PET Analysis of the Effect of Season,

Seasonal Affective Disorder and Light Therapy

on Serotonin Transporter Binding

By

Andrea Elizabeth Tyrer

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Pharmacology and Toxicology

University of Toronto

©Copyright by Andrea Elizabeth Tyrer (2017)

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PET Analysis of the Effect of Season, Seasonal Affective Disorder and Light

Therapy on Serotonin Transporter Binding

Doctor of Philosophy

2017

Andrea Elizabeth Tyrer

Department of Pharmacology and Toxicology, University of Toronto

ABSTRACT

Seasonal affective disorder (SAD) is characterized by recurrent major

depressive episodes during fall-winter with remission in spring-summer. SAD has an

annual prevalence of 1-6% with higher rates at more extreme latitudes. However,

there is limited understanding of its neuropathophysiology as no brain biomarkers

have been identified that are associated with SAD. Interestingly, serotonin transporter

binding (5-HTT BPND), an index of 5-HTT levels, varies seasonally in health; a finding

replicated by four independent research groups. The 5-HTT has a key role in affect-

regulation, given its involvement in serotonin reuptake. Yet, there have been no

longitudinal studies of seasonal fluctuation in 5-HTT BPND in SAD. Accordingly, we

investigated seasonal change in 5-HTT BPND in the anterior cingulate and prefrontal

cortices (ACC and PFC, respectively), using positron emission tomography (PET) in

SAD and health, scanning across summer and winter. Greater seasonal change in 5-

HTT BPND was observed in SAD relative to health.

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Additionally, there has been no investigation of deliberate light exposure upon

5-HTT BPND in the human brain despite evidence from previous PET studies, in

health, of an inverse correlation between sunshine duration and 5-HTT BPND. Thus, in

our second and third studies, we examined the effect of light therapy during fall-winter

on 5-HTT BPND in the ACC and PFC, in health and SAD, respectively, PET scanning

before and after treatment. In study two, we observed a decrease in 5-HTT BPND in

the ACC following light therapy in health. In study three, light therapy reduced 5-HTT

BPND across all examined brain regions in SAD.

The results of these studies have two important implications. First, the seasonal

change in 5-HTT BPND in study one was comparable to the reduction in 5-HTT BPND

following light therapy in study three. Hence, changes in light exposure may represent

a sufficient environmental condition to account for seasonal variation in 5-HTT BPND in

SAD. Second, the results of studies two and three demonstrate that light therapy

reaches a therapeutic target relevant to prevention and treatment of SAD. Overall, we

have identified a biomarker affected by season, associated with SAD pathophysiology

that is involved in treatment response to light.

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DEDICATION

In memory of Laura Kaitlin Peters whose life and struggle inspired this work.

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ACKNOLWEDMENTS

Firstly, I would like to extend my most heart-felt thanks and gratitude to my

primary Ph.D. supervisor Dr. Jeffrey Meyer whose mentorship has been invaluable to

both to my academic and personal growth. I met Dr. Meyer in early fall 2011 whilst

completing an honours specialization in physiology and psychology at the University

of Western Ontario. It had been my long-term goal to pursue translational research in

the field of mood/anxiety disorders, with particular focus on receptor-ligand

neuroimaging and I was excited to have the opportunity to participate in a project with

the potential to have real world impact. Although I had been trained in pre-clinical

research as an undergraduate, Dr. Meyer was always patient and willing to teach me

the basics of how to run a clinical study, of the mathematical modelling underlying

brain imaging techniques and to share his knowledge of how to best prepare findings

for publications in high-impact journals and at conferences. Furthermore, although I

did not arrive in his laboratory as the most confident trainee, he understood my

commitment to high quality research and my drive for success – to this end he

encouraged me to be confident in my own results, better my public speaking skills and

reminded me to always be optimistic and solution-oriented in the face of challenge. He

believed that I could be successful even when I doubted my own abilities. I have been

lucky to have such a Ph.D. mentor and I owe my skills, expertise and current position

to both his dedication and the excellent training environment in his lab.

I would also like to thank Drs. Robert Levitan and Jose Nobrega. Dr. Levitan’s

expertise in the field of seasonal affective disorder was invaluable in informing the

direction for this series of studies, guiding me toward relevant literature and

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encouraging me to present at conferences geared toward to seasonal research. Dr.

Nobrega’s input regarding interpretation of my PET findings, suggestions for statistical

analyses and possible extensions of this research to preclinical animal models were

always welcome and allowed me to challenge any preconceived notions I may have

held regarding my work.

