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ISBN 978-952-62-1958-5 (Paperback)ISBN 978-952-62-1959-2 (PDF)ISSN 0355-3191 (Print)ISSN 1796-220X (Online)

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

U N I V E R S I TAT I S O U L U E N S I SACTAA

SCIENTIAE RERUM NATURALIUM

OULU 2018

A 719

Antti Flyktman

EFFECTS OF TRANSCRANIAL LIGHT ON MOLECULES REGULATING CIRCADIAN RHYTHM

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF SCIENCE

A 719

AC

TAA

ntti Flyktman

ACTA UNIVERS ITAT I S OULUENS I SA S c i e n t i a e R e r u m N a t u r a l i u m 7 1 9

ANTTI FLYKTMAN

EFFECTS OF TRANSCRANIAL LIGHT ON MOLECULES REGULATING CIRCADIAN RHYTHM

Academic dissertation to be presented with the assent ofthe Doctoral Training Committee of Technology andNatural Sciences of the University of Oulu for publicdefence in the OP auditorium (L10), Linnanmaa, on 30June 2018, at 12 noon

UNIVERSITY OF OULU, OULU 2018

Copyright © 2018Acta Univ. Oul. A 719, 2018

Supervised byProfessor Seppo SaarelaDoctor Satu MänttäriProfessor Markku Timonen

Reviewed byProfessor Abraham HaimDoctor Burkhard Poeggeler

ISBN 978-952-62-1958-5 (Paperback)ISBN 978-952-62-1959-2 (PDF)

ISSN 0355-3191 (Printed)ISSN 1796-220X (Online)

Cover DesignRaimo Ahonen

JUVENES PRINTTAMPERE 2018

OpponentProfessor Jari Karhu

Flyktman, Antti, Effects of transcranial light on molecules regulating circadianrhythm. University of Oulu Graduate School; University of Oulu, Faculty of ScienceActa Univ. Oul. A 719, 2018University of Oulu, P.O. Box 8000, FI-90014 University of Oulu, Finland

Abstract

Light acts as the most important regulating and entraining factor of the mammalian circadianrhythm. This rhythm has evolved to set phases, in which different physiological and behavioralevents occur at the right time of the day to synchronize the organism. The mechanism of lighttransduction via eyes to the brain and its effects on circadian rhythmicity is well known. Yet, it hasalso been shown that light is able to penetrate the skull bone directly, but it is still unknown,whether transcranial light is able to affect molecules regulating circadian rhythmicity.Monoamines and especially opsins have been shown to act as important regulators in circadianrhythmicity. Both group of molecules can mediate the effects of light on regulation andentrainment.

In this thesis, mice and hamsters have been illuminated transcranially and the expression ofthree different opsins and the concentrations of several monoamines have been measured. Theanimals were illuminated under anesthesia either just after the onset of the light period or just afterthe beginning of the dark period. The opsin expression in rodent brain were measured by westernblot and the monoamine concentrations from mouse brain, plasma and adrenal gland weremeasured by HPLC.

It was observed that both opsin expression and monoamine concentrations can be influencedby transcranial illumination. The effect varied depending on the studied molecule, tissue and timeof illumination. The findings of this study demonstrate that opsins, which are considered to be themost important molecules regulating circadian rhythmicity, can be directly and specificallyaffected not only via the eyes but also by light illuminated through the skull. Furthermore,monoamine production can be altered in both the central nervous system and the peripheral tissuesby transcranial illumination. This thesis demonstrates an alternate pathway for circadianentrainment and regulation by light involving specific molecular mediators such as opsins andmonoamines.

Keywords: circadian rhythm, monoamines, opsins, transcranial light

Flyktman, Antti, Kallon läpi annettavan valon vaikutus vuorokausirytmiikkaasääteleviin molekyyleihin. Oulun yliopiston tutkijakoulu; Oulun yliopisto, Luonnontieteellinen tiedekuntaActa Univ. Oul. A 719, 2018Oulun yliopisto, PL 8000, 90014 Oulun yliopisto

Tiivistelmä

Valo on tärkein yksittäinen tekijä nisäkkäiden vuorokausirytmiikassa. Tämä rytmi on kehittynytajoittamaan fysiologiset ja käyttäytymiseen perustuvat ilmiöt tapahtumaan oikeaan aikaan vuo-rokaudesta. Valosignaalin välittyminen silmien kautta aivoihin ja sen vaikutukset vuorokausiryt-miikkaan ovat hyvin tunnetut ja paljon tutkitut, mutta vielä on epäselvää, pystyykö kallon läpiannettava valo samaan, vaikka valon on osoitettu pystyvän läpäisemään nisäkkäiden kallon.Monoamiinit ja opsiinit ovat molekyylejä, jotka ovat tärkeässä roolissa vuorokausirytmiikan sää-telyssä, ja molempien ilmeneminen on riippuvainen valon määrästä.

Tässä väitöskirjassa valotettiin hiirien ja hamstereiden aivoja korvan kautta annettavallavalolla ja mitattiin kolmen eri opsiinin ekspressiota sekä monoamiinien määrää. Eläimiä valotet-tiin nukutuksessa joko valojakson alussa aamulla tai valojakson päätyttyä illalla. Opsiinien eks-pressio aivoissa mitattiin western blot -menetelmällä ja monoamiinien HPLC-menetelmällä.

Tuloksista huomattiin, että sekä opsiinien ekspressioon että monoamiinien pitoisuuksiin voi-daan vaikuttaa suoraan kallon läpi annettavalla valolla. Valohoidon vaikutus riippui tutkittavastamolekyylistä, kudoksesta ja valohoidon ajankohdasta. Näiden tulosten avulla pystyttiin osoitta-maan, että opsiinien, jotka ovat tärkeimpiä molekyylejä vuorokausirytmiikan säätelyssä, määräävoidaan manipuloida myös kallon läpi annettavan valon vaikutuksesta. Lisäksi kallon läpi annet-tavalla valolla voidaan vaikuttaa monoamiinien pitoisuuksiin sekä keskushermostossa että muis-sa kudoksissa. Tämä väitöskirja antaa tärkeää tietoa vuorokausirytmiikkaa säätelevistä molekyy-leistä ja osoittaa, että niihin pystytään vaikuttamaan myös muuten kuin silmien kautta annetta-valla valolla.

Asiasanat: monoamiinit, opsiinit, transkraniaalinen valo, vuorokausirytmi

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Acknowledgements

I would like to thank my supervisors Seppo Saarela, Satu Mänttäri and Markku Timonen, who have been guiding me through my doctoral studies. Seppo, you introduced me to this subject and offered me the possibility to do my PhD on opsins and transcranial light. I’m truly grateful for that. Satu, you have been my supervisor since my bachelor’s degree, and I can’t imagine better help for all three theses. You introduced me to western blotting and every single time I had troubles with the method, you always gave me good advice for troubleshooting. Besides the lab work, you gave me encouraging feedback on my writing, so I had little more energy to continue revising my text. Markku, although it was hard to reach you, I got probably the best writing tips from you. Every time we spoke, I had the feeling that I was the most important person taking your time at that moment. I will also remember couple phrases you used several times during our meetings: “This is how Benno has taught me” and “Since I’m only a doctor…”. Those gave me the impression that I’m not only your student, but an equal member of your scientific team. You gave me the opportunity to start my PhD studies with a grant from Northern Ostrobothnia Health District. In this regard I would like to thank Alfred Kordelin foundation and Valkee Inc. for granting my studies.

This thesis would not have been possible without the input of Juuso Nissilä, the father of the idea that mice can also be illuminated transcranially. I have always admired your enthusiasm towards science and especially neurology. You also introduced me to Valkee and were good advisor at the early stages of my thesis, when I needed assistance.

I am very grateful to our laboratory technicians Marja-Liisa Martimo-Halmetoja, Minna Orreveteläinen, Matti Rauman and Jorma Toivonen. Marsa and Minna, without your help many theses would not have been completed. You were of invaluable help during every phase of my laboratory work. Matti, you made my study design possible with modifying the Valkee headset. Every unit would need as good technical assistance as you gave us. Jorma, your work as an animal keeper was somewhat underrated, but not for me. Without you my hamster studies would not have succeeded.

My life would be totally different if I would have never entered the guild room. I have found so many good friends there, and shared memorable times over the years. Especially I would like to thank Jusku and Inka who have influenced my life so much. Jusku, I still don’t know, how you do it, but you understand me so well. There is no topic I couldn’t talk with you. Inka, I have valued your friendship a lot. You have always been there to listen to my troubles and I have shared the deepest

8

thoughts with you. Also, I have had the possibility to follow few of my friends doing their PhD simultaneously to me. Tuomo, Vekku, Kaisa and Nelli, you all have helped me in your own unique way in conducting my studies and you have shared your feelings in different moments throughout the doctoral studies. You all have been great friends outside the university too. When working as a member of the guilds board, I had the privilege to get to know two efficient ladies and one gentleman, Mari, Ansku and Timo. Your way of handling things and the ability to take responsibility made a huge effect on me and we became good friends while working together. And Nico, a fellow Milan fan. It became soon clear that you make a good addition to the group of biology students. I’m not sure would we be such good friends without the 40+h trip to Tallinn, but I’m glad we made it. It’s a calming thought that besides science we can always talk football too. Besides these friends, there are a lot other person with whom I have shared memorable moments during my studies, thank you all.

I would also like to thank Sanni, for sharing all these similar thoughts and feelings during the studies. It has helped a lot that you have faced the same situations and problems as I did. It’s almost surprising how similar feelings we have had towards our theses. Also, as we both are animal physiologists, we understand little better the theoretical and the experimental part of our work than others.

Antti and Anssi, the godfathers of Enni and my best mans, you have been my friends for such a long time. I remember when we once laughed that we all choose such a different field after high school, but still stay together as good friends. But still we share fundamental similarities, which keeps our good friendship going on. Thank you for everything we have shared together.

I want to thank my family: mom, dad and my sister Helena. You always have let me do the things I wanted to do. Although you have questioned some things, I have done, you have let me do them as I liked them to do.

And last, but definitely not the least, I have to mention the two most important persons in my life, Johanna and our little daughter Enni. Johanna, I could not imagine better person to share my everyday life with. You tolerate my flaws and encourage me to engage in every single thing I decide to do. You are truly an amazing woman, and a great mother. I love you. Our little wonder Enni, what a girl. You have made sure that at home I don’t have to think about work related stuff, and I love it. It has been my privilege to watch you grow, and this thesis has felt as a little thing compared to that lifelong duty.

