Circadian Rhythms, Photoreception And Sleep

Question 1

Circadian entrainment and free run

Answer 1

Entrained state - regular 24 hrs sleep wake cycle, body temp trough pattern near end of sleep
Free running - longer than 24 hrs but relative pattern but shifts slowly, body trough temp changes so onset of sleep
Entrained - reverts and trough drifts until reaches normal again

Conclude: synchronising impact of light and dark on sleep cycle and body temp, ~24 hr cycle so daily rhythms

Question 2

Internal desynchronisation

Answer 2

Period length
Amplitude
Phase

Period length a lot longer so rhythms that usually are in the same period length with a fixed phase relationship relative to eachother now drift relative to eachother
So multiple time keeping sources in our body

Question 3

Suprachiasmatic nucleus of the hypothalamus

Answer 3

Main site of central time keeper in brain
Contains molecular oscillators and synchronising intracellular peptidergic signalling (VIP, AVP)
Lesion of SCN abolishes circadian rhythms of physiology and behaviour
Disrupt expression of clock genes in SCN neurons abolishes rhythms
The SCN receives light input from retina

Question 4

Light signalling

Answer 4

Light
Photoreceptors buried in ONL
Rods and cones signal to bipolar cells and then ganglion cells that collect into optic nerve
Pore arrangement so breaches light sensitive layer to leave the eye and go to brain

Question 5

Vertebrate light signalling

Answer 5

Ciliary vertebrate photoreceptor rod or cone
Hyperpolerised so transmitting signals when there’s no light
Cation channels open so depol so higher rate of transmitter release in the dark

Light - cation channels close in response to redopsin and leads to hyperpol so drop in membrane potential

Glutamate signals to bipolar cells. Some bipolar cells are excitatory and some inhibitory
Dark - excitatory bipolar cells due to release of glu, glu in ganglion off cell and cause an action potential
Light - inhibitory bipolar cells due to disinhibition, release glu on ganglion on cells causing AP

Question 6

Retinal ganglion cells and visual pathways

Answer 6

Info sent to visual cortex, superior caliculus for eye movement, dorsal lateral “connected” nuclei
Info from right eye goes to left side of brain and vice versa

Downstream of bipolar cells are ganglion cells and project laterally. Different types exist based on how they project

Question 7

Retinal ganglion cells indirectly connect to pineal gland via SCN mediated pathway

Answer 7

Retinalhypothalamic tract (Glutermetergic) to SCN
Pathways to super cervical ganglion in brainstem or spinal cord
NA to pineal gland which produces melatonin in light inhibited manner (produced during dark)

Question 8

Melatonin synthesis

Answer 8

Retina connects to SCN to PVN to upper thoracic cord the SCG that connect to the pineal gland via NA
NA bind to alpha and beta receptors on pineal sites which catalyse enzymatic reaction that turn tryptophan to serotonin to melatonin
Melatonin released into blood stream

Question 9

Melatonin level measurement

Answer 9

Control group - high at night
Blue light - delay of melatonin next day, Acute effect direct loss of melatonin (intrinsically photosensitive retinal ganglion cells?)
Green light - delay of melatonin next day, Acute effect is delay (cones)

Light is a phase resetting cue
Eyes have blue light photoreceptors that don’t act in same way as green light photoreceptors

Question 10

Visually bind sleep wake cycle

Answer 10

Both have 24 synchronised sleep wake cycle
Body temp rhythm synchronised in one individual and not in the other

Non 24 hr body temp - no ERG, no visually evoked potential, no pupillary reflex eg congenital glaucoma, would drift if not socially synchronised

24 hr body temp - no erg, abnormal vep, pr intact eg inherited mitochondrial optic neuropathies

Question 11

Ciliary vs rhabdomeric pathways

Answer 11

Different secondary messengers
Ciliary hyperpolerises
Rhabdomeric depolarises

Question 12

Ciliary pathway

Answer 12

Light on retina
Redopsin senses light that signals to G protein which activates cyclic GMP phosphodoesterase
Turns cyclic GMP to 5’ GMP so loss of cyclic GMP
Closing of CNG cation channels
So hyperpol

