Some non-mammalian circadian organisation: Silk moths
- Like drosophila, eclose at a particular part of the day/night cycle
- Persists in DD therefore clock controlled
- Where is the clock? Truman removed brain at pupal stage led to eclosed at random times.
- May mean brain was clock or you have destroyed the clock's ability to communicate and perform behaviour
- Truman (1970): transplant brain between two species that had different eclosure rhythms.
- Resulted in exchanging rhythms.
- This confirms clock is in the brain bc if it was just the mechanism, swapping would not change timing.
- Activity rhythms persist in DD (wheel running)
- Where are the pacemakers?
- The eyes? When removed eyes, they free run therefore clock not in the eyes
- The brain? remove optic lobes led to arhythmic therefore likely that the clock is in the brain
- Page (1982) transplant: noticed that only 1 optic lobe (left/right) needed for rhythmicity
- Two optic lobes, two clocks
- Put two cockroaches on different light cycles from birth 11:11LD or 13:13LD
- Period differences of 1h can be seen in DD months after entrainment to these cycles
- This is called after-effects
Circadian organisation: avian systems
- 1. Michael Menaker finds that a brain site is responsible for entrainment of the sparrow clock.
- 2. Sue Gaston finds that the pineal gland is important for expressing overt circadian rhythms.
- 3. Natille Zimmerman transfers the phase of a rhythm along with a pineal graft.
- 4. Joe Takahashi finds that the SCN is required for expressing overt circadian rhythms.
1. 1967 Michael Menaker finds that a brain site is responsible for entrainment of the sparrow clock.
- Got blind sparrows to entrain to light cycles
- Blind sparrows free ran
- Removal of feathers from the head lead to ability to entrain again
- Injecting ink under the scalp blocked entrainment
- Discovery of deep brain photoreceptor/ extra-ocular photoreception
1968 Sue Gaston finds that the pineal gland is important for expressing overt circadian rhythms.
- Pineal gland produces melatonin
- Taking away pineal gland led to arhythmic sparrow behaviour which suggests it may be clock
- But then giving strong light, was able to have cycle again
- Placed in dark, arhythmic again
- Masking: this behaviour may be called masking- not circadian but instead a response to external environmental cues
- BUT because it gradually goes arhythmic, suggests something else is going on too
3. 1969 Natille Zimmerman transfers the phase of a rhythm along with a pineal graft.
- Translated pineal gland resulted in the PHASE of the donor tissue being expressed in the host.
- Showed that is was the clock itself
4. 1982 Joe Takahashi finds that the SCN is required for expressing overt circadian rhythms.
- Electrical ablation of the sparrow suprachiasmatic nucleus (SCN) results in loss of free running rhythms
- Both the SCN and pineal play a role in the expression of avian circadian rhythms in DD.
Circadian organisation Avian systems
- Light input in through eyes/head to SCN
- SCN transmits its time of day information to pineal gland
- Pineal gland releases melatonin to tell SCN what time day it is
The Suprachiasmatic Nucleus (SCN)
How do we know it's the clock?
- The mammalian biological clock
- 1. Location: in an ideal position to get light information, so can be synchronized to the 24 hour light-dark cycle.
- Direct projection from the retina (retinohypothalamic tract: RHT).
- 2. Ablation: removing the clock eliminates rhythms.
- 3. Isolation: SCN rhythms persist after removing inputs in vitro
- 4. Explantation: SCN remains rhythmic in vitro
- 5. Transplantation: putting an SCN into an arrhythmic animal brings back rhythms.
- in an ideal position to get light information, so can be synchronized to the 24 hour light-dark cycle.
- Direct projection from the retina (retinohypothalamic tract RHT).
- Right at the optic chiasm where light information crosses
- Light info goes to both SCN
- Removing clock eliminates rhythm
- Stephan & Zucker 1972: SCN lesions eliminate activity and drinking rhythms.
- Moore 1972: SCN lesions eliminate corticosterone rhythms
- Inouye & Kawamura (1979): SCN 'islands' remain rhythmic- shows that does not require input to show rhythm
- 4. Explanation: Green & Gillette 1982: Took out SCN. Remains electrically active in vitro, 24h rhythm
- Ralph & Menaker 1988: found mutant hamsters that had 22hr clocks and when bred, showed 20h, 22h, 24h. Showed that the clock could be genetically passed on.
