Test 2 Review Flashcards
Aplysia californiana rhythmic behaviour
diurnal, moves from rock-home and returns late in the day, eye removal changes daily behaviour
Aplysia californiana compound action potentials
frequency of CAPs in the optic nerve is rhythmic - low at night, and rises rapidly at dawn, correlated with motion of organism
Aplysia californiana compound action potentials manipulations
remains rhythmic in constant dark, reset by light pulses in subjective night
Bulla gouldiana rhythmic behaviour
nocturnal, at the end of the night, no activity, near dawn, a strong peak of activity
Bulla gouldiana compound action potentials
frequency of CAPs in the optic nerve is rhythmic - low at night, and rises rapidly at dawn - Hyperpolarized at night = no CAPs; Depolarized in the day = CAPs
Bulla gouldiana compound action potentials manipulations
CAP frequency remains rhythmic in constant dark - CAP rhythm can be reset by light pulses in the subjective night
comparison between bulla and aplysia
if you shine light on Bulla, it’ll stop moving; if you shine light on Aplysia, it’ll start moving, almost identical in their circadian rhythm and living behaviour, The cells of Aplysia is distributed everywhere in retina, but in Bulla, the cells are
lined up in organized fashion, very convenient for research
inducing phase shift in bulla
low sodium pulse in one eye
bulla - basal retinal neurons
clock cells - can be taken out and still synchronize circadian
rhythm by themselves, could entrain to light/dark cycles, and oscillate in constant darkness with a consistent intrinsic period
light effects on bulla BRN’s
Light causes depolarization of the cell membrane of BRN, and depolarization opens up calcium channel, therefore an increase in firing frequency and depolarization of the BRNs
during the day, but the opposite at night
effects of BRN depolarization/hyperpolarization
Depolarization at night causes light-like phase shifts, hyper polarization causes phase shifts during the day, and hyperpolarization plus light blocks light-induced phase shifts
bulla optic nerve
light on optic nerve causes depolarization, low extracellular Na causes hyper polarization (delay), high extracellular K causes depolarization (advance), membrane part of clock system
effects of calcium on light pulses in bulla
block the effect of light by lowering calcium in sea water, can even get the reverse effect of the shift, since the Ca2+ concentration gradient gets reversed and the flow at calcium channel goes toward the opposite direction
effects of calcium on phase shift in bulla
Ca gets eyes excited, Ca also keeps K channels closed so can get lots of APs and depolarize cell in subjective day, taking calcium away chronically, you change the period completely, lowering extracellular calcium causes phase shifts in the day and blocks light-induced phase shifts at night
eye electrical potential in bulla
The two eyes’ electrical potential is coupled and the two eyes work together to create response to the brain
membrane oscillation summary in bulla
Starting with a hyperpolarized membrane at night, Near dawn, a potassium channel closes, membrane to starts to depolarize resulting in calcium entering the cell during the day. As calcium builds up inside the cell, it turns on a pump that begins to remove calcium and another pump that opens potassium channels further hyper polarizing the cell.
light induced calcium fluxes in bulla
A light-induced calcium flux early in the night will delay the removal of calcium (phase delay). A light-induced calcium flux later in the night will start the build up of calcium earlier (phase advance)
silk moth eclosion
eclose at a particular part of the day/night cycle: persists in DD ∴ clock controlled
Results of Trumans experiments on silk moths
transplanted brains between two different species, found that eclosion depends on donor brain
cockroach clock localization
Remove eyes: free run – Clock not in the eyes - Remove optic lobes: arrhythmic - Likely that the clock is in the brain. Low temperature pulse confirms two pacemakers, one in
each optic lobe. Pacemaker coupling takes time to re-entrain the contralateral pacemaker.
