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156 Cards in this Set

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Are reproductive cycles dependent on the same clock as circadian rhythms?
If so, what entrains them?
Rhythms seen to be linked with menstrual cycle, as evidenced by:
Risk behavior (lowest at ovulation)
Food cravings
Rectal temperature
Mood
STILL NOT SURE ABOUT WHAT ENTRAINS THE REPRODICTIVE CYCLES
Differences between estrous and menstrual cycles.
ESTROUS = Mating behavior confined to late follicular phase {most mammals}
-Behavioral Estrus: Lordosis
-Daily LH pulsativity: Ready each day to begin estrus, but Not until coupled with proper hormonal profiles (E) will LH surge and result in estrus (ovulation)
-estrus cycle is determined by both circadian rhythms and physiological parameters.



MENSTRUAL = Mating behavior can occur throughout cycle {primates} & vaginal bleeding due to decrease in sex steroids
Do tidal and lunar rhythms exist?
NEED TO FIND OUT
How is LH rhythm set in a LD cycle?
By onset of activity and NOT photic cues, as described below:
Onset of activity (a circadian output) is best predictor of onset of behavioral estrus. In rats/hamsters: estrus 1-4 hr before activity begins
Even in LL when activity free-runs, estrus free-runs exactly _______
1-4 hrs prior to the start of activity. This would indicate that estrus consists of a one-day clock cycling four times, but do not rule out the possibility of a circaquadrian clock
In terms of GnRH and circadian signaling, Drugs don’t affect______, but do prevent _________.
Drugs don’t affect GnRH neurons capability of releasing, but do prevent circadian signal from reaching GnRH neurons.
Drugs don’t affect GnRH neurons capability of releasing, but do prevent circadian signal from reaching GnRH neurons.
What evidence do we have that this is true? (3 things)
Block LH surge on day of proestrus with drugs



Delay surge by 24 hours (not 96 hrs)

or

Reinstate LH surge by electrical stimulation of hypothalamus
Describe the nature of estrus's free running properties.
Estrus free-runs : Always with τ = 4 x τACTIVITY


Also, FSH and LH surges have fixed relationships to activity.
What does D20 do to the activity tau and estrus cycle tau?
D2O lengthens τ of both activity and estrus cycle
What does D20 do to the relationship of FSH and LH (surges) to the estrus cycle?
The relationship is maintained
What is tau?
the period of a circadian rhythmn
What does D2O in water do to the period of activity and to the period of lordosis?
D2O in water: increases period in both activity and period of lordosis onset
What do SCN lesions do to the activity rhythm and the estrous cycle?
SCN lesions disrupts both activity rhythm and estrous cycle
How might you alter estrous cycle length?
Pheromones from female conspecifics can alter estrous cycle length
How long is the Melatonin rhythm during estrous cycle of rat?
4 days
“Estrous cycles
do not occur in nature”

What reasons may support this statement?
need to find out
Discuss the relative importance of circadian rhytmnicity to estrous and menstrual cycles.
Circadian rhythms may not be as important to menstrual cycles as they are to estrous cycles


Menstrual cycles are unstable…not like clockwork
What evidence do we have that GnrH pulsivity may be regulated by a clock?
SCN lesions = no GnRH/LH/FSH pulse
Hypothalamic sections in vitro secrete GnRH in rhythmic pulses
Which of the following are thought to be zeitgebers for the menstrual cycle:
a)LD cycle
b)moon cycle
The moon is not a zeitgeb for menstrual cycle.
I DON'T THINK THAT THE LD CYCLE IS A ZEITGEBER EITHER, BUT I AM NOT SURE. HERE'S WHAT THE NOTES SAY:
Women given melatonin during light hours showed no change in LH pulsitivity…may increase pulse amplitude
Women given melatonin during light hours showed what change in LH pulsitivity?
Women given melatonin during light hours showed no change in LH pulsitivity…may increase pulse amplitude
Discuss Melatonin and Menstrual Cycle
Even in LL or DD, nocturnal decline in GnRH electric signals persists

Women given melatonin during light hours showed no change in LH pulsitivity…may increase pulse amplitude

GnRH pulsivity essential but…
Duration of menstrual cycle & timing of major events depends on status of ovary.
How is the menstrual cycle controlled?
GnRH pulsivity essential but…
Duration of menstrual cycle & timing of major events depends on status of ovary.
What period of time is represented by the term "infradian" ?
greater than 1 day
What are some of the exogenous influences on the menstrual cycle?
Stress: Intense Exercise, Fasting, shift work schedule
Season (certain monkeys)
Drugs
Pheromones
What is one of the principal things influencing cycle length in women?
Pheromones
Translocation from one tidal situation to another results in ....
Translocation from one tidal situation to another results in a rapid adoption of new tidal period
Normally tidal animals that happen to establish residence in nontidal habitat display only daily rhythms in lab. Can they dev tidal rhythms?
Normally tidal animals that happen to establish residence in nontidal habitat display only daily rhythms in lab, but they can adopt tidal rhythms!
Does changing LD cycle affect the tidal rhythms animals influence by both light and tides?
Constant conditions: bi-modal rhythm will free run with period greater than 24.8 hr