18.8.2017 Antti Flyktman

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Abbreviations

5-HT serotonin

A adrenaline

BMI body mass index

CNS central nervous system

CONT control

DA dopamine

EL evening-light

ELISA enzyme-linked immunosorbent assay

GABA gamma-aminobutyric acid

HPLC high-performance liquid chromatography

ipRGC intrinsically photosensitive retinal ganglion cell

L-DOPA L-3,4-dihydroxyphenylalanine

MAO-A monoamine oxidase A

ML morning-light

mRNA messenger ribonucleic acid

NA noradrenaline

OPN3 encephalopsin

OPN4 melanopsin

OPN5 neuropsin

RHT retinohypothalamic tract

SCN suprachiasmatic nucleus

SDS sodium dodecyl sulphate

UV ultraviolet

10

11

List of original papers

This thesis is based on the following three manuscripts including two peer reviewed

publications, which are referred throughout the text by their Roman numerals:

I Flyktman A, Mänttäri S, Nissilä J, Timonen M & Saarela S (2015) Transcranial light affects plasma monoamine levels and expression of brain encephalopsin in the mouse. The Journal of Experimental Biology 218: 1521-1526

II Flyktman A, Jernfors T, Mänttäri S, Nissilä J, Timonen M & Saarela S (2017) Transcranial light alters melanopsin and monoamine production in mouse (Mus musculus) brain. Journal of Neurology Research 7(3): 39-45

III Flyktman A, Mänttäri S, Nissilä J, Timonen M, Yamashita T, Shichida Y & Saarela S (2018) The effects of transcranial light illumination on opsin expression in the Djungarian hamster (Phodopus sungorus) brain. Manuscript.

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13

Table of contents

Abstract

Tiivistelmä

Acknowledgements 7 

Abbreviations 9 

List of original papers 11 

Table of contents 13 

1  Introduction 15 

1.1  Circadian rhythm..................................................................................... 15 

1.2  The mammalian brain ............................................................................. 18 

1.2.1  The hypothalamus and the suprachiasmatic nucleus (SCN) ......... 19 

1.2.2  The cerebellum ............................................................................. 20 

1.2.3  The cortex ..................................................................................... 21 

1.3  Photoreception ........................................................................................ 21 

1.4  Opsins ..................................................................................................... 25 

1.4.1  Encephalopsin (OPN3) ................................................................. 27 

1.4.2  Melanopsin (OPN4) ..................................................................... 27 

1.4.3  Neuropsin (OPN5) ........................................................................ 28 

1.5  Monoamines ............................................................................................ 30 

1.5.1  Serotonin ...................................................................................... 30 

1.5.2  Dopamine ..................................................................................... 32 

1.5.3  Noradrenaline and adrenaline ....................................................... 34 

2  Aims of the study 37 

3  Materials and methods 39 

3.1  Study species ........................................................................................... 39 

3.1.1  Mouse ........................................................................................... 39 

3.1.2  Djungarian hamster ...................................................................... 39 

3.2  Study design ............................................................................................ 40 

3.3  Molecular methods .................................................................................. 42 

3.3.1  Western blot .................................................................................. 42 

3.3.2  HPLC ............................................................................................ 43 

3.3.3  Melatonin and cortisol determination ........................................... 43 

3.4  Statistical analyses .................................................................................. 44 

4  Results 45 

4.1  Opsins ..................................................................................................... 45 

4.2  Monoamines ............................................................................................ 46 

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4.3  Body masses and BMIs ........................................................................... 47 

5  Discussion 49 

5.1  The opsins ............................................................................................... 49 

5.2  Monoamines ............................................................................................ 52 

5.3  The connection between opsins and monoamines .................................. 54 

5.4  Weight, length and BMI .......................................................................... 55 

6  Conclusions and future prospects 57 

References 59 

Original papers 69 

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1 Introduction

As long as there has been life on planet earth, light has been an essential factor for

all living organisms. Above all, sun light is needed for photosynthesis, a

phenomenon that produces oxygen. Apart from photosynthesis, light is needed for

regulating various cycles that are essential for the life cycle of an organism. Such

things as growing season, reproductive cycle and migration are included to these

cycles and they all are dependent on the photoperiod. Although the events are

endogenous, light is a reliable agent to keep them synchronized with the right time

of the year or day.

Light is the most important regulating agent in rhythmicity. In vertebrates, the

rhythms – with different lengths – include circannual, infradian, circadian and

ultradian rhythms. The circannual (approximately one year), infradian rhythm

(between a year and a day) and ultradian (under 24 hours) rhythms are important

for animal homeostasis. This thesis concentrates on the circadian rhythm (form

Latin circa: about, diem: a day). The circadian rhythm includes events that occur

approximately every 24 hours. As other rhythms, the circadian rhythm exists

without environmental cues, therefore being endogenous. In this thesis, the focus

is on the properties of the circadian rhythm, what kind of effects light has on it, and

which structures and molecules are regulating the rhythm.

1.1 Circadian rhythm

All organisms need the endogenous ability in adapting to varying environmental

conditions, especially the changing light conditions. When the intensity of light

alters, during dawn and dusk, organisms need to respond to it by adjusting their

physiological and behavioral events. Biological rhythms are found in all taxons,

including mammals. Animal rhythmicity may be divided into several rhythms of

different lengths. The study of biological rhythms, which includes e.g. annual,

seasonal, and daily rhythms, is called chronobiology.

The most interesting part of chronobiology from the point of view of this thesis

is the mammalian circadian rhythm. The circadian system is evolved to set phases

at which physiological and behavioral events occur in a 24-hour period. This 24-

hour period can be roughly divided into two, usually unequal length, sections: the

dark phase and the light phase. Since night and day have differing demands

regarding the behavioral events (such as feeding and sleeping) of an animal,

activities of different systems have to be tuned regularly. Furthermore,

16

physiological processes, like hormone secretion, differ depending on the time of

the 24-hour period. One of the characteristics of circadian rhythm is that it appears

even without environmental cues, zeitgebers (Roenneberg et al. 2003). Therefore,

the rhythm is under genetic control, e.g. rodents have special clock genes to

maintain the rhythm (Ralph & Menaker 1988, Vitaterna et al. 1994). Although the

rhythm works without environmental cues, it should be entrained regularly to keep

it synchronized with the external 24-hour clock (Bellingham & Foster 2002). If all

external zeitgebers are absent, the animal enters a free-running mode, which varies

between species and is less or more than 24 hours (Campbell & Murphy 1998). If

the endogenous rhythm is shorter than 24 hours, which usually occurs in nocturnal

animals, the phase of the rhythm advances and different events occur earlier daily.

However, if the endogenous rhythm is longer than 24 hours, usually occurring in

diurnal animals, the phase delays and the physiological and behavioral events

appear later daily (Roenneberg et al. 2003).

As mentioned above, without zeitgebers, the circadian rhythm free-runs, and

the length of its period is less or more than 24 hours (Campbell & Murphy 1998).

Since the rhythm is not entirely synchronized with the solar day, it must be

entrained in order to work optimally. Although there are some other external signals,

such as temperature and feeding patterns, the most common zeitgeber is the amount

of light. Other signals are also required for entrainment, but they usually interact

with light (Schibler et al. 2003). Especially dawn and dusk – when the light

intensity changes significantly at a short notice – are the key points for entrainment.

For animals, there is a certain threshold, at which the response occurs. For example,

dim light does not necessarily affect a response, but bright light does. The threshold

intensity is also dependent on the time of the biological day of an animal

(Roenneberg et al. 2003, Duffy & Czeisler 2009). The entrainment is most

sufficient when the animal is most sensitive to it. For example, humans are much

more sensitive to light stimulus in their biological night than during the biological

daytime (Campbell & Murphy 1998).

As a sign of the importance of light in entrainment, many animals – especially

rodents – become active or inactive just depending on the lighting conditions

(Aschoff & Vongoetz 1988). As a natural continuum, Beersma et al. (1999) showed

that the light history has clear effects on the patterns of entrainment and helps to

tune the system to its environment. Light is a key factor in entrainment, since it

affects the organism in two different ways: indirectly through the circadian system

or directly through mechanisms that differ from the circadian rhythm, such as mood

and alertness (Stephenson et al. 2012). Considering entrainment, light is generally

17

less effective in phase shifting during the subjective day and most effective in

circadian resetting during the subjective night (Roenneberg et al. 2003). Regarding

the effects of temperature on entrainment, light exposure after lowest core

temperature (late night) causes an advance in the circadian system and light

exposure before the lowest core temperature (early night) causes delay in the

circadian system (Roenneberg et al. 2003, Stephenson et al. 2012).

Generally, the period of the diurnal animals is usually longer than 24 hours,

which means that if entering the free-running mode, the phase would be delayed.

The usual way of resetting the system is by a small phase advance, e.g., light

exposure in the late night, every day. For nocturnal animals, whose rhythms are

usually shorter than 24 hours, entrainment is achieved by a daily phase delay shift.

The strength of synchronizing signal also differs depending on the circadian period.

If the period of an animal is close to 24 hours, it needs a weaker synchronizing

signal than an individual, whose period is further away from 24 hours (Duffy &

Czeisler 2009).

When the light signal reaches the eye, it is gathered and transmitted into the

hypothalamus, where the endogenous biological central clock or the master clock

is located. The master clock gets endogenous information from the so called

peripheral slave clocks laid in every compartment or even almost in all animal cells.

Clocks maintain their owner’s circadian rhythms such as feeding and sleeping

which are synchronized by the master clock (Schibler & Sassone-Corsi 2002). The

suprachiasmatic nucleus (SCN), or master clock, is the timekeeping system, which

maintains the circadian system even if external pacemakers are absent. The clock

includes molecules, especially opsins, which keep the clock near 24 hours in

mammals. External stimuli, usually light intensity, change the conformation of

these molecules in the appropriate stage of the circadian cycle (Toh 2008). After

the light information reaches the SCN, the circadian signal is transmitted to tissues

via humoral and neuronal signals. This enables the adjustment of metabolism and

body functions to the current time of the day. Master clock and peripheral clocks

share similar mechanism leading to synchronization (Poletini et al. 2015). The main

difference is that peripheral clocks lose their rhythm more easily if there are

disturbances (Yamazaki et al. 2000). It is interesting to note that the central clock

is almost only entrained by light, but the peripheral clocks are influenced by

temperature, feeding and hormones as well (Poletini et al. 2015).

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1.2 The mammalian brain

The brain is an organ, which is the center of the nervous system in all vertebrates

and many invertebrate animal species. It is located near the sensory organs, such as

eyes, ears, nose and mouth. As a sing of its importance, for example, the human

brain weights only about 2% of the total body weight, but consumes 20% of the

energy produced. In mammals, the brain is the most important regulatory center. In

addition to circadian rhythmicity the brain regulates several behavioral and

hormonal patterns, e.g. feeding and sleep-wake cycle (Bear et al. 2016). In this

chapter, the role of the mammalian brain in controlling circadian rhythmicity will

be presented. The hypothalamus, the cerebellum and the cortex are introduced more

closely, since they were specifically targeted in this study and were served as tissues

for biochemical analysis in this thesis. These three tissues have been selected due

to their importance in regulating mammalian circadian physiology. The

hypothalamus is the main regulatory center, also in rhythmicity (Ralph et al. 1990),

but the cerebellum (Paulus & Mintz 2016) and the cortex (Rath et al. 2013, Rath et

al. 2014) are shown to affect circadian rhythmicity also. The location of these three

tissues in the rodent brain are presented in Figure 1.

