“Sleep and Circadian Rhythms in Research: Why should we care?”

31.1.2019 University of Oulu3R-seminar: Study design

Henna-Kaisa Wigren, PhDSleep Team HelsinkiSleepWell Research programFaculty of MedicineUniversity of Helsinki

What is a circadian rhythm?What is sleep?

Effects on experimental outcomes

Overview

“Time is nature's way of keeping everything from happening at once ” (Woody Allen)

12h to 12h light dark cycle

-energy availability

Why do we have biological clocks?

• Anticipation of environmental events-Adaptation tool

• Particularly important for primitive organisms

CIRCADIAN RHYTHM

CIRCADIAN RHYTHM-Is it truly intrinsic?

→ Free running rhythm(∼24h)

-amplitude-phase-period

ZEITGEBERS = light, food, temperature, social cues

* Variability in period length in inbred laboratory mouse strains

CIRCADIAN RHYTHM

Arrhythmic →

-Where is the clock?SCN= suprachiasmatic nucleus

Rhythmic →

-synchronized by external light

days

Suprachiasmatic nucleus –the master clock

Moore et al. 1972

CIRCADIAN RHYTHM-What is the clock/how does it work?

Gene mutants in Drosophila (Konopka and Benzer 1971)

Tau < 24 h

Tau > 24 h

Molecules of the rhythm - drosophila

E-box Per gene

Tim geneE-box

Nucleus Cytoplasm

CLK

TIM/PER complex

CYC

CYC CLK

OrthologsFly MammalianCLK ClockCYC BMAL1TIM CRYDBT CK1δ and CD1ε (casein kinases)

Nobel prize laureates 2017

• Jeffrey C. Hall; Brandeis University, Waltheim• Michael Rosbash; Brandeis University, Waltheim

• Michael W. Young; Rockefeller University, New York

• Cloning of the drosophila PER gene – both groups• Description of the negative feed-back loop – Rosbash/Hall• Timeless gene identification/Young• Double time (kinase CKI)/Young

CIRCADIAN RHYTHM-How is light entraining the clock

Wave length (nm)

Ab

sorb

anss

i

Cones

Rods Melanopsincells

-retinal GANGLION cells-no (major) role in vision-directly connected to the SCN

-Light: inhibition of melatonin secretion

-Dark: melatonin secretion

in the retina

Berson et al. 2002

Retinohypothalamic tract: photic entrainment

Pineal gland: Melatonin ↓

Blue light

Pupillary reflex

Non-visual effects of light

Peirson et al., 2018

The organization of the circadian system

Stenvers et al., 2019: Circadian clocks and insulin resistance. Nature Reviews Endocrinology15:

Peripheral clocksHow are all theseclocks synchronized?

Clock gene expressions havedifferent phases in peripheral tissues/cells

Entrainmnet by e.g. food?

Phase response curve

CT 12CT 0 CT 24

+

-

7.00 19.00 01.00 7.00

From: Fernandez et al., Dysrhythmia in the suprachiasmatic nucleus inhibits memory processing (2014) Science 346:6211

Circadian outputs

Cortisol

Melatonin

• Sleep/wake cycle• Melatonin rhythm• Temperature rhythm• Cortisol rhytm

Melatonin• The night/dark hormone• Secretion stops in light• Conveys light information to the gonadal axis

• Used as a cue in annual reproduction:- day length

• Pinealectomy prevents annual reproductioncycles

• Timed melatonin administrations restore it

Serotonin → N-acetylserotonin (AANAT)→ Melatonin (HIOMT)

Loss of melatonin from most laboratory mouse strains

Genetic variation of melatonin productivity in laboratory mice under domesticationTakaoki Kasaharaa,1, Kuniya Abeb, Kazuyuki Mekadac, Atsushi Yoshikic, and Tadafumi Katoa,1, 6412–6417 | PNAS | April 6, 2010 | vol. 107 | no. 14

-Does not affect circadian locomotor behavior-all mice are able to free-run andentrain to light

-Testis development promotedin MEL (-) mice-Attenuated by exogenousmelatonin

From: Genetic variation of melatonin productivity in laboratory mice under domesticationTakaoki Kasaharaa,1, Kuniya Abeb, Kazuyuki Mekadac, Atsushi Yoshikic, and Tadafumi Katoa,1, 6412–6417 | PNAS | April 6, 2010 | vol. 107 | no. 14

Light, circadianrhythms and experimental desing

-the widely used laboratory rodents are nocturnal!

