker of the circadian rhythms by transplanting the Tau mutant SCN into a receiver.
This led to a change of rhythms to 20 hours.
They also did the reverse and found that tau mutants could regain a normal rhythm after a WT SCN transplant.
This shows that the SCN carries all the timing information needed to drive internal rhythms.
18
Q
What did the 1994 Sehgal & Young study discover?
A
They found the binding partner for period in drosophila; it was called timeless.
19
Q
What did the 1997 Takahashi study reveal?
A
The first CLOCK gene found in a mammal.
They found mutating this gene strongly changed the circadian phenotype.
(Did this using mutagenesis in which you mutate all genes of a single type and see if there are phenotypic effects on the rhythm).
20
Q
What did Hogenesch find in 1998?
A
He identified the Bmal1 and Npas2 genes which are part of the positive arm of the circadian clock.
They initiate the clocks’ start.
21
Q
What did Kloss, Price and Young find in 1998?
A
They identified a Kinase called double time (in drosophila).
This was important because Per and Cry were repressors so they couldn’t start the clock.
To reset the clock, Per and Cry needed to be degraded - which is does by Kinase Mediated Phosphorylation.
22
Q
What did Menaker and Takahashi discover in 2000?
A
That the gene behind the tau mutation was Caesin Kinase 1 epsilon (CK1e) which was the ortholog of double time in drosophila.
This is amazing because it shows that the mammalian and drosophila clock use the same system.
23
Q
What are the 2 genes used in the negative arm of the mammalian and drosophila clock?
A
Mammalian: Period and Cryptochrome.
Drosophila: Period and Timeless.
24
Q
What are the 2 alternative names for BMAL1?
A
ARNTL or MOP3
25
What is another name for NPAS2?
MOP4
26
What are the 6 methods used to search for clock genes, starting from least to most modern?
Mutagenesis and behavioural tests in flies and mice.
Cloning of homologous genes.
Searching for interacting partners of known clock proteins.
Accidental Discoveries
Genome-wide RNAi/compound screening in cells.
Machine learning.
27
Which were the main clock genes discovered using the cloning of homologous genes?
mCrys
mPers
27
Which were the main clock genes that were discovered using mutagenesis and behavioural tests in flies and mice? (6)
dPeriod
Timeless
mClock
Doubletime
Cycle
Fbx13
28
Which were the main clock genes found by searching for interacting partners of known clock proteins?
TIME for PER
BMAL1 for CLOCK/NPAS2
29
What are the two main genes that were found by "accidental" discovery?
CK1epsilonTau.
DBP.
30
Detail briefly the discovery of the first clock gene.
It was Period in _drosophila_ by Konopka and Benzer, 1971.
They used actograms and found 3 mutant lines:
Per0 = arrhythmic.
PerS = short rhythmicity.
PerL = long rhythmicity.
31
What is the difference in the amount of Period genes in _drosophila_ and mammals?
_Drosophila_ only have 1 period gene.
Mammals have three mutations:
Per1
Per2
Per3
32
What did the study into mPer and their KO's reveal?
METHODS:
- Had a WT mouse, a mPer1 KO, a mPer2 KO and a mPer3 KO.
- Tested their phenotype via actograms in sleep wake and then free running in darkness.
RESULTS:
- mPer1/2 lead to a gradual loss of rhythmicity (mPer2 KO is quicker to lose it).
- mPer3 is important in timing sleep but not in rhythms - it's KO led to normal rhythms.
33
What did the Double Mutant mPer study show? Describe it and draw conclusions.
METHODS:
- Researchers used compound mutations to cross different combinations of mPer:
- mPer1/mPer3
- mPer2/mPer3
- mPer1/mPer2
- Used light/dark cycle and then constant darkness and measured behaviour with actograms.
RESULTS:
mPer1/mPer3:
- Gradual Loss of Rhythmicity
mPer2/mPer3:
- Gradual Loss of Rhythmicity.
mPer1/mPer2:
- IMMEDIATE Loss of Rhythmicity.
CONCLUSION:
- The inclusion of Per3 into a cross with Per1/2 doesn't change anything; their rhythms are the same in KO with or without Per3.