Fig. 1. The picture of the rodent brain (modified from Nissilä et al. 2012) indicating the

locations of the hypothalamus, the cerebellum and the cortex as outlined. The vertical

lines indicate the places for histological samples.

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1.2.1 The hypothalamus and the suprachiasmatic nucleus (SCN)

The hypothalamus is a tissue in the brain, which contains many nuclei that have

various functions. It is located under the thalamus and just above the brain stem

(Fig. 1). Although the hypothalamus is small, it influences many essential

behavioral, endocrine and autonomic functions in the body (Belk & Borden 2009).

It plays a key role in maintaining the whole body internal balance, also known as

homeostasis. The hypothalamus is also the link between the endocrine system and

the nervous system, and it is responsible for production of many essential hormones

and other chemical substances that control different cells and organs. The

hypothalamus uses set points in relation to blood pressure, body temperature or

lighting conditions in order to regulate body systems. It receives input from the

organism and makes changes if anything differs from the set points.

The part of the hypothalamus that is responsible for the regulation of the

circadian rhythm is the SCN, the so called circadian master clock. According to

generally accepted theory regarding mammalian phototransduction, the light

information from the retina reaches the SCN mostly via the retinohypothalamic

tract (RHT) (Bellingham & Foster 2002). The SCN is located in the anterior part

of the hypothalamus and besides entraining rhythmicity, it controls molecular and

physiological rhythms including sleep and alertness, physical activity, hormone

levels and body temperature (Toh 2008). The structure of the SCN is complex,

which may be due to its many different tasks in regulating body physiology and

rhythmicity (Morin et al. 2006). Although the ability of SCN to act as a circadian

clock was demonstrated earlier, its importance in circadian rhythmicity was first

discovered by Ralph et al. (1990) when they created a hamster line with a specific

mutation affecting circadian rhythms. This mutation reduced the period of circadian

rhythm from about 24 h to about 22 h in heterozygotes and about to 20 h in

homozygotes. After SCN transplantation, the normal duration of the circadian

rhythm was restored.

The circadian rhythm is set, when the SCN neurons entrain the circadian

rhythm according to certain light-dark cycles. Each of them operates autonomously

and generates its own electrical potential. The SCN is also able to sense the

environmental timing cues and different hormonal responses, sustaining the

circadian rhythm (Bernard et al. 2007). This happens via regulating circadian

expression of the genes in peripheral tissues through a combination of neural

pathways, neurohormones and neuropeptides. In lower vertebrates, the circadian

rhythm is sustained not only through the RHT, but also directly through the

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geniculohypothalamic tract from the intergeniculate leaflet of the thalamus and

serotonergic input from midbrain raphe neurons (Toh 2008). Besides photic cues,

these pathways outside the RHT may allow for the non-photic entrainment cues to

the SCN.

In addition to gathering light information, the SCN projects it to other brain

parts. Thereby projected light information modifies wakefulness, thermoregulation

and feeding, memory and learning, mental performance and endocrine secretion

(Toh 2008). The SCN receives serotonergic input from the median raphe nucleus

of the midbrain. Selective elimination of serotonergic input to the SCN is able to

amplify circadian responses to light. Retinal ganglion cells synthesize serotonin

receptors in the retina and ship them to SCN where they function as presynaptic

inhibitory receptors. Activation of these receptors modulates the response of the

SCN to photic input (Pickard & Sollars 2010). In this thesis the main focus is on

the SCN, which is the most important structure in circadian rhythmicity.

1.2.2 The cerebellum

The cerebellum is a region in the brain that is especially important for motor control.

Anatomically, the cerebellum is located under the cerebrum, behind the brain stem

(Fig. 1), and it is attached to the bottom of the brain. The cerebellum is made of

two hemispheres and it is divided into two compartments, the cortex and the deep

nuclei. The cortex contains Purkinje cells, which project through an inhibitory

system, to the deep nuclei. The deep nuclei then send information to other brain

regions (Raymond et al. 1996). The cerebellum receives input signals from the

sensory systems and other parts of the brain to regulate motor control (reviewed by

D'Angelo et al. 2011). The cerebellum regulates voluntary movements such as

posture, balance, coordination and speech. This regulation may be mediated by low

frequency oscillation, which enables the communication between cerebellum and

other brain parts (D'Angelo et al. 2009). Although the cerebellum constitutes a

relatively small portion of the brain mass, of note is that it contains half of the

brain’s neurons (Raymond et al. 1996).

The interest for this thesis is due to the fact that the cerebellum has been shown

to contain circadian clocks (Rath et al. 2012, Rath et al. 2014). Different clock

genes are shown to have rhythmic patterns within mouse cerebellum. Although

these circadian clocks have their own distinct rhythms, they may be controlled by

the SCN (Rath et al. 2012, Rath et al. 2014, Paulus & Mintz 2016). The significance

of SCN regulation is shown by the fact that the rhythm of the cerebellar clock is

21

delayed 5 hours compared to that of the SCN, thereby suggesting a master-slave

relationship between these two tissues (Rath et al. 2012, Rath et al. 2014).

1.2.3 The cortex

The third tissue of interest for this thesis is the cerebral cortex. It is a thin tissue

under the skull, and has two types of cortices, the primary cortices and the

association cortices (Fig. 1). The primary cortices have areas that receive sensory

input involving e.g. limb or eye movement, where the association cortices are

responsible for more complex functions such as memory, language and emotions.

The layered structure allows cortical areas to connect more easily with each other

and maintain the normal body functions (Shipp 2007).

It has been demonstrated that the cortex is under the control of SCN in

circadian rhythm regulation. Similar clock genes, which have been found in the

SCN, are also expressed in the rodent neocortex. To indicate the importance of the

SCN, these clock genes are incapable to work properly in cortex without intact

SCN (Rath et al. 2013, Rath et al. 2014). Furthermore, as a sign of its association

in circadian rhythmicity, encephalopsin (OPN3), a mediator of circadian rhythm

regulation, has also been found in the cortex (Blackshaw & Snyder 1999).

1.3 Photoreception

As mentioned above, light is the most important regulating factor in the timing of

many physiological and behavioral events of an animal. In this chapter, the focus

is on the mechanism, how light information is gathered and processed in vertebrates.

The event, where light stimulus is gathered and transmitted forward, is called

photoreception. It can be divided in two parts: visual and non-visual photoreception.

Eyes are the primary structure for photoreception, and inside the eyes, there

are two different pathways, visual and non-visual photoreception pathways. Visual

photoreception is the part in light detection that leads to vision and depends on

visual photoreceptors, which are called rods and cones. These photoreceptors are

located in the retina, the sensory part of the eye. Rods are the dominant light

receptors in mammals. They gather light information through the photopigment

rhodopsin, which (as other opsins) consists of an opsin protein and a retinal part

that absorbs light. Rod cells are most effective in dim light conditions. When the

retinal of the rod cell absorbs light, it is transmitted to ganglion cells. The ganglion

cells further transmit light information via the optic nerve to the visual cortex of

22

the brain. Cone cells absorb light most effectively in well-lighted conditions, and

they are responsible for color vision. There are different types of cone cells, all

acting most effectively at different wavelengths and thereby absorbing different

colors (Hunt et al. 2009, Kefalov. 2012).

Besides visual photoreception, non-visual photoreception is an event that can

process light information without leading to vision. In the eye, a key point for

gathering photons for both visual and non-visual photoreception, is formed by the

intrinsically photosensitive retinal ganglion cells (ipRGCs), which are connected

and form a syncytium of photosensitive and nonphotosensitive neuronal cells in the

retina (Berson et al. 2002, Hankins et al. 2008, Pickard & Sollars 2010). Unlike

rods and cones, which hyperpolarize in response to light, ipRGCs depolarize and

trigger action potentials, which run into the brain. While cones and especially rods

are easily activated, the light reaction of IpRGCs is less sensitive, and the response

can be very slow in dim light. Although the stimulation is slow, ipRGCs are capable

to absorb a single photon. After light absorption, it can take several seconds to reach

peak response and the response may continue for minutes after the end of the

stimulus. The low photon capture rate allows the majority of photons to pass by the

inner retina to interact with rods and cones and thereby not degrading the visual

image (Bailes & Lucas 2010, Pickard & Sollars 2010). IpRGCs transmit

information to the circadian master clock by activating melanopsin (OPN4), and

the ability for this kind of phototransduction is the most striking feature of these

cells. As a sign of their importance for phototransduction, many animals have

melanopsin-positive cells across the retina with only little differences in

distribution (Hattar et al. 2002, Pickard & Sollars 2010).

23

Fig. 2. Illustration of the phototransduction mechanism in the mammalian eye (modified

from Shichida & Matsuyama 2009) with the ganglion cells and the beginning of the RHT

highlighted.

Non-visual photoreception is a part of circadian rhythmicity. As a regulator for the

circadian rhythm, the brain utilizes two specialized tissues, the RHT and the SCN.

The RHT is a part of nervous system, and has is evolved to transmit light

information collected by the ipRGCs (Fig. 2). After ipRGC activation, the RHT is

also activated. After activation, the light information is transmitted via RHT, which

traverses along the optical nerve, to the master circadian clock located in the SCN

of the hypothalamus (Moore & Lenn 1972, Zele et al. 2011). The RHT is distinct

from other neurons in the eye, and the majority of its nerve endings are located in

the SCN (Hannibal 2002). The RHT is represented in Figure 3.

24

Fig. 3. Generalized illustration of mammalian optic nerve (modified from Sancar 2004)

indicating the route for the RHT via the retina and the SCN.

Although the eyes are the primary tissue for photoreception, some regions in the

brain are also capable to gather light information directly. Deep brain

photoreception or extraocular photoreception is an event where light information is

collected by non-image forming tissues outside the eyes and delivered to the SCN.

Deep brain photoreception has been best characterized in non-mammalian

vertebrates, and it is shown to occur at several sites, most importantly in the pineal

gland. The pineal gland is connected to the parietal eye in lower vertebrates and is

shown to control the circadian rhythm (Eakin 1973). It has earlier been shown that

OPN4, which is a key molecule for transmitting light information, has been found

in the pineal gland of birds where it also entrains the circadian clock (Bailey &

Cassone 2005).

Deep brain photoreception is well known in non-mammalian species. However,

light is also able to enter the brain through the skull bone in several mammalian

species, including rodents (Brunt et al. 1964). It is known that extraocular

photoreceptors can mediate circadian regulation, and are apart from the ocular light

receptors, which regulate vision (Campbell et al. 2001). Furthermore, Benoit (1964)

showed that the avian brain has photoreceptors. While the removal of eyes did not

eliminate testicular growth of ducks, a black cloth placed around their heads

prevented the corresponding response. This indicates that the brain contains

extraocular photoreceptors. Later, Lisk and Kannwischer (1964) showed that the

direct stimulation of SCN by light could regulate the estrous cycle, which was

commonly thought to be regulated through the eyes only. In mammals, light may

affect the brain neurotransmitter release independently by acting on other structures

25

than the retina(Wade et al. 1988). This indicates that like in other vertebrates, the

mammalian brain has deep brain photoreceptors too. In humans, light is able to

penetrate the skull and activates several areas in the cerebral cortex (Starck et al.