Courtesy of prof. Tobler,University of Zurich

The effect of light:

-Light induces sleep in the nocturnal mice!

-Light induces activity in the diurnal zebrafish

Muindi et al., Retino-hypothalamic regulation of light-induced murine sleep (2014). Frontiers in systems neuroscience B:135

→ Many light sensors throughout the zf brain

In Fernandes et al., Enlightening the brain: Linking deep brain photoreception with behavior and physiology (2013). Bioessays35(9):775-9

Kaitlin H. Snider , Kyle A. Sullivan , and Karl Obrietan (2018)Circadian Regulation of Hippocampal-Dependent Memory: Circuits, Synapses, and Molecular Mechanisms. Neural Plasticity

Circadian consequences→ on performance in behavioral tests

Circadian consequences→ on resting energy expenditure

Zitting et al., 2018, Human Resting Energy Expenditure Varies with Circadian Phase, Current Biology

Consequences of compromised circadian rhythms to health

Wt -mouse

Obese,Metabolic disease

Clockless-mouse

arrhythmic

Lean,Healthy

Amandine Chaix, Terry Lin, Hiep D. Le,Max W. Chang, Satchidananda Panda, Cell Metabolism 2018

Take home advices • Always control light conditions!

• no light exposure at the wrong time• measure light intensity• aim at performing behavioral tests at a proper time

• Control sample timing!• Particularly for strongly circadian modulated measures• To get actual circadian profile (samples every 2nd hour throughout

the 24h x 2)

• Make sure the animals are fully adapted to the light conditions before starting experiments

• 1 day/hour of circadian rhythm

* There is a circadian component in most measures!

Information available for circadian regulated variables• A reference human circadian transcriptome atlas: (Ruben, M. D. et al. A database of

tissue-specific rhythmically expressed human genes has potential applications in circadian medicine. Sci. Transl Med. 10, eaat8806 (2018).

• A primate diurnal transcriptome atlas : Mure, L. S. et al. Diurnal transcriptome atlas of a primate across major neural and peripheral tissues. Science 359, eaao0318 (2018)

• Dyar, K. A. et al. Atlas of circadian metabolism reveals system-wide coordination and communication between clocks. Cell 174, 1571–1585 (2018).

• Hughes et al., Guidelines for Genome-Scale Analysis of Biological Rhythms (2017) Journal of Biological Rhythms, 32: 380-393

• Wang et all., Nuclear Proteomics Uncovers Diurnal Regulatory Landscapes in Mouse Liver. Cell Metab. 2017 Jan 10;25(1):102-117.

•• Timed administration of drugs without changing dosage can improve potency of anti-

inflammatory drugs and pharmacological targeting of circadian clock is a new approach to treat cancer (see for example: Winter, C. et al. Chrono-pharmacological targeting of the CCL2-CCR2 axis ameliorates atherosclerosis. Cell Metab. 28, 175–182 (2018).

SLEEP

crustaceans

cuttlefish

mammals

birds

reptiles

fish

Arthopoda

Nematode

insects

Mollusca

c. elegans

VertebratesInvertebrates

SLEEP(/SLEEP-LIKE STATE) IS HIGHLY CONSERVED

~ 20 hThe brown bat

During the day

(nocturnal)

~ 8 h

Human

During the day

(diurnal)

~ 14 hThe Lion

Noon, midnight

(crepuscular)

~ 4 hGiraffe

At night

(diurnal)

~ 12 hRat

During the day

(nocturnal)

-ecological niche/life style, age, food availability, environment, temperature, energy need, breathing

The duration and timing of sleep is variable

Between species:

Within species:

The duration of sleep is variable

humansfruit flies

Long-sleepers

Short-sleepers

-No differenceIn life span!

In Harbison et., 2017: Selection for long and short sleep duration in Drosophila melanogaster reveals the complex genetic network underlying natural variation in sleep. PLoS Genet. 2017 Dec 14;13(12)

Examples of unilateral sleep:

Too hot to sleep? Sleep behaviour and surface body temperature of Wahlberg's Epauletted Fruit Bat.(2015). Downs et al. PLoSOne

All animals sleep, no matter how difficult it may be

left

right

hemisphere

Unihemispheric slow wave sleep in the Amazonian dolphin, Inia geoffrensis (1987). Mukhametov L.M. Neurosci. Lett. 79, 128-132

-Evidence that birds sleep in mid-flight (2016). Rattenborg et al., , Nat. Communications

The Frigate bird

”If sleep does not serve an absolutely vital function,Then it is the biggest mistake the evolutionary process has ever made” (Allan Rechtschaffen 1971).