I would like to thank everyone at the CAMH Research Imaging Centre for

making this series of work possible. Peter Bloomfield and Dr. Pablo Rusjan were

kindly willing to lend their time explaining PET physics or the fundamentals of kinetic

modelling. Laura Nguyen, Alvina Ng, Hillary Bruce and Anusha Ravichandran helped

me to book PET/MRI scan slots when I had participants urgently in need of seasonal

or post-treatment scanning, while Dr. Cynthia Xu and Laura Miler kindly stepped in to

accompany a participant to a scan when I had double-booked myself. I would also like

to thank Dr. Alan Kahn for his willingness to provide medical coverage for my PET

scans, for his advice regarding medical school post-Ph.D. and for allowing me to

shadow him during patient assessments for the last 2 years of my Ph.D.

Lastly, I would like to thank the people who made these four years at CAMH

truly special. My conversations with Dr. Vivien Rekkas regarding the difficulties

inherent in research were invaluable and she was always available to listen and

provide advice. I would especially like to thank Dr. Andre Ciré whose support,

kindness, patience and understanding enabled timely completion of this dissertation,

even while he also stressed the need for an occasional break from writing. Lastly, I

would like to thank my family, in particular, my parents and sister, Dr. David Tyrer,

Nancy Tyrer and Rosalyn Tyrer for their support during this training.

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Table of Contents

1 INTRODUCTION ....................................................................................................... 1

1.1 Statement of Problem ......................................................................................... 1

1.2 Statement of Purpose of the Study and Objective ............................................... 5

1.3 Rationale and Statement of Research Hypotheses ............................................. 7

1.3.1 Study 1: The Effect of Season on 5-HTT BPND ............................................. 7

1.3.2 Study 2: The Effect of Light Therapy on 5-HTT BPND in Health .................... 9

1.3.3 Study 3: The Effect of Light Therapy on 5-HTT BPND in SAD ..................... 12

1.4 Overview of the Serotonin System .................................................................... 14

1.4.1 The Raphe Nuclei ....................................................................................... 14

1.4.2 Serotonin Biosynthesis, Neurotransmission, Recycling and Degradation .. 16

1.4.3 The Serotonin Transporter (5-HTT) ............................................................ 18

1.5. Seasonality and the Serotonin System ............................................................ 22

1.6 Seasonal Affective Disorder .............................................................................. 26

1.6.1 Definition ..................................................................................................... 26

1.6.2 Neuropathophysiology of SAD .................................................................... 27

1.6.2.1 Role of Serotonin Physiology in Seasonal Behaviour .............................. 27

1.7 Light Therapy .................................................................................................... 32

1.8 Summary of Hypotheses ................................................................................... 36

2 MATERIALS AND METHODS ................................................................................. 38

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2.1 Rationale For Use Of [11C]DASB Positron Emission Tomography .................... 38

2.2 Study 1: The Effect of Season on 5-HTT BPND ................................................. 41

2.2.1 Participants ................................................................................................. 41

2.2.2 Image Acquisition and Analysis .................................................................. 44

2.2.3 Statistical Analysis ...................................................................................... 46

2.3. Study 2: The Effect of Light Therapy on 5-HTT BPND in Health ....................... 47

2.3.1 Participants ................................................................................................. 47

2.3.2 Study Design .............................................................................................. 48

2.3.3 Image Acquisition and Analysis .................................................................. 50

2.3.4 Statistical Analysis ...................................................................................... 52

2.4 Study 3: Effect of Light Therapy on 5-HTT BPND in SAD ................................... 54

2.4.1 Participants ................................................................................................. 54

2.4.2 Treatment Protocol ..................................................................................... 56

2.4.3 Image Acquisition and Analysis .................................................................. 58

2.4.4 Statistical Analysis ...................................................................................... 60

3 RESULTS ................................................................................................................ 62

3.1 Study 1: The Effect of Season on 5-HTT BPND ................................................. 62

3.1.1 Rationale .................................................................................................... 62

3.1.2 Effect of SAD and Severity Category on % Δ 5-HTT BPND ......................... 63

3.1.3 Effect of SAD and Severity Category on Δ 5-HTT BPND ............................. 65

3.1.4 Relationship of Symptoms to % Δ [11C]DASB 5-HTT BPND ........................ 67

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3.2 Study 2: The Effect of Light Therapy on 5-HTT BPND in Health ........................ 70

