Circadian Rhythms, Photoreception And Sleep Flashcards
Circadian entrainment and free run
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
Internal desynchronisation
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
Suprachiasmatic nucleus of the hypothalamus
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
Light signalling
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
Vertebrate light signalling
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
Retinal ganglion cells and visual pathways
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
Retinal ganglion cells indirectly connect to pineal gland via SCN mediated pathway
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)
Melatonin synthesis
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
Melatonin level measurement
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
Visually bind sleep wake cycle
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
Ciliary vs rhabdomeric pathways
Different secondary messengers
Ciliary hyperpolerises
Rhabdomeric depolarises
Ciliary pathway
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
Rhabdomeric pathway
Animals mainly
Redopsin senses light
GQ protein
Phospholipase c
Gating of cation channel (TRP) so depol as na+ and ca2+ in
Negative feedback
Vertebrate pigments include both ciliary and rhabdomeric opsins
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
Circadian photoreception in mammals
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
Melanopsin
Photopigment
Non image forming functions eg circadian rhythm
Melanopsin is expressed in a small number of retinal ganglion cells
Ganglion cell layer
Closer to inside of the eye than rod and cone cells
ipTGC projections
Connect to
OPN Olivary pretexts nucleus
d/vLGN dorsal/ventral lateral geniculate nucleus
IGL inter geniculate leaflet
SCN suprachiasmatic nucleus
Melanopsin impacts pupillary reflex
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
Visual acuity
Ability to see individual grey and black lines
Eventually lose ability to see the lines based on frequency and contrast
Melanopsin supports pattern discrimination
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
Melanopsin impacts light mediated circadian phase resetting
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
Melanopsin impacts circadian photoentrainment
All photoreceptors removed
Display innate period length not 24 hrs so shift in sleep wake cycle
Transgenic mice expressing receptor for diphtheria toxin in ipRGCs: selective ablation of ipRGCs by injection of diphtheria toxin
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
Season affective disorder
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
5 different types of ipRGCs
Projections
Response to stimulation (conductance)
M1 fast onset, slow offset, sensitive
M2-5 slow onset, slow offset, less sensitive
IpRGC Brn3b negative
M1 Brn3b negative
Projects to SCN
Rest circadian rhythm
m1 Brn3b positive and negative similarity
High melanopsin expression
Sensitive, fast onset, slow offset
Dendrites in OFF layer of IPL
Selective ablation of Brn3b+ ipRGCs
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
The peri habenular thalamic area links non visual light input to mood
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
SCN structure
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
Molecular circuits of the circadian clock
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
1994 - identification of circadian mutant mouse
Mutant called clock and result of large ENU screen for circadian mutants
Clock runs slow in this mutation
Clock mutant
Lacks q rich activation domain
RNA Polymerase 2 cannot bind
51 aa deleted
CLOCK interaction with BMAL1
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
CLOCK and BMAL1 dimerise through PAS domains and bind to DNA promoter e-box
E box - CACGTG
BMAL1 knockout nice are anything in DD
Light dark cycle can drive behaviour independently of BMAL1
3 distinct per genes in mammals
Per 1,2 & 3
Per 1 and 2 knockout mice have aberrant rhythms
Per 3 knockout have good rhythms with slightly shorter periods
Light indices mPer
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
How do SCN get light info
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
2 cryptochromes in mammals
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
Both mCRYs are rhythmic in the SCN and retina
Protein level
Lights on no CRYs
2 hrs after lights off - CRYs detected with antibodies
Both mcry genes are rhythmically controlled by CLOCK/BMAL1
Cytochrome knockouts cause alterations in period and/or arrhythmicity
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
CRY proteins and PER proteins repress CLOCK/BMAL1 activation
Reporter assay in tissue culture cells
Measure: luciferase activity
Transferred with CLOCK & BMAL1 results in boost of expression
PER & CRY repress
Tau mutant hamsters
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
What does the tau mutation effect
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
Casein kinase 1 epsilon binds and phosphorylates PER protein
Tau mutant shows normal binding but reduced in vitro kinase activity
Way more phosphorylation so weaker kinase
Per1 mRNA rises earlier and falls earlier in tau mutant hamsters than wild type
Due to shortened period length
Contrasting phenotypes of tau and CK1 epsilon null
Tau mutant qualitatively alters CK1 epsilon function
Knockout gene so period lengthened
Also CK1 delta in genome
Mutant and wild type counteract eachother
Tau specifically destabilises PER 1/2 protein
MRNA relatively unaffected
Accumulation ok but nuclear clearance of PER protein accelerated in tau mutant
Tau summary
Altered substrate specificity to PER1/2
Destabilise sites phosphorylated
Stabilising sites targeted less and so accelerated nuclear clearance
Familial advanced phase sleep syndrome
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
Mutant analysis in mouse PER2
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)
FASPS in summary
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
Interlocked loop leading to rhythmic expression of BMAL1
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
Sleep controlled by clock and homeostat
Build up sleep debt when awake
Pay back sleep debt when asleep
Circadian cycle coincides
What is sleep
Altered consciousness
Reduced movement and responsiveness
Change Typical posture
Homeostatic regulation
Daily rhythmicity
Loss of muscle tone
Rapid eye movements in REM
Measure sleep through electrode (Electroencephalogram)
High frequency low amplitude = wake and REM sleep
High amplitude low frequency = non REM sleep
Electroencephalogram
Neurons not synchronised not much pattern
Synchronised then waves seen
EEG during 1st hr of sleep
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
NREM sleep
Reduced physiological activity
Shift to parasympathetic activity
Thermoregulation maintained
Sleep cycle
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
Polysomnogram of REM sleep
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
Suppression of somatosensory response and muscle relaxation during REM sleep
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
Brain areas responding to wake
Tubero- mammillary nucleus of hypothalamus (TMN) - histolergic
Locus coeruleus- NE
Raphe nuclei - 5HT
Cholinergic nuclei - ACh
Lateral hypothalamic area - orexin
Brain regions responding to NREM
Ventrolateral preoptic nucleus (VLPO) - GABA
sciences other NTs relating to wake
Brain regions responding to REM
VLPO - GABA
LDT - ACh
PPT - ACh
Why do we need sleep
Sleep is necessary
Skin lesions
Swelling of paws
Loss of motor control
Loss of EEG amplitude
Stomach ulcers
Respiratory symptoms
Cognitive impact on sleep disruption
Innattention
Changes in cortical EEG responses
Slower computational speed
Impaired verbal fluency
Reduced creativity
Reduced abstract problem solving
Learning issues
Lower IQ
Theory: clean toxins out of brain
Sleep triggers increased drainage of the brain
AB peptides flushes more during sleep
Theory: sleep unclutters the brain
Eliminate unnecessary connections
Challenge connections and those connected to pre existing circuits survive
Dendritic spines reduced (seen experimentally)
Synaptic homeostasis hypothesis
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
Experimental evidence for synaptic homeostasis hypothesis
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
How does synaptic homeostasis hypothesis work
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
Sleep
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
Criticism if synaptic homeostasis hypothesis
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?
