Lecture 6 - Circadian Rhythms Flashcards
What is a sleep cycle?
24 hours with cues
What time period will a circadian rhythm be maintained in even in the absence of light and how was this discovered?
24 hr cycle; in a study in which they removed light cues they found that the sleep and wake period for humans was 25-26 hours and 23 hrs and they showed a shift in people would stay up later each day and wake up later; but when they reintroduced light cues there was entrainment
What to have in addition to our daily cycle of sleep and wake which is also controlled by our circadian rhythm?
melatonin levels
body temp
serum cortisol levels
Where is the central clock?
the SCN or suprachiasmatic nucleus (above the optic chiasm)
What three pieces of evidence proved that the SCN is the master clock?
- neurons in the SCN fire APs in a 24 hour rhythm and isolated SCN neurons continue to keep this rhythm (meaning this occurs intrinsically)
- ablation of the SCN disrupts wake/sleep cycle in mice
- transplanted SCN confers time keeping of host to recipient (if you transplant the SCN of a mouse on a different time cycle to another cycle you can change the rhythm of the mice)
How was it proven that isolated SCN neurons maintain 24hr circadian rhythm?
-used calcium indicator like Fura2
-put a calcium indicator into the cell and could measure the calcium firing throughout the day and they found that the calcium firing would go up and down depending on the time of day in a circadian rhythm
What did the APs show from the isolated SCN neurons?
that they would fire in a circadian rhythm in a 24 hr rhythm and if you put TTX it would stop, but then wash it away it would restore
Why are hamsters good to study for circadian rhythms?
they are nocturnal and they run at night
In mutant flies that had a disrupted circadian rhythm what was the mutant gene found and what did they show?
the per gene was mutant (for period) and they showed no rhythm in darkness
Mutations in the per gene in rodents also found what to be disrupted?
the circadian rhythm; there is another gene as well which is called Clock - need to note that the WT have a rhythm which is less than 24 hours in constant darkness and the mutant has a cycle of 24 hrs mutation in clock animals still maintained circadian rhythm
What does the per gene a part of?
the clock in the SCN
What does the per gene encode?
a transcription factor
What is a transcription factor?
a protein that can turn on genes and causes them to be transcribed to RNA
How many distinct per genes are in some species that have similar functions?
2-3 distinct per genes
What length of a cycle does the per protein and per mRNA have?
a 24 hr cycle
The level of the per mRNA and per protein are found to vary at different times throughout the day?
the per mRNA is highest in the day and the proteins levels are delayed by 6 hours so they peak at dusk
When a per promoter was driving the fluorescent gene Luc bioluminescence what was observed?
-that it gets brighter and dimmer throughout the day in the same circadian rhythm over seven days
What does the promoter upstream of the genes do?
binds a transcription factor Eric h then turns on a gene by promoting its transcription from DNA to mRNA
How many different gene can a transcription factor affect?
the expression of many genes and can bind to many genes
What are transcription factors where are they made and how do they get to the nucleus?
small proteins which are made in the cytoplasm and translocated to the nucleus
What is the molecular clock or transcriptional network made of?
4 transcription factors that act as dimers in two transcriptional networks to generate a 24 hour cycle of transcription
How do per (+cry) turn off?
they turn themselves off which takes some time since the mRNA need to make protein which needs to enter the nucleus and takes 6 to 12 hours
-they inhibit their transcription by taking off clock and bmal
What turns the per gene back on?
clock +cycle (bmal)
note that cycle is the fly version of bmal
What is a quick summary of the molecular clock or transcriptional network?
-DNA when transcribed is going to get mRNA and make the per protein’ there is another similar gene called cry which also gets transcribed and is made in the cytoplasm and they both gets translocated into the nucleus; various ways that you can make a clock and how you can make it out of genes and DNA and cells and there is a negative feedback that is critical to the clock so that the per protein once it makes it into the nucleus turns off its transcription so you Strat having less per and then something else then turns back on the per which is the clock and cycle which are transcription factors which bind to the per gene and turn per back on (ignore Tau in the diagram)
What is the circadian rhythm in per mRNA and protein levels?
per mRNA levels grow during the day and the protein levels peak at night which means that when per enters the nucleus it blocks its own expression and these changes happens in both diurnal and nocturnal animals
How do we do from oscillations in level of per to daily oscillations in electrical activity? What are four proposed mechanisms?
ion channel expression distribution or functional properties must change
- synthesis and degradation of ion channels
- trafficking
- distribution clustering or decluttering of ion channels
- phosphorylation to activate the ion channels
How is the clock entrained?
light will come in through the eye through non image forming retinal ganglion cells or ipRGCs and this will change the activity of the SCN which will then input to our internal organs
Can the clock be reset instantaneously?
the clock cannot be reset instantaneously and it can only be tweaked one hour or so every day
What does light entrainment observed in the absence of rods and cones indicate?
that there is a third class of photoreceptor in the eye in addition to rods and cones and that is melanopsin and it is a light sensitive receptor
Where was melanopsin first found and also later found?
first found in frog skin and it is found in the mammalian retina in RGCs
How were melanopsin expressing cells identified?
LacZ reporter which is blue which was expressed under the melanopsin promoter and found melanopsin expressing RGC
What does each sleep stage have and provide and example of this in the awake state versus stage IV?
each stage has a specific wave form
the awake state is less synchronized activity while stage IV is due to synchronized activity due to all cells firing at the same tome
What is the relationship between the ascending brainstem-reticular activating system and the thalamus and sleep?
the ascending brainstem reticular activating system inhibits the thalamus which causes or induces sleep