2012, Persinger et al. 2013, Sun et al. 2016). It has been hypothesized that deep

brain photoreception in mammals may be evolutionary relict. During the nocturnal

bottleneck, the deep brain photoreceptors were moved into the eye to maximize the

light sensitivity (Heesy & Hall 2010), and can now be found in both eyes and the

brain. One evidence indicating this change and evolutionary development is that

opsins, which are presented in the next chapter, are also found in the mammalian

brain (Blackshaw & Snyder 1999, Nissilä et al. 2012).

1.4 Opsins

The opsins are molecules most importantly responsible for visual and non-visual

phototransduction. They are old and form a very wide spread G-protein coupled

protein family with a molecular mass of 30–55 kDa, and are mostly found in the

retina and the brain (Porter et al. 2012). The non-visual opsins were rediscovered

in the 1990’s (Max et al. 1995). They exist in all vertebrate groups and are widely

expressed in all photosensitive tissues. Therefore, non-visual opsins, along the

clock genes, have an important role in synchronizing the body functions to the

ambient photoperiod (Poletini et al. 2015).

The function of opsins can be divided in two parts: the absorption of light and

G-protein activation. The common structure for all opsins is the seven

transmembrane helices that are involving in several signaling functions. Their

difference to other G-protein coupled receptors is that opsins have lysine in the

seventh transmembrane helix, which allows the connection to the chromophore

(Schertler 1998). The opsins become photosensitive by attaching the chromophore.

With this event, the conformation of the chromophore changes from the cis- to the

trans-isomer, and the light energy is transduced into chemical-free energy (see Fig.

4). The energy is used for conformational changes in the protein to activate the G-

protein coupled receptor. The opsins are key molecules in detecting and

transmitting the light information to orchestrate different biological phenomena

(Shichida & Imai 1998, Porter et al. 2012).

26

Fig. 4. Structures of opsins and the chromophore retinal (modified from Terakita 2005).

a) The secondary structure of bovine rhodopsin with highly conserved amino acid

residues indicated with dark grey. b) A plot on the cis-trans photoisomerization. The

transformation changes from cis to trans isoform by light is indicated by the boxes. In

the 11-cis isoform the two hydrogens are between the carbon atom 11 and carbon atom

12 on the same side of the double bond, whereas in the all-trans isoform the hydrogens

are on the opposite sides of the molecule.

Opsins can be divided in visual and non-visual opsins depending on the ability to

carry photoreception. Visual opsins can further be subdivided in cone opsins (cone

cells, underlie color vision) and rhodopsin (rod cells, underlie twilight vision).

Cone opsins have four different subgroups depending on their absorption spectra.

To confirm the long evolutionary history of opsins, lower invertebrates have some

non-visual opsin genes, which belong to the same subfamily as the vertebrate visual

opsins (Terakita 2005). This thesis is focusing on three non-visual opsins,

encephalopsin, melanopsin and neuropsin, all of which are shown to play a role in

27

circadian rhythmicity (Ruby et al. 2002, Koyanagi et al. 2013, Buhr et al. 2015)

and are also found in the mammalian brain (Blackshaw & Snyder 1999, Nissilä et

al. 2012, Yamashita et al. 2014b, Nissilä et al. 2016).

1.4.1 Encephalopsin (OPN3)

Encephalopsin (OPN3) was first discovered by Blackshaw and Snyder (1999), and

was the first opsin specifically expressed in the mammalian brain. OPN3 has the

highest homology with vertebrate pineal and retinal opsins, which have been shown

to play an important role in phototransduction to the brain. More accurately, OPN3

is highly expressed in the paraventricular nucleus of the hypothalamus and the

medial preoptic area. Both tissues are involved in encephalic phototransduction

(Blackshaw & Snyder 1999). Besides its widespread distribution in rodents such as

the mouse, human OPN3 is also found in several other tissues (Halford et al. 2001).

This is not a surprise, since rodent and human OPN3 show different characteristics

at gene level (Kasper et al. 2002), and OPN3 homologs can act as light sensors in

various tissues (Koyanagi et al. 2013). For light transmission, OPN3 activates a G-

protein-coupled receptor after light administration. It is capable to form blue-light

sensitive pigments, with an absorption maximum of around 465 nm (Sugihara et al.

2016).

Although the OPN3 was already found in 1999, the first detection in the brain

was made at the mRNA level (Blackshaw & Snyder 1999). Thus, it was still unsure

whether OPN3 acts as a functional protein in the cells. Significantly, Nissilä et al.

(2012) later found out that OPN3 protein is also expressed in the cortex, the

cerebellum and the hypothalamus in mouse brain. The finding of Nissilä et al. (2012)

was of great impact and importance, since mRNA expression does not necessarily

correlate with the existing protein levels (Greenbaum et al. 2003), although

regarding OPN4, the expression levels of protein and mRNA showed similar

patterns (Hannibal et al. 2013).

1.4.2 Melanopsin (OPN4)

Before OPN3, another opsin namely OPN4 was found in melanophores, brain and

eye of the African clawed frog (Xenopus laevis) (Provencio et al. 1998). Since it is

isolated from a melanophore cDNA library, this opsin is called melanopsin. After

its discovery, OPN4 has been the most intensively studied opsin, because of its light

transmitting characteristics.

28

Although OPN4 is a vertebrate opsin, it shares more common features with

invertebrate opsins. For example, as Drosophila rhodopsin, OPN4 is a bi-stable (it

has both ultraviolet (UV) sensitive and light sensitive states) molecule (Koyanagi

et al. 2005). In non-mammals, OPN4 is expressed in both ocular and non-ocular

photoreceptors, while in mammals, the main site for OPN4 expression is being

formed by the retinal ganglion cells, which projects to the SCN via the RHT (Fig.

3). Therefore, it is thought that inside the ganglion cells, OPN4 works as the most

important regulator of the circadian rhythm and photoperiodic responses

(Provencio et al. 1998), although the circadian clock works without OPN4 (Panda

et al. 2002). The latter finding was confirmed by Ruby et al. (2002); they showed

that OPN4 is not essential for light transmission, despite of being an important

photoreceptor. Ruby at al. (2002) found that light pulse during the dark phase

induced phase delays in wild and OPN4 knock-out mice. Furthermore, the light in

normal light-dark cycle entrained the knock-outs too, and the rhythm period did not

differ in total darkness between the wild type and OPN4 knock-outs. Additionally,

the finding that visual photoreceptors can support non-visual photoreception, if

OPN4 expression is reduced, confirms the conclusions of Ruby et al. (2002) and

Hankins et al. (2008).

As mentioned, OPN4 is capable of absorbing both, visual and UV wavelengths.

The blue light wavelengths excite OPN4 most effectively; the absorption maximum

of OPN4 is 420–480 nm, depending on the source (Newman et al. 2003, Melyan et

al. 2005, Walker et al. 2008). The peak sensitivity correlates well with non-image

forming responses – such as photoentrainment – in natural circumstances for

instance by prolonged light exposure, when rods and cones are already adapted.

Furthermore, the highest sensitivity of sleep-wake cycle to light exposure is at 460–

480 nm. Therefore, OPN4 is capable of modulating sleep in both constant and

variable lighting conditions (Tsai et al. 2009).

Besides the effects in the central nervous system (CNS), OPN4 has a regulating

role in the peripheral tissues too, especially in the blood-vascular system. Sikka et

al. (2014) showed that functional OPN4 receptors are found in blood vessels of the

mouse. Additionally, the activation of OPN4 by blue light leads to vasorelaxation

in the mouse aorta.

1.4.3 Neuropsin (OPN5)

The most recently discovered opsin studied in this thesis is neuropsin (OPN5). It

was first characterised 2003in mice and humans (Tarttelin et al. 2003). The

29

expression of OPN5 is neuron specific. Therefore, it is called neuropsin. Since

OPN5 is expressed in both neural retina and retinal pigment epithelium in the eye,

Tarttelin et al. (2003) hypothesized that it may have some function in light detection.

Their hypothesis was later proven right by other studies (Nieto et al. 2011,

Yamashita et al. 2014b, Buhr et al. 2015).

Besides retina, OPN5 is expressed in the brain – specifically the preoptic area

of the hypothalamus – and testis (Yamashita et al. 2014b). In birds, OPN5 is found

in the retina, pineal and paraventricular organ in the hypothalamus, where it may

be a part of controlling system in seasonal reproduction cycle (Nakane et al. 2010,

Yamashita et al. 2010).

As other opsins, the OPN5 also binds to the G-protein, especially the Gi

subtype of G-protein (Koyanagi & Terakita 2008, Yamashita et al. 2010). Although

OPN5 belongs to the opsin superfamily, it shows significant differences compared

to other opsins (Tarttelin et al. 2003). The distinctive feature of OPN5 is the

sensitivity towards UV light. Another interesting difference in OPN5 compared to

other opsins studied in this thesis is that OPN5 binds 11-cis-retinal because it is less

sensitive to light when binding to the all-trans-retinal form of the chromophore. By

binding the cis-retinal form, OPN5 can be more sensitive to short wavelength light,

and it is therefore considered to constitute a very important entraining agent in the

retina (Buhr et al. 2015).

While OPN3 and OPN4 are mostly visible light absorbing photopigments,

OPN5 is subdivided in different classes depending on their molecular properties.

Mammalian OPN5 is a bistable photopigment and its distinctive feature is the

ability to absorb both, UV-light and visible light, with a peak sensitivity in the UV-

light region (Kojima et al. 2011). The two forms are interconvertible by light

irradiation. The 11-cis-retinal state is capable of absorbing UV-light at wavelength

360 nm and the all-trans-retinal absorbs visible light in wavelength 469 nm

(Terakita 2014). When OPN5 is active, it binds visible light, and couples with the

G-protein. OPN5 is also able to activate G protein in UV-light, but this activation

is lost after yellow-light irradiation (Yamashita et al. 2014b). Chicken OPN5 has

similar properties including UV-light absorption, but it also may function as a

chemoreceptor using the all-trans-binding agonist in non-photoreceptive organs

(Ohuchi et al. 2012). Unlike other lower vertebrates, ray-finned fishes (Lepisosteus

oculatus) have a mammalian OPN5-like molecule. As the mammalian and chicken

OPN5, this protein absorbs UV-light when binding to the 11-cis-retinal.

Interestingly, this OPN5 form is not able to bind all-trans-retinal, and therefore it

is only suited for UV-light detection (Sato et al. 2016).

30

The ability of OPN5 to absorb short-wavelength light is important for

entrainment (Buhr et al. 2015). The ex vivo retinal circadian rhythm of the mouse

is most sensitive to short-wavelength light, and the retinas that were lacking OPN5

were unable to photoentrain even when other visual functions seemed to be normal.

Additionally, the corneal circadian rhythm is also regulated by functional OPN5.

Although these results sound very promising, it is not yet known, how OPN5 works

at the brain level when synchronizing biological rhythms in humans or other

mammalian species.