3.2.1. Rationale ................................................................................................... 70

3.2.2 5-HTT BPND Decreases in the ACC Following Light Therapy in Winter ...... 70

3.3 Study 3: The Effect of Light Therapy on 5-HTT BPND in SAD ........................... 74

3.3.1 Rationale .................................................................................................... 74

3.3.2 Global Reduction in 5-HTT BPND Following Light Therapy in SAD ............. 75

4 GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS ................ 78

4.1 General Discussion of Results .......................................................................... 78

4.1.1 Study 1: The Effect of Season on 5-HTT BPND ........................................... 78

4.1.2 Study 2: The Effect of Light Therapy on 5-HTT BPND in Health .................. 82

4.1.3 Study 3: The Effect of Light Therapy on 5-HTT BPND in SAD ..................... 86

4.2 Summary of Findings and Conclusions ............................................................. 92

4.3 Recommendations for Future Studies ............................................................... 98

4.3.1 Differentiating the Effects of Mood vs. Seasonality on 5-HTT BPND in SAD 98

4.3.2 Generalizability of Findings to “Seasonal Bulimia Nervosa” ..................... 104

4.3.3 Neurobiology Underlying the Effect of Season and Light on 5-HTT BPND 109

REFERENCES ......................................................................................................... 114

LIST OF PUBLICATIONS ......................................................................................... 133

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LIST OF ABBREVIATIONS

5-HIAA 5-Hydroxyindoleacetic Acid

5-HT 5-hydroxytryptamine; Serotonin

5-HTP 5-Hydroxytryptophan

5-HTT Serotonin Transporter

5-HTT BPND Serotonin Transporter Binding (non-displaceable)

[123I]ADAM (123)I-labeled 2-((2-((dimethylamino)methyl) phenyl)thio)-5-iodophenylamine

β[123I]CIT 2β-carbomethoxy-3β-(4-iodophenyl)tropane)

[11C]DASB 11C-labeled-3-amino-4-[2-dimethylaminomethyl- phenylsulfanyl]benzonitrile

[11C]HOMADAM C-11-labeled N,N-dimethyl-2-(2'-amino-4'- hydroxymethylphenylthio)benzylamine

[11C]MADAM 11C-labeled- N-dimethyl-2-(2-amino-4 methylphenylthio)benzylamine

[11C]McN5652 11C-(+)-6-(4-Methylthiophenyl)-1,2,3,5,6,α10β- hexa-hydropyrrolo[2,1-a]isoquinoline

Aβ β-Amyloid

ACC Anterior Cingulate Cortex

AADC Aromatic Amino Acid Decarboxylase

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ATD Acute Tryptophan Depletion

BDI Beck Depression Inventory

BN Bulimia Nervosa

CAMH Centre for Addiction and Mental Health

Cl- Chloride Ion

CNS Central Nervous System

DAS Dysfunctional Attitudes Scale

DAT Dopamine Transporter

DBS Deep Brain Stimulation

DMH Dorsal Medial Nucleus of the Hypothalamus

DRD4 Dopamine Receptor D4 Gene

DRN Dorsal Raphe Nucleus

DSM-IV-TR Diagnostic and Statistical Manual of Mental Disorders Version IV – Text Revision

GC-MS Gas Chromatography–Mass Spectrometry

HRRT High-Resolution Research Tomograph

IGL Intergenticulate Leaflet

K+ Potassium Ion

MAO-A Monoamine Oxidase A

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m-CPP meta-Chlorophenylpiperazine

MDD Major Depressive Disorder

MDE Major Depressive Episode

MIP Mood Induction Procedure

MPA Medial Preoptic Area

MRI Magnetic Resonance Imaging

MRN Median Raphe Nucleus

Na+ Sodium Ion

Na+/K+ ATPase Sodium-Potassium Pump

NET Norepinephrine Transporter

NPAS Neuronal PAS Domain-Containing Protein Gene

NSAID Nonsteroidal Anti-Inflammatory Drug

NSS Neurotransmitter Sodium Symporter

p38MAPK p38 Mitogen-Activated Protein Kinase

PET Positron Emission Tomography

PET-CT Positron Emission Tomography - Computed Tomography

PER3 Period Circadian Protein Homolog 3 Gene

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PFC Prefrontal Cortex

RGC Retinal Ganglion Cell

ROI Region of Interest

SAD Seasonal Affective Disorder

SCID-I/II Structured Clinical Interview for Axis I/II Disorders

SCN Suprachiasmatic Nucleus

SIGH-ADS Structured Interview Guide for the Hamilton Depression Rating Scale with Atypical Depression Supplement

