Lectures 1-3 Flashcards
(134 cards)
1
Q
What two overarching levels of processes that the body clock impacts?
A
Physiological and Cellular.
2
Q
What are some examples of the physiological processes that the body clock impacts? (6)
A
Sleep/Wake
Body Temperature
Cardiac Output
Hormone Secretion
Metabolism
Immune Response
3
Q
What are some examples of the cellular processes that the body clock impacts? (6)
A
Cell Cycle
DNA damage repair
Energy storage/release
Detoxification
Excitability
Communication
4
Q
What are the two major types of diseases that disruption of the body clock impacts?
A
Neurological/degenerative and Inflammatory/metabolic.
5
Q
What are some examples of neurological/degenerative diseases that are associated with disruption of the body clock? (6)
A
Sleep Disorders
Bipolar
Schizophrenia
Alzheimer’s
Parkinson’s
Huntington’s
6
Q
What are some examples of inflammatory/metabolic diseases that are associated with disruption of the body clock? (5)
A
Diabetes
Asthma
COPD (Chronic Obstructive Pulmonary Disease)
Arthritis
Cancer
7
Q
What are some modern lifestyle influences that can disrupt the body clock?
A
Shift work
Inappropriate light exposure
Altered eating habits
Jet Lag
8
Q
Summarise why organisms developed ways of biological timing.
A
There are many selective pressures that occur cyclically due to the earth and its inherent characteristics.
Thus the organisms needed to develop a system that allows them to not only adapt but thrive to these.
Therefore, it makes sense to create a system to monitor these changes.
9
Q
Give examples of some of the earths cyclical selective pressures. (7)
A
Sunlight
Temperature
Climate
Food Availability
Predation
Social Interaction
Sexual Pressure
10
Q
Give 3 examples of biological timing in organisms. (C, P, D)
A
Cyanobacteria - they express cyclical daily expression of a gene.
Plants - even in the dark they express their cyclical leaf movements usually present in light.
Drosophila - they have pronounced daily activity despite a lack of external timing factors; even their egg hatching has a rhythm.
11
Q
Define and give an example of ultradian rhythms.
A
Typically <20h (hours, minutes, seconds).
Pulsatile hormone release and/or sleep stages.
12
Q
Define and give an example of circadian rhythms.
A
Daily 20-28 hours
Body temperature and/or sleep/wake.
13
Q
Define and give an example of Infradian rhythms.
A
Typically >28 hours.
Oestrous Cycle (also circalunar) and/or seasonal breeding (also circannual).
14
Q
Define and give an example of Circalunar rhythms.
A
Monthly
Oestrous cycle.
15
Q
Define and give an example of Circannual rhythms.
A
Seasonal
Seasonal Breeding.
16
Q
What are the 4 major properties of a circadian clock?
A
It’s a self-sustained oscillation.
It drives rhythmic outputs.
It’s relevant to the environment.
It’s responsive to the environment.
17
Q
Why is the circadian cycle a clock and not an hourglass?
A
An hour glass needs some kind of input to restart the cycle every time it ends.
A clock is self sustained and runs continually.
18
Q
What does it mean that a circadian clock needs to be relevant to the environment?
A
It needs to be time coherent/relevant to the external environment (24 hours) sot heat the behaviour is expressed when needed.
19
Q
What are zeitgebers and what does this term mean? List some examples. (5)
A
They are the external environmental stimuli that input the internal clocks, guiding their accuracy.
It means ‘Time Givers’.
Light, food, exercise, temperature, social interaction.
20
Q
What are the 5 major key terms of circadian rhythms? What do they mean?
A
Period - clock speed (e.g., 24 hours for Humans).
Amplitude - magnitude difference between peak and trough.
Phase - temporal alignment relative to environment. (Can be light or another rhythm).
Entrainment - stable relationship with environmental signals (zeitgebers).
Free Running Rhythm - natural function of the clock when entrainment is removed.
21
Q
What is an actogram?
A
An actogram is a plot that shows rhythms in biological variables throughout the day.
Traditionally, actograms describe phases of activity and rest, but they have also been used to visualize rhythms in protein phosphorylation, gene expression, and hormone secretion.
Actograms are commonly used to study the underlying circadian rhythms of animals in fields such as ecology, reproductive biology, and sleep medicine.
22
Q
What is ‘double plotting’ in actograms and why is this used?
A
It is when you take the same actogram, lower it one level and shift it to the left of the original plot.
When dealing with shifts in cycles, at some point the time will go ‘off the edge’ and start on the other side.
By having a double plot, it allows you to see this shift over a 48 hour period compared to a 24 hour period (for example) so you can see the continual shift in a simpler way.
23
Q
When are mice most active? What happens to mouses locomotor activity in a dark ONLY cycle? Why is this?