HYPOTHETICAL DETERMINANTS OF SLEEP

ENVIRONMENTALFACTORS

INTRINSICNEEDS

SLEEP

NERVOUS SYSTEM/BRAIN

Septal Cholinergic Neuromodulation Tunes the Astrocyte-Dependent Gating of Hippocampal NMDA Receptors to WakefulnessPapouin T, Dunphy JM, Tolman M, Dineley KT, Haydon PG. Septal CholinergicNeuromodulation Tunes the Astrocyte-Dependent Gating of Hippocampal NMDAReceptors to Wakefulness. Neuron. 2017 May 17;94(4):840-854.e7.

To SLEEP OR NOT TO SLEEP?

NO SLEEP

Short-term consequences-cognitive performance↓-mood ↓-apetite for high energy food ↑-immune activation

Acute (predatory) stressReproductive succesForaging/FeedingAddiction

StarvationExtreme temperatures

SLEEP

Short-term outcome- Survival ↑- Fitness ↑

Long-term consequences-Chronic inflammation-Congnitive malfunction-Chronic disease-Death

TORPORHIBERNATION

To SLEEP OR NOT TO SLEEP?

Acute (predatory) stressReproductive succesForaging/FeedingAddiction

StarvationExtreme temperatures

SLEEP

NO SLEEP

Short-term outcome- Survival ↑- Fitness ↑

long-term outcome- Survival ↓

- Fitness ↓

TORPORHIBERNATION

RECOVERYSLEEP- Brain Slow waves

Harmfull consequences-cognitive performance-health

Measuring sleepin the lab

Sleep is characterized by:

• Diminished contact with environment – potentially dangerous• Specific sleep posture• Immobility• Closed eyes

• Diminished/lacking muscle tone

• Diminished reactivity for stimulation• Specific brain activity as measured using EEG• Sleep homeostasis

Assessment of motor activity

• Actigraphy• Running wheel• Beam breaking• Video

Running wheel, rodents

Behavioural monitoring

Infrared beam braking flies

Lethargus is a sleep-like state in C. elegans

Raizen et al., Nature 451, 569-572(31 January 2008)

WARNING

Immobility is ≠ sleep !

Real-life example: Sensory tresholdmeasurementin zebra fish larvae

Aho et al., 2017: Homeostatic response to sleep/rest deprivation by constantwater flow in larval zebrafish in both dark and light conditions. JSR

Automated analysis from video recording: Motor activity of zebra fish larvaein different light conditions

Dark LightDark Light

Larval zebrafish chambers

High-speed camera• 1000 images / s

Water pumps

Zebrafish sleep research system

Aho, et al., 2017, J Sleep Res. 2017 Jun; 26(3):394- 400

Aho, et al., 2017, J Sleep Res. 2017 Jun; 26(3):394- 400

Measuring sleep withEEG

Electric activity in the brain

Summation of extracellular field potentialsin vertically oriented pyramidal neurons

Action potential

NEURON

+++

++

EEG recording

Slow oscillation – UP and DOWN

SWS= slow wave sleep/delta waves

Vyazovski et al. 2012 Nat Rev Neuroscience

Delta waves

BEHAVIOUR BRAIN STATE

EEG EMG

WAKE

NREM

REM

MUSCLE TONE Temperature

↑

↑

↑

↑

POLYSOMNOGRAPHY

atonia

SLEEP ARCHITECTURE

hypnogram

Chemelli et al., Cell, 1999

Chronic/long term sleep studies• Tethered recordings:

• Chronic implantation• Single housing • Habituation to handling/chamber/recording

cables (minimum 1 week)

• Telemetric/wireless recordings• Chronic implantation• Allows group housing• No need to extensively habituate the

animals for handling • Less discomfort• Allows true long term studies• + movement and temperature

+Longitudinal study design: no need for separate control groups(reducing variability + number of subjectsneeded)

Alternatives

• In vitro/in toto studies• Animals needed to provide the tissue• How to define sleep?

• Modeling studies (are as good as the input data)

• Effective use of existing data/information• Databases• Publication of negative results!• Standards in methodology across labs

• Less-invasive methods• Behavioural monitoring, telemetry

Sleep on a dish?

Sleep homeostasisSleep deprivation experiments

- minimum amount of sleep is essential

- If wakefulness is prolonged / sleep is prohibited lost sleep must be recovered by sleeping (recovery sleep)

- The amount and depth of recovery sleep is dependent on previous waking

HOMEOSTATIC REGULATION

The two-process model of sleep regulation (Borbely, 1982).