- 5. Transplantation: Martin Ralph 1990: transplanted mutant SCNs. Showed that transplantation produces donor rhythms in the host
The SCN has a heterogeneous organisation
- Different jobs inside the SCN
- Light info enters core first then exchanges info with shell
- Retinohypothalamic tract (RHT) releases glutamate and PACAP into the SCN
- SCN also receives serotonin from DRN & MRN (brain stem)
- IGL: intergeniculate leaflet also inputs to SCN through GHT (geniculohypothalamic tract) NPY and GABA
Behavioural rhythms are not controlled by direct neural connection
- Experiment which encapsulated SCN in polymer, which allowed signals to diffuse through small pores did not allow for neural connections.
- Something diffuses out of SCN that drives time not neural connection
Possible diffusible signals from the SCN that inhibit locomotion
Some signals from SCN may inhibit locomotion, which could be related to time
- Epidermnal growth factor (EGF) receptor signalling: anything that binds to this seems to inhibit activity rhythms
- They injected a drug to see its effect
Prokineticin 2 signalling: when injected during the night, no activity
Light input to the clock
Entirely through eyes
Non visual photoreception-
What is evidence for a novel photoreceptor system?
- The type of light matters in the circadian system.
- Looked at how different types (wavelengths) of light suppresses melatonin at night.
- Found that peak melatonin suppression peak at around 460nm (blue)
- However visually, cone and rod photoreceptors are more sensitive to green and yellow light
- Researchers from that 0.3% of all ganglion cells have this opsin called melanopsin evenly spread throughout the retina.
- Melanopsin in some retinal ganglion cells- these cells have direct input into certain areas of the brain eg. SCN, subcortical areas.
- These are called:
- Intrinsically-photosensitive retinal ganglion cells (ipRGCs) that contain melanopsin have a dense projection to the circadian clock (SCN)
These cells have direct input to a lot of areas in the brain- clock, leaflet, hebbinula
- Hebbinula: more active and larger in people who are depressed. Seen as a pessimism region, when activated changes your expectation of success and failure.
- Light decrease activation of hebbinula
- SSRIs increase responsiveness to light.
- Therefore maybe increased light responsiveness from SSRIs leads to decreased activation of hebbinula, this process may be through ipRGCs cells that transmit info to hebbinula.
It's not all about the melanopsin
- Photic circadian entrainment is abolished in mice without rods, cones, and melanopsin.
- All these are important because rods and cones still indirectly feed to clock
Effect of melanopsin in polymorphism
- Graph shows pupil dilation in dark, then red then dark then blue then dark
- Pupil does not go back to full dark size
- This is because once melanopsin is turned on (through blue light), it is not easy to turn off.
- Therefore level of melanopsin in a person can be measured through this test.
- Blue lines are ones with less melanopsin.
effects from blue light emitted from phone
Clock genetics early milestones
- Bunning (1935). Drosophila raised in constant light were arrhythmic for 30 generations. Synchronized rhythms resumed immediately after return to constant dark.
- Pittendrigh (1960). Different species have different free running periods
- Feldman & Hoyle (1973) Frequency (frq) mutants discovered in the bread mold, Neurospora crassa
- Konopka & Benzer (1979) First bonafide clock mutants induced by x-ray mutagenesis in Drosophila
An autoregulatory feedback loop creates a 24h rhythms within cells
- A negative feedback loop
- This is how 24hr rhythms are regulated
- Positive elements turn on expression: clock & BMAL
- Negative elements turn off expression: period & chryptochrome
Phase shifting at the clock gene level
- Through the intracellular transduction pathways, the photic signal eventually causes the increase in per (period) transcription
- If per is about to become active spontaneously, then the effect of light will be to initiate this step earlier = phase advance
- If per activity is being inhibited, then the effect of light will be to delay the switching off of the gene = phase delay
Rhythmic clock gene expression can be seen in most tissues
We are made of clocks with different inputs, outputs and physiological consequences