Mid Term 2:
Circadian rhythm models:
Organismal biology Clock biology
• behavior/physiology (mammals) • behavior/anatomy (non-mammalian
• seasonality (birds, mammals,
plants) • genetics (insects, mammals, plants,
• migration/orientation (birds,
crustaceans) • cell biology (molluscs, protists)
• reproduction (mammals, insects) • entrainment mechanisms
(mammals, birds, fungi)
• health, emotionality, learning,
performance (human beings, animal
Aplysia californiana & Bulla gouldiana
diurnal, eye removal changes daily behavior
frequency of compound action potentials (CAPs) in the optic nerve is rhythmic - low at night,
and rises rapidly at dawn
CAP frequency remains rhythmic in constant dark (circadian)
CAP rhythm can be reset by light pulses in the subjective night
Remove the eyes and Bulla and Aplysia are arrhythmic behaviorally in constant conditions.
Recordings of electrical activity from the eye are made using suction electrodes attached to the
cut end of the optic nerve in vitro.
The isolated eyes will survive in an artificial seawater bath for up to 10 days.
Compound action potentials are produced by basal retinal neurons.
Illumination of only BRNs in Bulla produces only CAPs on the optic nerve. Illumination of only the visual retina produces only small, asynchronous activity in the
The asynchronous activity is the discharge of the visual retinal ganglion cells.
Reduced eye still produces
Circadian photoreceptor localization
Current injection causes phase shift
Changes in membrane potential cause phase shifts
Low calcium blocks light-induced phase shifts
Low calcium lengthens period
Bulla Gouldiana Circadian System CAPs are produced by about 100 basal retinal neurons (BRNs) in each eye. The cells are
electrically coupled, so discharge is synchronous.
The rhythm in CAP frequency is driven by a rhythm in membrane potential (blue line) in the
BRNs. Hyperpolarized at night = no CAPs; Depolarized in the day = CAPs
Depolarization can be accomplished by light, injection of depolarizing current, or by switching to
a high potassium bathing medium.
Light induces phase shifts at night
Light depolarizes the membrane
Depolarization at night causes light-like phase shifts
Hyperpolarization can be accomplished with current injection or switching to a high sodium
Hyperpolarization plus light blocks light-induced phase shifts
Hyperpolarization causes phase shifts during the day
Lowering extracellular calcium causes phase shifts in the day and blocks light-induced phase
shifts at night.
The membrane oscillation in Bulla
1. Starting with a hyperpolarized membrane at night
2. Near dawn, a potassium channel closes
3. This causes the membrane to start to depolarize
4. Depolarization results in calcium entering the cell during the day
5. As calcium builds up inside the cell, it turns on a pump that begins to remove calcium.
6. Calcium also acts to open other potassium channels which helps to make the cell more
hyperpolarized. 7. A light-induced calcium flux early in the night will delay the removal of calcium (phase
8. A light-induced calcium flux later in the night will start the build up of calcium earlier
Note that it is possible to design an oscillating cycle using only the membrane mechanisms.
However, additional evidence suggests the existence of a nucleus driven oscillator.
Different organisms have been exploited in studies of circadian rhythms for various reasons.
The snails are useful because their cells tend to be large and identifiable in the nervous system.
They can be found in the same position in each animal, and they are large enough that
electrophysiological experiments can be performed. This means that the clock can be studied
effectively at the cellular level.
However, snails are not the best animals for studies of behavioral rhythms nor for genetics. For
these, the insect have been useful. Also, many insects are models of endocrine function and
environmental control of development.
• Like drosophila, eclose at a particular part of the day/night cycle: persists in DD
\ clock controlled
• Question: where are the pacemakers?
• The brain
• Truman: removed brain at pupal stageÞeclosed at random times.
• The silkmoths have economic as well as scientific value. The clock or the hands of the clock
are located in the brain as indicated by brain lesion studies.
• Truman: transplants between two species (Antheraea pernyi & Hyalophora cecropia).
• Eclosion “gated” for different times of day:
o pernyi : late in the day o cecropia : early in the day
• Donor brain determines eclosion timing of the host
o (remember Ralph and the tau hamster SCNs??)
o Conclusion: clock is in the brain of the silkmoth.
To distinguish between the hands of the clock and the clock itself, transplant studies were
performed. A feature of the overt rhythm that distinguished the donor from the host was
transferred along with the brain transplant.
(A–E) Double-plot activity histogram (A; day 145–156) andχ 2-periodogram analysis (B; day
150–154; Sokolove significance line=SSL=χ2 for P=0.01) of another optic lobe-less cockroach
(animal ID 13/21; Table 2) shows circadian wheel-running activity (τ=20.8 h) in constant
darkness 148 days after the transplantation. The solid line at day 153 indicates computer failure.
(C) The rhythm scan periodogram plot (Qp/χ2) over the complete length of the wheel-running
recording (day 1–167) detects rhythmic peaks in consecutive 10-day-χ2-periodograms
(rhythmicity) before removal of the remaining optic lobe (day 19–39) and after the
transplantation (day 143–151). s.p.l. = single periodogram length. Additionally, rhythmic activity can be seen in the two activity plots, which show the averaged locomotor activity ± S.D. of the
animal during the course of a circadian day before (D; day 27–39) and after (E; day 150–154)
Activity rhythms persist in DD (wheel running)
Where are the pacemakers?
Remove eyes: free run
Clock not in the eyes
Remove optic lobes: arrhythmic
Likely that the clock is in the brain
Cockroaches are great for behavioral studies. Removal of the optic lobes results in arrhythmicity
in DD. Therefore either the clock or its hands are in the OL.
Terry Page: transplant study
long noticed that only one optic lobe (left/right) needed for rhythmicity.
– Two optic lobes, two clocks
Put two cockroaches on different light cycles: DD & LL (Aschoff’s rules)
Cockroaches in DD had short period
Cockroaches in LL had long period
Take one optic lobe out of LL animal and put it in the DD animal
Displayed two rhythms: long and short
• Each optic lobe receives input from an eye • Transect connection between optic lobes and light pulse one eye
One optic lobe phase shifted
will see two rhythms
Conclusion: the clock/pacemakers are in the optic lobes
• A working period gene is required for overt expression of rhythmicity
• “Genetic mosaics can be produced that restrict this expression to specific tissues and cell
• The period gene may be switched off in all tissues except the dorsal lateral neurons in the
brain, and rhythms will still be produced.
Conclusion: the clock/pacemakers are in the dorsal lateral neurons.
Bulla & Aplysia
rhythms at the cellular and biochemical levels
specialized oscillator cells that are photosensitive
rhythm in membrane potential drives output
changes in membrane potential critical for entrainment
calcium influx regulated by membrane potential is the