Negative feedback

Question 13

Rhabdomeric pathway

Answer 13

Animals mainly
Redopsin senses light
GQ protein
Phospholipase c
Gating of cation channel (TRP) so depol as na+ and ca2+ in

Negative feedback

Question 14

Vertebrate pigments include both ciliary and rhabdomeric opsins

Answer 14

Only a subset used for visual photoreception
In vertebrates - only ciliary opsins in visual photoreception

We have melanopsin in ganglion cells for non visual photoreception and is a rhabdomeric opsin

Question 15

Circadian photoreception in mammals

Answer 15

Separable from vision but require the retina
Requires intrinsically photosensitive retinal ganglion cells which contain circadian blue light photipigment melanopsin, connect rods and cones to SCN and are selectively spared in mitochondrial optic neuropathies
Melanopsin act through rhabdomeric rather than ciliary pathway
Seasonal affective disorder associated with mutations in melanopsin gene

Question 16

Melanopsin

Answer 16

Photopigment
Non image forming functions eg circadian rhythm

Question 17

Melanopsin is expressed in a small number of retinal ganglion cells

Answer 17

Ganglion cell layer
Closer to inside of the eye than rod and cone cells

Question 18

ipTGC projections

Answer 18

Connect to
OPN Olivary pretexts nucleus
d/vLGN dorsal/ventral lateral geniculate nucleus
IGL inter geniculate leaflet
SCN suprachiasmatic nucleus

Question 19

Melanopsin impacts pupillary reflex

Answer 19

Mop -/- results in reduced pupillary reflex but not accent as other photoreceptors
Triple knock out plus knock out of rods and cones results in no pupillary reflex
Carbachol tests muscles ability to contract which it can do it is really signalling

Question 20

Visual acuity

Answer 20

Ability to see individual grey and black lines
Eventually lose ability to see the lines based on frequency and contrast

Question 21

Melanopsin supports pattern discrimination

Answer 21

Swim test of mice with platform based on ability to see patterns
Visual water test
No rods, cones or melanopsin then can’t do it
Melanopsin but not rods or cones they can still see some difference
So melanopsin dependent behaviour

Optokinetic tracking test
Watch mice head movement
No contribution of melanopsin

Question 22

Melanopsin impacts light mediated circadian phase resetting

Answer 22

Light pulses of different strengths at night will shift its phase
Running on wheel

Melanopsin null still had phase resetting but reduced in level of phase resetting so sensitivity reduced

Question 23

Melanopsin impacts circadian photoentrainment

Answer 23

All photoreceptors removed
Display innate period length not 24 hrs so shift in sleep wake cycle

Question 24

Transgenic mice expressing receptor for diphtheria toxin in ipRGCs: selective ablation of ipRGCs by injection of diphtheria toxin

Answer 24

Instruct ipRGCs to commit suicide through expression of the toxin
So kill retinal ganglion cells
Visual cliff test, still good result
Pupillary reflex completely gone because photoreception not computed from both melonopsin or rods and cones
Act like in constant darkness all the time

Question 25

Season affective disorder

Answer 25

3% in uk Low mood, loss of pleasure/interest Tiredness Difficulty concentrating Treatment with daylight/blue light Most efficient wave length 470 nm Post illumination pupil response to blue light is affected in SAD SAD vs nondepressed (220 altogether) SAD = 7 we’re homozygous for melanopsin P10L allele 5.6 x increased risk of SAD Healthy have earlier bedtime in short days, later in long days

Question 26

5 different types of ipRGCs

Answer 26

Projections Response to stimulation (conductance) M1 fast onset, slow offset, sensitive M2-5 slow onset, slow offset, less sensitive

Question 27

IpRGC Brn3b negative

Answer 27

M1 Brn3b negative Projects to SCN Rest circadian rhythm

Question 28

m1 Brn3b positive and negative similarity

Answer 28

High melanopsin expression Sensitive, fast onset, slow offset Dendrites in OFF layer of IPL

