Wk 3: Neurobiology of circadian rhythms

• 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

• 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.

• 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

• 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

• Translated pineal gland resulted  in the PHASE of the donor tissue being expressed in the host.
• Showed that is was the clock itself
• 

• 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.

• 
• 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 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

• 
• 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

• 
• 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

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

Entirely through eyes

• 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.

• 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

• 
• 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

• 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

• 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
• 

• 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
• 

We are made of clocks with different inputs, outputs and physiological consequences