sleep and circadian rhythms

What is sleep?

• State of reduced physical activity and responsiveness 

• An altered form of consciousness 

• Homeostatic process associated with anabolism  

• Daily rhythm  

• Highly conserved across species 

What is not sleep

• Dreaming —> occurs during sleep in only a subset of animal species  

• Circadian rhythm —> they regulate processes such as sleep, but sleep itself is not a rhythm and not a clock

Recording sleep

• Can be recorded with EEGs non-invasively 

• Records the brains electrical activity  

• The different electrodes placed on the scalp at standardised positions generate a pattern which represents the synchronous activity of many neurons  

• The more neurons that fire synchronously, the clearer and more defied the wave pattern will be 

EEG and sleep phases

• Sleep is made up of a cycle which is made of different phases 

• Lasts less than 2 hours so most people have several sleep cycles a night  

• Starts with awake phase, with beta waves, then progressing through several non REM phases progressing from theta to delta waves 

REM sleep

• Another sleep phase but is well studied and so has its own category  

• Eyes move rapidly under the eyelids during REM 

• Most of dreaming occurs, specifically lucid dreams 

• Minority of sleep phase and conserved through different ages  

• Known disturbances which are characterised by effects on the REM phase:  

  • Muscular atonia – muscles not responding to thoughts  

• Sleep paralysis is when people wake up during the REM phase but muscular atonis is still engaged 

• People prone to have hallucinations during sleep paralysis 

Why do we need sleep?

• The fact that its conserved suggests that there must be a reason for sleeping  

• Sleep disruption has many consequences:  

• Inattention  

• Impaired cognition  

• Reduced learning capacity 

• What happens when we sleep – common hypotheses 

• Clearance of metabolites in the brain – specifically deposits of amyloid beta, linking insomnia as a risk factor for dementia 

• During sleep the brin prunes unnecessary synapses 

• Sleep homeostasis: argument that sleep restores the baseline that becomes elevated during wakefulness. Synaptic strengthening during wake leads to cellular costs and systemic consequences. Evidenced by several empirical observations: changes in brain wave patterns during NREM sleep and reduced evoked potentials. But this is not a universal mechanism that holds true for all species and does not account for the existence of REM sleep and so is limited. 

• Hypothesized to be a consequence of a complex nervous system 

What time does our body want to go to sleep?

• Variation between individuals, most people find that halfway through their sleep will fall between the hours of 2 and 4am.  

• During teenage years, tend to go to bed later and when you get older tend to go to bed earlier 

• Not only does sleep time, but also temperature fluctuates during sleep – starts dropping when our body anticipates to go to sleep 

 

Do we have a clock inside our body?

• Wanted to challenge the hypothesis that there is a circadian clock in the late 1930s  

• Believed that the sleep wake pattern was dependent on light input 

• thought that if they locked themselves inside a cave where sunlight could not reach, they could live a longer day 

• Even without light exposure, external time cues, their bodies showed a consistent 24 hour rhythm 

What is a circadian rhythm?

• Circadian refers to a rhythm that lasts approx. 24 horus  

• Other rhythms: Infradian rhythms last for longer than 24hours, ultradian rhthms means shorter than a day 

• Circadian rhythms are autonomous and continue in the absence of external cues (planetary cycles) 

• Continue even in the presence of free running conditions (dark-dark)  

• However, they can be entrained by exogenous cues called zeitgebers and these include:  

  • Light  

  • Temperature  

  • Humidity  

  • Sound (in some conditions)  

• When external cues are present, we refer to zeitgeber time (ZT) and we describe their internal phase using circadian time (CT)  

Independent evolution across all life forms

• Most organisms have circadian rhythms, and can even be found in plants 

• Being able to anticipate predictable environmental changes provides a strong evolutionary advantage 

  • Example: if bacteria undergoes mitosis during the dark phase, it reduces the chance of UV exposure mutations 

• Origins of rhythm different but are all a molecular clock – conserved between mammals and fruit flies but starts to vary in organisms like bacteria 

How can we measure circadian rhythms?