SIGH-SAD Structured Interview Guide for the Hamilton Depression Rating Scale with Seasonal Affective Disorder Supplement

SLC6A4 Solute Carrier Family 6 Member 4 Gene

SNRI Serotonin–Norepinephrine Reuptake Inhibitor

SPAQ Seasonal Pattern Assessment Questionnaire

SPECT Single-Photon Emission Computed Tomography

SPVZ Sub-Paraventricular Zone

SRTM2 Simplified Reference Tissue Method 2

SSRI Selective Serotonin Reuptake Inhibitor

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TPH2 Tryptophan Hydroxylase 2

TSPO Translocator Protein

VAS Visual Analogue Scale

VNTR Variable Number Tandem Repeat

WHO World Health Organization

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LIST OF TABLES

Table 1-1 Negative correlations between average sunshine duration,

day length in Toronto, Ontario, Canada and brain 5-HTT BPND

Table 1-2 Seasonal fluctuation in 5-HTT BPND as replicated by four

independent research groups

Table 2-1 Comparison of PET and SPECT Radiotracers for the 5-HTT

Table 2-2 Participant Demographic Characteristics (Study 1)

Table 2-3 Participant Demographic Characteristics (Study 3)

Table 3-1 Group differences in seasonal percent change in 5-HTT BPND

Table 3-2 Group differences in seasonal percent change in 5-HTT

BPND in participants undergoing [11C]DASB PET in spring-

summer and fall-winter

Table 3-3 Group differences in 5-HTT BPND values in participants

undergoing [11C]DASB PET in spring-summer and fall-winter

Table 3-4 Correlations between seasonal percent change in 5-HTT

BPND and Seasonal Pattern Assessment Questionnaire

Global Seasonality Score

Table 3-5 Correlations between seasonal change in 5-HTT BPND values

and Seasonal Pattern Assessment Questionnaire Global

Seasonality Score

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Table 3-6 Correlations between seasonal percent change in 5-HTT

BPND and domains of the Seasonal Pattern Assessment

Questionnaire

Table 3-7 Group differences in brain 5-HTT BPND values in healthy

subjects undergoing [11C]DASB PET in winter (n=10) and fall

(n=9) following light therapy and placebo conditions

Table 3-8 Group differences in sleep parameters in healthy subjects

undergoing [11C]DASB PET in winter (n=10) and fall (n=9)

following light therapy and placebo conditions

Table 3-9 Change in 5-HTT BPND values in SAD participants before and

after light therapy

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LIST OF FIGURES

Figure 1-1 A schematic summary of the reciprocal connections

between the suprachiasmatic nucleus and the midbrain

dorsal raphe nucleus and median raphe nucleus

Figure 1-2 The Serotonin Biosynthesis Pathway

Figure 1-3 Organization of the human serotonin transporter gene

(SLC6A4)

Figure 1-4 A schematic of the membrane-bound 5-HTT protein

Figure 1-5 Architecture of Human 5-HTT as visualized by X-ray

crystallography

Figure 1-6 A scanning electron micrograph immunostained

for the 5-HTT

Figure 1-7 A scanning electron micrograph immunostained for the

5-HTT of proximal axon bundles near the DRN

Figure 1-8 Reciprocal peaks and troughs of brain 5-HTT BPND and

duration of sunshine as measured by [11C]DASB PET in

88 healthy participants

Figure 1-9 The effect of light on mood in seasonal depression

Figure 1-10 A comparison of the efficacy of morning versus evening light

therapy for treatment of seasonal affective disorder

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Figure 3-1 Seasonal percent change in serotonin transporter binding

potential as measured in healthy volunteers and moderate

and severe SAD participants across 8 brain regions

Figure 3-2 Seasonal change in serotonin transporter binding

potential as measured in healthy volunteers and moderate

and severe SAD participants across 8 brain regions

Figure 3-3 Positive correlations between magnitude of seasonal change

in serotonin transporter binding and SAD severity (as

measured by the Seasonal Pattern Assessment

Questionnaire) in the ACC and PFC

Figure 3-4 Serotonin transporter binding potential as measured across

conditions (light therapy versus placebo) in 6 brain regions

of interest in healthy volunteers during the winter

Figure 3-5 Serotonin transporter binding potential measured across

conditions in 7 brain regions of interest in SAD participants

before and after two weeks of daily morning light therapy

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1 INTRODUCTION

1.1 Statement of Problem

In 2008 the World Health Organization (WHO) identified Major Depressive

Disorder (MDD) as the leading cause of death and disability in moderate to high

income nations indicating that new treatment and prevention methods are needed

(Mathers et al., 2008). Seasonal affective disorder (SAD) is a subtype of MDD

characterized by recurrent major depressive episodes (MDEs) which occur fall-winter