Question 29

Selective ablation of Brn3b+ ipRGCs

Answer 29

Only left with negative pathway IpRGC > SCN projections mediate the impact of light/dark in learning and LTP 7 hr light 7 hr dark Morris water maze Novel object recognition long term potentiation Light pulse induction but not rhythmicity

Question 30

The peri habenular thalamic area links non visual light input to mood

Answer 30

Elevates mood in light T7 LD cycles impact Sucrose preference test Tail suspension tail Forced swim test Circadian rhythms in pHb Dependent on Brn3b+ ipRGCs But bilateral pHb inactivation takes away negative impact of t7 light/dark cycle But activation of pHB makes mice depressed even in normal sleep wake cycle

Question 31

SCN structure

Answer 31

Core - input from retina, VIP emntrainment function/synchronisation, melatonin, feedback from arousal centres, release GABA, VIP and GRP to shell part Shell - pacemaker function, outputs GABA, AVP and PK2 to sPVZ and DMH resulting in secretion of molecules, arousal centres, neuro endocrine cells, pre autonomic neurons

Question 32

Molecular circuits of the circadian clock

Answer 32

Heterodimer of CLOCK & BMAL1(negative feedback loop by targeting BMAL) Helix pas domain transcription factors Bind to target sequence e box Catalyses induction of many CLOCK control genes (CCGs) Per1/2, cry1/2 have transcribed Translated and assemble tripartite complex with kinases which phosphorylate per and cry components Regulate stability and subcellular localisation Once there’s enough, enters nucleus where per and cry inhibit BMAL CLOCK complex by binding to stop promoter binding Time delay between transcription of period and cryptochrome and negative feedback on BMAL1CLOCK complex

Question 33

1994 - identification of circadian mutant mouse

Answer 33

Mutant called clock and result of large ENU screen for circadian mutants Clock runs slow in this mutation

Question 34

Clock mutant

Answer 34

Lacks q rich activation domain RNA Polymerase 2 cannot bind 51 aa deleted

Question 35

CLOCK interaction with BMAL1

Answer 35

BMAL looks like CLOCK but missing c terminal activation domain So activation done by the complex is done by CLOCK mutant clock = no activation domain so complex doesn’t work

Question 36

CLOCK and BMAL1 dimerise through PAS domains and bind to DNA promoter e-box

Answer 36

E box - CACGTG

Question 37

BMAL1 knockout nice are anything in DD

Answer 37

Light dark cycle can drive behaviour independently of BMAL1

Question 38

3 distinct per genes in mammals

Answer 38

Per 1,2 & 3 Per 1 and 2 knockout mice have aberrant rhythms Per 3 knockout have good rhythms with slightly shorter periods

Question 39

Light indices mPer

Answer 39

Per 1 and 2 expression in the SCN Phase of activity jumps if you give light pulses and then leave in darkeneds Phase delay Per 3 nearly no effect Before CT 16 can get phase dealt After CT 18 get phase advance Per 1 and 2 are negative feedback molecules Enhance in early night, alread inhibited BMAL so inhibition prolonged and delayed of phase Late, ascending per 1 and 2 so advance because even more

Question 40

How do SCN get light info

Answer 40

Retinal hypothalamic tract Axons of ipRCGs (M1 Brm3 negative) Express glutamate and PACAP Work on NMDAR, L type voltage gated sodium channels and PAC1R (AC or PLC) CAMK from NMDAR and L type > CREB phosphate PAC1R > AC or PLC > CREB phosphate To resetting of circadian oscillator

Question 41

2 cryptochromes in mammals

Answer 41

One in drosophila - blue light photoreceptors 2 in mammals - transcriptional represses Not athologues but homologues Vertebrates don’t have drosophila cry All came from DNA repair All use pterin and flavin cofactors

Question 42

Both mCRYs are rhythmic in the SCN and retina

Answer 42

Protein level Lights on no CRYs 2 hrs after lights off - CRYs detected with antibodies Both mcry genes are rhythmically controlled by CLOCK/BMAL1

