S1 N1: 1:00-2:00Scribe: Sally Hamissou

Wednesday, March 11, 2009Proof: Sunita Jagani

Dr. LesterCircadian RhythmsPage1 of 7

Abbreviations: NE= nor epinephrine, ACH= acetylcholine, RF= reticular formation, SCN= suprachiasmatic nucleus, AP= Action Potentials

Circadian Rhythms

  1. Circadian Rhythms [S1]
  2. Exam- if anyone wants to talk to him about the grades privately, drop him an email. He’s had some more challenges, if you want to check your test, email Jen and get them to Lester quickly so he can change the scores.
  3. This is a neuroscience behavior topic because things have been worked out at the level of gene regulation all the way up to behavior.
  4. Nice way of looking at how neuroscience is gene to behavior.
  5. For medical students, it is a common topic that comes up on their boards all of the time.
  6. Today’s lecture is in two parts: we will discuss the clock itself, where it is and how if functions, and we need to understand that. Then we will talk about basic properties and mechanisms underlying sleep.
  7. Functional neuroanatomy.
  8. Biological Clocks & Sleep [S2]
  9. Learning Objectives [S3]
  10. Understand the concept of free-running & entrained pace-makers
  11. Clock that we all have is an entrained pacemaker.
  12. Basic concept: clock is ticking over but looses track of time with respect to world turning if we don’t reset it and tell it what time it is every morning.
  13. Its is a clock that runs a little bit slow or fast, it is a little off and needs to be reset.
  14. Discuss the anatomical location of the clock and its associated light-entraining pathways
  15. We will watch a movie on how the clock works
  16. Clock is contained within a small nucleus within hypothalamus and is contained within every cell in nucleus. Each cell has its own clock. When putting the cell together they work as one and their output is the main output of the clock.
  17. Sleep is one of the topics that is not understood but is clinically important. Sleep disturbances is another reason people come and see their doctors.
  18. Explain how activation-repression loops within single cells can generate continuous cycling output in terms of changes in AP firing rates
  19. Discuss physiology & pharmacology of REM versus non-REM sleep
  20. Pacemakers [S4]
  21. Why do we need a clock?
  22. We have the generic animal that needs to know what time of day it is, what time of year it is, regulate activities, what stage of life we are in. We all have clocks that run on different scales.
  23. The circadian clock is a master clock that sends out signals that regulates other behaviors. It regulates sleep (it doesn’t control sleep) and regulates timing of sleep. It might regulate metabolic pathways during different times of the day, you may want to up or down regulate enzymes.
  24. It’s a clock that regulates behaviors.
  25. Learning Objective #1 [S5]
  26. Understand the concept of free-running & entrained pace-makers
  27. Main Features of Rhythms [S6]
  28. How people that work on clocks plot their data based on experiments.
  29. They tend to plot it one day at a time over a number of days. Each of the bars represents one day of measurement.
  30. This represents 24 hrs from beginning to end, a normal real day.
  31. Blue is when the animal is awake, red is when the animal is asleep.
  32. Over a number of days when we observe behavior, we usually observe hamsters as models, and we monitor activity or non-activity.
  33. This is wake and sleep in humans because we follow the same pattern. It is the start of the day when we wake up, and it roughly occurs at the same time every day.
  34. In green part, we turn out all of the lights and the animal is in total darkness. Still has sleep wake but now it starts happening at different times of the real day. This animal starts to wake up later and later each day. From the pattern, you can see that the clock is a little longer than 24 hours because each day we add a couple hours until it wakes up. It’s clock is free running—if not regulated by environmental signals, it would run longer than 24 hours.
  35. Then have light/dark cycle again that resets to 24 hours and we all have that clock.
  36. General organization [S7]
  37. Overall idea: free running clock that controls various behaviors, it regulates when you wake up or go to sleep.
  38. It can be entrained or regulated by light.
  39. In constant darkness it would free run but in light-dark cycles like we have normally, we could train it to correspond to natural day and night cycles.
  40. Learning objective #2 [S8]
  41. Discuss the anatomical location of the clock and its associated light-entraining pathways
  42. Where is the clock? [S9]
  43. Hypothalamus controls motivated behaviors and is important for maintenance of internal environment in the face of changing conditions in the internal or external environment.
  44. Different nuclei have different functions and control different things- feeding, temperature regulation, etc.
  45. Above the optic chiasm (SCN)
  46. Points out optic chiasm, nucleus sits ventrally in hypothalamus right above optic chiasm
  47. If we take a coronal section midline here is the SCN- bottom left figure
  48. SCN is necessary…… [S10]
  49. SCN ablation:
  50. Results in a loss of circadian rhythms
  51. We need to know that this is the area of where the clock is and there is substantial proof of this.
  52. With the SCN, people have shown that is not only necessary, but it is sufficient. It contains all elements of the clock.
  53. Same experiment as before light-dark cycle, animal has nice normal cycle. In total darkness, clock free runs and is slow.