A
During the night (nocturnal).
It will begin to phase advance (shift to the left) due to a free running rhythm.
This is because the internal circadian rhythm of a mouse is faster than 24 hours.
Therefore, with no zeitgebers, it will begin to get up earlier and sleep later, locomoting within that period.
24
Q
When are Rhabdomys’ most active? What happens to a Rhabdomys’ locomotor activity in a dark cycle? Why is this?
A
During the day (Diurnal).
It will begin to phase advance (shift to the left) due to a free running rhythm.
This is because the internal circadian rhythm of a Rhabdomys is faster than 24 hours.
Therefore, with no zeitgebers, it will begin to get up earlier and sleep later, locomoting within that period.
25
When are Degus' most active? What happens to a Degus' locomotor activity in a dark cycle? Why is this?
At dawn and dusk (Crepuscular).
It will begin to phase delay (shift to the right) due to a free running rhythm.
This is because the internal circadian rhythm of a Degus is slower than 24 hours.
Therefore, with no zeitgebers, it will begin to get up earlier and sleep later, locomoting within that period.
26
If you were to be given the actogram of an average human living their life, would you be able to discern their circadian rhythm with confidence? Why?
No.
Their life is guided by a myriad of complex zeitgebers; day light, alarm clocks, different eating times, socialising, travelling etc.
Without the _absence_ of zeitgebers, you can't tell with confidence the rhythm; they are entrained to the environment around them.
27
What is Social Jet Lag?
It is the difference in your sleep/wake between your cycle when you are working and have an external responsibility to get up and your natural time of waking on your free time (e.g., on the weekends).
28
Why are there seasonal changes in a humans sleep/wake cycle?
Solar time doesn't change; solar noon is always at the same absolute time.
However, local times changes (Summer and Winter time changes).
This means that the local environment has changed, e.g., work always starts at 9am but this changes relative to solar noon with these clock changes.
Therefore, depending on the season, your sleep/wake shifts in response to these external stimuli.
29
How does the body adaptations of Jet Lag take effect?
Activity patterns gradually shift across time zones as you acquaint yourself with the new environment.
30
Why can Jet Lag be so impactful on a person?
Every aspect of your bodies' physiology is effected by the change in time, hence it can be quite intense/strenuous to feel these effects.
31
What are the two axis of the circadian rhythm that external clocks set?
The time they are set on/occur on and the speed that they occur.
32
Describe *Part 1* of the WT vs WT/tau vs tau/tau mouse experiment on the limits to entrainment.
METHODS:
- A classic light/dark cycle then dark only cycle experiment (tested circadian rhythms in both conditions).
- A WT mouse was used with a clock of ~23.7 hours.
- A tau/tau mouse was used with a clock of ~20 hours. (tau mutation causes the speeding up of internal rhythms).
- A WT/tau mouse was bred that had a clock of ~22 hours.
- Exposed each to light/dark then dark only and used actograms to see what happened.
RESULTS:
- WT mouse would be entrained in the light/dark to be active only at night.
- It would phase advance in dark only.
- tau/tau mouse would not entrain in light/dark and would free run due to its clock being to fast to phase delay.
- would free run in darkness too.
- WT/tau would entrain in light/dark, however, with an abnormal phase (waking up slightly too early and sleeping too late; was awake partially for light).
- WT/tau would free run in dark only.
THIS SHOWS:
- There is a limit to whether an animal can entrain depending on how close its internal clock is to the external stimuli.
33
What are the limits to entrainment?
Generally, clocks can only speed up or slow down by 2-3h per day.
The range of differences in entrainment was a function of the differences in the intrinsic period and environment and results in two different altered phases:
If the intrinsic period is faster, there is an earlier phase.
If the intrinsic period is slower, there is a later phase.
34
Describe *Part 2* of the WT vs WT/tau vs tau/tau mouse experiment on the limits to entrainment.
METHODS:
- Researchers introduced a 50/50 split of WT to tau/tau mice in a field, wild type environment for a year.
- They left them to live life and wanted to see the effect of the tau/tau mutation and it's prevalence in this population.
RESULTS:
- By the end of the year, natural selection had resulted in a reduction of the tau allele to only 0.2.
- This shows the intense selection against it.
- Likely due to predation, breeding (biological and temporal availability).
35
Where are the 'body clocks'? How do they work (simple statement).
Individual cells across the body are clocks; most cells have them.
They work by a process of gene expression and degradation.
36
What is the basic process of the ACTIVATION of the molecular clock? Use the mouse as an example.
Two genes bind together, BMAL1 and CLOCK, both of which are transcriptional activators.
This means they drive the expression of genes, specifically period and cryptochrome.