Question 43

Cytochrome knockouts cause alterations in period and/or arrhythmicity

Answer 43

Phenotype of circadian rhythmicity Knock out both CRY 1 and 2 = arrhythmic mouse Knockout CRY1 = fast running clock Knockout CRY2 = slow running clock So complementary functions and affects

Question 44

CRY proteins and PER proteins repress CLOCK/BMAL1 activation

Answer 44

Reporter assay in tissue culture cells Measure: luciferase activity Transferred with CLOCK & BMAL1 results in boost of expression PER & CRY repress

Question 45

Tau mutant hamsters

Answer 45

NOT AD TAU short and stable 22 hr cycle not 24 Bred and made homozygous for tau and now has 20 hr cycle Hamsters don’t have convenient hamster, expensive etc Point mutation of arginine to cysteine

Question 46

What does the tau mutation effect

Answer 46

May effect the catalytic site and substrate affinity Homologise of fruity gene - double time Authogue of double time is Casein kinase 1 epsilon and delta

Question 47

Casein kinase 1 epsilon binds and phosphorylates PER protein

Answer 47

Tau mutant shows normal binding but reduced in vitro kinase activity Way more phosphorylation so weaker kinase

Question 48

Per1 mRNA rises earlier and falls earlier in tau mutant hamsters than wild type

Answer 48

Due to shortened period length

Question 49

Contrasting phenotypes of tau and CK1 epsilon null

Answer 49

Tau mutant qualitatively alters CK1 epsilon function Knockout gene so period lengthened Also CK1 delta in genome Mutant and wild type counteract eachother

Question 50

Tau specifically destabilises PER 1/2 protein

Answer 50

MRNA relatively unaffected Accumulation ok but nuclear clearance of PER protein accelerated in tau mutant

Question 51

Tau summary

Answer 51

Altered substrate specificity to PER1/2 Destabilise sites phosphorylated Stabilising sites targeted less and so accelerated nuclear clearance

Question 52

Familial advanced phase sleep syndrome

Answer 52

Advance of 4-6 hrs relative to controls Single gene trait Mutation in hper2 Point mutation in PER2 gene at site phosphorylated by Caseinkinase1 epsilon Serine changed to glycine (S662G) Fast running clock also seen in flies Deficient phosphorylation of hPER2

Question 53

Mutant analysis in mouse PER2

Answer 53

Cellular clocks show the FASPS phenotype Expression of FASPS or mut7 PER2 in tissue culture results in reduced stability and early nuclerat clearance (through cychloeximide which is an inhibitor of translation)

Question 54

FASPS in summary

Answer 54

Mutated phosphorylation sites in FASPS destabilises PER2 protein and advanced nuclear clearing Resembles tau mutant and tau kinase shows specifically reduced activity for FASPS site Another FASPS pedigree identified T44A mutation in casein kinase 1 epsilon which reduced kinase activity

Question 55

Interlocked loop leading to rhythmic expression of BMAL1

Answer 55

BMAL1 rhythmically expressed under control of RORs and REV-ERBs (REVs negative regulators, RORs positive regulators) E box controlled so simultaneously expressed with PERs and CRYs REV-ERBs dominant so repress expression of BMAL1 so ends up out of phase REV-ERBs turned over RORs take over and so out of phase of PER and CRY etc

Question 56

Sleep controlled by clock and homeostat

Answer 56

Build up sleep debt when awake Pay back sleep debt when asleep Circadian cycle coincides

Question 57

What is sleep

Answer 57

Altered consciousness Reduced movement and responsiveness Change Typical posture Homeostatic regulation Daily rhythmicity Loss of muscle tone Rapid eye movements in REM

Question 58

Measure sleep through electrode (Electroencephalogram)

Answer 58

High frequency low amplitude = wake and REM sleep High amplitude low frequency = non REM sleep

Question 59

Electroencephalogram

Answer 59

Neurons not synchronised not much pattern Synchronised then waves seen

Question 60

EEG during 1st hr of sleep

Answer 60

Progress to higher amplitude lower frequency waves Awake beta waves Stage 1 theta waves Stage 2 theta waves Stage 3 theta waves Stage 4 delta waves Then REM sleep