- Thus showing that Per3 is not important for rhythms.
- Per1 and Per2 must have a partial redundancy and are VITAL as losing them means immediate loss of rhythm.
34
Describe the study into the Clockdelta19 mutant.
METHODS:
- Planned to screen mice constantly until they found one with a mutation in clock gene.
- Took 200 mice to find a mutant that they wanted.
- Mutated a single allele or both alleles to see what happened.
RESULTS:
- WT mouse has 23.6 period.
- A single allele mutation in Clockdelta19, the period is longer at 24.8 hours.
- When both alleles are mutated then it has an even longer period of 27.1.
CONCLUSION:
- This occurs because CLOCK loses the ability to activate downstream genes but it can still bind to BMAL1 but there is NO transactivation domain present.
35
Describe the finding about the difference in Clock KO and mutations.
Researchers thought that if you knock out CLOCK then you would get a strong phenotype, due to the results of the single and double allele mutations in clock changing rhythmicity.
However, if you knock out CLOCK, the animals have almost perfect rhythmicity.
This shows a big differences between mutant and knock out.
Knock Out will have other genes 'step in' and compensate for it.
In this case, it is Npas2 as it can equally bind to Bmal1 and activate the transmembrane domain.
In the mutant, the Clock gene can still bind to Bmal1 but it cannot activate downstream genes due to lack of transactivation domain.
Now, Npas2 cannot do anything as it isn't filling the EMPTY role of CLOCK; it's not able to preferentially step in.
36
What did the Clock Shock Experiment show us?
That if you remove BOTH Clock and Npas2 then you lose rhythmicity.
You cannot afford to lose both to maintain rhythms.
37
What compensates for loss of Clock in peripheral circadian oscilators?
Npas2.
38
What are the two most important take aways from the 'Clock Shock' Paper?
There can be important differences between a mutant gene product (Clockdelta19) and the absence of a gene product (Clock-/-).
Clock and Npas2 are redundant in the SCN and peripheral tissues.
39
What did the study into Cry1 and Cry2 KO mice reveal?
When free running in darkness:
- Cry1 KO leads to a shorter periodicity.
- Cry2 KO leads to a longer periodicity.
If both Cry1 and Cry2 are KO then there is an immediate loss of rhythm.
This study shows that they are essential for circadian clock function but they also have some redundancy.
40
What is arguably the most important clock gene in the circadian rhythm? Why?
BMAL1.
There is NO redundancy and the KO leads to immediate loss of rhythmicity.
41
What is the importance of CK1epsilon and what is it?
It is an important kinase that regulated clock pace; it targets Per protein for degradation.
It's also the ortholog of doubletime.
42
Briefly outline the finding of CK1epsilon tau mutant effect on the clock.
Tau mutant had an internal rhythm of ~4hrs faster than the WT.
Thought that this was a LOF of the enzyme.
Researchers asked: how could a kinase LOF speed up the clock? If anything it would lead to a longer clock because it would inhibit the breakdown of the negative arm proteins (Per and Cry).
Then a later study came.
43
Describe the PART 1 findings of the Meng et al., 2008 into tau mutations in mice.
Created a tau mutation version of the hamster in a mouse as they are easier to study .
Found that a SINGLE base pair mutation changed the whole rhythm to 20 hours.
Shows the power of a tau mutation.
44
Outline the PART 2 Meng et al., 2008 study into tau mutant PER2::Luc mice.
METHODS:
- Used a PER2::Luc (luciferase) Tau mutant mouse.
- Tracked the rhythm of the molecular clock in the SCN via PER2 gene expression.
RESULTS:
- Found that the molecular clock is accelerated in the Tau mouse, not just the phenotypic rhythm.
- This can be seen in the bioluminescent expression of Luciferase.
45
Outline the RESULTS of the PART 3 Meng et al., 2008 study into tau mutant PER2 degradation in mice.
If there is a tau mutation, the degradation of PER2 is faster.
Therefore it is a GOF mutation because the enzyme has become MORE active.
