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Neuro 4623 - Obrietan Lecture #1-4
Terms in this set (47)
Where is the SCN found? How many neurons are there? What type of neurons are they? Is there a large difference between a mouse SCN and a human SCN?
-found just above the optic chiasm in the diencephalon. Lateral to the 3rd ventricle
-about 8-10,000 neurons
-GABAergic neurons, GABA is principle NT
-similar SCN's, found in the same region with same amount of neurons
How does the SCN during the day compare to the SCN during night? How is the firing rate affected?
-high levels of metabolic activity during the day, radioactive glucose show very prominent SCN during the day
-such little metabolic activity during the night, pretty much disappears, shows such little circadian behavior
-higher firing rate during the day compared to night
How does a lesioned SCN compare to an intact one in terms of locomotor activity?
-lesioned SCN results in a complete loss of consolidation of locomotor activity, very speratic
-intact SCN shows clear rhythms
In an experiment, the old SCN was lesioned and another SCN was implanted into the host from a donor. How were rhythms affect in each step? What does this imply about how the SCN works in the body? How was this clearly shown when the SCN was put in a meshwork of plastic and not allowed to come in contact with the host brain?
-rhythms were lost when lesioned, when re-implanted, rhythms subtly reappeared.
-this works because the SCN controls rhythms based on hormonal release, doesn't require distinct synaptic circuitry
-when it was out in plastic, saw hormones release but synaptic connections not reinstated
Synaptic SCN connections within the SCN itself is why it's functional. How do individual neuron rhythms compare to SCN rhythms as a whole?
-SCN has beautiful 24 hr robust rhythms, but when SCN neurons are isolated they retain their timekeeping capability but it's not as robust/consistent
What was found to actually control the oscillatory behavior? What was the first experiment that proved this?
-degradation and express of genes (transcriptional vs. translational expression)
-tested firing properties of the optic nerve in bubble shell gastropods
-blocked translation (protein synthesis), this stopped the clock
How does a reduced rate of protein synthesis affect a clock's tau? How does blocked protein synthesis affect a clock's tau?
-reduced rate results in a reduced tau (period).
-blocked synthesis will stop the clock and it'll resume at a different phase
Is rhythmic hormone expression also regulated by transcription/translation? (AVP)
yes, seen in release and expression of AVP(vasopressin). Greater during the day than at night.
Explain the basic transcriptional feedback loop.
Oscillator will produce positive elements that express clock genes. The clock genes will express negative elements that block the positive elements from expressing clock genes.
What is ENU screening? How does it work?
-inject N-ethyl-N-nitrosourea, this shit will trigger the transversion of A's to T's
-1 out of every 1000 As gets turned into Ts
-this will mess up the functionality of the gene and eventually the phenotypical behavior
-then you can go backwards and locate the genes that cause those changes
What mutated gene led to total arrythmicity? Homozygous or heterozygous? Where was this gene located and what did the mutation cause?
-CLOCK gene on chromosome 5
-Mice homozygous for it were arrythymic
-Mice that heterozygous will have a period of more than 24 hrs
-was a single A to T mutation
-protein makeup was changed
What type of transcription factor does the clock gene code for? How does it activate? Where does it bind to DNA?
- a basic helix-loop-helix (bHLH)
-these two transcription factors will dimerize and coil/bind together
- DNA binding domain will attach to DNA at the "E-BOX" motif (CACGTG)
Do bHLH transcription factors often homodimerize or heterodimerize? What was found to be CLOCK's binding partner?
Contrast forward genetics with reverse genetics. Give example of each.
Forward: attempt to find the genetic basis of a phenotype or trait (e.g., ENU screen)
Reverse: seeks to find what phenotypes arise as a result of particular gene (e.g., via KO technology)
After they knocked out BMA1, what did they find? How did they knock it out?
-knocked out the HLH part so it couldn't bind to CLOCK
-Animal went completely arrhythmic, actogram looked like that of a lesioned SCN.
Describe the behavior of the KO BMAL1 mice. What does this indicate about the importance of rhythms?
