tim

Question 1

discuss photoperiod

Answer 1

• daylength
• used by animals to predict time of year in order to predict for the season
• LH hormone changes throughyear to change the size of testis for mating
• some animals use cells other than eye to detect light; birds use hypothalamus

Question 2

discuss rhythms in humans

Answer 2

• changes in body throughout the day
• 3.30pm=best reaction times
• 9.00pm=melatonin secretion begins
• 2.00am=deepest sleep
• 7.30=melatonin secretion stops
• different chronotypes exist due to genetics; people have different preferences for sleeping time and wakeness
• chronotype changes with age; earlier activeness

Question 3

how is the molecular basis of daily rhythms studied

Answer 3

• rats/mice
• running wheels with sensors on alert us when animal is awake and active
• actograms produced
• french researcher ran an experiment on himself; 2 months underground

Question 4

describe rodent sleep/wake cycle

Answer 4

• active and night and not active during light conditions due to evolution
• absence of light causes the cycle to continue but it takes slightly less than 24 hours
• a small amount of light is needed to synchronise the cycle (20mins); entrainment
• in humans a similar thing happens but cycle gets slightly longer (free running cycle)
• light pulse which shifts the cycle backwards (early pulse) or forwards (late pulse) are known as Zeitgebers

Question 5

describe rhythm graph terminology

Answer 5

peak: highest point on line
period: distance between peaks
amplitude: number of peaks

Question 6

define circadian rhythm

Answer 6

-period of approx 24 hours
-persists in different conditions (absence of darkness, tempertaure changes)
-capable of entrainment (resynchronisation)
(if criterea not met then the rhythm may be a daily rhythm)

Question 7

what is the biological mechanism of timekeeping

Answer 7

• SCN in the anterior hypothalamus is vital
• those with disrupted SCN have unusual sleep/wake patterns
• transfering SCN (superchiasmatic nuclei) from tau mutant hamsters (22 hour period rather than 24) caused the wild type hamsters to have a 22 hour cycle
• SCN generates the rhythm, not just a relay station
• SCN cell removal and experimentation in vivo was done in an electric chamber; circadian rhythm maintained (glucose staining done too)
• similar experiments done on “electrode array boards”
• per gene is key to timekeeping
• chemical mutagenesis of drosophila created individuals with altered circadian rhythm (time of eclosion) when the period (per) gene was mutated
• per sequencing used to demonstrate its expression all over the body (high expression in brain)
• theory that Per gene acts alone by causing negative feedback (cytopalsm expression then movement to nucleus and act as a TF) is incorrect
• Per doesnt have a DNA binding motif
• model cant explain why the cycle takes 24 hours
• tim gene is involved by forming a heterodimer with per, but other are invovled
• those drosophila with mutated tim cant keep time
• per does have a PAS domain so can bind to tim
• time is degraded by light so explains how entrainment can occur
• tim doenst have a DNA binding motif
• Clock gene is involved and is the missing link
• ENU chemical mutagenesis of mice have a free running period
• clock has a DNA binding domain so can bind to tim and per genes (at the E-box) when combined with CYC as well as PAS domain to bind to TIM/PER proteins which have entered nucleus
• doubletime protein (DBT) explains why the cycle takes 24 hours
• DBT phosphorylates cytoplasmic PER causing degradation, however this cannot occur when TIM builds up and binds to PER (this complex enters nucleus and degradation takes 10 hours)
• other molecules are important too
• CRY photopigment identified

Question 8

what are the differences between mammals and drosophila in terms of timekeeping

Answer 8

• mammals have 3 per genes (mPer1, mPer2 and mPer3) where as drosophila have 1
• in mammals, per concentration is highest during the day, but concentration in drosophila is highest at night
• drosophila have cycle but mammals have Bmal1
• the mechanism of Tim in drosophila is unclear because it doesnt form a dimer with Per (cry does instead)
• doubletime exists in drosophila but casein kinase 1 epsilon exists in mammals

Question 9

discuss het lag and entrainment

Answer 9

• feeling of jet lag occurs due to unsynchronised rhythm
• lots of travel and shifting of your rhythm has negative effects on human health
• melatonin pills and eye masks can trick your circadian rhythm into readjustment
• pharmaceutical research needed to help pilots and those who need to do a lot of travelling

Question 10

investigations have been done with identify which genes have expressional changes when changing time zones, how can gene expression be quantified

discuss the results of an experiment which measured expressional changes

Answer 10

• grind up tissue and and quanitfy mRNA
• cDNA created and applied to microarrays
• mice SCN extraction was done in the dark and in mice who experienced light 1 hour before death
• genes where downregulated in mice experiencing light
• only those genes a 1.5 fold upregulation/downregulation and those with characteristics commmon to entrainment genes (camp response elements close to TATA box) were investigated (too many genes to look at otherwise
• entrainment occurs due to light pulses which cause Ca2+ influx and then Per1 expression (CREB becomes phosphorylated, its activator CRTC becomes dephosphorylated and binds to CREB, and the complex then binds to Per1 and Sik1 genes promoting transcription)
• when SIK1 protein has accumulated, it deactivates CRTC, which now cant interact with CREB, which becomes inactivating, stopping Per1 transcription (prevention of entrainment)

Question 11

how can entrainment been achieved more quickly

Answer 11

RNAi technology was used to inactivate SIK1; a 2 hour phase shift occurred (very quick compared to normal)

Question 12

describe what the experiments which reported Per2 expression showed

Answer 12

• Knock-in mice with a fusion gene (per2 transcribes with luciferase); when cells bathed with luciferin then florescence indicates where Per2 is expressed
• per2 is not only expressed in the SCN but other cells like lungs, liver, muscle etc
• non SCN cells display a rhythm in terms of Per2 expression but the rhythm is uncoordinated between different cells
• the cells in the SCN are do coordinated expression; florescene moves from one part to another indicating specialsied role
• the SCN is thougtht to be a master clock; it is signalled the light presence status and passes signals to periphral tissues (slave clocks)
• master clock needed to to sustain rhythm across body
• unknown how the SCN passes signals to other tissues but it is known that neuronal connections to the SCN exist and diffusbale signals are also used (replacing lesioned SCN eith healthy SCN caused normal rhythms despite having no neuronal connections. perhaps the animals behaviour effects how the rhythm becomes synchronised

Question 13

are rods and cones need for entrainment

Answer 13

• no
• irRGC cells (intrinsically photosensitive retinal ganglion cells) are needed, which use melanopsin
• blind people are often capable of light entrainment
• melanopsin knonkouts cant be entrained

Question 14

discuss how bird reproductive systems have a circadian rhythm

Answer 14

• system responds to day length change
• birds have 2 SCNs, with rhythms being generated in the medial SNC rather than the visual SCN (per2, per 3 and bmal1 only detected in the medial SCN
• when given 6 hours of light followed by a 15 min burst of light at different times during the night, testis developed differently depending on the time of the burst