The CK1epsilon tau mutation targets Per2 only, not Cry1; it doesn't matter which mutation you introduce, the degradation of Cry1 was not increased.
46
Outline the results of the Meng et al., 2010 study into inhibition of casein kinase 1 enzymes.
There is a dose dependent response between the amount of CK1delta INHIBITOR and the length of the clock.
*You need CK1delta for clock rhythm to be maintained.
47
What are the two nuclear hormone receptors responsible for regulating BMAL1?
RORs and REV-ERBalpha/beta.
48
What is RORs?
A nuclear hormone receptor that regulates BMAL1
49
What is REV-ERBalpha/delta?
A nuclear hormone receptor that regulates BMAL1
50
Describe the process of the Transcriptional Translational Feedback Loop.
In the Morning:
Clock and BMAL1 (the positive arm) bind to the E-box (enhancer box) found in the promoter regions of Per and Cry.
Resulting in Per and Cry being transcribed into proteins, from which they come together and bind to dimerise.
In the Evening:
They come back to the nucleus and they bind to Clock and BMAL1 and form a protein complex.
From here, this supresses the ability of Clock and BMAL1 to activate downstream genes and you then close the loop.
For the next day to start, the repressors, Per and Cry, have to be degraded and this is by kinase mediated phosphorylation.
For Per, it's CK1.
For Cry, it's GSK3beta and AMPK.
After these are degraded, the next cycle will start again.
To make this more robust, BMAL1 is fine-tuned via the nuclear hormone receptors.
51
Name 5 pieces of evidence that support the TTFL model.
Mice lacking functional Pers, Crys, Clock/Npas2 or Bmal1 are arrhythmic.
Mutations affect cycle length (e.g., CK1, Clock).
CLOCK and BMAL1 increase Per/Cry/Rev-erb-a/b expression; PER and CRY inhibit their expression.
CK1 activity determines clock pace.
Rhythmic expression in the SCN and peripheral tissues/cells.
52
What was found about rhythmicity of Bmal1, Per2 and Rev-erba using luciferase labelling in cultured cells?
There was a self-sustained and cell-autonomous circadian clock.
(They all showed strong rhythmicity).
53
What's an experimental method to track molecular rhythms over the period of days?
Tag the clock proteins with VENUS fluorescent tag.
54
Where is the location of the SCN?
It's in the very ventral hypothalamus above the optic chiasm.
55
What are the two main peptides found in the SCN?
Arginine Vasopressin (AVP)
Vasoactive Intestinal Polypeptide (VIP)
56
What is AVP?
Arginine Vasopressin
57
What is VIP?
Vasoactive Intestinal Polypeptide
58
What is GRP?
Gastrin Releasing Peptide
59
Briefly outline the Core and Shell model of the SCN.
The Core receives input from the retinohypothalamic tract.
The core expresses VIP cells and GRP cells that have inputs throughout the SCN and project to areas such as the Shell.
The Shell area has outputs from the SCN and expresses AVP clock cells which further project out from the SCN as the main signal output.
60
Briefly outline the Herzog et al., 2004 study into individual neurons having different periods in Rats and Mice.
METHODS:
- Wanted to test whether the synced periodicity is maintained when the cells are dispersed and when in SCN slice.
- Homogenised SCN cells AND took slices of SCN and measured their rhythms.
RESULTS:
- In slices, rats and mice largely have the same 24 hour rhythms when connected within the SCN - these rhythms are consistent across cells as they fire at the same time.
- When homogenised you see a wide range of periods of the oscillation (roughly forming a ND with 20h and 28h being the very most tails).
61
What is the difference in the rhythms of synchronised and desynchronised SCN neurons?
(E.g., SCN slice or Homogenised)
Synchronised neurons have a coherent and stronger amplitude rhythm.
Desynchronised neurons have a less coherent and weaker amplitude rhythm.
62
Where and how is VIP expressed in the SCN?
The cell bodies are located in the ventral area of the SCN above the optic chiasm (the location of retinal input).
Their fibres extend across the SCN and other nearby areas.
63
What is true about VIP expression across mammals?
It is expressed ventrally.
64
What is VPAC2?