with full remission in the spring-summer (Rosenthal et al., 1984). The annual

prevalence rate of SAD is estimated at 1 to 6 percent, with higher rates occurring at

more extreme latitudes (Magnusson, 2000). For example: Haggarty et al., reported a 7

percent rate of SAD and 22 percent rate of MDE in a Northern Canadian sample,

while, in Alaska, Booker et al., found that 9 percent of individuals surveyed met criteria

for SAD (Booker et al., 1992; Haggarty et al., 2002). Furthermore, SAD poses a heavy

burden on both sufferers and the healthcare system as, 40 percent of cases progress

to spontaneous non-seasonal MDD and amongst the subtypes of MDD, SAD has the

highest frequency of MDEs being almost yearly; for instance, it is not atypical for

individuals with SAD to experience greater than 9 seasonal MDEs by the third to

fourth decade of life (Faedda et al., 1993; Lam et al., 2006; Modell et al., 2005;

Schwartz et al., 1996). Those with SAD also tend to be heavy users of an already

burdened healthcare system, as they typically present with somatic symptoms such as

fatigue, lethargy, bingeing, weight gain and hypersomnia that require costly

investigation (Eagles et al., 2002). As such, in primary care settings, SAD is frequently

misdiagnosed and only fifty percent of specialist referrals are psychiatric in nature

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(Eagles et al., 2002). There is additional reason to study the seasonal impact upon

mood since 25 percent of healthy individuals experience seasonally related changes

in mood, energy, appetite and sleep that affect daily functioning (i.e. subsyndromal

SAD) (Chotai et al., 2004; Kasper et al., 1989; Okawa et al., 1996; Perry et al., 2001;

Rosen et al., 1990). Given the high prevalence of SAD, its role in predisposing to

MDD and the common problem of impaired function from seasonal variation in mood

there is an urgent need for research to identify biological mechanisms relevant to

understanding illness pathology so as to better develop strategies for illness

prevention and treatment.

Light therapy is an evidence-based first-line treatment for SAD with a similar

efficacy to that of antidepressant treatment (Lam et al., 1995; Lam et al., 2006;

Moscovitch et al., 2004). There have been several advances in the technique of light

therapy, insofar as it is well accepted that a light intensity of 10,000 lux is superior to

3000 lux, and that treatment in the early morning has greater efficacy as compared to

evening administration (Lewy et al., 1998; Terman et al., 1990). However, at present,

there is no consensus as to the mechanism by which light therapy exerts its

antidepressant effects and 45 percent of SAD cases do not adequately remit after this

treatment, indicating a need for improvement (Lam et al., 2006). Some theories as to

the mechanism for light exposure in regards to ameliorating seasonal depression

during winter include circadian phase advance, suppression of melatonin and

enhancing resilience against tryptophan depletion (Lam et al., 1996b; Lewy et al.,

2006; Lewy et al., 1987; Neumeister et al., 1997). However, it is generally agreed that

these mechanisms are still under investigation and the means by which light therapy

exerts its antidepressant effects is an on-going area of research.

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Advances in mental health research are needed to improve mental health care

and research utilizing neuroimaging techniques is integral to this forward progress.

Receptor-ligand neuroimaging studies (i.e. positron emission tomography, PET; single

photon emission tomography, SPECT) provide a means to visualize and quantify the

neuropathophysiology of mental illness in vivo in the living human brain. In

combination with information about illness phenomenology, such findings can be used

to better understand illness pathophysiology and to determine novel biological targets

for treatment or prevention. A significant body of research has focused upon

investigation of monoaminergic systems (i.e. serotonin, dopamine, norepinephrine) to

identify biomarkers associated with neuropsychiatric illness so as to better understand

the etiology and pathophysiology of such disorders. In regards to SAD, there is most

evidence of seasonal rhythmicity within the serotonin (5-HT) system, and thus it is a

promising candidate for study in regards to understanding the neuropathophysiology

of this disorder and to explore possible avenues by which to improve existing

strategies for prevention and treatment of SAD (Levitan, 2007).