LD cycle: bi-modal rhythm with period exactly 24.8 hr







LL LD


If daily influence prevent from being circadian, then tidal influence prevented from being circatidal
Can tidal rhythms be entrained by LD cycles?
Expose penultimate crabs to 6.2 hr : 6.2 hr L:D cycle


No phase change of circatidal activity

TO ME, IT DOESN'T LOOK LIKE TIDAL RHYTMS CAN BE ENTRAINED BY LD CYCLES BUT THE NEXT PART OF THE NOTES STATES THE FOLLOWING:If both tidal and light cycles affect rhythm
What do D2O and EtOH do to the period of tidal rhythm & activity?
D2O and EtOH lengthen period of tidal rhythm & activity
If both tidal and light cycles affect rhythm…

these chemicals will alter rhythms identically. What does this indicate?
This is evidence for a single master clock
What aspects of tide produce entrainment?


Inundation?




Temperature Variation?
Inundation?

Expose arrhythmic crabs to 10 sessions of 12.4 cycle (6.2 hr in water : 6.2 hr in air)

Afterwards…activity rhythm not entrained.

Temperature Variation?

Expose crabs to same sessions EXCEPT now water was cold and air was warm.

Afterwards…activity peaks same as times of cold inundation. Entrainment!!







What aspects of tide produce entrainment?


Final experiment:

Forget inundation. 10 sessions of 12.4 hr cycle (6.2 hr in warm air: 6.2 hr in cold air)

Afterwards…ENTRAINMENT!!!

TEMPERATURE ENTRAINS TIDAL RHYTHMS
What aspects of tide produce entrainment?


Anything Else?

YES! Alternate intervals of high and low ambient atmospheric pressure resulted in entrainment!

Lastly, mechanical agitation can also entrain tidal rhythms in some cases
When do rhythms first appear developmentally?
Rhythms detected in the SCN during last 2-3 days of fetal life in hamsters and rats;
Metabolic rhythm as indicated by measuring uptake of radiolabeled 2DG

SCN becomes rhythmic before it receives retinal input
When do circadian rhythms first assume adult form?
NEED TO FIND OUT
Are central mechanisms regulating circadian rhythms in place before effector pathways?
NEED TO FIND OUT
Are rhythms innate or dependent on some sort of rhythmic input?
NEED TO FIND OUT
Does maternal input regulate the phase of their offspring’s rhythms, and if so, does such communication occur post- or prenatally?
NEED TO FIND OUT
Discuss the development of the SCN
Light induction of c-fos in hamsters’ SCN is first seen at postnatal day 4
Rhythms detected in the SCN during last 2-3 days of fetal life in hamsters and rats;
Metabolic rhythm as indicated by measuring uptake of radiolabeled 2DG
Conclusion: SCN becomes rhythmic before it receives retinal input
Is exposure to environmental cycles required for development of a circadian oscillator?
1. Experimental observations:
Evidence that period length of circadian clock is largely genetically determined

2.Deguchi (1978): Asked whether clock that controls pineal NAT rhythm is set independently of environmental light or only set after newborn is exposed to LD cycle:
Measured pineal NAT rhythm in adult rats in LD, DD, and blinded:
Established regulation by internal biological clock
Showed that NAT rhythm of population phase delayed by about 3h/week when animals are in DD (so tau must be close to 24.5h)
Placed pregnant rats in opposite LD cycles, then put mother and her pups into DD on day of birth---NAT rhythms of pups in phase with those of mothers in each case

Deguchi considered possibility that pups perceived LD cycle before birth (by light penetrating abdominal wall of mother)---to test this:
He put adult pair of rats in DD 10 days before mating and keep mother in DD during pregnancy and after birth of pups
Measured pups' NAT at 23 days of age
Phase of rhythm delayed several hours as compared to previous study---could be because mother was being delayed from time she went into DD and she entrains pups