-BMAL1 KO pups were much smaller and their mortality rates were extremely high. By 50 weeks all the mice were dead. They were almost half the weight of the normal mice
-affects reproduction, can't breed, infertile
-Circadian timing affects virtually all aspects of physiology and health (not just sleep/wake and locomotor)
What are the two positive elements that drive gene expression for circadian timing ?
CLOCK and BMAL1
How were fruit flies used to identify the period gene? What method was used to get to this result? How was their locomotor activity?
-Used forward mutagenesis to mutate fruit fly genes
-went through and found the phenotype they were -looking for, cloned and found Period1
-eclosion of fruit flies is a rhythmic thing, but there was a subset of flies that didn't follow this rhythm, they were arrhythmic and so was their locomotor activity.
Describe period 1 expression in the SCN.
During circadian day they are really high levels of expression, vice versa during night
Are period 1 oscillations regulated by CLOCK and BMAL1? If so, then explain why that is so? Experimentally, how did they figure this out?
-identified 3 EBOX motifs in the regulatory site of the per1 gene
-fused firefly luciferase gene with the period 1 gene
-they then altered levels of CLOCK and BMAL1, identified different amounts of light that correlated with different levels of transcriptional activity.
In BMAL1 KO mice, how was mPer1 signal different?
-You KO BMAL1, you KO the per gene
-BMAL1 (and Clock) drive rhythmic Period1 expression!
-KO mice showed no expression of per during the subjective day or during the subjective night
What made scientists believe that period1 was a part of the feedback loop? What part is it actually? How is it expressed?
-Per1 gene has two identical protein interaction domains with the clock gene
-It is a negative elements
-BMAL1 and CLOCK bind to EBOX motif at the regulatory region of the per1 gene`
In the Period1-luciferase reporter gene assay, when the concentration of Period1 was increased, what happened?
-there was a reduced capacity of CLOCK and BMAL1 to stimulate luciferase expression
-Period1 blocked its own expression
How many mPer genes are there? Which ones play a role in regulating expression? Was circadian oscillation of mPer1 and mPer2 different across subjective day and night?
3 genes, only 1 and 2 play a role in circadian oscillation
-both had the same oscillation concentrations, high expression during the day and little expression during night.
KO (reverse) technology was used to KO Per 1 and KO Per2 and test locomotor rhythms, what were the results?
- Both Per1 KO and Per2 KO mice went completely arrythmic.
Where is the majority of Per1 and Per2 located? Why is that an issue for figuring out the negative feedback loop?
-majority is located in the cytoplasm and never enters the nuclues. But somehow Per1 and Per2 are translocating into the nucleus to inhibit their own expression.
What was found to be the binding partner of Per1 and 2? What are characteristics of these? What happens when they are knocked out in mice?
-these are flavoproteins and they are sensitive to blue light so when a blue light is shined on it, it'll change structure
-KO mice seemed normal (visually), however the circadian rhythm was completely disrupted.
-CRY1 KO mice had got a shorter tau, CRY2 KO mice got a slightly larger tau
-When both KO, went completely arrythmic
Was there rhythmic expression in the SCN of CRYs? What was observed when the CRY concentration was increased?
Yes, higher in day and lower at night. When concentration was increased there was an increased suppression of CLOCK and BMAL1.
How do butterflies use cryptochromes to navigate? What three things do butterflies integrate to do this?
-Butterfly cryptochromes detect magnetic fields-a key navigation strategy for their seasonal migration
-polarization of light, magnetic fields from north and south pole, and circadian timing system
How did Dr. Menaker figure out the tau phenotype? What protein did this alter? and what does this protein do?
-noticed one of his mice had a really short tau (20 hours)
-located tau gene by mutation, found it in the Casein Kinase 1 epsilon gene, codes for he protein CK1
-CK1 phosphorylates Per1 3 times and tags it with ubiquitin so then it undergoes degradation
When does CK1 come in action?
When the per concentration reaches a certain threshold. Degrades at a precise time for a 24 hr period
How does rate of phosphorylation affect the tau? Is CK1 the only enzyme that does this?
slower turnover ->long tau
rapid turnover -> shorter tau
-no, there are numerous enzymes
So, why does the Tau mutation result in a shorter rhythm?