The VPAC2 receptor is a G-protein-coupled receptor (GPCR) that specifically binds to vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP).
65
Where is VPAC2 Receptor Expressed?
It's expressed throughout the brain; a fair amount throughout the thalamus.
But it is MOST densely expressed throughout the SCN - mainly in the Shell area.
66
What is another name for VPAC2 receptor?
VIP2
67
Name two types of neurons that express VPAC2. How do we know this?
AVP and VIP neurons - mainly expressed in the Shell area.
If you co-stain for AVP or VPAC2 you will see that they are co-expressed
68
What has VIP-VPAC2 signalling been implicated in? How is this said to work?
Photic entrainment of the clock via the SCN.
The retina sends photic information via the RHT to VIP neurons which project throughout the SCN and entrain other neurons on time of day.
These more robust clocks then produce the outputs.
69
Summarise 4 main points about the neuroanatomy of VIP-VPAC2 signalling.
VIP produced mostly by neurons in the ventral SCN.
VIP neurons receive photic input from the RHT.
VIP axons innervate many SCN neurons as well as neurons in other brain areas.
VPAC2R present in many SCN neurons, including those that make VIP and AVP.
70
Outline the results of study into wheel running behaviour of Wild Type and VIPR2-/- Mice.
METHODS:
- Took a WT and VIPR2-/- mouse.
- Exposed them to ~14 days of light/dark and then constant darkness.
- Recorded activity and made actograms.
RESULTS:
- You can see usual phenotypical behaviour in the WT in which they are free running in constantly darkness <24 hours.
- In VIPR2-/- mice, they seem perfectly entrained to light/dark cycle but they are arrhythmic in constant darkness - thus they are not entrained.
- Negative masking is occurring in which they do not move during the day due to risk of predation but they are actually awake - so it looks like it has a rhythm.
71
What was found by the Cutler et al., (2003) study into the firing rates of SCN cells from WT and VIPR2-/- mice in vitro?
METHODS:
- Firing rates is one of the outputs of the SCN.
- Took WT and VIPR2-/- Mice.
- Recorded from a slice of SCN to see activity.
RESULTS:
- In WT there were peaks at around ZT8.
- In VIPR2-/- there were no peaks; the firing rate is abolished.
72
Outline the study and results into the molecular clock in live SCN slices *in vitro* using per1::EGFP in WT, VIPR2+/+ and VIPR2-/- mice.
METHODS:
- Used Mice
- Fluorescently labelled Per gene using EGFP (Green Fluorescent Protein).
- Watched the rhythm in activation in Per in WT, VIPR2+/+ and VIPR2-/-
RESULTS:
- In WT you see a normal oscillation of per activation.
- In VIPR2+/+ and VIPR2-/- you still see oscillating activation of neurons but it's at a much lower rate.
- ALSO, the overall activation of the SCN is dampened.
73
What is the result of VIP-VPAC2 signalling absence on SCN neurons? Why is this?
Both the amplitude and the Synchrony are impaired - there is still an oscillation but dampened and the individual phases are no longer entrained therefore the whole SCN was less rhythmic.
This is because there are less connections between neurons so you lose the coherence between their firing.
74
What 3 major disruptions occur due to the absence of VPAC2R signalling?
Locomotor activity rhythms are disrupted.
*In vitro* firing rate rhythm SCN neurons is damped and appears arrhythmic.
Both the synchrony and amplitude of SCN neuronal oscillators are reduced.
75
What is AVP and what are its receptors?
Arginine Vasopressin.
V1a and V1b.
76
Outline the Yamaguchi et al., (2013) study that shows what happens in mice lacking AVP receptors.
METHODS:
- Took WT and V1a/V1b knockout mice and entrained them to a standard light dark cycle.
- Then advanced them 6 hours to simulate jet lag.
- After a week they shifted the cycle back 6 hours.
- Recorded activity and presented in actograms.
RESULTS:
- WT entrained to starting cycle.
- Then, in the 6 hour advance, it took them about 6 days to entrain.
- Once pushed back to 6 hours before, it also took them 6 days to reentrain.