He also used mothers from original LD cycles that were 180 degrees out-of-phase with each other
If pups were switched between the two types of mothers on day of birth, their pineal NAT rhythms were intermediate to the phases of unswitched pups---This an important observation in that it indicates both prenatal and postnatal influence of mother rats on circadian phase of pups' rhythms
By using blinded mothers, he showed that pups could be synchronized to LD cycles by 10 days after birth
Deguchi (1978): Asked whether clock that controls pineal NAT rhythm is set independently of environmental light (no clock) or only set after newborn is exposed to LD cycle (presence of light, so clock required). What did he find?
1. Established regulation by internal biological clock
Showed that NAT rhythm of population phase delayed by about 3h/week when animals are in DD (so tau must be close to 24.5h)
2. Deguchi considered possibility that pups perceived LD cycle before birth (by light penetrating abdominal wall of mother)---to test this:
He put adult pair of rats in DD 10 days before mating and keep mother in DD during pregnancy and after birth of pups
Measured pups' NAT at 23 days of age
Phase of rhythm delayed several hours as compared to previous study---could be because mother was being delayed from time she went into DD and she entrains pups
3. He also used mothers from original LD cycles that were 180 degrees out-of-phase with each other
If pups were switched between the two types of mothers on day of birth, their pineal NAT rhythms were intermediate to the phases of unswitched pups---This an important observation in that it indicates both prenatal and postnatal influence of mother rats on circadian phase of pups' rhythms
4.By using blinded mothers, he showed that pups could be synchronized to LD cycles by 10 days after birth
What indicates that there are both prenatal and postnatal influence of mother rats on circadian phase of pups' rhythms?
He also used mothers from original LD cycles that were 180 degrees out-of-phase with each other
If pups were switched between the two types of mothers on day of birth, their pineal NAT rhythms were intermediate to the phases of unswitched pups---This an important observation in that it indicates both prenatal and postnatal influence of mother rats on circadian phase of pups' rhythms
What did F.C.Davis discover during his work with hamsters?
Time of postnatal development of overt rhythm does not necessarily denote time of development of pacemaker's rhythmic activity

Pacemaker likely becomes rhythmic before overt rhythms can be detected
If litter of hamsters raised in dim LL (after mother pregnant also in dim LL), their activity rhythms are approximately coincident with each other and with mother's activity rhythm---

If pups were cross-fostered at birth to a new mother, the pups’ rhythm phase was closely related to the phase of their biological mother rather than their foster mother---
This suggests that the mother entrains the pups’ rhythms before they are born

Activity onset for pups on day of weaning was close to that of mother's, suggesting entrainment by mother at sometime before weaning

Unlikely that pups set mother's phase because her phase accounted for by initial entrainment to L:D and then free-runs through pregnancy and lactation

Pups of SCNx mothers did not exhibit synchronous rhythms (though each pup had its own rhythm)
Shows that SCN of mother required for phase setting (synchrony) of pups, but not required for development of pup rhythms in general
This is true if SCNx performed at day 7 of pregnancy, but not if done on day 14

Daily injections of melatonin in SCNx mother hamsters could result in synchronous phases of pups.
Mother’s melatonin rhythm might be involved in synchronizing the rhythms of her pups

Reppert et al.: 2-DG was used to assess fetal SCN rhythms in late gestation.
Pinealectomy, adrenalectomy, or hypophysectomy (removal of pituitary) failed to block transmission of maternal temporal cues

Entraining signal: Timed daily prenatal injections of melatonin are sufficient to entrain the phase of the pups’ rhythms

Timed daily prenatal injections of SKF38393 (a dopamine agonist) are also sufficient to entrain the pups’ rhythms postnatally.
Pups of SCNx mothers did not exhibit synchronous rhythms (though each pup had its own rhythm). What does this demonstrate?
Shows that SCN of mother required for phase setting (synchrony) of pups, but not required for development of pup rhythms in general
This is true if SCNx performed at day 7 of pregnancy, but not if done on day 14
If pups were cross-fostered at birth to a new mother, the pups’ rhythm phase was closely related to the phase of their biological mother rather than their foster mother---what does this suggest?
This suggests that the mother entrains the pups’ rhythms before they are born
Activity onset for pups on day of weaning was close to that of mother's, suggesting entrainment by mother at sometime before weaning
Unlikely that pups set mother's phase because her phase accounted for by initial entrainment to L:D and then free-runs through pregnancy and lactation
Does the Time of postnatal development of overt rhythm denote time of development of pacemaker's rhythmic activity?
No. Time of postnatal development of overt rhythm does not necessarily denote time of development of pacemaker's rhythmic activity
If litter of hamsters raised in dim LL (after mother pregnant also in dim LL), what are their activity rhythms like?
If litter of hamsters raised in dim LL (after mother pregnant also in dim LL), their activity rhythms are approximately coincident with each other and with mother's activity rhythm
If pups were cross-fostered at birth to a new mother, is the pups’ rhythm phase was closely related to the phase of their bio mom or foster mom?
biological mother rather than their foster mother---
This suggests that the mother entrains the pups’ rhythms before they are born
Activity onset for pups on day of weaning was close to that of mother's. What does this suggest?
Activity onset for pups on day of weaning was close to that of mother's, suggesting entrainment by mother at sometime before weaning
What is phase setting?
What does experiemental evidence regarding phase setting tell us about phase setting?
synchrony.SCN of mother required for phase setting (synchrony) of pups, but not required for development of pup rhythms in general
How could you induce synchronized phases in pups whose mom has had scn lesioned?
1.Daily injections of melatonin in SCNx mother hamsters could result in synchronous phases of pups.
What are the entraining signals, prenatally and post-natally?
Timed daily prenatal injections of melatonin are sufficient to entrain the phase of the pups’ rhythms