Terry Page transplant study results
noticed that only one optic lobe (left/right) needed for rhythmicity. Put two cockroaches on different light cycles: DD & LL (Aschoff’s rules)
- Cockroaches in DD had short period
- Cockroaches in LL had long period
Took one optic lobe out of LL animal and put it in the DD animal, displayed both rhythms
drosophila period gene localization and proof
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
drosophila period gene
required for overt expression of rhythmicity
moth clock localization
brain
cockroach clock localization
optic lobes
common approaches to discovering clock localization
transplantation, lesioning, transections
clock proteins
TIM, PER, CK1
clock genes
doubletime, tim (timeless), per (period), CLK, CYC, CRY, JET, Clock, TOC
effects of Ca removal in bulla
take Ca out, but put it back in before subjective
night, nothing happens. but if its out past start of
day, it causes delays in the subjective day
holistic eye rhythm
2 clocks operating: one generates holistic eye
rhythm — communication with outside world at
membrane, other clock that we can’t see
bunning - clock genetics
Drosophila raised in constant light were arrhythmic for 30 generations. Synchronized rhythms resumed immediately after return to constant dark.
pittendrigh - clock genetics
Different species have different freerunning periods.
konopka and benzer - clock genetics
First bonafide clock mutants induced by x-ray
mutagenesis in Drosophila - pers (short), perl (long), per0 (arrhythmic)
gene discovered in neurosporra crassa
Frequency (frq)
per gene
on X chromosome, so recessive mutations on autosomes not expressed, causes mRNA translation of the PER protein, which has (-)ve feedback on per gene transcription
Tim+Per
Tim and Per oscillate, produce dimeric complexes, dimer can re-enter the nucleus, acts as a negative transcription factor to shut down the production of tim and per mRNA
doubletime
novel Drosophila clock gene that regulates PER protein
accumulation, encodes a protein closely related to human casein kinase 1ε, mRNA oscillations precede the protein oscillations by about 6 hours
tim
protein is rapidly degraded by light, resetting of oscillation requires rapid change of protein TIM (degradation), no Tim in subjective day — light has no effect on system
- at beginning of night when Tim and Per being produced, light pulses
degrade Tim (and Per), producing a delay in the oscillation
- Will advance system into next cycle if light pulse goes on during the
transcription, removing transcription and its product
physiology of light pulse induced phase delays
at beginning of night when Tim and Per being produced, light pulses degrade Tim (and Per), producing a delay in the oscillation, will advance system into next cycle if light pulse goes on during the transcription, removing transcription and its product
CLK and CYC
discovered in mouse nucleus, Centre of 2 main oscillations that deal with clock-controlled processes including Tim and Per; one oscillation is the rhythmic regulation of per and tim, the other is rhythmic regulation of CLK and CYC
CRY
photoreceptor for non-vertebrate, conserved
throughout all phylogeny, interacts with light and then tim,
JET
when activated, interacts with light and degrades tim resulting in phase shift (non-vertebrates)
molecular mechanisms for locating the clock
Immunocytochemsistry, In-situ hybridization, Manipulate expression of genes
Dorsal and ventral lateral neurons
optic lobe genes important for rhythm generation, dorsal genes control beginning of day, ventral genes control end of day
recent milestones of clock genetics
Discovery of the tau mutation in hamsters, Cloning of the first mammalian clock gene - clock, mutation at clock produces a long circadian period (29 hr) and arrhythmicity in the homozygote, Identification and cloning of the tau gene in hamsters
principle of synteny
used to scan human genome for clock genes
casein kinases
Ck1,2 - involved in control of cell morphology
Ck1ε – responsible for the short period tau mutation in hamsters, and doubletime in drosophila
ASPS in humans
Loss of Ck1ε phosphorylation site on mPer2
ASPS in transgenic mice
Mutation on ck1δ
clock controlled genes
ROR (+) and REV-ERB (-) feedback on BMAL1
BMAL1
drives production of other genes like per and cry
mammalian clock loop 1
(-)ve feedback path where the PER and CRY proteins suppress the transcription of the per and cry genes
mammalian clock loop 2
(+)ve regulator of per and cry transcription, but is also (-)ve feedback through rev-erba and bmal1
CK1δ and CK1ε
regulate per and cry buildup
Dexras1
appears to regulate the relative impact of GLU/PACAP and NPY input to the clock genes.