• In humans: 

  • Trackers of sctivity with cameras 

  • Temperature 

• In mice and flies:  

  • Activity and rest cycles  

  • Can be measured with running wheels in mice and tubes measuring activity with IR sesnors in flies 

• This information is compilated into an actogram and can be analysed precisely with statistics 

 

• Can also analyse the expression of proteins throughout the day with  

  • GFP tagging  

  • Westerm blot  

  • qPCR  

• but the issues associated with this is that you ned to perform an extraction at each time point which can be difficult and expensive 

Drosophila period

• first component of molecular clock  

• when dawn has just passed it is the safest time for flies to hatch – provides a way to measure the circadian rhythms 

• Mutants which had a mutation in a gene called period, has no eclosion (hatching) rhythm à this mutation now known as per01  

• Other mutants identified such as pers which has a shorter subjective day of around 19 hours and is caused by the S589N substitution  

• The third mutant (perl) had longer subjective days with a circadian rhythm of roughly 28 hours, aused by a V243D mutation 

Mammalian Pers

• Flies have only one period, but we have 3  

• The endogenous period of a mouse is slightly shorter than 24 hours, but when per 1 or 2 is mutated, this becomes arrhythmic 

• However, for period 3, when mutated, can still be rhythmic 

• Period 1 and 2 important for the circadian clock, while period 3 contributes to the peripheral clocks  

Period expression oscillates

• Period cycles during the day confirmed with qPCR or mRNA extracts (protein rhythms follow the mRNA pattern) 

• Pattern related to the circadian time: 

  • During the light, period accumulates  

• During the night period peaks and then drops 

• How can a protein like this oscillate? Due to the control of cryptochrome  

Cryptochrome

• Behaves exactly like period 

• Blue light sensitive protein in drosophila and plants, but CRY1 and CRY2 (mammalian cryptochrome) is insensitive to blue light 

• Nonetheless cycle with almost identical rhythms to period  

• Binds to period forming a heterodimer  

CRYs required for rhythmicity

• Partially overlapping role 

• Animals which lack CRY1 or 2 still have rhythms, although these may be shifted. For complete arrythmia, both CRY proteins must be lost. 

• This shows us that period can bind to both cryptochrome proteins and is likely that when one is lost, period will bind to the other 

• Specific neurons have specification of CRY1 or 2 and will determine the phenotype of the mutatnts —> different responsibility of the CRY proteins  

Role of the complex

• 2 different mutants discovered: CLOCK and BMAL1 which resulted in arrhythmia in absence of external conditions 

• These two proteins found to have basic helix loop helix motif which allows them to bind to a vary specific E box DNA sequence  

• Proteins can also bind to each other  

• Clock and BMAL1 makes a dimer and recognises DNA sequences upstream of genes period, cryptochrome and other circadian controlled genes (up to 600) 

• Forms the basis of molecular clock and I a negative feedback transcriptional loop 

The molecular clock

• Activity of cock bmal1 heterodimer controls period and cryptochrome expression  

• This heterodimer of period and cry inhibits clock and bmal1 heterodimer, which downregulates their expression  

• Cry/per is eventually degraded, allowing clock/bmal1 to bind and increase their expression 

• Other genes which are controlled by clock and bmal1 expression finetune components of the molecular clock by:  

  • Finetuning the affinity of period and cry  

  • Change the post-translational modification which makes them more/less prone to binding 

  • Nuclear entry is an important gating system à circadian clocks can be induced in cells which don’t have one by inserting clock components into the nucleus of cells 

  • Modifying cryptochrome and period stability 

Per stability and degradation

• Period and cryptochrome subjected to multiple phosphorylation events, some of which promote degratdation and others enchance stability  

• Kinases which have been discovered:  

  • Casein kinase 2 

  • DBT – known in humans as casein kinase 1 epsilon (promotes degradation of period) 

  • Slimb  

Casein kinase 1 epsilon

• Mutated CK1e and have a much shorter lifetime of per proteins due to the tau mutation which makes the kinase more active