Timed daily prenatal injections of SKF38393 (a dopamine agonist) are also sufficient to entrain the pups’ rhythms postnatally
How do DA agonists facilitate the entrainment process? Is this similar to the entraining mechan that occurs with light?
Similar to light, DA agonists induce c-fos expression in the SCN
Is DA the neurotransmitter that mediates the ability of light to set the phase of fetal circadian rhythms?
yes, it appears that way.

Pregnant rat dams were injected with a DA-agonist or saline at different phases of the day
Results indicated that the DA-agonist and not saline were able to set the phase of the pups’ circadian locomotor activity rhythm postnatally in DD.
Inference is that DA is involved, as suspected previously
A Duke University Medical Center study has shown that exposing babies born before 31 weeks of gestation to what helps them?
cycled light. It helps them grow faster.
The study identifies no short-term advantages to keeping infants in total near darkness -- the standard practice with many infants.
Discuss the respective lengths of pre term and full term pregancies.
baby is considered pre-term if born before 37 weeks gestation.
A full-term pregnancy is 37 to 40 weeks gestation
How can the SCN maintain a ~24h rhythm?
And be responsive to the phase shifting effects of light?
NEED TO FIND OUT
the first genetic links to circadian rhythms were found in the fruit fly Drosophila melanogaster. What was the gene that they discovered?
per
Discuss the schedule of Drosophillia. What type of clock does it have?
Like many other organisms, the fruit fly Drosophila melanogaster operates on a 24-hour schedule maintained by environmental input to an internal body clock
Describe the molecular mechanisms by which the drosophillic clock operates
The molecular basis of the clock relies on oscillations in the activation of particular genes at certain times of the day.

**Key feature of these molecular oscillations is a negative feedback loop in which the protein products of genes actually turn off production of more protein
Drosophilia experiences molecular oscillations is a negative feedback loop (in which the protein products of genes actually turn off production of more protein). This can occur in all cells, but the highest concentrations of the essential molecules are found
where?
in lateral neurons of the central nervous system.
These lateral neurons, or pacemaker cells, are the Drosophila equivalent of mammalian neurons in the suprachiasmatic nucleus.
The negative feedback loop that forms the basis of the Drosophila molecular clock occurs at the level of ...
gene transcription
Describe the negative feedback loop in drosophillia. Include a description of the per gene.
The negative feedback loop that forms the basis of the Drosophila molecular clock occurs at the level of gene transcription.

The activated period (per) gene in the nucleus of the cell is seen transcribing messenger RNA (mRNA) molecules. As the animation begins, per mRNA moves to the cytoplasm, where ribosomes translate the mRNA into PERIOD (PER) protein molecules. Some PER molecules (shown in pink) degrade shortly after synthesis; others (shown in red) are stable and accumulate in the cytoplasm.

PER protein levels reach a maximum during the middle of the night. At that point, the stable PER molecules enter the nucleus. Inside the nucleus, the PER protein inhibits transcription of its own gene. The per gene turns black to indicate that transcription is repressed.

As the sun rises, PER molecules become susceptible to degradation (shown in pink). Over the course of several hours, all PER protein disappears. In the absence of PER, transcription of the per gene begins again.
Where is the per gene located and what does it do?
The per gene is located in the nucleus of the cell. It transcribes per mRNA.
Where does translation of per mRNA into per protein occur?
in the cytoplasm, on a ribosome.
Discuss what happens to PER proteins after they have been formed.
Some PER molecules (shown in pink) degrade shortly after synthesis; others (shown in red) are stable and accumulate in the cytoplasm.

PER protein levels reach a maximum during the middle of the night. At that point, the stable PER molecules enter the nucleus. Inside the nucleus, the PER protein inhibits transcription of its own gene. The per gene turns black to indicate that transcription is repressed.