- V1a/V1b knockout entrained after almost one day.
- *NOT negative masking because when in darkness they free ran very quickly - showing their clock is quickly adaptable.
77
What happens to Per1/Per2 clock gene expression in the SCN of mice lacking AVP receptors?
They rapidly phase shift to changes in the light-dark cycle - quicker than WT.
78
What happens to Bmal1/Dbp clock gene expression in the Liver of mice lacking AVP receptors?
They rapidly phase shift to changes in the light-dark cycle - quicker than WT.
79
Outline the study into the effects of CHX on SCN rhythms in mice lacking AVP receptors vs WT.
METHODS:
- Used Per::Luc
- Introduced CHX (a protein inhibitor) to WT and V1a/V1b-/- mice.
- Recorded the expression of proteins for up to a long period afterwards to notice the effect.
RESULTS:
- In the presence of CHX, rhythms in protein expression are abolished.
- In WT mice, once CHX is washed off, the rhythms are restored back to the phase and amplitude pre-CHX introduction.
- In V1a/V1b-/- mice, once CHX is washed off, the phase is no longer coherent and the amplitude doesn't recover as much as before.
- This suggests they are more sensitive to perturbations and have a weaker clock as a result.
80
Outline what the pharmacological blockade of AVP receptors showed about the genetic model of AVP receptor KO.
METHODS:
- Due to potential confounds of Genetic modification they use WT and used Pharmacological interventions to mitigate this potential effect.
- Introduced AVP receptor blockers.
RESULTS:
- When given the antagonist, it shows a quicker entrainment to the new cycle.
- This showed a dose response: the larger the dosage of AVP blocker, the quicker the entrainment.
- Showed the same effect as AVP KO mice studies.
81
Summarise the 5 major points we know about AVP neurons and their involvement in the circadian system.
AVP and its receptors (V1a/V1b) are expressed in the SCN.
AVP neurons are more prevalent along medial aspect of the SCN.
Mice lacking both V1a and V1b reset more rapidly to changes in the light-dark cycle.
AVP signalling via these receptors ordinarily acts like an intrinsic 'brake' on the circadian system to stop it shifting too quickly.
Interneuronal signal serves to resist external perturbance of the circadian clock.
82
Outline **PART 1** of the Maywood et al (2011) study into SCN grafts in VIP deficient mice.
**METHODS:**
- Used a VIP-/- mouse line.
- Took a brain slice with PER2 bound to luciferase.
- Grafted some WT mouse brain without luciferase so all the luminescence comes from the host.
- Looked to see what would happen to the rhythms.
**RESULTS:**
- VIP-/- mouse before graft loses rhythms.
- Adding WT graft restores rhythms.
- ONLY works with SCN tissue grafts.
- *If you use mutant mice like tau then the rhythm length will be that of the donor.
- DONOR GIVES RHYTHM.
83
Outline **PART 2** of the Maywood et al (2011) study into SCN grafts in VIP deficient mice.
METHODS:
- Put a membrane between the VIP-/- 'Host' brain slice and the Graft slice (no PER2:Luc).
- Used membranes ranging between 2-10kDa to see what was likely causing the restoration of rhythms.
RESULTS:
- A bigger membrane will give a restoration; too small and it will barely restore.
- VIP is ~3kDa so a likely candidate.
- However, if you increase the membrane past 10kDa you get an even larger amplitude.
84
Outline the study into Vip2r-/- and WT grafts - what occurs when you KO the receptor for VIP and introduce a WT graft?
METHODS:
- Take a Vip2r-/- mouse line, a VIP receptor KO.
- This mouse cannot detect the VIP if present.
- Add a WT graft and see what occurs.
RESULTS:
- There is a partial restoration of rhythms.
- Thus there might be another influence aside from VIP that restores function.
85
Outline the study into VIP-null, VPAC2-null GRP and AVP receptor inhibiting and the effects on rhythms.
METHODS:
- Took VIP-/- and VPAC2-/- mouse lines.
- Looked at BBR2 (GRP) and V1a/V1b (AVP) receptors
- Applied pharmacological blockades to inhibit these receptors.