After deprivation:

Process C behaves like a clock - it continues without need for resetting;

Process S behaves like an adjustable hourglass (filled more by increasedwakefulness).

Recoverysleep

Sleep deprivation to study sleep homeostasis

Prolongedwaking

Recoverysleep

1.5 h3.0 h

6.0 h

SLEEP DEPRIVATION(SD)

Lights on (CT1-CT12)

Novel object

-even relatively short periods of prolonged waking induce recovery sleep!-take into account when designing routine behavioral screens!

Sleep is extremelysensitive to stress and adversity

•Long habituation period is needed to get sleep back to baseline after stressful events like handling/operations etc.

•On the other hand good sleep can alleviate pain/discomfort/stress!

If experimentation must be performed during the inactive/sleeping period, be aware of the effects of sleep deprivation!

•Attention ↓•Learning ↓•Anxiety ↑•Pain threshold ↓•Temperature regulation ↓•Circadian effects

Sleep is a sensitive marker of early life adversity!

Early life experience Sleep changes

suspectibility todepression/anxiety

depression-stress/maternal separation-insufficient maternal care-sleep deprivation

impairments in memory&learning

Immature brain Mature braindeveloping brain

prolongs corticosterone secretion in response to novelty, increases locomotorreactivity, impairs cognitive functioning.

Cross-fostering model Sleep disruptions

Santangeli et al., 2016,Sleep and behavior in cross-fostering rats: Developmental and sex aspects. Sleep. 2016; 39(12):2211-2221

• Light and the laboratory mouse (2018) Stuart N. Peirson, Laurence A. Brown, Carina A. Pothecary, Lindsay A. Benson, Angus S. Fisk, Journal of Neuroscience Methods 300 (2018) 26–36

• Light and Cognition: Roles for Circadian Rhythms, Sleep, and Arousal. .Fisk AS, Tam SKE, Brown LA, Vyazovskiy VV, Bannerman DM, Peirson SN.(Front Neurol. 2018 Feb 9;9:56)

• Novel concepts in sleep regulation. Wigren and Porkka-Heiskanen. (Acta Physiol (Oxf). 2018 Apr; 222(4)

Further reading

Acknowledgements:

Tarja Stenberg (Porkka-Heiskanen)Senior PI of the team

Funding: Academy of Finland and past EU framework programs

�“Sleep and Circadian Rhythms in Research: Why should we care?”�OverviewSlide Number 3Why do we have biological clocks?CIRCADIAN RHYTHMCIRCADIAN RHYTHMCIRCADIAN RHYTHMSuprachiasmatic nucleus – the master clockCIRCADIAN RHYTHMMolecules of the rhythm - drosophilaNobel prize laureates 2017Slide Number 12��Non-visual effects of lightSlide Number 15Peripheral clocksPhase response curveSlide Number 18Slide Number 19Melatonin�Loss of melatonin from most laboratory mouse strainsSlide Number 22Light, circadian�rhythms and �experimental desingSlide Number 24The effect of light:Circadian consequences� on performance in behavioral testsSlide Number 27Consequences of �compromised circadian rhythms to healthSlide Number 29Take home advices Information available for circadian regulated variables�SLEEPSLEEP(/SLEEP-LIKE STATE) IS HIGHLY CONSERVEDSlide Number 34Slide Number 35Slide Number 36Slide Number 37HYPOTHETICAL DETERMINANTS OF SLEEPSlide Number 39To SLEEP OR NOT TO SLEEP?To SLEEP OR NOT TO SLEEP?Measuring sleep�in the labSleep is characterized by:Assessment of motor activityRunning wheel, rodentsInfrared beam braking fliesSlide Number 47WARNINGReal-life example: �Sensory treshold �measurement �in zebra fish larvaeSlide Number 50Slide Number 51Slide Number 52Measuring sleep with�EEG� Electric activity in the brainSlow oscillation – UP and DOWN��SWS= slow wave sleep/delta wavesSlide Number 56SLEEP ARCHITECTUREChronic/long term sleep studiesAlternativesSleep on a dish?Sleep homeostasisSlide Number 62The two-process model of �sleep regulation (Borbely, 1982). Sleep deprivation to study sleep homeostasisSleep is extremely�sensitive to stress �and adversitySlide Number 66Slide Number 67Sleep is a sensitive marker of early life adversity! Further reading