As the sun rises, PER molecules become susceptible to degradation (shown in pink). Over the course of several hours, all PER protein disappears. In the absence of PER, transcription of the per gene begins again.
When are per protein levels the highest?
the middle of the night
What happens at night, when per protein reaches its highest levels?
the stable PER molecules enter the nucleus. Inside the nucleus, the PER protein inhibits transcription of its own gene.
What happens between the middle of the night and the time that the sun rises (such that per starts getting transcribed again)?
As the sun rises, PER molecules become susceptible to degradation (shown in pink). Over the course of several hours, all PER protein disappears. In the absence of PER, transcription of the per gene begins again.
Describe the process by which the per gene is activated.
For the per gene to be transcribed, two proteins, CYCLE (CYC) and CLOCK, must bind to a DNA region called the E-box in the per gene promoter.
Describe the condition of the following early in the night (before mid night)
A) cyc and clock
b)per gene
At night, the CYC/CLOCK complex is bound to the promoter, and the per gene is transcribed.
Describe what happens with cyc and clock in the middle of the night (ie, when transcription is repressed).
Transcription is repressed when PER protein molecules interact directly with the CYC/CLOCK complex.
Discuss what happens with cys and clock when the sun rises.
After the sun rises, however, PER molecules degrade, thereby releasing the repression of the CYC/CLOCK complex. As a result, per gene transcription resumes.
What do the per gene and the timeless gene have in common?
Like the per gene, transcription of the timeless (tim) gene is activated by the proteins CYC and CLOCK
TIM and PER form complexes in the cytoplasm. What is the advantage of this?
TIM and PER proteins bind to one another to form a heterodimer (a molecule formed by joining two nonidentical molecules). The formation of a complex with TIM protects PER from rapid degradation.
Describe how the tim/per complex interacts with cyc/clock (to repress transcription of tim and per genes).
PER/TIM complexes enter the nucleus, where they directly interact with CYC/CLOCK complexes. This interaction represses transcription of the tim and per genes
Discuss what happens with tim, per, cyc, and clock as the sun rises.
As the sun rises, light causes rapid degradation of TIM.
Without TIM as a stabilizing partner, PER also degrades.
The repression of CYC/CLOCK is thereby released, and
transcription of tim and per genes resumes.
Discuss how per and tim are degraded.
The Drosophila doubletime protein, which is found both in the cytoplasm and nucleus, is homologous (evolutionarily closely related) to mammalian casein kinase 1 epsilon.
Kinases are enzymes that add phosphate groups to molecules.
What does the addition of phosphate groups to the PER protein do to its activity?
The addition of phosphate groups to the PER protein accelerates its degradation
What degrades PER?
What degrades TIM?
doubletime
cytochrome

As PER protein is synthesized in the cytoplasm,
doubletime causes the degradation of PER proteins

Activated cryptochrome interacts with TIM, causing it to degrade
What degrades PER?
As PER protein is synthesized in the cytoplasm,
doubletime causes the degradation of PER proteins
What degrades TIM?
Activated cryptochrome interacts with TIM, causing it to degrade
What is one reason that per and tim form a heterodimer?
PER proteins that have formed complexes with TIM proteins are resistant to degradation by doubletime
What represses the transcription of per and tim genes?
PER/TIM complexes enter the nucleus and some interact with CYC/CLOCK, resulting in the repression of transcription of the per and tim genes.
what are some examples of clock genes?
cytochrome, timeless, per
The cytochrome protein degrades TIM. What activates the cytochrome protein?
As the sun rises, light causes a conformational (shape) change in the cryptochrome protein, thereby activating it
Activated cryptochrome interacts with TIM, causing it to ...
degrade.

Without the stabilization provided by TIM, PER proteins become susceptible to degradation by the doubletime protein in the nucleus
The degradation of PER/TIM results in the occurance of what two processes?
1. release of repression
2. transcription of the per and tim genes
Mutant doubletime does what to the period?
A specific mutation in the doubletime kinase molecule results in a fruit fly with a period of about 28 hours. Watch this animation display how a less-effective doubletime molecule results in a lengthened period
Mutant, less-effective doubletime molecule results in a lengthened period. How does this happen?
Doubletime phosphorylates PER monomers (single molecules) in the cytoplasm, resulting in PER degradation. This process in the mutant is less effective than that in the wild type (shown by the mutant doubletime "firing twice" to result in PER degradation, while wild type doubletime "fires" only once). Nonetheless, the accumulation of PER/TIM heterodimers in the cytoplasm is comparable to that observed in wild-type flies. (A heterodimer is a molecule formed by joining two nonidentical molecules.)
PER/TIM heterodimers block transcription. Light induces a conformational change in cryptochrome. Cryptochrome degrades TIM.

At this point, the timing of PER degradation differs in the mutant (left screen) and wild type (right screen). Mutant doubletime degrades PER but at a slower rate than that of wild type. As a result, the per gene's release from repression occurs later in the mutant; in turn, per and tim genes are activated later. The resulting effect on the fruit fly's circadian rhythm is a lengthened period.
Discuss the clock genes and the process by which they work. (starting with light to the retinal ganglion cells).