- Introduced a WT graft and looked to see the effect on restoration.
RESULTS:
**GRP:**
- VIP-null mouse, there was no difference in restoration.
- VPAC2-null mouse (removing ability to detect VIP) if you block GRP signalling, you see a reduction in restoration.
- GRP can be a limiting factor ONLY in the absence of VIP mediated signalling.
**AVP:**
- In VIP null, there is a reduction in restoration, showing AVP signalling is also important for regulating the rhythms in the clock.
86
What is BBR2 a receptor for and where is it located?
GRP in the SCN.
87
What does the removal of VIP signalling either through VIP-/- or Vip2r-/- result in? What does this imply?
A dampening of SCN oscillations.
This implies a strong role for VIP in maintaining synchrony in the SCN.
88
What effect do WT grafts have on Vip2r-/- hosts (VIP blind) and what is the effect of inhibiting GRP or AVP signalling on these effects?
WT grafts can partially restore rhythms in Vip2r-/- hosts.
This partial restoration is blunted when either GRP or AVP signalling is inhibited.
89
What is the proposed hierarchy of maintaining rhythmicity in the SCN?
VIP > AVP > GRP
90
Name 3 examples of changes to VIP and AVP neurons that results in a phenotypic change in humans.
Number of VIP neurons in the human male SCN declines with age. Circadian rhythms in sleep-wake change as we age.
Number of AVP and VIP neurons in human SCN is reduced in people who have had Alzheimer's disease. Disruptions of sleep-wake are typical of AD.
Genetic mutations that alter the Vipr2 gene may increase risk of developing schizophrenia. SZ patients often present with sleep-wake alterations.
91
Outline **PART 1** the Brancaccio et al., (2019) study into the role of astrocytes in SCN communication.
METHODS:
- Used Cry KO mice which are arrhythmic due to this lack of gene.
- Try to reinitiate cry in these animals.
- Do this in JUST neurons or JUST astrocytes using a 'genetic switch' that allows them to do this.
- See the effect on rhythms.
RESULTS:
- To turn them on in neurons restores the rhythms.
- If this is done in astrocytes it also restores function.
92
Outline **PART 2** the Brancaccio et al., (2019) study into the role of astrocytes in SCN communication.
METHODS:
- Given the previous results that showed initiating cry in neurons AND astrocytes restored rhythms, they now wanted to see what happened in behaviour.
- Mice that lack CRY will be arrhythmic.
- Injected them with viruses that would restore CRY into a small subset of SCN cells.
RESULTS:
**Neurons:**
After the surgery, they free run with a long period, typical of cryptochrome animals.
**Astrocytes:**
- After the surgery, they free run despite not having functional neurons.
- The periods of the free running astrocyte clock is slightly shorter than the one in neurons.
93
What process do cells use throughout the brain and body to maintain an autonomous molecular clock?
Transcriptional Translational Feedback Loops (TTFL)
94
What does TTFL stand for?
Transcriptional Translational Feedback Loops
95
Briefly describe how the circadian clock operates.
Highest activity in SCN will be mid/late parts of the day (when properly entrained.
PER and CRY hybridise into a dimer.
They will accumulate in the early parts of night and repress the BMAL1 and CLOCK complex.
Therefore, the lowest RNA will be in the mid part of the night.
Then the protein will be lowest in the morning and the repression as a result, restarting the clock.
*There is a difference in the expression of the RNA and the protein, of course, so it's important to look out for this in the literature.
96
When do the following mRNAs or Proteins peak and when are they at their lowest?
per/cry mRNA
PER/CRY protein
PEAKS:
per/cry mRNA - mid-late day.
PER/CRY protein - early night.
Lowest:
per/cry mRNA - mid part of the night.
PER/CRY protein - minimal early day.
97
What are the implications of functional redundancy between the "core" molecular clock genes?
Redundancy gives you robustness; If you have a mutation in one clock gene that occurs, you have others that can cover that function too.
98
Which of the "core" mammalian molecular clock genes could be considered most essential?
BMAL1.
99
Outline the differences between 2 gene KO's between Per1, Per2 and Per3 and what this shows us.