Among its functions as a circadian rhythm modulator, OPN5 positive neurons

occur in the paraventricular organ that mediate seasonal reproduction cycles at least

in birds (Nakane et al. 2010). They are most likely able to use also short-wavelength

light in their natural habitats, because blue and UV-light regulate photoperiodism

and circannual rhythm in mammals and insects.

1.5 Monoamines

In this chapter the focus is on serotonin and the catecholamines dopamine,

noradrenaline and adrenaline, which all have been well studied and are shown to

play a role in regulating circadian rhythmicity. Monoamines are neurotransmitters

that contain an amino group that is connected by two carbon chain to an aromatic

ring (Fig. 5 and Fig. 6). The monoamines are regulating processes such as memory,

emotions, arousal and rhythmicity.

1.5.1 Serotonin

Serotonin (5-hydroxytryptamine) is one of the four monoamines studied in this

thesis. Unlike the other studied monoamines (dopamine, noradrenaline and

adrenaline), serotonin is formed by a different synthesis pathway (Fig. 5). Its origin

is the amino acid tryptophan that is ingested in the diet. Even though serotonin and

catecholamines are synthesized from different amino acids to derive from, they

share similar biochemical features (Rapport et al. 1949). Serotonin synthesis begins

with the enzyme tryptophan hydroxylase, which converts tryptophan into 5-

hydroxytryptophan and 5-hydroxytryptophan is then decarboxylated to serotonin.

The concentration of serotonin is directly proportional to the amount of tryptophan

available for synthesis (Sanchez et al. 2008). Serotonin is found mostly in the

gastrointestinal tract, the blood platelets and the CNS. In the CNS, serotonin

regulates almost all mammalian behavioral processes, although only less than one

31

in a million CNS neurons produce serotonin (Gershon & Tack 2007, Berger et al.

2009). In the brain, serotonin is most abundantly expressed in the raphe nucleus, a

structure regulating sleep-wake states. Here the serotonergic neurons are the main

neuronal components and are a serotonin source for the rest of the brain (Hornung

2003). Serotonin regulates almost all behavioral events, such as mood, sexuality,

memory and appetite.

Fig. 5. The synthesis of serotonin from the amino acid tryptophan (modified from

Mueck-Seler & Pivac 2011). The boxes show the substitutes in the molecules where the

chemical reactions affect.

In circadian rhythmicity, the wide expression levels of serotonin receptors inside

the SCN, and the fact that raphe nucleus projects to the hypothalamus, shows that

serotonin activity has a regulatory role in processing the photic information in the

circadian system and therefore is an entraining agent (Rea et al. 1994, Amir et al.

1998, Deurveilher & Semba. 2008). Although serotonin transmits the photic

information, its rhythm is generated endogenously. Dudley et al. (1998) showed a

nadir in the late midday followed by a sharp increase in serotonin release in

14L:10D light dark cycle in the hypothalamus. Although the rhythm of serotonin is

endogenous, its secretion peaked at the dark/light-transition and the output declined

to basal levels during the night. As a sign of the importance in circadian rhythmicity,

32

the serotonin secretion patterns were similar in total darkness, with increased

secretion at the beginning of a subjective night. Additionally, serotonin shows

similar secretion patterns in different brain regions (Sanchez et al. 2008).

1.5.2 Dopamine

Differing from serotonin, dopamine is derived from tyrosine and belongs to the

catecholamines. Catecholamines share all the same structure of a benzene ring with

two hydroxyl groups attached to it, an ethyl chain and an amino group (Fig. 6).

Dopamine can be synthetized from tyrosine and phenylalanine, as phenylalanine

can be converted to tyrosine by hydroxylation. Tyrosine itself can then also be

hydroxylated to form L-3,4-dihydroxyphenylalanine (L-DOPA). With a

decarboxylation, L-DOPA is transformed into dopamine. These events of dopamine

formation occur in the brain and in the medulla of the adrenal gland. In the brain

and the central nervous system, dopamine functions as a neurotransmitter. Outside

the nervous system, dopamine serves as a local chemical messenger (Wurtman &

Fernstrom 1975).

33

Fig. 6. Catecholamine synthesis. The synthesis of catecholamines from the amino acid

tyrosine (modified from Kuhar et al. 1999). The boxes mark the places in the molecules

where the chemical reactions affect.

34

Dopamine affects the circadian rhythmicity by synthesis in the CNS. The retina, as

discussed earlier, constitutes a very important place for regulating circadian

rhythmicity. Dopamine and melatonin are key molecules for modulating the

circadian rhythmicity.. While melatonin is mostly expressed during the night, the

peak expression time of dopamine is in the daytime (Mendoza & Challet 2014). In

the retina, dopamine regulates the circadian rhythm also by altering melanopsin

mRNA expression (Sakamoto et al. 2005). Dopamine has been shown to affect the

circadian rhythm in the retina and in the brain. In the rat brain, the circadian rhythm

of dopamine can be dependent or independent of the environmental lighting

conditions, as demonstrated in different brain tissue. For example, in the striatum,

light was shown to affect the circadian rhythm of dopamine, while in the nucleus

accumbens the circadian rhythm of dopamine was independent of light (Castaneda

et al. 2004).

Even if the effects of light on dopamine levels are still unknown, dopamine

may have a role in regulating circadian rhythmicity. Few studies have shown that

dopamine receptors are found in the SCN (Sleipness et al. 2007, Mendoza &

Challet. 2014). The fact that dopamine is an important neurotransmitter especially

in the prenatal stage and mother’s dopamine secretion also signals the daytime

period to the fetus underline the importance of this catecholamine in circadian

rhythm control (Viswanathan & Davis 1997), although the sensitivity of the clock

to dopamine declines after birth (Weaver & Reppert 1995). Apart from the circadian

effects, the dopaminergic system in the brain can influence motor functions,

motivation and drug intake (Mendoza & Challet 2014).

1.5.3 Noradrenaline and adrenaline

As dopamine, noradrenaline and adrenaline belong to the catecholamine group and

they both are synthesized from precursor dopamine (Fig. 6). Noradrenaline and

adrenaline are synthesized in the medulla of adrenal gland or postganglionic cells

of the sympathetic nervous system and they can function as neurotransmitters

(inside the nervous system) or hormones (through blood circulation). Adrenaline is

derived from noradrenaline with an addition of a methyl group to the amino

terminal end. Before this, a hydroxyl group is attached to the ethyl chain of

dopamine to form noradrenaline (Axelrod 1974).

Along with dopamine and adrenaline, noradrenaline is the most important

neurotransmitter in the sympathetic nervous system. It is widely expressed in

almost all brain regions (Marzo et al. 2009). Together with adrenaline,

35

noradrenaline affects the flight-or-fight response under stress conditions when

necessary through the sympathetic nervous system. Their main role is to increase

the amount of available chemical energy for immediate use (Haller et al. 1997,

Libersat & Pflueger 2004), and the levels of noradrenaline and adrenaline are

altered significantly during stress and arousal (Kvetňanský 1973, Sanchez et al.

2003).

Adrenaline and noradrenaline exhibit a circadian rhythm, at least outside the

nervous system, with an increased expression during the dark time in 12L:12D

cycle. When the circadian phase is disturbed, the expression of noradrenaline and

adrenaline is altered in the peripheral tissues. After adapting to the new rhythm, the

levels of catecholamines return to normal (Sudo & Miki 1995). At the brain level,

noradrenaline levels remain constant regardless of the photoperiod even though

expression of dopamine alters. This implies that noradrenaline mainly affects the

rhythmicity of the peripheral tissues and dopamine is the dominant catecholamine

in regulating brain rhythmicity (Miguez et al. 1995).

36

37

2 Aims of the study

The circadian rhythmicity and the opsin protein family are both well studied

subjects in physiology and neurology. It has also been known for decades that light

is capable to penetrate the mammalian skull. Until now, there is hardly any

information combining these important aspects of photobiomodulation. This

doctoral thesis provides information on how light administrated through the skull

(transcranial light) affects molecules that regulate the circadian rhythmicity in

rodents. The study hypotheses are:

1. Transcranial light is able to alter opsin expression in the rodent brain.

2. The response in opsin expression varies between the times of the day when

light exposure affects the brain of mice.

3. The response in opsin expression varies between the times of the day when

light exposure is applied to hamsters.

4. Monoamine production is altered in both peripheral tissues and brain tissues of

transcranially illuminated mice and the response in monoamine expression is

also dependent on the time of the light exposure.

38

39

3 Materials and methods

3.1 Study species

3.1.1 Mouse

Mouse (Mus musculus) is a widely used test animal in laboratory studies. It is a

nocturnal animal and has a clear circadian rhythm (Chang et al. 2002). We used

total adult C3H/HeNHsd rd/rd male mice (Harlan Laboratories, Venray, The

Netherlands) in the substudies I and II. Male mice were used, since their hormonal

control of the reproductive cycle is less dominant than in female mice. All the mice

were 8–10 weeks old at the beginning of the study.

3.1.2 Djungarian hamster

The Djungarian hamster (Phodopus sungorus) is a photoperiod species and

therefore an optimal test animal for this kind of studies. Hamsters have several

behavioral and physiological adaptations to changes in photoperiod, e.g. changes

in sleep deprivation, melatonin levels and body weight depending on the annual

photoperiod (Hoffmann 1973, Palchykova et al. 2003). We used 24 (11 female, 13

male) Djungarian hamsters in the substudy III. All the hamsters were adults, i.e.,

10–12 weeks old, at the beginning of the study (Cantrell & Padovan 1987).

40

3.2 Study design

This study was approved by the Finnish national committee for animal

experimentation (Licence number ESAVI/567/04.10.03/2012). The study design

was similar between the studies. All animals were kept in a 12L:12D

light:dark -cycle, lights on at 7 a.m., lights off at 7 p.m. Before each study, mice

and hamsters were randomly assigned to three groups: control group (CONT),

morning-light group (ML) and evening-light group (EL). The animals were

weighted three times a week. Transcranial light was given via ear canals for four

weeks, five times a week (Monday to Friday). Light treatment was given under

anaesthesia (Isofluran, anaesthesia 3%, maintenance 1,5%) for eight minutes per

mouse. The ML group was exposed to light stimulus just after the beginning of the

light period (7–9.30), and EL group was exposed to illumination just after the end

of the light period (19–21). Rodents are nocturnal animals, so their resting state

begins when the light period begins and their active state begins when the dark

period begins. The timeline of the progression of all the studies is represented in

Figure 7.

Fig. 7. A timeline on the progression of the study.

41

Fig. 8. The photograph of the study design shows the setup of the mouse headset

during transcranial light illumination.

For transcranial light illumination, we used modified Valkee NPT 1000 headset

(Fig. 8, Valkee Inc., Oulu, Finland). The intensity of the light in the Valkee headset

was 2,00 x 10*15 photons/cm2/s. The Valkee NPT 1000 has two intensity peaks:

first with wavelength of approximately 450 nm and second with wavelength of

approximately 550 nm (Paper I, Fig. 3). There is no significant heat effect caused

by the headset, since only a small, non-measurable fraction of the mechanical heat

is produced by the headset (less than 14.7 mW) and then conducted into the ear

canal. Additionally, in test phase, we measured the temperature of the ear before

and after light illumination. We found that there was at most a 0,1 °C difference

between the measurements (data not shown).