The serotonin transporter protein (5-HTT), a component of the serotonin

system, is involved in clearance of extracellular 5-HT from the synaptic cleft and

regulates magnitude, duration and termination of serotonergic neurotransmission

(Murphy et al., 2004). Serotonin transporter binding potential (non-displaceable) (5-

HTT BPND) equals the ratio of specifically bound to free and non-specifically bound

radioligand in tissue at equilibrium, an index of serotonin transporter protein (5-HTT)

levels, that is commonly used in neuroimaging studies (Meyer, 2007). Interestingly,

there is particularly strong evidence from neuroimaging studies of seasonal change in

levels of this brain protein in health, with higher 5-HTT BPND observed in winter

4

relative to summer; a finding that has been replicated by four independent research

groups. (Buchert et al., 2006; Kalbitzer et al., 2010; Praschak-Rieder et al., 2008;

Ruhe et al., 2009). Yet, there are key components missing from current evidence

connecting seasonal changes in mood and behaviour to seasonal variation in 5-HTT

levels and environmental factors such as light exposure.

Previous neuroimaging studies of seasonal variation in 5-HTT BPND utilized

retrospective data from healthy participants and were cross-sectional in design. As

such, there have been no longitudinal neuroimaging studies investigating the effect of

season on 5-HTT BPND in a clinical sample of individuals, such as those with SAD, a

clinical population that displays marked seasonal changes mood and behaviour,

relative to healthy volunteers. This is a key issue because there have been no brain

biomarkers identified that are associated with SAD and thus, there is limited

understanding of the neuropathophysiology of this illness. Furthermore, the efficacy of

light therapy of SAD, is only 55 percent and the mechanism by which light therapy

exerts its therapeutic effects remains unclear. Notably, two of the aforementioned

studies using high quality imaging methods and large samples found an inverse

relationship between duration of daily sunlight and 5-HTT BPND, indicating this

biomarker is sensitive to light exposure (Kalbitzer et al., 2010; Lam et al., 2006;

Praschak-Rieder et al., 2008). However, there have been no neuroimaging studies

evaluating the effect deliberate light exposure upon 5-HTT BPND in human brain in

either health or in SAD. Therefore, an investigation of the effect of light therapy upon

5-HTT BPND would be valuable in order to determine the feasibility of developing a

brain biomarker for light therapy so as to improve response to this treatment.

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1.2 Statement of Purpose of the Study and Objective

There is ample research in support of seasonal rhythmicity of the serotonin

system, 5-HT itself has a key role in modulation of behaviours and affective states that

change seasonally and there is evidence of aberrant function of the serotonin system

in SAD. The 5-HTT is particularly important for further study in SAD given that this

brain protein has a key role in clearance of extracellular 5-HT and 5-HT depletion is

associated with depressive symptoms (Bel et al., 1992, 1993; Delgado et al., 1990;

Jennings et al., 2006; Leyton et al., 2000; Mathews et al., 2004; Shen et al., 2004;

Young et al., 1985). In addition, there is consistent evidence from neuroimaging

studies, using both PET and SPECT technologies, of seasonal fluctuation in 5-HTT

BPND, an index of 5-HTT levels, in health, with marked elevation in fall-winter relative

to spring-summer (Buchert et al., 2006; Kalbitzer et al., 2010; Praschak-Rieder et al.,

2008; Ruhe et al., 2009). To elaborate, in regards to seasonal variation of 5-HTT

BPND, neuroimaging studies of reasonably large sample size consistently reported this

finding in a high proportion of brain regions sampled: a [11C]DASB PET study of 88

subjects in Toronto, Canada, found seasonal variation in [11C]DASB 5-HTT BPND

across all examined brain regions including the prefrontal cortex, anterior cingulate

cortex (PFC and ACC, respectively) and hippocampus (Praschak-Rieder et al., 2008).

Similarly, an independent [11C]DASB PET study of 57 participants in Copenhagen,

Denmark found evidence of seasonal fluctuation in 5-HTT BPND in three of four brain

regions with significant change in the caudate and putamen, a trend in the thalamus

and no effect in the midbrain (Kalbitzer et al., 2010). Two additional studies in

Amsterdam, Netherlands and Hamberg Germany, using lesser quality technology,

applying [123I]β-CIT SPECT and [11C]McN5652 PET, respectively, investigated the

6

thalamus and midbrain and both found seasonal variation in 5-HTT BPND in the

midbrain (the Amsterdam study included both healthy and non-SAD MDD patients)

(Buchert et al., 2006; Ruhe et al., 2009). However, as previously stated, there has

been no longitudinal study investigating the effect of season on 5-HTT BPND in SAD

as compared to health and thus, the magnitude by which this brain protein fluctuates

across seasons in this clinical population relative to health is unknown. Accordingly,

the objective of study one was to use [11C]DASB PET to determine and compare the

magnitude of change in 5-HTT BPND in healthy individuals and those with SAD using a

within-subject design across winter and summer seasons.