***GOOD GENERAL OVERVIEW
Light activates subset of retinal ganglia cells
Photic input reaches SCN via RHT

Activates SCN cells via glutamate

Ligand binding activates several pathways ultimately leading to gene transcription
“Clock” genes form a feedback loop that self regulates the oscillator

Central oscillator regulates peripheral function through additional gene transcription, changes in synaptic activity, and/or hormonal mediated signaling

Activation of the PER and TIM promoters increases production of mRNA
Ribosomes translate the mRNA producing protein that dimerizes and re-enters the nucleus thereby completing the feedback loop
Describe how the clock genes function in mamals. How is this different from the mechanism of operation in flies?
Mamals don't have timeless, they have B mal.

Flies have tim/per dimers, while mamals have timeless/bmal
Discuss the components of the oscillator that are associated with the mamalian clock.
Negative Loop
Transcription of Period (mPer1-3) and Cryptochrome (mCry1-2) are regulated by the transcription factors BMAL1 and CLOCK
As mPER and mCRY are translated the reenter the nucleus and inhibit BMAL1/CLOCK

Positive Loop
mPER2 contributes to the transcription of Bmal1 180° out of phase with mPer/mCry
An increase in Bmal1 = more BMAL1/CLOCK, thereby resetting the cycle
Discuss the "negative loop" associated with the mamalian clock.
Transcription of Period (mPer1-3) and Cryptochrome (mCry1-2) are regulated by the transcription factors BMAL1 and CLOCK
As mPER and mCRY are translated the reenter the nucleus and inhibit BMAL1/CLOCK
In the mamalian neg feedback loop, as mPER and mCRY are translated the reenter the nucleus and inhibit...
BMAL1/CLOCK
In mammals, the transcription of period (per) and cytochrome (CRY) are regulated by what two transcription factors?
Transcription of Period (mPer1-3) and Cryptochrome (mCry1-2) are regulated by the transcription factors BMAL1 and CLOCK
Discuss the positive loop in mamals.
Positive Loop
mPER2 contributes to the transcription of Bmal1 180° out of phase with mPer/mCry
An increase in Bmal1 = more BMAL1/CLOCK, thereby resetting the cycle
per and tim genes are transcribed during ...
the day
Are rods and cones needed to provide input to the clock?
no - Knockout mice (cl) lacking UV and “green” cones are still able to phase shift
Other studies have shown that a retinal degeneration (rd) mutation which lacks rods is also able to phase shift
In terms of clock input, Light stimulates retinal ganglion cells (RGC’s) which project to the ___ via the ___.
Light stimulates retinal ganglion cells (RGC’s) which project to the SCN via the RHT
What is the photopigment that is thought to be involved in the circadian system?
Melanopsin
Retinal ganglion cells have their optimal response at wavelengths similar to entrainable wavelengths. Describe how they respond to 1) prolonged light stim
2) brief light stim
Responds “sluggishly” but continually to prolonged stimulation
Responds poorly to brief light stimulation
Optimal response at wavelengths similar to entrainable wavelengths
Discuss the link between RHT and the oscillator
Release of Glutamate from RHT terminals may activate different signaling pathways depending on the time of release
Describe what happens in
early subjective night.
Phase delay
NMDA receptor
Intracellular Ca+2 leading to CREB activation
Describe what happens in late subjective night.
Phase advance
Nitric oxide (NO) production leading to CREB activation
Does NO exhibit rhythmicity?
NO was found to be rhythmically active as measured by production of oxidized product
Does Nitric Oxide Synthase (NOS) exhibit rhythmic variation?
eNOS and nNOS are present in SCN cells (astrocytes shown here)
However they were not found to undergo rhythmic variation
NOS activation is thought to be mediated by what?
glutaminergic activity at the NMDA receptor
Compare the NOS activity of animals sacrificed during a period of photic stim (LS) to those who were in DD.
Animals sacrificed during a period of photic stimulation (LS) had greater NOS activity than animals in DD
What effect does time of stimulus (light pulse)have on NOS expression in animals in DD?
DD animals presented with a light pulse had elevated NOS expression regardless of time of stimulus
Consider NOS and phase shifting. What does administration of a NOS INHIBITOR do to the phase?
Administration of a NOS inhibitor (L-NAME) prevented phase delays or advances