The accumulation of these two genes' RNA leads to their protein synthesis in the cytoplasm.
These two bind and dimerise, from which the dimer translocate to the nucleus.
37
What is the basic process of the REPRESSION of the molecular clock? Use the mouse as an example.
The Period and Cryptochrome dimer translocate to the nucleus and then inhibit the action of BMAL1 and CLOCK.
This leads to their transcription of period and cryptochrome RNA, so per and cry decrease.
This leads to a reduction in per and cry proteins, thus leading to the lack of inhibitory action from them.
From which the cycle can start again at activation.
38
How long does the cycle of activation and degradation in the molecular clock take? Why is this salient?
It takes roughly 24 hours and is responsible for a lot of the rhythms in cell function, hence it is the basis of the circadian cycle.
39
What is extraordinary about the molecular clocks appearance between organisms?
There are many unique examples of where it evolved separately, showing its fundamental nature to biology on this planet; if it developed many times independently then it must be crucial.
E.g.:
Cyanobacterial clock
Insect Clock
Fungal Clock
Mammalian Clock.
40
Under what circumstances might organisms not have a biological clock?
If they live in environments where there are no reliable zeitgebers to provide info on the cycles occurring.
E.g., living underground, living in the arctic circle.
41
Give 3 examples of animals without functioning biological clocks. Why is this the case?
Svalbard Ptarmigan, Svalbard Reindeer - both of these live in the artic circle in which there is either no sunset or no sunrise, hence there is no consistent light stimulus to entrain a cycle to.
The Somalian Cave Fish lives in complete darkness in water in caves, even in the presence of light dark cycles it doesn't entrain.
Each of these do _have_ a clock but mutations present in it mean that they don't function.
42
What keeps all these clocks in line with the environment? Where is it located?
The Suprachiasmatic Nucleus (SCN) in the hypothalamus.
It's located above the optic chiasm, where the optic nerves cross within the Retino-Hypothalamic tract.
43
What are some mechanisms that the SCN uses to control the clocks across the body?
Neural signals, hormonal signals, cortisol.
It also acts indirectly on activity, feeding and body temperature.
44
What are the two main factors that dictate the impact of light on the circadian rhythm?
Timing of Light
Availability of Light
45
What is photoentrainment?
The fact that the circadian system is tuned to ensure robust alignment to the environmental light/dark cycle.
46
Outline the effect of time that which light is presented in a *nocturnal* rodents cycle and why.
Light introduced in the **early** part of the night:
There is a phase delay in the cycle because the circadian system will compute that the day lasted longer than it had expected; it should wait later for night to come and it to be active.
Light introduced in the **late** part of the night:
There is a phase advance in the cycle because the circadian system will compute that the night was shorter than expected; it should wake up earlier and sleep earlier to avoid being awake in the day.
47
What is a phase response curve (PRC)?
It is used to show the relationship between circadian time and the phase shift (in hours) elicited by an external stimulus such as light.
This can be used to show us what effect the stimulus has on the shifting of the circadian cycle (whether it be phase advance or delay) and where any 'dead zones' are.
47
What is a dead zone in a PRC?
It is a period of circadian time in an animals cycle in which introduction of a zeitgeber will not cause a phase shift of any kind.
47
What are the differences in phase shifts in mice and hamsters? Why?
Mice often show much larger delays and smaller advances.
This is because they have a fast running clock, thus they need to delay more often.
Hamsters often show much larger advances and smaller delays.
This is because they have a slow running clock, thus it needs to advance more often.
48
What can the multiple difference in availability of light between midday and midnight be?
Ambient illumination can be a billion (10^9) times higher at midday than midnight.
49
Describe the study into the effect of the intensity of light on hamster activity rhythms.
METHODS:
- Hamster wheel running in constant conditions.
- Light pulses applied during late subjective night at varying intensities starting from dim and increasing sequentially to bright light.
- Wanted to see the impact on activity onset changes post light introduction.
RESULTS:
- When it was dim, nothing happened to the animals.
- As it gets brighter you start to see a phase advance.
- At the highest intensity of light you saw the largest phase advance.
Therefore, the brighter the light, the larger the advance.
50
What did plotting the results of "The effect of the intensity of light on hamster activity rhythms." show us that was an important finding?
When plotting the Log(Photons) you can see that phase advances track the increase of available light very closely.
This is an important finding because it didn't align with out previous understanding.
So we now know that circadian photoentrainment relies upon accurate quantitative measurement of environmental light.
51
What was the problem of using retinal photoreceptors (rods and cones) to explain the mechanism underlying photoentrainment?
Given that circadian photoentrainment relies upon accurate quantitative measurement of environmental light, the rods and cones are not sufficient to supply this information.
Rods are fully saturated at very low levels of light.