After four weeks of transcranial light treatment, the animals were sacrificed by

cervical dislocation. The length and body mass were measured, and samples of

hypothalamus, cerebellum, cortex, adrenal gland, plasma, liver and retina were

collected between 8:00 and 14.00 and stored at -80 °C. Body mass indexes (BMI)

were calculated using the general formula for BMI determination: mass (as

kilograms) / length2 (as meters).

42

3.3 Molecular methods

3.3.1 Western blot

Western blot is widely used to determine the amount of proteins in tissue samples.

Western blot combines the high definition of electrophoresis with the ability of

sodium dodecyl sulphate (SDS) to break down the proteins into polypeptide chains.

Proteins are separated by molecular weight and by gel concentration (Burnette

1981). In this thesis, the molecular weights of opsins were between 35 and 55 kDa,

and we used a gradient gel, which concentration of SDS was 4–12%.

The total amount of proteins in brain, retina and liver samples were determined

according to Bradford (1976). Homogenization buffer, containing the protease

inhibitors leupeptin, pepstatin and PMSF, was added to the samples and all samples

were homogenized. After homogenization a fraction of each sample was taken for

the total protein determination and diluted with 0,9% NaCl. The dilution for

hypothalamus samples was 1:100, cerebellum 1:100, liver 1:200 and retina 1:20.

The absorbances of blanks (0,9% NaCl), controls (BSA diluted in 0,9% NaCl) and

samples were determined using a microplate reader.

After total protein determination, the samples were diluted either in ratio of 1:1

(OPN5, cerebellum), 1:5 (OPN3 and OPN5, hypothalamus) or 1:15 (OPN4). Equal

amount of protein was loaded in a separating gel and separated electrophoretically.

The separated proteins were transferred to a nitrocellulose membrane according to

the method of Towbin et al. (1979). The transfer was made either over the night (for

mice samples) or in 4 hours (for hamster samples).

After transferring the membranes were blocked with non-fat milk powder (for

mice samples) or in Amresco 10x blocking solution (for hamster samples). After

washing, the membranes were incubated in primary antibodies for OPN3 (45 kDa),

OPN4 (53 kDa) and OPN5 (40 kDa). OPN3 (1:500), OPN4 (1:500) and OPN5

(1:750 for hypothalamus samples and 1:500 for cerebellar samples) antibodies were

incubated for 2 h at room temperature (for mice samples) or overnight (17 h) at

+8°C (for hamster samples). After incubation membranes were washed and

incubated with secondary antibody (1:3000 for all opsins) for 2 h at room

temperature. After secondary antibody incubation, the protein bands were detected

and their intensities compared to the background were analyzed with the VersaDoc

imaging system (Bio-Rad).

43

3.3.2 HPLC

High-performance liquid chromatography (HPLC) is used to identify, separate and

quantify different compounds in a solution. It has become one of the most popular

methods for chemical analytics and it has many applications in the field of

chemistry and biology. In HPLC, the sample is dissolved into a moving phase that

is forced through a stationary phase. The compounds in the sample interact

differently with the stationary phase, causing different retention times at the

specific flow rate to separate the different molecules. This allows for a detection of

the compounds as they flow out the column.

We used HPLC to determine the monoamine concentrations in the mouse brain

(cortex, hypothalamus and cerebellum) as well as in adrenal glands and plasma.

The method for HPLC is described in more detailed by Nieminen et al. (2004). The

plasma samples were deproteinized before injection into the HPLC autosampler.

3.3.3 Melatonin and cortisol determination

The enzyme-linked immunosorbent assay (ELISA) is a biochemical technique,

where antibodies and colour change are used to determine the amount of specific

substances in the samples. In the ELISA method, the antigens of the samples are

attached to the surface. Then an antibody is linked with the antigen of the protein.

An indicator enzyme is attached to this antibody-antigen-combination, and forms a

measurable compound with a reaction, usually colour change. The concentration of

the final compound reflects the concentration of the original antigen (Engvall &

Perlmann 1971, Van Weemen & Schuurs 1971).

Melatonin concentrations in blood plasma samples were determined by ELISA

with a specified commercial kit for mouse melatonin. This was done according to

the instructions of the kit. Controls and samples were made as duplicates and

absorbances were read at 450 nm using a microplate reader. The sensitivity for

melatonin was 1,0 pg/ml.

Cortisol concentration in blood serum samples was also assayed by ELISA with a

commercial kit for human cortisol. This was done according to the instructions of

the kit. The cortisol kit was found suitable for mouse serum samples after test

measurements. Calibrators, controls and samples were made as duplicates, and

absorbances were read at 450 nm using a microplate reader. The sensitivity for

cortisol was 0,4 µg/dl, and calibrators were used to validate the method.

44

3.4 Statistical analyses

In all sub studies, the values are expressed as mean ± standard error (S.E.). The

statistical analyzes were made using the R 3.1.2 program. Before any statistical

analyzes, it is ensured that the samples were normally distributed in order to select

the proper statistical test. For the amounts of opsins, monoamines, melatonin and

cortisol, one-way ANOVA was used to compare the differences between groups. If

there was a significant change, pairwise comparisons between the test groups and

control group were made using the Tukey aposterior test. Length, weight and BMI

comparisons were also made with one-way ANOVA and Tukey test. Weight change

comparisons within groups were made using a paired t-test.

45

4 Results

4.1 Opsins

The levels of opsins were studied in hypothalamus and cerebellum tissues with

western blot method. Generally, transcranial light illumination had a significant

effect on OPN3 expression in both studied tissues and in both mice and hamsters

(except hamster hypothalamus). Interestingly, the time of the light illumination had

different effects on OPN3 expression, especially in the mouse. Summary of the

opsin results are presented in table 1.

Table 1. The table summarizes the statistically significant effects of transcranial light

administration on opsin expression, showing whether the light illumination has

increased (↑), decreased (↓) or had no effect (-) on opsin expression in hypothalamus

and cerebellum with mice and hamsters when compared to controls (ML = morning-

light group, EL = evening-light group; OPN3 = encephalopsin, OPN4 = melanopsin,

OPN5 = neuropsin).

hypothalamus cerebellum

ML EL ML EL

OPN3

mouse ↑ ↓ ↓ ↓

hamster - - - ↑

OPN4

mouse ↑ ↑ - ↑

hamster - - - -

OPN5

hamster - - - -

The time of the illumination had a significant effect on mouse OPN3 in the

hypothalamus. The amount of OPN3 increased in the ML group (P < 0,001) and

decreased in the EL group (P < 0,05) when compared to controls (Paper I, Fig. 1A).

Although hamsters showed a similar pattern, there were no statistically significant

changes when the test groups were compared to controls (Paper III, Fig. 1A).

The amount of cerebellar OPN3 decreased significantly in both ML group

(P < 0,001) and EL group (P < 0,05) in mice (Paper I, Fig. 1B), and increased in

the EL group (P < 0,05) in hamster (Paper III, Fig 2A).

46

The amount of OPN4 in mouse hypothalamus increased in both ML group

(P < 0,001) and EL group (P < 0,01) (Paper II, Fig. 1A). No differences were seen

in the hamsters when compared to controls (Paper III, Fig. 1B)

The amount of OPN4 increased in the EL group (P < 0,01), but no changes

were seen with the ML group in mouse cerebellum. Furthermore, there was a

significant difference (P < 0,05) between ML group and EL group in mice (Paper

II, Fig. 1B). In hamsters, no changes were seen in OPN4 expression (Paper III, Fig.

2B).

The expression levels of OPN5 were studied in hamsters, because of the

photosensitivity of the species. Although both hypothalamus and cerebellum

samples were blotted, only the hypothalamus samples gave measurable results

(Paper III, Fig 1C). In the cerebellum, half of the optical densities of the samples

(4 out of 8) were too low to be measured. The sensitivity of the densitometer was

too low to identify all optical densities.

4.2 Monoamines

Monoamine concentrations in all studied tissues were measured in mice with the

HPLC method. The summary of results is shown in Table 2.

Table 2. The table shows the statistically significant changes in monoamine

concentrations in cortex, plasma and adrenal gland in the test groups when compared

to controls (ML = morning-light group, EL = evening-light group; 5-HT = serotonin, DA

= dopamine, NA = noradrenaline, A = adrenaline). Hypothalamus and cerebellum

exhibited no difference in either test group comparing to controls. 0 = not measurable

cortex plasma adrenal gland

ML EL ML EL ML EL

5-HT ↓ - - - 0 0

DA - - ↑ - ↑ ↑

NA - - - - ↑ ↑

A 0 0 - ↓ ↓ ↓

The amount of serotonin in the cortex was significantly lower in the ML group mice

compared to controls (P < 0,01). There were no statistically significant differences

seen in any other experimental group monoamines compared to the controls (Paper

II, Fig. 2); there was no detectable amount of adrenaline in the brain samples.

Plasma dopamine concentration was significantly higher in the ML group

compared to controls (P < 0,01), but no differences were seen in the EL group

47

compared to controls (Paper I, Fig. 2A). There was no measurable amount of

dopamine in the control group. The adrenaline concentrations in EL group plasma

were significantly lower compared to controls (P < 0,05), but no differences were

seen for ML group and controls. Serotonin and noradrenaline showed no

differences in any test group when compared to controls (Paper I, Fig. 2A).

In the adrenal gland, dopamine concentrations were significantly higher in both

ML group (P < 0,001) and EL group (P < 0,01) mice. Also, noradrenaline

concentrations were significantly higher in both test groups (P < 0,001) when

compared to controls. Additionally, the amount on adrenaline decreased in both ML

group (P < 0,001) and EL group (P < 0,001) mice. There was no detectable amount

of serotonin in the adrenal gland samples (Paper I, Fig. 2B).

4.3 Body masses and BMIs

The body masses of the mice increased significantly during the study within the

groups in all study groups; there were no significant differences between groups at

the beginning of the study. The average mass of the EL group was significantly

lower (P < 0,05) at the end of the study compared to control group. The average

length of the EL group was also smaller (P < 0,05) compared to control mice at the

end of the study. There was no change in the BMIs between controls and test groups

at the end of the study (Paper I, Table 1). In hamsters, no changes were observed

either in the weights, lengths nor BMIs in or between the groups (start and end

weights or weights, lengths and BMIs; Paper III, Table 1).

48

49

5 Discussion

5.1 The opsins

Earlier, it was already known that OPN3 is found in the mammalian brain at both

mRNA (Blackshaw & Snyder 1999) and protein level (Nissilä et al. 2012).