The inactive isomer (D-NAME) had no effect

This indicates that NOS may play a role in behavioral phase shifting
How is the MAPK pathway activated?
The MAPK pathway is thought to become activated by the influx of calcium that is produced following glutaminergic stimulation
MAPK pathway is activated by light in a phase-dependent manner. Where does this occur?
in the SCN
MAPK pathway is activated by light in a phase-dependent manner in the SCN, and regulates the activation state of what entity?
MAPK regulates the activation of CREB.
What triggers the phosphorylation of CREB?
Light is known to trigger CREB phosphorylation
What induces MAPK activation?
Light
If you block the MAPK pathway, what happens to light-induced phase shifting?
Blocking the MAPK pathway prevents light induced phase shifting
Briefly summarize the molecular basis of circadian clocks.
The circadian clock perceives light by activation of a subset of retinal ganglion cells that project via the RHT to the SCN
Glutamate release triggers multiple signaling pathways that lead to gene transcription
Clock genes are self regulating through positive and negative feedback loops that make up the oscillator
The oscillator can regulate peripheral function through changes in synaptic function, additional gene transcription, and hormonal regulation via the pineal
What is the pathway by which non-photic info is conveyed to the circadian clock.
Intergeniculate leaflet (IGL) projections to the SCN are essential from conveying non-photic information to the circadian clock.
Give 2 examples of non photic entrainment.
In rats, neuronal electrical stimulation, paired with light, was able to reset the pacemaker.
Also in rats, water restriction at the onset of dark (active phase) lead to increased pineal melatonin release.
List the types of non-photic cues
Circadian Control of Feeding
Food Entrainment
Environmental change
Locomotor activity
Social synchronization
Discuss circadian control of feeding.
SCN controls timing of feeding at several sites.
SCN sends efferents to all hypothalamic nuclei
Disruption of ARH-PVN axis perturbs temporal pattern of feeding.
SCN directly innervates NPY, galanin, and POMC) neurons in the ARH
SCN projects to the LH
LH lesions increase nocturnal eating
Lesions of the PVN induce hyperphagia and disrupt the normal day-night pattern of food intake and blunt daily cort rhythms
Lesions along the ARH-PVN axis pertub timely release of orexigenic and anorexigenic agents resulting in changed daily patterns of food intake.
Orexigenics = NPY, glanin, and POMC show daily rhythms in blood
Lesions along the ARH-PVN axis do what?
Lesions along the ARH-PVN axis pertub timely release of orexigenic and anorexigenic agents resulting in changed daily patterns of food intake.
Orexigenics = NPY, glanin, and POMC show daily rhythms in blood
What are the orexigenics?
Orexigenics = NPY, glanin, and POMC show daily rhythms in blood
SCN sends efferents to ....
all hypothalamic nuclei
Disruption of ARH-PVN axis perturbs what?
The temporal pattern of feeding
SCN innervates....
SCN directly innervates NPY, galanin, and POMC) neurons in the ARH

(I think the oxygenics).
SCN projects to the ....
LateralHypothalamus
Discuss the effects of LH lesions, PVN lesions, and lesions along the ARH-PVN axis.
LH lesions increase nocturnal eating
Lesions of the PVN induce hyperphagia and disrupt the normal day-night pattern of food intake and blunt daily cort rhythms
Lesions along the ARH-PVN axis pertub timely release of orexigenic and anorexigenic agents resulting in changed daily patterns of food intake.
Orexigenics = NPY, glanin, and POMC show daily rhythms in blood
Discuss the effects of LH lesions
LH lesions increase nocturnal eating
Discuss the effect of lesioning the PVN
Lesions of the PVN induce hyperphagia and disrupt the normal day-night pattern of food intake and blunt daily cort rhythms
Discuss the effects of lesioning along the ARH-PVN axis.
Lesions along the ARH-PVN axis pertub timely release of orexigenic and anorexigenic agents resulting in changed daily patterns of food intake.
Orexigenics = NPY, glanin, and POMC show daily rhythms in blood
The daily rhythms of cort readily entrain to food intake patterns. Since this is the case, what would you predict would happen if food is restricted?
Restriction of food access shifts the peak to the new food intake time
What types of things can be entrained by food availability?
Hormones and other signals associated with the control of ingestion can be entrained by food availability.
A change in meal time will effect....
Diurnal rhythm of leptin is shifted by a shift in meal-time.
Same is true for hypothalamic NPY mRNA, POMC mRNA, galanin mRNA, serum leptin levels and adipocytes leptin mRNA.
Can Feeding affect Circadian Hormone Rhythms?
yes
What is the mechanism of action for the Food Entrainment? (I don't think/not sure if this is same as FEO. FEO is in rats).
We don't know the mechanism of action for this clock, but it seems to act somewhat independently of the SCN
Discuss the food entrainment as it relates to food restricion.
Food-restricted animals can anticipate a brief daily phase of food availability, even when kept under constant lighting conditions.