  54. But if you lesion SCN, its activity pattern is broken up and doesn’t regulate its daily function. What that tells you is light is necessary to make clock entrain it and run at a consistent time each day.
  55. Clock still works in absence of light but if you destroy the clock then you can’t regulate the overt mechanisms, and behavior can’t be entrained or regulated normally.
  56. …and sufficient [S11]
  57. In this experiment, they took the animals that had a mutation in one of the genes that regulates the clock, and it makes the clock run at a different speed.
  58. This wildtype animal has a clock runs at 24 hours and runs freely in respect to light-dark cycles. But this one run runs fast and we expect slope to go in a opposite direction
  59. If area is sufficient, we should be able to take cell out of one animal and switch their clocks, which is what they did.
  60. SQ: When you do the switch, was the one that was mutant restored to the normal 42 hour? A: Yes. For the wild type, in darkness still runs at 24 hours and this one runs faster at 20 hours. If you take out the cells and put it in another one it completely switches the behavior of the animals. Everything that is the clock is in that nucleus and the properties of the clock are there.
  61. Learning objective #3 [S12]
  62. Explain how activation-repression loops within single cells can generate continuous cycling output in terms of changes in AP firing rates
  63. Like a lot of the things we have talked about in the hypothalamus in general, there is a theme about feed back loops of activity.
  64. We will talk about them in respect to sleep and wakefulness. We have these ongoing loops cortical down to subcortical regions and back up through the thalamus to the cortex. The loops are what keep us conscious and make us ready to act and aware. If we have different amount of loop activity, we are almost unconscious drifting off somewhere.
  65. If you give painful stimulus that is salient, it will knock you into gear again and wake you up.
  66. Think about RF and parts of the brainstem that kicks us into action.
  67. There are loops that control consciousness and there are loops in cells. It makes sense because if we have a clock, something going up comes back down, and that may be what keeps track of time and now we know that that is true.
  68. The output of the cell is a change in AP firing frequency.
  69. If through neurons, change in firing rate in cells. Mechanism within cell that loops and makes the neuron fire more or less.
  70. Important point is that the firing is not the key regulator, it is the output. The key regulator is the gene transcription and translation of protein synthesis. That is what cycles and that cycling influences the firing of the cell.
  71. SCN neurons are oscillators [S13]
  72. Cycling rhythm is in every cell
  73. Individual SCN neurons:
  74. Take SCN and you disassociate every cell, look at them individual and look at firing rates
  75. In daytime, the cells fire more 5-10 Hz. At night, drops close to 0 Hz. Here we are looking at firing rate of the cells.
  76. Top figure: Within 24 hour period, there is high and low high and low, it is reasonably regular. Each cell (not talking to each other) all have a rhythm and are not synchronized and are out of phase with each other. All of the cells would cancel each other out and would not be a good clock.
  77. Coupled to generate a uniform rhythm of electrical firing
  78. When they are put together in nucleus they have gap junctions between them and GABA, which regulates synchronicity.
  79. Few thousand cells follow this nice rhythmic behavior- that is the basis of the output of the clock.
  80. What drives the rhythmic firing? [S14]
  81. What drives the rhythmic firing? Control gene transcription and protein synthesis.
  82. He will not ask for the names of the genes involved, but some are really obvious. For instance, clock is really obvious. A lot have the three letter names ie PER for period. Just know that there are cycling periods of high gene transcription.
  83. Bottom figure: When we measure the mRNA and how much has been transcribed of a particular gene, there is a lot transcribed during day and not much at night.
  84. So this is the fundamental control at this level.
  85. Right graph: There is a feedback loop, these go up and down and what you can see is that the proteins that are synthesized from these messages lag behind a little bit. So as the message goes up and it takes a while for protein to be synthesized, and the protein goes down after the message goes down.
  86. How do we link the end of one cycle to the beginning of one cycle? Because that gives you fluctuating rhythm. How we do that is by using the product to feedback and turn something off. There are more complicated loops but we just need to understand the basic one.
  87. Activation-repression loops [S15]
  88. You have this gene that needs to be transcribed, transcribe it, make protein, the protein interacts with other proteins, feedbacks and inhibits/turns off transcription, and now we need something to turn it back on again. Luckily the proteins, they have a limited life span and as soon as they are degraded again, they start transcribing, and as long as it happen in the 24 hour cycle, you are okay and it should keep going.
  89. Animation [S16]
  90. HHMI animation. Understand this is the basic drive of feedback loop.
  91. The BMAL and clock that are positive activators of set of 5 all 3 PER genes and cryptochrome genes are under regulation of clock based on genetic experiments.