Cones are responsible for day time colour vision and have a mechanism to actively adapt their sensitivity to the available light.
Thus neither of them can provide a holistic measurement of the relative light levels between night and day time.
52
Why is cone adaptation beneficial?
It allows cones to detect fine details across a wide range of light intensities.
53
Briefly describe the studies into circadian photoentrainment in sparrows that revealed extraocular photoreceptors.
METHODS:
- Took Sparrows and Enucleated them (removed their eyes)
- Put them in light dark cycle to see their phenotypic response.
- Shifted the light dark cycle to see the effect of this too.
RESULTS:
- Despite the lack of eyes, the birds followed a light dark cycle, displaying a typical circadian rhythm.
- When light shifted, so did their rhythms; they were able to regulate to the external light stimuli.
Therefore there must be extraocular photoreceptors to track this.
54
Briefly describe the studies into circadian photoentrainment in sparrows that *tested* the location of the extraocular photoreceptors.
METHODS:
- Took Sparrows and Enucleated them (removed their eyes)
- Put them in light dark cycle to see their phenotypic response.
- Injected a C.B dye under the skin of their scalp, preventing light entering into their skull.
RESULTS:
- Birds followed the rhythms precisely and were fine doing this whilst enucleated.
- After the injection they began to free run, showing that the location of the photoreceptors were somewhere in the brain.
55
Where are there photoreceptors in non-mammalian animals?
In the Pineal Gland and the Parietal Eye.
(It's a lot more confusing to understand their light perception as they can detect light without eyes).
56
What is true across ALL mammals when they are enucleated?
They cannot photo entrain; all aspects of circadian photoreception must originate in the eye.
Therefore it is much easier to identify their location of photoreception.
57
What are the 8 types of cells, from deepest out, in the retinal structure?
Choroid
Rod
Cone
Horizontal Cell
Bipolar Cell
Amacrine Cell
Ganglion Cell
Axons to Optic Nerve
58
Outline the mice study into whether rods and cones alone act as the circadian photoreceptor.
METHODS:
- Found mice that had a naturally occurring mutation in the phosphodiesterase B enzyme that kills the rods and cones (cones die because they also rely on rods for survival).
- They crossed this with another mouse with a toxin that kills cones leaving NO rods or cones.
- Tested whether they can still synchronise even thought they are fully blind.
RESULTS:
- They were able to respond to light as a normal mouse would showing that the photoentrainment mechanism was working well.
- Hence, it is NOT these two photoreceptors that are responsible for photoentrainment in the circadian cycle.
59
What type of retinal cells project to the SCN?
Intrinsically Photosensitive Retinal Ganglion Cells (ipRGCs).
~1% of the RGC's from the retina project to the SCN (these are the ipRGCs).
60
By which tract do which cells from the retina project to the SCN?
ipRGCs by the retinohypothalamic tract.
61
What does ipRGC stand for?
Intrinsically Photosensitive Retinal Ganglion Cells.
62
How were the cells that project to the SCN identified?
A retrograde tracer was used from the SCN travelled back down the retinohypothalamic tract to ~1% of RGCS - The ipRGCs.
63
Outline the study of responses of SCN-projecting RGCs in rats.
METHODS:
- Once the ipRGCs had been identified they could see their reaction to light.
1 - Tested the response to light at 'daylight' intensities.
2 - Tested the responses once removed from the retina
3 - Tested the relative activation in response to different wavelengths of light.
RESULTS:
1 - These cells exhibit an excitatory response to light at 'daylight' intensities (less sensitive than rods and cones).
2 - Responses remain when removed from retina (only SCN projecting RGCs).
3 - Found that they were MOST sensitive to light in 'cyan' part of the spectrum ~480nm (different to rods and cones).
Thus, the retinal neurons that project to the SCN are ipRGCs.
64
Briefly describe Rod/Cone Phototransduction.
Rhodopsin or Cone opsin is the photopigment in rods or cones respectively.
It's a 7 transmembrane domain receptor (GPCR) which has the ligand 11-cis retinal.
11-cis retinal is an inverse agonist in which binding inhibits the receptor.
When a photon of light hits 11-cis retinal, it activates into all-trans retinal and is released from the opsin, thus activating it and causing downstream cascades.
65
What was the opsin found in the ipRGCs when looking for the one responsible for circadian photoentrainment?
Melanopsin
66
What is the role of LacZ in circadian research?
In scientific research, the LacZ gene is often used as a reporter gene in studies of photoreception.
Researchers attach LacZ to a photoreceptor gene's regulatory sequence.
When the photoreceptor gene is active, LacZ is expressed, producing β-galactosidase.