Increased locomotor activity, body temperature and plasma CORT are seen after about a week of food restriction.
Seen in rats, squirrel monkeys, rabbits, and the marsupial kowari
What is a stronger zeitgeber for orexigenic agents - the LD cycle or food availability?
Food availability is stronger zeitgeber for orexigenic agents than is the LD cycle
With food restriction, rhythms become uncoupled from other circadian rhythms except for one type of rhythm, which is not uncoupled from circadian rhythms. Which type of rhythm is this?
the rhythm of hypothalamic leptin mRNA.
circadian oscillators occur in the peripheral organs such as liver, heart, and pancreas. Do these organs have clock genes?
Yes. These organs contain clock genes such as Period genes (per1, per2, per3) and cryptochrome gene (Cry1.
What is unique about the expression of the peripheral clock genes (as compared to the expression of SCN genes)?
Under normal conditions the expression of these peripheral clock genes phase-lag expression of these genes in the SCN by several hours
Does Restriction of food availability to the day time in nocturnal rodents do anything to the peripheral clock genes? If so, what?
Restriction of food availability to the day time in nocturnal rodents induce a phase resetting of gene expression of peripheral clock genes
Describe the features of the Food Entrainable Oscillator in Rats.
Persistence. Free-running activity pattern coexists with a food-entrained component in constant lighting conditions.
Limited range of entrainment (like photic entrainment).
Coupling relations to the SCN. FEOs can be readily dissociated from the SCN oscillators, via food restriction experiments, and are behaviorally expressed in the presence or absence of the SCN.
Discuss the persistence feature of the Food Entrainable Oscillator.
Free-running activity pattern coexists with a food-entrained component in constant lighting conditions.
Discuss the limited range of entrainment feature of the food entrainable oscillator.
(like photic entrainment).
Coupling relations to the SCN. FEOs can be readily dissociated from the SCN oscillators, via food restriction experiments, and are behaviorally expressed in the presence or absence of the SCN.
Discuss the "coupling relations to the SCN" feature of the FEO in rats.
Coupling relations to the SCN. FEOs can be readily dissociated from the SCN oscillators, via food restriction experiments, and are behaviorally expressed in the presence or absence of the SCN
which is the stronger zeitgeber, food or light?
food is not as strong a zeitgeber as light. Light Entrainable Oscillators (LEO) will continue to function under LD cycles or in constant dark (free-running) in the presence of an FEO.
Food restriction does 3 things. What are they?
Food restriction inhibits cellular function and inhibits activity of SCN and reduces vasopressin release.
Does SCNx impact food-entrained cycles?
SCNx has limited effect on food-entrained cycles.
Why is it difficult to distinguish between LEO and FEO effects?
There may be 2 distinct oscillators that entrain circadian rhythms independently.
BUT, when there is a light cycle present, non-photic effects on circadian rhythm become less clear, and therefore more difficult to isolate and study
Describe the linkage between the FEO and the liver.
The liver of a rat that has been entrained to a regular feeding schedule shows “anticipatory activity” in metabolism, even when removed from the rat. This presumably acts to optimize nutrient processing to opportunistic feeding patterns. Liver energy change does not show significant modifications before feeding, but shows a decrease after feeding.


The liver is linked to the FEO via endocrine signals. Corticosterone and glucagon increase and insulin decreases before food access times in food restricted rats.
Why is it hard to do food depravation studies on hamsters?
food-deprivation studies tend to fail because hamsters can not opportunistically eat large quantities at once. Instead, hamsters hoard food.
Is food hoarding pattern in hamsters analogous to FEO in rats?
Daily hoarding opportunity can entrain the SCN-based light-entrainable pacemaker. There is not a separate food-hoarding pacemaker, like the FEO.
Removal of the SCN disrupts hoarding rhythm, though some of this rhythm remains intact. Perhaps this is due to output from extra-SCN pacemaker cells that are usually controlled by the SCN.
Interspecific differences exist between rat and hamster food entrainment.
Cage changes induce phase shifts. Describe the above statement and detail why we think that this is the case.
Syrian hamsters were allowed to free-run for 23 days, then removed from their cages and given new, clean cages.

Clean cages either phase advance or delay, depending on where the hamster was in his particular activity rhythm. For example, a new cage presented a few hours before activity onset yielded a phase advance.
Entrainment may have more to do with arousal than changes in environment itself.
Cycles of low and high temperatures have been shown as zeitgebers in some animals, but not in others. Describe.
Cycles of low and high temperatures have been shown as zeitgebers in lizards, pocket mice, house sparrows, and some bats, but not in golden hamsters, flying squirrels, other mouse species, or squirrel monkeys.
Did temp entrainment work very well in pigtail macques?
Temperature fluctuation did not work well as a non-photic zeitgeber. Few monkeys entrained at all, and they only did so temporarily.
This could be due to the slow nature of the temperature changes in the animal room. Light cues are much more discrete stimulus.