Light therapy is an evidence-based, front-line therapeutic for seasonal

depression and there have been several advances in regards to optimizing this

technique since its initial application for treatment of SAD by Rosenthal et al., in 1984

(Lam et al., 1999; Rosenthal et al., 1984). It is now well accepted that a light intensity

of 10,000 lux is superior to 3000 lux, and that treatment in the early morning has

greater therapeutic efficacy as compared to evening administration (Lewy et al., 1998;

Terman et al., 1990). However, 45 percent of SAD cases do not remit following light

therapy indicating need for improvement and there is no consensus as to the means

by which this treatment ameliorates seasonal depressive symptoms, further

complicating its development (Lam et al., 2006). Interestingly, both preclinical rodent

studies and PET investigations report a reduction in 5-HTT binding concomitant with

an increase in light exposure, and blockade of the 5-HTT has been shown to be

important for antidepressant response (Kalbitzer et al., 2010; Meyer et al., 2001;

Meyer et al., 2004b; Praschak-Rieder et al., 2008; Rovescalli et al., 1989). Taken

together, these results suggest that there may be a relationship between light

7

exposure, 5-HTT binding and mood. As such, the objectives of studies two and three

were to evaluate the effect of light therapy on 5-HTT BPND in health and in SAD,

respectively, using [11C]DASB PET.

1.3 Rationale and Statement of Research Hypotheses

1.3.1 Study 1: The Effect of Season on 5-HTT BPND

Although information about SAD is accumulating, a critical gap is the lack of

direct brain investigations of this illness, thus, there have been no brain biomarkers

identified for this neuropsychiatric illness. Biological abnormalities of SAD include, in

winter, increased duration and/or delay of nocturnal melatonin secretion, blunted

norepinephrine, cortisol and prolactin response to challenge with the non-selective

serotonin receptor agonist meta-Chlorophenylpiperazine (m-CPP), and decreased rod

sensitivity to light as measured by flash electroretinography (Lavoie et al., 2009;

Levitan et al., 1998; Schwartz et al., 1997; Wehr et al., 2001). Vulnerability markers

include altered polymorphism frequencies of clock genes NPAS (neuronal PAS

domain-containing protein) and PER3 (period circadian protein homolog 3), as well as

a variable number tandem repeat on exon 3 of the DRD4 (dopamine receptor D4)

gene (Johansson et al., 2003; Levitan et al., 2006). However, given the substantial

burden of SAD, its high prevalence, and the lack of knowledge regarding the brain

abnormalities associated with this disorder, there is a clear need to identify

neurochemical and neuropathological markers associated with SAD.

One strategy for selecting a target to investigate in SAD is to choose a

functionally relevant brain marker that is sensitive to seasonal effects in healthy

humans. Once a biomarker has been chosen, the magnitude of seasonal change in

8

the target of interest can be examined in SAD relative to health to determine if

seasonal rhythmicity differs between populations, indicating a possible biological

abnormality associated with illness pathology. Brain markers influenced by season in

health include greater striatal L-Dopa uptake in the fall and winter, decreased 5-HT1A

receptor binding in limbic regions in winter, altered whole brain 5-HT turnover and

greater 5-HTT BPND in the fall-winter as compared to spring-summer (Buchert et al.,

2006; Eisenberg et al., 2010; Kalbitzer et al., 2010; Lambert et al., 2002; Praschak-

Rieder et al., 2008; Ruhe et al., 2009; Spindelegger et al., 2012). However, as

previously stated, evidence is most consistent in regards to seasonal variation of 5-

HTT BPND in health, as such findings have been reported by four independent

research groups with reasonably large sample sizes (Buchert et al., 2006; Kalbitzer et

al., 2010; Praschak-Rieder et al., 2008; Ruhe et al., 2009). Notably, the 5-HTT is also

an important target for controlling affect given that polymorphisms in the 5-HTT

promotor region are associated with risk of developing MDD, medications that

influence the 5-HTT influence both cognitive recall of emotionally valent material as

well as negative cognitive interpretations of life events; and that overexpression of 5-

HTT in regions controlling affect are associated with depressive behaviors in rodents

(Caspi et al., 2010; Harmer et al., 2004; Line et al., 2014; Meyer et al., 2003; Mouri et

al., 2012).