This enzyme can then be used to track the expression pattern of the photoreceptor gene, often by staining tissues (e.g., retina) with a substrate like X-gal, which turns blue when cleaved by β-galactosidase.
Therefore, LacZ helps researchers visualise and study where and when photoreceptor genes are active.
67
Outline the details of study into whether melanopsin is the circadian photoreceptor.
METHODS:
- The researchers created a melanopsin promotor tau:lacZ transgenic mouse.
- *tau is a component of microtubules and lacZ encodes b-galactosidase that digests X-gal and creates a blue precipitate.
- *This means that any cell that expresses melanopsin would present a blue, showing where it is present in the brain.
- They then imaged the retina and the underside of the brain (so they could see the SCN) to see where melanopsin was expressed.
RESULTS:
- They found that melanopsin expressing RGCs form the retinohypothalamic tract and fairly selectively project to the SCN.
- This was shown by there being blue expression in some RGCs and the RHT & SCN being very blue.
68
What happened to melanopsin knock-out mice and their response to light? What does this show?
It was abolished, showing that melanopsin is necessary for ganglion cell photosensitivity.
69
Detail the study into melanopsin expression in Neuro2A cells and the effect it had.
METHODS:
- Expressed Melanopsin into Neuro2A which are not light sensitive (moues neuroblastoma).
- They then introduced 11-cis retinal and light to see if they could bring about depolarisation in these cells.
RESULTS:
- When retinal was introduced WITH light (not light alone) depolarisation was caused.
- This shows:
1 - Melanopsin is sufficient for photosensitivity.
2 - Melanopsin light detection is consistent with that of other known opsins.
70
Give 3 reasons why we can state that melanopsin is the photopigment of the ipRGCs.
Without melanopsin they do not respond to light.
Melanopsin can make other cells light-responsive.
Melanopsin is expressed in the human retina.
71
What do blind humans with rod/cone damage or RHT damage tell us about photoentrainment in humans?
By looking at the melatonin rhythms in these two types of patients we can see which of these two are more important for circadian rhythms.
Rod/Cone damage patients still can entrain their melatonin cycle, showing that these two are not required for photoentrainment.
RHT damage patients melatonin cycle free runs, showing that this prevents photoentrainment - they cannot detect the light available without an intact RHT.
72
Does melanopsin account for all aspects of circadian photoentrainment? Describe the results of a study to back your answer.
No, it doesn't.
Melanopsin knockout mice retain photoentrainment, therefore it's not required but it is sufficient.
73
Answer whether a mouse can photo entrain or not with the following genotypes:
Melanopsin Only:
Melanopsin Less:
Rod Only:
Cone Only:
Melanopsin Only: Yes
Melanopsin Less: Yes
Rod Only: Yes
Cone Only: No
74
What are the two parts of the photoreception system that combine to encode the full range of light intensities relevant to the clock?
Rods and Melanopsin.
Rods encode to a certain point from which they are saturated, from here melanopsin covers the rest of the range.
This allows a gradient understanding of appropriate timing across a large range of light intensities.
75
When are rods sufficient for 'accurate' photoentrainment? Why?
In a lab setting; they aren't testing night to day gradient understanding of timing, simply whether there is light or not.
So, if the light is brighter than the rods maximum saturation point, then the animal would consider it 'daytime'.
76
What do cones sense? How do they combine their activation to provide us information? What can we infer from this?
A wavelength of light, NOT COLOUR.
It is the combined activation of different cones and their relative intensity that provides us information about colour/colour sensitivity.
Therefore, colour processing occurs downstream of the cones.
77
State 3 facts about cone opsins.
All opsins are 'biased' towards certain wavelengths of light.
Sensitivity is determined by the opsins genetic sequence.
Most mammals posses two types of cone opsins: S and M (humans have L as well).
78
Why is colour useful for the circadian clock?
The colour of light, AKA the spectral composition, changes at different times of the day so this is important for discerning the time.
To tell the time of the day, which is the angle of the sun relative to your location, you can't only use light; if it was cloudy then how can you tell different levels of light?
Therefore, photoreceptors use the blue availability as this changes with the angle of the sun and is resistant to the objects in the way (such as clouds).
79
What are the main findings of the JW Mouland Paper on Cones and their relevance in Circadian Responses? (4)
Cone-derived blue:yellow colour signals influence circadian
entrainment in mice.
The blue colours associated with twilight suppress circadian
light responses.
Colour signals support circadian entrainment to low-amplitude
light:dark cycles.
Colour signals buffer the clock against cloud-related changes
in light levels.
80
What might be the role of colour signals in the mammalian clock?
They may help the mammalian clock to adjust for weather-dependent variations in brightness.
*The relative importance of rods, cones and melanopsin may vary across mammals.
81
Detail the difference between sources of light from the sun and light bulbs and how it will have different effects on the clock.
