The effects of ambient light in rodent research
Seppo Saarela
Seppo Saarela, Department of Genetics and Physiology, 2016
Laboratory animal maintenance
Common housing conditions
• 12L:12D, Ta = room temperature, 221C
Lights on 08:00, Lights off 20:00
• Long photoperiod 14L:10D
• Short photoperiod 10L:14D
Natural vs. artificial conditions for rodents
• Nocturnal (vs.diurnal) animal
• Light intensity?
• Spectrum?
• Dim-light, light pollution
Seppo Saarela, Department of Genetics and Physiology, 2016
Light signal, evolution
• Light is an important signal for living
organisms
• First living organisms
(procaryocytes and bacteria)
adapted to day/night –cycle
• Light is the ultimate factor for
seasonal acclimatization at high
latitudes
Seppo Saarela, Department of Genetics and Physiology, 2016
Electromagnetic Spectrum
Seppo Saarela, Department of Genetics and Physiology, 2016
Relative sensitivity
Human eye vs. plant response
Seppo Saarela, Department of Genetics and Physiology, 2016
Short history of chronobiology
300 BC
• Androstenes (amanuensis of Alexander the Great):
- plant leaves are open during the day and closed at night
1700’s
• de Mairan
- the first experiments → rhytmicity of leaves continues in
the dark (DD)
1900’s
• Karl von Frisch (Nobel 1973)
- experiments with minnows: a photosensitive organ in the
brain → the pineal gland
- nectar harvesting by bombus 16-18 CT
 endogenic clock
Seppo Saarela, Department of Genetics and Physiology, 2016
History 2
Lerner (1958), Axelrod (1960) (Nobel 1970)
• pineal melatonin
Jürgen Aschoff (1960’s)
• Tier Bunker –isolation experiments for 3-4
weeks
• Human sleep-wake rhythm and human
circadian rhythm of Tb → endogen rhythm
ca. 25 h)
• Free-running rhythm
Seppo Saarela, Department of Genetics and Physiology, 2016
History 3
1997
• Mammalian clock genes (clock, Per1, Per2 and Per3)
- expression of rhythms
1999-2001
• Plant photopigments
- phytochromes A ja B (phyA, phyB)
- cryptochromes (cry1, cry2)
 cover visual light spectrum (natural light in the
morning, at noon and in the evening)
Seppo Saarela, Department of Genetics and Physiology, 2016
Biological rhythms in mammals
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•
•
•
circadian (diurnal), 24 h (period)
seasonal (annual)
ultradian <24 h, infradian >24 h
ability to anticipate circadian and annual
rhythms developed, entrainable →
circadian clock
Seppo Saarela, Department of Genetics and Physiology, 2016
Requirements for biological clock function
1.
2.
3.
4.
5.
6.
Self-regulating
Resetable
Endogenic
Response to day/night-cycle
Response to Ta
Manipulatable by genetic and
biochemical methods
Seppo Saarela, Department of Genetics and Physiology, 2016
Location of biological clock
First known use 1955
•
•
•
Mammals
Birds
SCN = suprachiasmatic nucleus of the hypothalamus
Seppo Saarela, Department of Genetics and Physiology, 2016
•
Newman 1993
Rat brain
14C-2-deoxyglucose
uptake
A = day, B = night
Biological clock
Seppo Saarela, Department of Genetics and Physiology, 2016
Retinohypothalamic tract
Traditional paradigm
•Visual phototransduction
•Rhodopsin
•Melanopsin
•Spinal cord
Seppo Saarela, Department of Genetics and Physiology, 2016
Structure of retina
Melanopsin (OPN4) is expressed in ganglion cells
Seppo Saarela, Department of Genetics and Physiology, 2016
Actogram of feeding activity
 = 23 h 36 min
in free-running feeding rhythm
LL = continuous light
Phodopus sungorus
Seppo Saarela, Department of Genetics and Physiology, 2016
Puchalski, Saarela & Lynch 1996.