Additionally, it was well known that light is capable of penetrating the skull bone

(Brunt et al. 1964, Persinger et al. 2013, Sun et al. 2016), and stimulate the

hypothalamic neurons directly even in animals without eyes (Lisk & Kannwischer

1964). Still, until now, a causal relationship between opsins and transcranial light

stimulation has not been demonstrated. The study of Koyanagi et al. (2013) showed

promising results regarding from the point of view of the present study with the

finding that homologs of mammalian OPN3 are able to form functional

photopigments and sense light information in nonphotoreceptive tissues. The

results of this study regarding OPN3 and OPN4 expressions confirm their findings

and thereby support the hypothesis of this thesis, since it is shown here that direct

light stimulation through the skull bone alters opsin expression in the hypothalamus.

It seems that opsins may be affected by transcranial illumination and the route for

light information into the SCN is not restricted only to the RHT.

Although light stimulation changed OPN3 expression in the mouse

hypothalamus, the response in expression pattern was different when light was

administered at different times of the day. OPN3 expression increased when the

light was administered in the morning and declined when the mice were illuminated

in the evening. Since OPN3 can form functional photopigments outside the eye

(Koyanagi et al. 2013), the transcranial light could be able to directly stimulate the

OPN3 expression. Furthermore, it was earlier assumed that the deactivation of

OPN3 could act through melatonin signalling pathways, since its levels are high

during the dark phase. Melatonin has also been shown to regulate neurohypophysial

hormone release in the hypothalamus (Yasin et al. 1996), so it could regulate OPN3

expression. However, there is no evidence that melatonin is the only molecule

capable of regulating the circadian system (Yamazaki et al. 1999, Bromundt et al.

2014, Jurvelin et al. 2014), so inevitably there exist other pathways (than melatonin

signalling pathways) to modulate OPN3 expression in mammalian brain.

Theoretically, gamma-amino butyric acid (GABA), an abundant inhibiting

molecule in the nervous system, may be one key factor mediating OPN3 inhibition.

It has been shown that in the response to a light pulse, the GABAA receptor activity

50

increases in retinal and cortical cells of hamsters (Leszkiewicz & Aizenman 2003).

Therefore, if mice have similar response to an additional light stimulus at the end

of the light period, the GABA receptor activity may be increased. If so, the OPN3

activity in the hypothalamus of EL group mice may decrease.

In the mouse cerebellum, transcranial light decreased the OPN3 expression inh

both test groups. Again, OPN3 has previously been found at the level of rodent

cerebellum mRNA (Blackshaw & Snyder 1999) and at theprotein level (Nissilä et

al. 2012). In mice, GABA may be a signalling neurotransmitter to alter the OPN3

expression. The study of Wade et al. (1988) strongly supports this alternative. They

found that when white light was administered to the cerebral cortex, the expression

of GABA was increased. If the transcranial light treatment – used in this study –

had similar effects in the cerebral cortex, the increase in GABA expression may

lead to the inhibition in the OPN3 expression. Moreover, it is known that GABAB

receptors are functioning through G-protein-coupled receptors (Calver et al. 2003).

If true, GABA may compete with opsins for the same binding sites at a G-protein-

coupled receptor. Additionally, the cerebellum is considered to be the centre of

motor control (D'Angelo et al. 2011), and the reduced expression of OPN3

especially in the ML group may indicate, that the control of motor coordination is

also reduced. It seems to be important to stress the fact that mice are nocturnal

animals and that their resting period therefore begins in the morning.

Interestingly, hamster OPN3 expression in the cerebellum differed from the

mouse OPN3 expression in transcranially illuminated mouse. The ML group

showed a small, although non-significant increase in OPN3 expression, but the

levels of the EL group were already significantly increased compared with the

controls. The study of Wade et al. (1988) suggests that if the light intensity is high

enough, the activity of GABA is suppressed. If the hamster brain is more sensitive

to light or the transfer of light information is more efficient than in the mouse brain,

the activity of GABA may decline and the amount of OPN3 could increase. A recent

hypothesis discusses the possibility of specialized myelinated axons that are able

to transmit light information (Kumar et al. 2016). If so, we have to consider the

possibility that these axons in the hamster brain are more efficient or more abundant

when compared to the mouse brain and that they are able to transmit light

information to the cerebellum effectively. Moreover, considering the motor control

the increased expression of OPN3 may reflect a gain in motor control (D'Angelo et

al. 2011), since hamsters like mice are also nocturnal animals, and their active

period begins in the evening.

51

The transcranial light illumination affected also the expression of mouse OPN4

in the hypothalamus. The expression levels increased in both test groups. The most

studied and the best known route for light information into hypothalamus is through

the eyes (Hattar et al. 2002, Bailes & Lucas 2010), but OPN4 has been found to be

expressed in the brain level too (Provencio et al. 1998, Bailey & Cassone 2005,

Nissilä et al. 2016). OPN4 is one of the most important molecules in regulating the

master clock in the SCN and entraining circadian rhythmicity (Panda et al. 2002,

Ruby et al. 2002, Panda et al. 2003, Panda et al. 2005). Additionally, light has been

shown to modulate directly the effects of OPN4 (Tsai et al. 2009). Therefore, these

results support the literature and it seems that transcranial light illumination is

capable to entrain the circadian system by OPN4 activation in the hypothalamus

regardless of the illumination time. These remarkable results have been obtained,

although the spectrum of light source used was not optimal for targeting the specific

absorbance maximum of OPN4 at 476–484 nm (Torii et al. 2007, Bailes & Lucas

2013). Nevertheless, the light used was able to induce significant alterations in

OPN4 expression and therefore sufficient to modulate OPN4 photobiomodulation.

The time of the transcranial illumination had a significant effect on OPN4

expression in the mouse cerebellum. Although, the OPN4 expression in the ML

group showed no difference, the EL group OPN4 expression increased when

compared to controls. Also, the expression of the EL group was significantly higher

when compared to the ML group. OPN4 is found in the vertebrate brain also at the

protein level (Bailey & Cassone 2005, Nissilä et al. 2016), but direct effects of

transcranial light and cerebellar OPN4 have not been studied earlier than in this

thesis. It is known, that cerebellum regulates the circadian rhythm in rodents

through clock gene activation (Rath et al. 2012, Rath et al. 2014), and OPN4 resets

the circadian rhythm through the activation of clock gene Per1 (Yamashita et al.

2014a). Thus, there is a possibility that OPN4 activation affects the circadian

rhythm through Per1 or other clock gene activation in the cerebellum. Furthermore,

the amount of OPN4 mRNA in the brain in avian species is shown to be highest in

the late subjective night (Bailey & Cassone 2005). The results in this mice study

supports this view, since they are nocturnal animals and their late subjective night

is in the evening. Again, the possible effect of specialized optic fibres facilitating

the transmission of light information to the brain could explain the direct effects of

transcranial light application to the brain on OPN4 expression as observed in this

study (Kumar et al. 2016). If they have an effect, the transcranial light may be

directly transmitted into the cerebellum through these fibres too.

52

It was surprising not to find any significant differences between the groups

regarding OPN5 expression, since the other studies demonstrated an effect of

opsins in response to transcranial light administration. Furthermore, OPN5 has also

been shown to be capable to transmit the light signal (Nakane et al. 2010). However,

it has been shown that there is only 25–30% amino acid identity between OPN5

and other opsins (Tarttelin et al. 2003), so its ability to transduce photic information

may be lower in comparison to OPN3 and OPN4. One reason, why the transcranial

light application did not evoke any significant changes in OPN5 expression, may

be the fact that OPN5 is more sensible to UV-light and therefore may therefore be

incapable of transmitting transcranial light information to the brain or least to the

hypothalamus (Yamashita et al. 2010). Additionally, OPN5 is a bistable molecule

and can bind both 11-cis and all-trans retinal forms of the chromophore. The 11-

cis form absorbs UV-light and all-trans form is the visible light absorbing form.

UV-light transforms OPN5 to the all-trans form whereas yellow light transforms

the OPN5 molecule back into the 11-cis state (Yamashita et al. 2014b). The fact

that OPN5 is most active in UV-light and that only yellow light is capable of

transforming OPN5 back to the 11-cis form may indicate that the blue-enriched

light used for transcranial illumination is incapable of activating OPN5.

5.2 Monoamines

At the brain level, cortical serotonin levels in the ML group decreased after light

illumination, other tissues showed no changes when compared to controls. It has

been shown that the concentrations of serotonin are regulated by monoamine

oxidase A (MAO-A), which is a key molecule in monoamine degradation (Cases et

al. 1998). Therefore, if the activity of MAO-A increases, the amount of serotonin

decreases and vice versa (Holschneider et al. 2000). Although non-significant,

there was a similar pattern seen in cortical noradrenaline and dopamine

concentrations with reduced levels of these monoamines being detected after light

exposure. Since mice are nocturnal animals, and the ML group is illuminated in the

beginning of their resting state, the changes in serotonin levels may also reflect a

disturbance of circadian rhythm regulation (Iritani et al. 2006). Furthermore, the

sample collection time has to be taken into account here, since the samples were

harvested in the morning or early afternoon. Several studies show that serotonin

levels peak in the brain level either at the end of a light period or during the dark

phase (Dudley et al. 1998, Barassin et al. 2002, Sanchez et al. 2008).

53

As hypothesized, transcranial light has been demonstrated to be able to affect

the monoamine production in both plasma and the adrenal glands. In the plasma,

the levels of dopamine increased in the ML group mice and the adrenaline levels

of the EL group mice decreased. It has been shown that light affects the dopamine

concentrations in the mouse nervous system (Pozdeyev et al. 2008), but the effect

of transcranial light on plasma and peripheral tissues has never been investigated

before. On the contrary, the importance of light on rat adrenaline concentrations

has been demonstrated over 20 years ago (Sudo & Miki 1995). In urine, the

adrenaline concentration is higher during the dark phase compared to light phase.

There is a similar pattern emerging from the findings of this thesis, because the

amount of adrenaline was highest in the control group and significantly lower in

the EL group, which had the longest exposure to light during the day. Additionally,

anaesthesia itself increased the amount of adrenaline (data not shown), but light

illumination was able to reduce its levels and thereby even reverse the effect of

anaesthesia. The effect of transcranial light on the adrenaline levels thus appears to

be highly specific.

The increase in dopamine concentration in the ML group mice may be

attributed at least to some degree to non-specific stress factors caused by the

experiment (Feenstra et al. 2000). Dopamine plays an important role in mediating

reactions to stress, and can thereby help animals to adapt to stressful situations

(Nieoullon & Coquerel 2003). The transcranial light illumination caused a delay in

the beginning of the resting state of the mice, which may have stressed the animals.

Furthermore, the levels of dopamine in the control group plasma were undetectable.

This may be caused by the effects of isoflurane anaesthesia. Anaesthesia inhibits

dopamine uptake and release (Tsukada et al. 1999, Shahani et al. 2002), and

therefore less dopamine is secreted into the blood circulation. Interestingly,

transcranial light seems to be able to increase the amount of dopamine in the blood

and can thereby even reverse the effect of anaesthesia. The strong effect of

transcranial light on dopamine is thus quite remarkable.

In the adrenal gland, dopamine and noradrenaline levels increased and

adrenaline levels decreased in both test groups. It has been shown that

noradrenaline secretion can be enhanced by hypothalamic stimulation (Gordon &

Bains 2005). Therefore, transcranial light illumination may be a similar enhancing

element able to enhance noradrenaline concentration in the adrenal gland.