Sunlight and a light bulb both produce white light.
However, the sun shows a gradient of ALL wavelengths whereas a light bulb has (usually) 3 very sharp peaks at different wavelengths.
But both their activation patterns lead to a human perception of light.
A lightbulb doesn't effect our clock as much because it has a different activation pattern then that of melanopsins (the regulator of time of day).
The short wavelength content from the sun activates melanopsin and hence regulates our circadian clock.
82
How do we know the SCN is the master clock? Use results of studies to back this claim.
Lesions made using electrical currents revealed that an animal INSTANTLY loses ALL circadian rhythms once the SCN is lesioned and begins to act very sporadically.
However, the critical experiments were ones in which the SCN of one animal was transplanted from one animal to the lesioned animal.
It didn't restore everything (due to severed downstream connections) but it could restore a lot of it's cycles and those cycles matched the rhythms of the donor animal.
83
What is the difference in sleep rhythms between humans with no damage to the SCN and those with SCN damage due to pituitary tumour?
Typical humans have a normal, consistent sleep pattern.
SCN damage due to pituitary tumour ends up having a very sporadic, constantly changing, short bursts of sleep.
They cannot set a rhythm.
This occurs in other animals too.
84
What are the differences and similarities between the Human and Mouse SCN?
They are about the same size (similarity) meaning that the mouse has a proportionally bigger SCN than a Human (difference).
85
Why does the SCN look dark compared to surrounding areas in a coronal section using Nissl stain?
Because the cells are very tightly packed within it.
86
Outline the details of the SCN Anatomy in the following categories:
- General structure:
- Dimensions:
- Size of Neurons:
- Neurons/Hemisphere:
- Neurotransmitter:
General structure:
- It's a small bilateral structure in the midline hypothalamus.
Dimensions:
- 300-400um in width
- 500-600um in height
- 800-1000um in length
Size of Neurons:
- Very small, (~8-12um in diameter)
Neurons/Hemisphere:
- 8000/10000, thus very densely packed.
Neurotransmitter:
- GABA, inhibitory (can also co-express a variety of neuropeptides).
87
What type of network is the SCN
Inhibitory.
88
Describe the experiment that led to the understanding that SCN **cells** are autonomous clocks.
METHODS:
- Researchers mutated a mouse so that one of the genes (Per2) would produce luciferase whenever it was expressed (fluorescence comes from this enzyme).
- Then they homogenised the cells and recorded the expression of this gene over a week (using the fluorescence).
RESULTS:
- The cells express the gene individually in a cyclical clock type pattern.
- They were NOT organised and instead had their own patterns sporadically (due to the homogenised nature destroying the connections between them).
Therefore, this shows us that they are autonomous clocks and it is the connections within the SCN that gives them their time coherence.
89
Fundamentally, what does the clock in the SCN cells regulate?
Ion Channel Expression.
90
How does the central loop of the molecular clock control the body clock?
The central loop is the core look from which auxiliary loops extend from.
These loops are 'turned' by the main cycle and lead to a cascading effect from the core loop molecular cycling.
This changes which genes are expressed at different times of the day across the system.
Which changes the expression of ion channels; changing the excitability of cells as a function of time of day.
91
Describe the results of the study that showed that SCN clock cells communicate internal timing signals.
Isolated SCN neurons and recorded from them using a multielectrode array and found that they fire independently but sporadically.
This is due to the homogenisation leading to the disruption of connection.
SCN neurons recorded from and intact SCN slide shows synchronous rhythmic activity.
This is because they are still connected and can coordinate.
92
What is fundamentally true about the firing of SCN cells across all animals no matter what type of behaviour they show between light/dark cycles?
The SCN cells fire during daylight hours.
93
What can the mouse and chipmunk show us about the consistency of SCN cell firing?
These animals are diurnal and nocturnal respectively, yet both their SCN cells fire during daytime.
Therefore, no matter the behaviour in response to light availability, this firing is consistent.
94
What is the most important input to the SCN?
Photic Input - Light.
95
What is fundamentally the same about the ipRGCs in the RHT and normal RGCs?
They have an excitatory downstream effect.
96
What two substances do ipRGCs produce and what type of substance are they?
Glutamate - an excitatory neurotransmitter.
PACAP (Pituitary Adenylyl Cyclase Activating Peptide) - a co transmitter.
97
What is PACAP? Why is it useful for studying the cells it's from?
Pituitary Adenylyl Cyclase Activating Peptide.
A co-transmitter created by ipRGCs - seemingly increase the effect of glutamate on downstream cells.
We aren't sure why it's so important but it can be used as a marker of these cells (Useful).
98
Outline the study into the anatomy of the retinohypothalamic tract (RHT).