If you KO Per1/3 or Per2/3 you still have some rhythms present.
If you KO per1/2 then the animals become instantly arrhythmic.
This shows the redundancy of Per3 and how Per1 and Per2 are way more important for maintaining rhythms.
100
Outline the differences of Cry1-/-, Cry2-/- and Cry1-/- Cry2-/- and what this tells us.
If you knock out Cry1 your rhythm speeds up.
If you knock out Cry2 your rhythm slows down.
If you knock out both then you become arrhythmic.
Shows that they are not entirely redundant and that they play different roles.
101
Outline the differences of Clock-/- and BMAL1-/- and what this tells us.
CLOCK:
If you knock it out CLOCK, Npas2 will compensate it and you will maintain rhythms.
Mutating clock means you lose your cycle - it's a GOF so you don't get Npas2 compensation.
If you KO then it can be compensated by Npas2.
BMAL1:
Seriously important in the cycle as -/- leads to immediate arrhythmicity.
102
What is responsible for signalling the degradation of the Per genes?
Casein Kinase 1 (epsilon/delta).
*There are other important genes in the clock that are not 'core' but they have a function in tagging Per and Cry to make sure they are broken down.
103
What is responsible for signalling the degradation of the Cry genes?
FBXL3/21
*There are other important genes in the clock that are not 'core' but they have a function in tagging Per and Cry to make sure they are broken down.
104
What is the purpose of the auxiliary loops in the circadian clock? Briefly explain the process as to why.
Stabilise oscillation and adding flexibility to clock controlled output.
105
Briefly explain an example process of auxiliary loops in the circadian clock.
BMAL1 and CLOCK drive the expression of Rev-erbα and RORα. Rev-erbα represses BMAL1 expression, while RORα activates it, creating rhythmic oscillations in BMAL1 levels.
This feedback loop helps orchestrate the timing of gene expression across the circadian cycle, allowing different processes to occur at specific times of day.
Approximately 10-15% of genes in any given tissue exhibit circadian oscillations, driven by a combination of the central SCN clock and local tissue clocks.
Different tissues have unique circadian profiles, with little overlap in the specific genes regulated in each tissue.
106
What is it about the SCN that makes it so critical for whole animal circadian rhythms?
Direct Retinal Input.
This is very important for the drive of the rhythm.
Projects to highly influential regions of the brain (e.g., hypothalamus).
Cells in SCN gene expression leads to direct opening of Ion Channels - being able to fire action potentials in relation to this timing.
107
What does the molecular clock *actually* coordinate rhythms in?
Membrane excitability and action potential firing.
This is what allows SCN cells to communicate time.
108
What does the timing of melatonin and cortisol peaks tell us about the SCN?
There must be something that tunes the timing of the SCN.
109
If the highest SCN population activity represents day time, how does it control the rest of the bodies rhythms independently?
The timing signals are differentially interpreted by downstream circuits to dictate physiological timing and local clock phase.
110
What has been suggested about SCN signals across animal types? What has been suggested about how this relates to different behavioural types such as nocturnal, diurnal etc?
There has been a suggestion that the SCN signals sent out that are the same across animal types.
It's suggested that there are inverted signals; nocturnal has an inhibitory and diurnal has excitatory neuron (for example)
This would mean that same signal can lead to opposite response.
111
What are two key factors that determine effect of light?
Time and intensity.
112
What is the phase response to light in a circadian rhythm at these different times of day: Early night, Late night, Mid day?
How do the responses relate to light intensity?
Early night: Delay
Late night: Advance
Mid day: No effect
Responses at a given point of the circadian cycle scales with light intensity.
113
How is circadian brightness defined?
Circadian 'brightness' signals come from rods and melanopsin via ipRGC projections to the SCN.
114
How do cone-derived colour signals modulate the response to brightness?
Colours associated with twilight ("bluer") evoke weaker responses than colours associated with daytime ("white-yellow").
*Understanding based on experiments in mice.
115
What is an important factor that might influence the photoreceptive signals relevant to the clock in different mammals?
Your lifestyle; photoreceptors will be tuned for different conditions (e.g., ground animals).