Question 61

NREM sleep

Answer 61

Reduced physiological activity Shift to parasympathetic activity Thermoregulation maintained

Question 62

Sleep cycle

Answer 62

REM periods 90-120 First REM period shortest Most REM sleep occurs late Most deep sleep (stage 3,4) early With age Similar amount of REM sleep Diminishing 3,4 sleep Increased sleep fragmentation

Question 63

Polysomnogram of REM sleep

Answer 63

Heart rate, respiration, EEG, neck muscles, penile responses Resembles wake state for brain activity, heart rate, respiration Diverges for Eye movement, muscle tone, thermoregulation, penile erection

Question 64

Suppression of somatosensory response and muscle relaxation during REM sleep

Answer 64

Inhibition of cells in dorsal column nuclei results in diminished response to somatic sensory stimuli resulting in inhibition of lower motor neurone and so paralysis Glu, 5HT and ACh All inhibited by GABA

Question 65

Brain areas responding to wake

Answer 65

Tubero- mammillary nucleus of hypothalamus (TMN) - histolergic Locus coeruleus- NE Raphe nuclei - 5HT Cholinergic nuclei - ACh Lateral hypothalamic area - orexin

Question 66

Brain regions responding to NREM

Answer 66

Ventrolateral preoptic nucleus (VLPO) - GABA sciences other NTs relating to wake

Question 67

Brain regions responding to REM

Answer 67

VLPO - GABA LDT - ACh PPT - ACh

Question 68

Why do we need sleep

Answer 68

Sleep is necessary Skin lesions Swelling of paws Loss of motor control Loss of EEG amplitude Stomach ulcers Respiratory symptoms

Question 69

Cognitive impact on sleep disruption

Answer 69

Innattention Changes in cortical EEG responses Slower computational speed Impaired verbal fluency Reduced creativity Reduced abstract problem solving Learning issues Lower IQ

Question 70

Theory: clean toxins out of brain

Answer 70

Sleep triggers increased drainage of the brain AB peptides flushes more during sleep

Question 71

Theory: sleep unclutters the brain

Answer 71

Eliminate unnecessary connections Challenge connections and those connected to pre existing circuits survive Dendritic spines reduced (seen experimentally)

Question 72

Synaptic homeostasis hypothesis

Answer 72

Sleep improves cognitive ability Synapses strengthen during wake Spontaneous firing during sleep weakens synapses selectively Eg Limits energy use, remove unnecessary info, restore memory/ learning capacity, limit cellular resources

Question 73

Experimental evidence for synaptic homeostasis hypothesis

Answer 73

No. Of dendritic spines and axon spine interface increase during wake and decreases during sleep (mice and flies) Evoked responses are lowered during sleep (electricity and mice) AMPA glu receptors increase during wake but decrease during sleep (as well as others) NREM sleep slow waves decrease in course of sleep NTs and BDNF concentrations lower during NREM Local NREM strength determined by plasticity use during wake “local sleep” during sleep deprivation Important during brain development

Question 74

How does synaptic homeostasis hypothesis work

Answer 74

Post synaptic protein, Homer, in many forms Tetrameric form - link metabolic glu receptors to calcium channels in er and activation Truncated version - sleep, evh domain and (coiled coil produced) , can’t make connection in postsynaptic density so less active state of neurons Homer 1A just EVH Homer long EVH and coiled coil Tetrameric form big

Question 75

Sleep

Answer 75

Increased 1A in post synaptic density Decrease mGlu-R signalling Arc May impact AMPAR similarly Synaptic weakening via GSK-3 beta Inactivated at S9 during wake

Question 76

Criticism if synaptic homeostasis hypothesis

Answer 76

Mechanisms not clear LTP during NREN in model of monocular deprivation Some arguements are species specific Other rational possible eg drainage of toxins via lymph like system, selective growth of glia Role of REM sleep?