METHODS:
- Injected the following two into the eye:
- Used Cholera toxin as a tracer to see where the retinal fibres input the SCN.
- Also used immunohistochemistry for PACAP-containing nerve fibres.
RESULTS:
- Both sides of the brain were strongly labelled for both fibre types - not usual for rodents.
- RHT Input is NOT uniform across the SCN.
- There is a stronger input to the ventral (core) rather than the dorsal (shell) part of the SCN.
99
Describe the Brown et al. (2011) study into _in vivo_ electrophysiological recording of mouse SCN.
METHODS:
- Multielectrode probe recording from the SCN of an anaesthetised mouse.
- The electrode has contact across the whole SCN.
- Light introduced and recorded the relative responses from different parts of the SCN.
RESULTS:
- When light was introduced and the firing rate of the cells increased.
- The biggest responses were elicited from cells at the bottom of the SCN: where there are more retinal inputs.
100
Describe the Hanna et al. (2017) research into _in vitro_ SCN slice recording of mouse SCN.
METHODS:
- Took _in vitro_ recordings of SCN slices.
- Introduced pharmacological activation of glutamate receptors via NMDA receptor activation and measured the results.
- They also took normal recordings and then introduced a glutamate inhibitor wash over the SCN slice and measured the effect of this on firing rate of the cells.
RESULTS:
- Drugs that activate NMDA (glutamate) receptors led to an increased firing rate.
- Firing rate before inhibitor introduction led to increased firing rate, once the glutamate inhibitor WAS introduced, the firing rate massively reduced, showing that the effect has gone.
Thus this effect is mediated via glutamate.
101
Outline the study into the effect of light input on SCN neurons state (rate of change and peak firing).
METHODS:
- _In Vivo_ tracking of SCN cells over a period of ~16 hours.
- Introduced oscillating light/dark cycles in hour periods (1 hour light, 1 dark).
- Looked to see how the basal firing rate changed throughout the 16 hour period and how light introduced in these different stages influenced the rate of change in the light response.
RESULTS:
Basal Firing Rate:
- During day time, firing rate is very high, during night time, firing rate is very low - presenting in a sinewave esc pattern.
Light Response Rate Of Change:
- During daylight hours, shining a light elicited a very small response.
- During night time hours, shining a response elicited a huge response.
- This is because during daytime, the basal rate is already quite high, so the additional light doesn't increase the rate of firing that much.
102
Explain how light alters circadian timing in a 'phase' dependent manner. What type of animals does this occur in?
(Very Oversimplified).
Light presented at Evening/Dusk delays clock - delays the decline of SCN activity (re-spikes it).
Light presented at Morning/Dawn advances clock - advances increase in SCN activity (spikes it early).
Light at Midday has no effect - SCN activity is already high.
Occurs in both nocturnal AND diurnal animals.
103
What is the second most important input to the SCN - what are both the names for it?
Non-photic stimuli - 'arousal inducing'.
104
What are the two main inputs into the SCN for non-photic stimuli?
The Intergeniculate Leaflet (IGL - part of the Thalamus)
The Median Raphe
105
What are the neurotransmitters of the IGL? What is the pathway called that inputs the SCN from the IGL?
GABA and NPY
The Geniculohypothalamic Tract (GHT)
106
What is the neurotransmitter of the Median Raphe?
Serotonin.
107
What are the three main neurotransmitters that are used to input the SCN from non-photic input centres?
What effect do they have on the SCN and why is this interesting?
GABA (From IGL)
NPY (From IGL)
Serotonin (From Median Raphe)
ALL of them have inhibitory effects on the SCN which is interesting because this is the opposite effect to light.
108
What is interesting about the inputs to the IGL and what does this suggest about its function?
It receives inputs from non-photic stimuli AND the retina.
This shows that it receives light information and arousal based stimuli, perhaps signifying that it has a processing role before projecting to the SCN.
*However, we know it's critical for arousal stimuli but we don't know the significance in relation to light yet.
109
Where do the NPY fibres from the IGL and the 5-HT fibres from the Raphe Nucleus project to in the SCN?
Why is this interesting?
They are most densely innervating the Ventral SCN (Core).
This is the area that receives retinal inputs too.
110
How does the introduction of non-photic stimuli influence the circadian cycle of a nocturnal rodent?
During Day: Inactive Phase
- You'll produce a phase advance in the clock.
- This will be reducing the SCN activity (it's high during day and these stimuli are inhibitory).
During Night: Active Phase
- There will be next to no effect.
- This is because SCN activity is already low so additional inhibition won't do much.
111
What is the difference between the phase response curves in nocturnal animals for photic and non-photic stimuli?
*Photic Stimuli:*
- Introduction of Bright Light at start-mid of active phase (early night) will cause a delay in phase.