Therefore photoreceptors will be adapted for those conditions.
Some animals won't be tuned for colour so that won't work that much.
116
Why does it matter which photoreceptors are involved in resetting the clock? Explain.
Because how do you define brightness without constant photoreceptors?
You need to understand which cones are present in each animal so that you can compare the relative effect of their activation.
If you want to make a light you need to tune it to the animals spectral sensitivity.
Melanopsin activation curve is constant between them, thus we can draw between species.
117
What are the three main measures of light in photometry?
Lux
Candelas
Lumens
118
Define Lux.
Lux is a unit of illuminance, measuring the total luminous flux (in lumens) falling on a surface per unit area (energy as a function of wavelength).
(SI stands for the International System of Units)
119
Define Candelas.
The candela (cd) is the SI unit of luminous intensity, which describes the perceived "brightness" of a light source in a particular direction, weighted according to human visual sensitivity.
(SI stands for the International System of Units)
120
Define Lumens.
The lumen (lm) is the SI unit of luminous flux, representing the total amount of visible light emitted by a source, weighted by the photopic luminosity function to reflect human visual sensitivity.
(SI stands for the International System of Units)
121
What is the brightest perceived wavelength? How was this found?
550nm - a light matching task.
122
What is the problem of measuring light in lux?
The photopic curve does not match up to melanopsin, thus measuring light in lux doesn't tell you how much it activates melanopsin and how this effects your circadian rhythm.
123
What is the difference in the spectral quality between sunlight and artificial light?
Both produce white light as detected by the human 3 cone system BUT artificial light has 3 peak wavelengths for each cone, whereas sunlight has a relatively flat activation across all wavelengths.
124
What is the difference in the melanopsin activation between sunlight and artificial light?
Sunlight directly stimulates melanopsin activation.
Artificial light has peaks in the tails of the melanopsin activation curves; it doesn't maximally activate it.
125
What is the new light measurement approach equivalent to photometry but for melanopsin?
Melanopic Equivalent Daylight Illuminance (Melanopic EDI)
126
What is Melanopic EDI?
This is the measurement that for any light source, the amount of daylight that would produce the same activation of melanopsin.
127
What did Brown (2020) reveal about the differences in using Photopic Lux vs Melanopic EDI in understanding wavelengths of light and their relation to melatonin supression?
METHODS:
- Exposed ps to different wavelengths of light measured in either photopic lux or melanopic EDI.
- Measured the melatonin suppression at each wavelength of light.
- Imposed all the results onto a log graph to see if there was a relationship/activation curve.
RESULTS:
Photopic Lux:
- There was no overlap in curves and it was quite scattered.
- Lux isn't good for circadian system.
Melanopic EDI:
- All the curves superimpose on themselves.
- We can now see the melanopsin activation caused by different wavelengths of light.
128
What is the relevance of colour to human circadian responses in ariticial light?
Adding or subtracting short wavelength ("Blue") light from a white source can change the melanopic EDI 2-3 fold.
129
What do changes in levels of short wavelength light in artificial lights have on the circadian system?
Increase Wavelength (Yellow/Red Light):
* Suppresses circadian response by reducing melanopsin activation, allowing melatonin production and promoting sleep.
Decrease Wavelength (Blue Light):
* Enhances circadian response by activating melanopsin, suppressing melatonin, increasing alertness, and delaying sleep.
130
Describe the Allen et al., (2018) study into new approaches for selectivity change melanopic content of visual displays.
**METHODS:**
- Used 2 projectors and changed how they worked so that they could manipulate the colours that they were emitting.
- One had RGB one had 2 wavelengths.
- So a total of 5 primary colours.
- This allowed them to make movies that looked the same; cone spectral perception was the same but the melanopsin activation was different.
- Had people watch this in the evening and measured their sleepiness and melatonin activation.
**RESULTS:**
- Watching the melanopsin low movies had an increase in sleepiness and increase in melatonin.
131
What is the overarching summary about the role of non-photic stimuli on entrainment?
They don't have a very strong effect; they can still be used to change your activity but they don't greatly change internal rhythms.
