Lecture 4 Flashcards
What is the circadian clock?
- Endogenous molecular pacemaker
- Drives daily rhythms that affect physiology, biochemistry, behaviour and other functions
- Expression of large parts of transcriptome and proteome shows daily oscillations
- Oscillations are translated to physiological and behavioural rhythms
- Small subset of evolutionary conserved genes drive oscillations and make core.
Evolutionary conservation of molecular clock
- First ‘clock’ mutants were isolated in Drosophila
- Later clock mutants were isolated in organisms like mice
- Mutants indicated genetic clock mechanism involved a network of interacting proteins
- Single cells hold complete network forming cell-autonomous clocks
- Central dogma - conserved mechanism of circadian clock is negative feedback loop
Locomotor activity in studying the circadian clock in Drosophila
Free run - Biological rhythm exists without any external input or cues
Entrainment - Biological rhythm synchronised to an external (light/dark cycle) oscillation.
Circadian clock can affect sleep cycles.
Experiments to test locomotor activity
- Fly placed in glass tube and IR beam is shone across the tube. Registers movement when fly crosses the beam and measured how many times every minute.
- Activity levels of animals in 30 minute intervals throughout day using zeitgeber time (h) (12h light, 12h dark).
Drosophila clock mutants
- Mutant Drosophila screens proved single gene mutations can affect circadian rhythms.
- Period was the first gene identified - per0 mutant alleles produced arrhythmicity, perL mutants produced long period rhythms (28hrs), and perS produced short period rhythms (19hrs)
- Per homologues have been identified in humans and mice
- Studies of genetically mosaic and transgenic Drosophila indicated circadian behavioural locomotor rhythms are controlled by 20-30 neurons.
What 4 properties define circadian rhythms?
- Oscillations persist, or free run, under constant conditions, indicating presence of self-sustaining clock.
- Clock-driven events recur approximately , but not precisely, every 24 hours.
- Rhythms entrained by sun-driven changes in light and temperature.
- Period of clock is remarkably stable over wide temperature range (temperature compensation).
Three parts of Circadian Rhythms
Organised into three parts:
Core clock - keeps time
Input pathways - synchronize clock to environment
Output pathways - Transmit information to temporarily organize behaviour and physiology
Negative feedback loop in Drosophila
Clock/cycle protein complex binds E-box elements in promoters of the period and timeless genes to allow for expression, forming mRNA and eventually proteins
Doubletime, Casein Kinase 2, and shaggy + PPA2 and PP1 modify PER and TIM proteins
PER and TIM dimerize and move to nucleus where CLK/CYC activity is repressed, inhibiting their own expression
PER and TIM bind E3 ubiquitin ligase SLIMB, leading to proteolysis.
How long does the negative feedback loop take in Drosophila
24 hours
Light in circadian feedback loop
Tim degraded by light
Triggered by light sensitive pathways that involve Cryptochrome (activated by light)
Without Tim, Per is unstable and degrades
Per-Tim complexes don’t accumulate and don’t enter nucleus
Allows for continued transcription of Tim and Per as Clk-Cyc not inhibited
How does light regulate adaptation of clock to environment
- Sun does not rise and set at same time each day of the year
- Zeitgebers harmonize clock neurons to environment (light, temperature, social cues)
- Light (most important zeitgeber), reaches clock neurons via photoreceptive organs containing rhodopsins - eyes, ocelli, and HB-eyelets
The role of PDF neurons
- Output transmitter of clock
- Coordinates and synchronises oscillations of clock neurons when external signals absent (an internal Zeitgeber).
- Phases oscillations of clock neurons and activity peaks to right time of day under LD
- Light-input and arousal factor of clock
- Under natural conditions, PDF may help system to adapt to seasonal variations in the photoperiod
Behaviours/physiology regulated by circadian clock
Sleep
Learning/memory
Chemosensation
Feeding and metabolism
Courtship
Immunity
Sleep regulation
Circadian - preferred bedtime
Homeostatic - Changes in sleep quantity/quality after sleep loss, performance impairment after sleep loss
After waking up, the desire and need to sleep increases throughout the day
Circadian regulation of sleep
- Circadian genes affecting timing of sleep and possibly homeostatic regulation
- Flies bearing dominant-negative or null alleles of circadian transcriptional activators Clk or cyc exhibit less overall sleep than wild-types.
- Molecular/cellular basis of mutant effects unknown
- Prolonged sleep deprivation in rats kills them
- cyc mutants are hypersensitive to these lethal effects
What has CRY1 variation been associated with?
A familial form of delayed sleep phase disorder, providing genetic underpinnings for ‘night owls’.
How to measure sleep in flies
- Activity monitor using IR beam
- Sleep in flies is 5 minutes of no activity and brain activity can be measured using probes
- Neurophysiology measured used electode and foam ball
Circadian regulation of sleep
- Activation of ILNv by selective channel expression - disrupts night time sleep, whereas ablation increases sleep.
- GABAergic inputs inhibit arousal promoting function of neurons, source of inputs remains unknown.
Light activates long ventral lateral neurons, releasing PDF onto short ventral lateral neurons
s-vLNs project to other clock neurons and brain regions such as ellipsoid bodies.
Both l and s-vLNs express GABAA receptors, enabling sleep-promoting neurons to supress wakefulness
Sleep control centres
Kenyon cells comprise mushroom bodies, fan-shaped body, and ellipsoid body
Represent major sleep-regulating centres in brain
Regulated by dorsal paired medial neurons and dopaminergic PPL1 neurons
Wake promoting circadian pathways
DN1pneurons receive inputs from the s-LNvs and relay time-of-day information to wake-promoting classes of neuropeptide-expressing neurons expressingDrosophilainsulin-like peptide 2 (Dilp2), co-expressing diuretic hormone 44 and SIFamide (DH44/SIFa) in the pars intercerebralis, and expressing leukokinin in the lateral horn (LHLK).
Convergence of circadian pathways onto EB
Pathways linking the clock neuron network to a homeostatic sleep center.
DN1ps project to tubercular-bulbar neurons (TuBu) within the anterior optic tubercle, which project to the EB ring.
Wake-promoting LNs project to dopaminergic PPM3 neurons, which also innervate the EB ring