- Introduction of Bright Light at mid-end of active phase (late night) will cause a phase advance .
*Non-Photic Stimuli:*
- Introduction of Arousing Stimuli during the inactive phase (early/mid morning) will cause a phase advance.
- Introduction of Arousing Stimuli during the active phase (early/mid night) will cause little to NO effect.
112
What is AVP? What type of compound is it?
Arginine Vasopression; a neuropeptide
113
What is mENK? What type of compound is it?
met-enkephalin; a neuropeptide
114
What is VIP? What type of compound is it?
Vasoactive Intestinal Polypeptide; a neuropeptide
115
What is GRP? What type of compound is it?
Gastrin-Releasing Peptide; a neuropeptide
116
What are the 4 main neuropeptides expressed in the SCN and which part are they expressed in?
What type of connections do they have within the SCN? (2)
Dorsal (Shell): Sparse Internal Connections
- AVP
- mENK
Ventral (Core): Strong Internal Connections
- VIP
- GRP
117
Outline the Core and Shell Model of SCN Organisation.
The premise is that there is a specialised core and shell subdivision within the SCN.
Ventral/Core Region:
- Receives Strong Photic and Non-Photic Input.
- Have weaker internal clocks.
- Has extensive connections to the shell.
- VIP/GRP are the neuropeptides mainly expressed here.
Dorsal/Shell Region: (Brings about effects of SCN)
- Has Weak Photic and Non-Photic Input.
- Have strong internal clocks.
- Has extensive output projections.
- AVP/mENK are the neuropeptides that project here.
118
Outline the 4 major points about the Ventral/Core Region of the SCN
Ventral/Core Region:
- Receives Strong Photic and Non-Photic Input.
- Have weaker internal clocks.
- Has extensive connections to the shell.
- VIP/GRP are the neuropeptides mainly expressed here.
119
Outline the 4 major points about the Dorsal/Shell Region of the SCN
Dorsal/Shell Region: (Brings about effects of SCN)
- Has Weak Photic and Non-Photic Input.
- Have strong internal clocks.
- Has extensive output projections.
- AVP/mENK are the neuropeptides that project here.
120
What are two studies results that indicate the Core/Shell model of the SCN is oversimplified?
AVP receptor knockout mice have enhanced phase setting.
VIP cells have robust rhythms and regulate aspects of downstream physiology.
121
What are the main Hypothalamic outputs of the SCN? (4)
Sub-paraventricular Zone (sPVN)
Paraventricular Nucleus of the Hypothalamus (PVN)
Medial Preoptic Nucleus (MPN)
Dorsomedial Nucleus (DMH)
122
What is the main SCN thalamic output pathway?
Paraventricular Nucleus (PVT)
123
What does the SCN Paraventricular Nucleus of the Hypothalamus (PVN) output pathway control? (3)
Melatonin
Cortisol
Autonomic Nervous System
124
What does the SCN Medial Preoptic Nucleus (MPN) output pathway control? (2)
Body Temp
Gonadal Steroids
125
What does the SCN Dorsomedial Nucleus (DMH) output pathway control? (2)
Arousal
Feeding
126
What does the SCN Paraventricular Nucleus (PVT) output pathway control? (2)
Behaviour
Cognition
127
What do traces of the following secretions in rats show us about circadian rhythms?
- Corticosterone
- Luteinising Hormone
- Melatonin
Each of them have their own pronounced circadian rhythms of when they are secreted.
Therefore, there is some mechanism to which the SCN uses to control the different timings of downstream systems.
These hormones can then influence clocks in different parts of the body and act as a clock for them.
128
Detail the melatonin secretion pathway. (6)
State whether each point in the pathways is excitatory or inhibitory.
ipRGCs (+)
SCN (-)
PVN (+)
Intermediolateral Spinal Cord (+)
Superior Cervical Ganglion (+)
Pineal Gland = Melatonin Secretion.
129
How do we know that melatonin doesn't control sleep?
Nocturnal animals secrete this at night whilst they are awake.
130
How does the activation or inhibition of the SCN relate to melatonin production?
SCN is stimulated by the presence of light and it has an INHIBITORY action in the melatonin secretion pathway.
Hence, if light is present and SCN is stimulated, there is LOW Melatonin secretion.
If light is NOT present and SCN is not active, there is high level of melatonin secretion.
131
What is melatonin fundamentally a signal of and why?
Time of day/year.
In Winter there are longer nights, so the SCN is active longer = your brain knows it's winter.
In Summer there are longer days, so the SCN is active less = your brain knows it's summer.
*This is how seasonal breeders know when to prepare.
132
When does corticosterone fire in both nocturnal and diurnal animals?
During the start of the active phase.