-tau mutation makes CK1 more effective so it can phosphorylate faster and increase turnover rate
How much of the genome is under control of a circadian clock? Where all does BMAL1 bind to?
-binds to 2,000 sites in the genome, and the top 200 are under the control of a circadian clock
-10% of genome is under circadian control
Where is Vasoactive Intestinal Peptide (VIP) produced? What are its many functions? What does it do in terms of binding?
-produced in gut, pancreas, brain (including SCN)
Functions: Vasodilation, lowers blood pressure, relaxes smooth muscles, regulates pituitary hormone release, SCN physiology
-will binds to two types of receptors and increase cAMP levels in addition to PKA.
The SCN has distinct (3) populations, inferring that it isn't homogenous, but each population has their own role. What are the two main populations and what properties do they have?
-Core neurons: express GABA and VIP and are non-rhythmic or weakly rhythmic.
-Shell neurons: express GABA and AVP and are robustly rhythmic-they are the main pacemaker cell populations in the SCN.
-Core neurons modulate activity of the shell neurons
A VIP receptor KO was test as well s a VIP KO in a population of mice and sleep/wake rhythms were measured. What were the results? What does this tell us about communication between shell and core neurons?
-Both the receptor KO and VIP KO showed really erratic behavior with no consolidation
- this tells us that VIP release from the core neurons plays an essential role in the generation of SCN circadian rhythms.
What did SCN slice recordings reveal for the VIP receptor KO mice?
-revealed that while the high amplitude rhythms were gone, the cells were still oscillating and their rhythms were super low and irregular.
Describe the intracellular VIP circuit? What are the three main roles of VIP?
-VIP is released from the core to the shell and sets the phase of the rhythm
- 1. Regulates the amplitude of the core clock
- 2. Wires together the cell autonomous oscillators
- 3. Creates a coherent output signal from the SCN clock
Pull it all together: How does VIP and shell neurons interact to control behavior?
-shell sends a signal in the from of clock control genes to the core , then VIP feeds back to the shell neurons and drives a higher amplitude oscillation. VIP amplifies the rhythmic transcription
How were intracellular calcium levels measured? Did they oscillate within the SCN?
-calcium reporters were fired into the SCN, once bound with calcium would fold onto itself and fluoresced. These levels exhibited rhythms in the SCN and were driven by VIP
Was cAMP levels found to oscillate too in the SCN? How were the regulated?
YES, and VIP REGULATES THIS TOO
When cAMP levels were disrupted, so were rhythms in the SCN. What two big things do cAMP (and calcium) regulate?
1. regulate the amplitude of SCN rhythms
2. regulate the synchrony of SCN oscillations
Well, what are the targets of Ca2+ and cAMP?
-Calcium: stimulates calcium kinases (PKC, CaMKs, MAPK)
-cAMP: Protein kinase A
Briefly describe the role of CREB in altering gene expression. Is it rhythmically regulated in the SCN?
-CREB plays a role in learning/memory
-CREB will bind to a cAMP response element on DNA, at the CRE site, and become phosphorylated. Serine 133 phosphorylation will activate CREB and allow it to bind to CBP thus allowing for DNA transcription
-yes it is rhythmically regulated
Are there more CRE sites or EBOX sites on the regulatory region of the Per1 gene? What does this tell us about CREB in relation to CLOCK and BMAL1? What happens if we inject VIP in SCN in terms of per1 expression?
More CRE sites than EBOX sites. CREB is also a regulator of per1 gene and will activate expression. If we inject VIP in the cell, per1 gene expression will increase.
From the VIP signaling pathway to CREB and then to period 1, describe the whole pathway.
VIP will be released form the core cells and binds to the shell cells (at 7MR) activate G protein which activated Adenylate cyclase and Phospholipase C. AC will produce cAMP and PLC will increase intracellular calcium. Ca2+ will activate Calcium kinases (PKA, MAPK, CaMKs) and cAMP will activate PKA. These kinases will phosphorylate CREB (a transcription factor). CREB binding protein is then recruited (in addition to a ton of other proteins). They all form a complex and then bind to the CRE site on Per1 and activate transcription.
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