J Biol Rhythms
Electrical activity of SCN neurons
•
•
•
•
Extracellular recording
from the SCN neurons of
brain slices in vitro
Neurons in the SCN of the
hypothalamus exhibit a
daily rhythm in
spontaneous electrical
activity
Phase shift of 8 h →
rhythm of spikes
disappeares
Reentrainment of a
circadian output to a
shifted light:dark cycle
commonly takes several
cycles
•Puchalski, Saarela & Lynch 1996
Seppo Saarela, Department of Genetics and Physiology, 2016
Peak time of hormone daily secretion
Binkley (1993) Experientia 49, 648-653.
Seppo Saarela, Department of Genetics and Physiology, 2016
Circadian rhythm of young rats
1) Rhythm is missing in
newborns
2) Sleeping & waking rhythm
unclear or mising
3) Rhythm is developing
slowly within 2-3 months
Scientific paper by Fred C. Davis et al.
For further reading see e.g. Wreshning, Dolatshad &
Davis (2014) Embryonic Development of Circadian
Oscillations in the Mouse Hypothalamus. J Biol Rhytms
29: 299-310.
Seppo Saarela, Department of Genetics and Physiology, 2016
Traditional hypotheses of light transmission
1. Visual transmission through optic nerve.
2. Intact eyes are needed to entrain many
physiological processes.
3. Extraretinal photoreceptors in mammals?
Extra-ocular photoreceptors in vertebrates.
• Karl von Frisch (1911), minnow skin
• Lisk & Kannwischer (1964) photosensitive
neurons in hypothalamus of enucleate adult rats
• Several reports 1980-1990, skin, peripheral
organs and CNS, birds, fish, amphibia, reptiles
• Photosensitive pineal in neonatal rat (Blackshaw
and Snyder 1997)
Seppo Saarela, Department of Genetics and Physiology, 2016
Melanopsin, OPN4
•
•
Initially identified in amphibians, reported in all
vertebrate classes
Resemble invertebrate visual opsins
•
Primary photoreceptor molecules for non-image
forming function, such as the photo-entrainment
of the circadian clock and pupillary constriction in
mammals
•
Participate also in visual tasks by regulating optic
inputs from photoreceptor cells during the day
Seppo Saarela, Department of Genetics and Physiology, 2016
Encephalopsins, OPN3
• In rodents brain and
testes
• Widely expressed in nonphotoreceptive and nonneuronal tissues
• Regulate photic
entrainment of peripheral
clocks
Seppo Saarela, Department of Genetics and Physiology, 2016
The Opsins
• Opsins belong to family A of GPCRs (G-proteincoupled receptor), heptahelical transmembrane
proteins
• GPCRs are biggest family of cell membrane receptors
• GPCRs respond to different ligands through different G
proteins
• Opsins use retinals as a ligand
• Rhodopsin evolved from a retinoid receptor acquired
the ability to covalently bind to to its ligand, allowing it
to evolve a photoreceptor molecule.
• Opsins went through a diversification process by
coupling with different G proteins → see subfamilies of
the phylogenetic tree
Seppo Saarela, Department of Genetics and Physiology, 2016
Vertebrate visual opsins
Visual opsins: 5 subgroups (4 cone opsins, 1 rod
opsin)
• S group cone opsins absorb UV or violet light
• M1 blue light
• M2 green light
• L red or green light
• Rod opsin, denoted Rh, absorbs green/blue light
Vertebrate visual opsins are also expressed in nonvisual photoreceptor cells such like pineal
photoreceptor cells
Seppo Saarela, Department of Genetics and Physiology, 2016
Summary
Scientist working with rodents should know that
1. Most rodents are nocturnal animals  To
turn the rhythm 180 for your convenience it
takes time 1 h/day.
2. Pay attention to spectrum and light intensity
(for mole 1 lux is bright light).
3. Rodents are sensitive to lights on during
dark phase. Avoid to enter in the dark room.
4. Circadian clock gets information also
through non-visual track.
5. There are photsensitive opsins in many
location in rodents brain.
Seppo Saarela, Department of Genetics and Physiology, 2016