  92. These are activators of all of the proteins and to simply the animation, it is reduced to PER and cryptochrome. Once the PER and cryptochrome genes are turned on, RMA accumulates and proteins are made. In this case, different dimers (PER dimers and cryptochrome dimers) can form different combinations with various PER and cryptochrome proteins. They then translocate into the nucleus where they interact directly with clock and DMAL to turn off the PER and cryptochrome gene.
  93. As time progresses, these negative factors turn over and are degraded and then the inhibition is relieved and activation begins and the start of cycle begins.
  94. He considered this to be a little bit more complicated than what he had said, but the basic idea is there is some sort of negative feedback.
  95. Clock genes drive oscillations [S17]
  96. Now we know the output pattern the change in AP firing of the cells, which follows the cycling of the genes and the gene products. So how are they coupled?
  97. Once you start patterns of gene translations and make proteins, those proteins can self regulate and activate other genes and turn on other things. One thing they can do is turn on and off potassium channels, which control membrane potential and hence control firing frequency of the cells.
  98. More K channels, firing frequency goes down and less K channels, firing frequency goes up.
  99. Output is not the main regulator, the K channels is important.
  100. So if that is true that the clock gene controls firing pattern, so if we get rid of the clock gene it should disrupt the firing pattern. So If we knock out clock gene (hetero or homo), nice regular firing pattern is eliminated when you knockout clock gene, we need those genes to control firing output pattern.
  101. Electrical oscillation is only output [S18]
  102. If it really was the firing pattern that regulates cycling and if we stop the firing pattern, it should disrupt the cycling.
  103. We can do that by putting TTX on them, which blocks Na channels and blocks Na AP. Increase firing during day, decrease at night. Now we can’t see output of cells because no AP.
  104. If we wash TTX off after a few days, firing pattern comes back and it is still as predicted if we hadn’t knocked it out. That means something was cycling underneath and we just knocked out the output. The gene cycling is still going on here even though we put TTX on and when we wash TTX on there, we can see that it is still there.
  105. BK channels….. [S219]
  106. Don’t worry about the details of this.
  107. Focus on this: the K channel, so normal animals without K channel. Normally, cells firing a lot during the day and not at night. If we knock out K channel, they don’t slow down at night. If there are no channels, then you can’t connect the clock to AP pattern.
  108. No output pattern, so animal can’t regulate its running on hamster wheel. Start as gene cycling, genes and proteins and that connects via changes in K channels, AP firing frequency tells other parts of the brain how to behave at different times of the day.
  109. Entrainment [S20]
  110. How is it reset? We don’t need to know anymore than what Dr. Gawne talked about- retinal ganglion cells project to optic chiasm to LGN
  111. But some find their way to SCN and they take light signal directly to SCN.
  112. Everywhere you read, people throw in jet lag. We need light and light will re-entrain your clock. We need light input to do this. Gulatamate and other NT can regulate it too.
  113. There are internal ways of regulating it. We probably all can alter our clocks a little bit in other mechanisms but light is the big one.
  114. Other mechanisms
  115. Melatonin - nighttime (pineal gland). Comes up because people take it if they have sleep pattern problems.
  116. Made in pineal gland and goes up at nighttime. May play a role in regulating the clock.
  117. Actually not so worked out in humans.
  118. If you have a pineal problem, you are still able to have sleep wake cycle. Pineal problems are more associated with sexual development and other things. We haven’t quite figured out the role of melatonin.
  119. SCN output mechanisms…. [S21]
  120. We understand how clock works and controls various behaviors and we always focus on sleep or arousal wake cycles.
  121. At different times of the day we turn things on and off.
  122. Examples….
  123. Temperature regulation
  124. Autonomic function
  125. Arousal – sleep
  126. Sleep characteristics [S22]
  127. Key thing about sleep is that it is an active state, different altered state of the brain. It is not everything turning off. Different things happen during sleep.
  128. More than one phase of sleep. We already know about some i.e REM sleep.
  129. It is associated with these things:Behavioral criteria
  130. Reduced motor activity and output
  131. We lie down and are more relaxed during sleep. So it is a good idea to lie down because you aren’t able to support yourself.
  132. Reduced sensory input- one of the controlling aspects of anatomy and sleep is thalamus, which is the gateway of sensory to get to the cortex, all except for olfaction. These conscious loops also control a level of arousal and awareness. So if we mess with thalamus, we will disrupt our state of consciousness.
  133. Altered state and alteration with decreased things: decreased motor output and decreased sensory input, and this tells you that you have done something to the loops: cortical to motor and back to sensory and these loops involve the thalamus.
  134. In book (only 2 pages so if you have the book it’s not bad) it tells you main players: thalamaocortical activity and brain stem.
  135. A case about a boy, who couldn’t sleep, could only sleep 2 hours a night.
  136. It is not good for you, since sleep is an active process it does something for you.