Additionally, dopamine concentrations were higher in the adrenal gland after

transcranial light stimulation and this biogenic amine has been shown to regulate

catecholamine release in the adrenal gland (Artalejo et al. 1985). These results

54

indicate that light illumination enhances adrenal dopamine concentrations and

since this monoamine can regulate noradrenaline and adrenaline formation,

concentration and release the specific changes in catecholamine formation and

release seen can be explained.

Transcranial light appears to have an effect on adrenal gland adrenaline

production. Adrenaline secretion is higher during the dark phase than during the

light phase (Sudo & Miki 1995). Since most of the bodily adrenaline is produced

in the adrenal gland and this production is strongly affected by transcranial light

illumination the greater differences between control group and test groups in the

adrenal gland as compared to circulating levels in the plasma are easily explained.

Elevated concentrations of dopamine and noradrenaline may also be to some extend

be caused by stress induced during the experiment (Feenstra et al. 2000, Tanaka et

al. 2000, Nieoullon & Coquerel. 2003).

A possible effect of transcranial light illumination on MAO-A could also

contribute to some findings observed on adrenal monoamines and circulating levels

of these chemical messenger molecules. The reductions seen in adrenal gland

adrenaline concentrations as well as those observed in the plasma after light

exposure may at least to some extend be driven by an enhanced activity of MAO-

A. MAO-A is a key molecule in monoamine degradation and therefore not only

serotonin, but also adrenaline may be consumed (Cases et al. 1998). Furthermore,

both behaviour and MAO-A activity can be altered by disturbing the SCN (Hampp

et al. 2008). It is reasonable to hypothesize that light can alter monoamine

concentrations in peripheral tissues like in the brain and will thereby have a specific

effect on the circadian rhythm. Anaesthesia may have significant effects on the

basal level of monoamines in the tissues. Dopamine and noradrenaline transporters

are shown to be inhibited with isoflurane anaesthesia, naturally leading to lower

levels of dopamine and noradrenaline release (Shahani et al. 2002). Since all groups

of animals were anaesthetized, but only two test groups (ML group and EL group)

were illuminated, the effect of transcranial light illumination has to be considered

specific and of such magnitude that it can even reverse the effect of anaesthesia on

dopamine and noradrenaline levels.

5.3 The connection between opsins and monoamines

This study design did not allow to test for the possibility that there was a causal

relationship between the detected changes in the opsins and the monoamines, since

this would require experiments on additional animals whose opsin protein

55

expression was silenced. Therefore, the connection between these two main

molecular mechanisms and mediators of photobiomodulation and possible

interactions between them may at this point of the investigation only be discussed

at a hypothetical level in the context of previous studies. In lower vertebrates,

endogenous dopamine in the retina has been shown to increase opsin expression

after light exposure during the daily dark phase (Alfinito & Townes-Anderson

2001). Dopamine increases opsin expression also, when administrated in the early

morning as demonstrated in experiments on the zebrafish (Li & Li 2003).

Furthermore, dopamine was shown to regulate opsin expression in mammals too

(Sakamoto et al. 2005). Considering the findings presented in this thesis,

transcranial light may stimulate opsin expression through the activation of

dopamine signalling. Dopamine signalling could have in turn an effect on opsin

expression and this could at least in part explain some of the observations presented

in this first pioneer study on transcranial light illumination. The interactions of

opsins and monoamines in orchestrating circadian rhythm regulation could thus

turn out to be quite complex.

When considering the inhibiting pathways linked to opsin and monoamine

synergy, the thalamo-cortical pathway may be a possible target. Brown et al.

(Brown et al. 2010) showed that melanopsin transfers light information to the

thalamo-cortical pathway that is involved in enabling various effects in the brain.

If melanopsin regulates this pathway, it may regulate the cortical functions by

lowering the serotonin production. Alike, lower adrenaline level in the adrenal

gland may be somehow linked to a reduced expression of OPN3. However, the

knowledge on the putative relationship between monoamines and opsins is still

insufficient, and the connection between these two important systems requires

further studies. The findings of this thesis can stimulate such future work and may

guide this experimentation.

5.4 Weight, length and BMI

Although morning bright light in humans reduced the amount of body fat and

appetite, light seems to have no effect in body mass in humans (Danilenko et al.

2013). These results confirmed similar patterns observed in animal experiments.

Actually, the weight of mice increased during this study within all study groups.

Light administration at night is shown to increase body mass of the mice (Fonken

et al. 2013) as this was the case in the EL group of this study. Light at night disrupts

circadian rhythmicity, inducing weight gain (Aubrecht et al. 2015). Since in this

56

thesis the mice were illuminated in the morning and in the evening, the light

administration may have disrupted the rhythm of mice in several ways. Since mice

are nocturnal animals, their subjective night is during the day and their activity

period is during the dark period. Therefore, especially the light administration in

the evening may disrupt the beginning of the activity period. Also, the weight of

hamsters changes significantly between different seasons (Steinlechner et al. 1983).

Thus, these animals may be more resistant toward weight changes induced by

transcranial light, and no significant changes in hamster masses were seen.

57

6 Conclusions and future prospects

In this thesis, the direct effects of light through the rodent skull on opsin expression

and monoamine production were studied for the first time. Opsins are a broad

family of transmembrane proteins that are reactive to light and thus can serve as

endogenous molecules in phototransduction. They are also a vital part of

endogenous systems regulating circadian rhythmicity. Monoamines affect many

events inside the body, including rhythmicity. The ability of light to penetrate the

skull and the fact that the brain is light-sensitive are well documented. Although

these core features of brain physiology are well-studied, the connection between

light transmitted through skull bone and light dependent as well as sensitive

molecules in the brain has not been studied earlier. The results – according to aims

of the study – show that 1) opsin expression in the brain and 4) monoamine

production in both the brain and in the peripheral tissues can be affected by

transcranial light. Additionally, the time of the day at which transcranial

illumination is given has different effects on both mice 2), and hamster 3) opsin

and 4) mice monoamine levels. Interestingly, the levels of both OPN4 and OPN3

were higher in mice compared to hamsters, although future comparative statistical

analyses have to confirm this.

These first findings can be put into a broader context. Because transcranial light

treatment affected molecules that are associated with circadian rhythmicity, they

may have enabled entrainment and regulation. It is reasonable to hypothesize that

with this study design the circadian rhythms of mice and hamsters were entrained.

The successful entrainment of circadian rhythmicity allows to target the key factors

that are affected by rhythm dysfunctions – such as seasonal affective disorder – and

thus this study indicates new possibilities for treatment of these conditions, for

instance by transcranial light. But with this study design and at this early stage of

animal experimentation, it is premature to arrive at any conclusions regarding the

treatment of humans affected by such conditions. Future research based on this

pioneer study will include behavioral tests and can therefore come to first

mechanistic insights on how transcranial light can successfully employed in

treatment of these conditions in humans.

All in all, this thesis only scratches on the surface and is a first step to

demonstrate the ability of light to regulate rhythmic actions through other pathways

than the eye and RHT. Herein, the different specific responses of opsins towards

light administration were studied for the first time at the protein level. Hannibal et

al. (2013) have showed that the levels of melanopsin at mRNA and protein level

58

show similarities in the rat retina. It would be of interest to confirm that protein and

mRNA expression correlate at the brain level and extend these observations to other

opsins as well, since their expression may be different (Greenbaum 2003).

Although both opsin and monoamine expressions were studied, due to the

experimental design, a causal relationship of opsins and monoamines has not been

determined with certainty. The focus of this study was on the circadian rhythm, but

animals have other rhythms too. Best known is the circannual rhythm, which is

regulated by the photoperiod, mostly by the length of the day. It would be of great

interest and importance to study whether transcranial light can also alter the

circannual rhythm and its regulation.

59

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Original papers

I Flyktman A, Mänttäri S, Nissilä J, Timonen M & Saarela S (2015) Transcranial light affects plasma monoamine levels and expression of brain encephalopsin in the mouse. The Journal of Experimental Biology 218: 1521-1526.

II Flyktman A, Jernfors T, Mänttäri S, Nissilä J, Timonen M & Saarela S (2017) Transcranial light alters melanopsin and monoamine production in mouse (Mus musculus) brain. Journal of Neurology Research 7(3): 39-45

III Flyktman A, Mänttäri S, Nissilä J, Timonen M, Yamashita T, Shichida Y & Saarela S (2018) The effects of transcranial light illumination on opsin expression in the Djungarian hamster (Phodopus sungorus) brain. Manuscript.

Reprinted with permission from The Company of Biologists (I) and Elmer Press

(II).

Original publications are not included in the electronic version of the dissertation.

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704. Hartikainen, Heidi (2017) Malice in Wonderland : children, online safety and thewonderful world of Web 2.0

705. Ventä-Olkkonen, Leena (2017) The characteristics and development of urbancomputing practices : utilizing practice toolkit approach to study public displaynetwork

706. Mononen, Jukka (2017) Korkeasti koulutettujen vammaisten integroituminenICT-alalle heidän itsensä kokemana : ”Älä anna muille etumatkaa!”

707. Tolonen, Katri (2018) Taxonomic and functional organization ofmacroinvertebrate communities in subarctic streams

708. Turunen, Jarno (2018) Responses of biodiversity and ecosystem functions to landuse disturbances and restoration in boreal stream ecosystems

709. Huusko, Riina (2018) Downstream migration of salmon smolts in regulated rivers :factors affecting survival and behaviour

710. Huusko, Karoliina (2018) Dynamics of root-associated fungal communities inrelation to disturbance in boreal and subarctic forests

711. Lehosmaa, Kaisa (2018) Anthropogenic impacts and restoration of boreal springecosystems

712. Sarremejane, Romain (2018) Community assembly mechanisms in river networks: exploring the effect of connectivity and disturbances on the assembly of streamcommunities

713. Oduor, Michael (2018) Persuasive software design patterns and user perceptionsof behaviour change support systems

714. Tolvanen, Jere (2018) Informed habitat choice in the heterogeneous world:ecological implications and evolutionary potential

715. Hämälä, Tuomas (2018) Ecological genomics in Arabidopsis lyrata : localadaptation, phenotypic differentiation and reproductive isolation

716. Edesi, Jaanika (2018) The effect of light spectral quality on cryopreservationsuccess of potato (Solanum tuberosum L.) shoot tips in vitro

717. Seppänen, Pertti (2018) Balanced initial teams in early-stage software startups :Building a team fitting to the problems and challenges

718. Kinnunen, Sanni (2018) Molecular mechanisms in energy metabolism duringseasonal adaptation : Aspects relating to AMP-activated protein kinase, keyregulator of energy homeostasis

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Antti Flyktman

EFFECTS OF TRANSCRANIAL LIGHT ON MOLECULES REGULATING CIRCADIAN RHYTHM

UNIVERSITY OF OULU GRADUATE SCHOOL;UNIVERSITY OF OULU,FACULTY OF SCIENCE

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