mammalian clock gene effects
remove clock, rhythmicity continues bc BMAL1 acts with it, mutant clock causes period change, remove BMAL1, no rhythmicity, remove CK1e, its role can be taken over by CK1d, remove per1: period lengthening, remove per2: period shortening
GABA
used for communication between cells of the SCN
non-mammalian physiological responses to light
light -> melanopsin > other light responses AND/OR light > photolyase/cryptochrome > DNA repair and circadian entrainment
mammalian physiological responses to light
light -> melanopsin > other light responses AND/OR light > glutamate > cryptochrome > DNA regulation and circadian entrainment
Cryptochrome and photolyase
used by bacteria to bring energy into circadian system, sensitive to UV and blue light; photolyase targets DNA, crytopchrome targets other proteins
cyanobacteria clocks
grow in 24 hr rhythm in constant light, divide every 8-10 hours
cyanobacteria clock mechanism
Ordered KaiC autokinase and autophosphatase activities drive the circadian oscillator
- KaiC autophosphorylates and dephosphorylates
- KaiA promotes phosphorylation
- KaiB inhibits KaiA
light-dependent cellular metabolism synchronizes the clock with local time
KaiC mutation
allows cells to maintain near 24-h time but prevents the ability to synchronize that timing with the solar day
acetabularia clock
nucleus determines phase but nucleus not required for rhythmicity, clock revolves around TOC gene - activates morning and inhibits night genes
seasonality determined by
acute observation, circannual clocks, photoperiodic time measurement
acute observation
plant blooming patterns (daffodil in spring), animals that hibernate (arctic ground squirrel)
arctic ground squirrel rhythmic behaviour
hibernates for 8 months, knows where food and neighbours are when wake up, goes through a number of arousals during hibernation, at one point they start to get cold and stop reacting to light, all emerge together (males emerge before females)
circannual clocks
annual rhythmicity - bird migration patterns, marmots weight fluctuation, human conception rates
photoperiodic time measurement
measures activity and length of day with respect to season - u shaped graph for chrysanthemum flowering (optimal 8-9 hours light/day), hyoscyanus flowers as days lengthen; butterfly diapause: metamorphosis interrupted when day lengthened for short cycle species
syrian hamsters seasonality
summer/winter physiology determined by light, testes increase in size in spring time - manipulating DL cycles can change physiology in lab
night break/resonance experiments results
Manipulate length of night - consistently putting light in the middle of subjective night that will cause phase shifts or adding more dark to night to change the zeitgeber cycle
T cycle
8 hr light, 16 hr dark for 24 hr cycle; add 12 more hrs dark for each 24 hr increment increase in cycle (8hr light, 28 hr dark for T cycle 48hrs); cycles that are a multiple of 24 have the light occurring always at the same circadian time
T cycles between 36-60 hours
have light pulses occurring in the middle of every other subjective night
External vs. Internal Coincidence in flowering plants
External coincidence: when light coincides with subjective night you get a photoperiodic response, could trigger a light sensing pathway to alter gene expression during the night
Internal coincidence: Phase relationship btw different processes changes at night, and its that coincidence that produces the photoperiodic response
roles of melatonin
transducing mechanism for photoperiodic time measurement, pinealectomy eliminates circannual rhythmicity in rodents and birds, melatonin affects rhythm generation by the SCN, melatonin synchronizes rhythms in birds and pups
in vivo Soay sheep model
circannual regulation of prolactin secretion derived from a pituitary-based timing mechanism
circannual rhythm generation
product of the interaction between melatonin-regulated timer cells and adjacent prolactin-secreting cells, which together function as an intrapituitary “pacemaker-slave” timer system