Given the consistency of seasonal change in 5-HTT BPND in health and the

importance of the role of 5-HTT in affect regulation, we first hypothesized that the

magnitude of seasonal variation in 5-HTT BPND in the PFC and ACC would be greater

in SAD compared to health. We prioritized the PFC and ACC as these regions had

considerable seasonal variation in previous study, contain structures with key roles in

9

mood regulation and cognitive processing of emotion, and are the regions for which 5-

HTT overexpression is associated with depressive behaviors (Line et al., 2014; Mouri

et al., 2012; Praschak-Rieder et al., 2008; Ressler et al., 2007). The second

hypothesis was that seasonal variation in 5-HTT BPND in the PFC and ACC would be

associated with severity of SAD symptoms. The rationale for the second hypothesis is

that SAD is well known to be a dimensional illness with a continuous distribution within

health (such that 25% of healthy individuals experience mild seasonal symptoms)

through to SAD of moderate to high severity (Bartko et al., 1989; Kasper et al., 1989;

Rohan et al., 2011; Terman, 1988); and in MDD the magnitude of brain biomarker

abnormalities is often correlated with severity, reflecting that MDD is a complex

neuropsychiatric illnesses for which any individual pathology is more likely to present

when MDD is more severe (Chiuccariello et al., 2014; Deschwanden et al., 2011;

Fujita et al., 2012; Meyer, 2012; Sanacora et al., 2004; Setiawan et al., 2015). We

also examined other structures such as the hippocampus, ventral striatum, thalamus,

dorsal putamen, dorsal caudate and midbrain as 5-HTT density is high in these

regions (Backstrom et al., 1989; Cortes et al., 1988; Laruelle et al., 1988).

1.3.2 Study 2: The Effect of Light Therapy on 5-HTT BPND in Health

There is reason to study seasonal impact on mood in healthy individuals who

have not yet developed SAD as 25 percent of healthy individuals experience

seasonally related changes in mood, energy, appetite and sleep that adversely affect

daily functioning during the winter months (Kasper et al., 1989). Interestingly, two

previous studies in healthy volunteers, including one from our laboratory, detected an

inverse correlation between duration of daily sunshine and 5-HTT BPND (Praschak-

10

Rieder et al., 2008; Kalbitzer et al., 2010). Thus, it was our intent to examine effect of

light therapy, a standard intervention for SAD, on 5-HTT BPND during fall-winter in

health to investigate the potential of 5-HTT BPND as a biomarker for light exposure and

to develop prevention strategies for new-onset SAD. We elected to first study healthy

volunteers (i.e. without the confound of SAD pathophysiology) with the intent to further

examine this effect in full-syndrome SAD in study three.

However, in regards to prophylactic interventions for new-onset SAD, direct

investigation of prevention strategies through clinical trials are both difficult and labor-

intensive since only a subset of individuals develop SAD. Similarly, direct investigation

of treatment strategies with large scale clinical trials is also challenging as these trials

must be oriented to the winter months. Hence a valuable intermediate approach is to

develop biomarkers to assess the effect of intervention strategies. The magnitude of

effect of the intervention strategy on the biomarker can then be applied to identify

therapeutics that should be carried forward in development. One candidate biomarker

for this approach is the serotonin transporter binding potential (non-displaceable) (5-

HTT BPND), as measured using PET with the radioligand [11C]DASB. Interestingly, in

four independent studies, 5-HTT BPND has been found to be elevated across multiple

brain regions in the fall-winter months compared to spring-summer in healthy

volunteers, suggestive of the sensitivity of this biomarker to seasonal effects (Buchert

et al., 2006; Kalbitzer et al., 2010; Praschak-Rieder et al., 2008; Ruhe et al., 2009). In

addition, in two of these previous studies of seasonal variation in 5-HTT BPND, using

[11C]DASB PET, an inverse correlation between duration of daily sunlight and 5-HTT

BPND was found, suggesting that this might be a promising biomarker for light therapy

(Kalbitzer et al., 2010; Praschak-Rieder et al., 2008).

11

The 5-HTT also has an important role in affect-regulation given that in humans,

lowering 5-HT via acute tryptophan depletion associated with low mood, particularly in

those vulnerable to developing MDEs such as those with family histories of MDE, or

past histories of MDE (Acta Psychiatrica et al., 2013; Delgado et al., 1990; Leyton et

al., 2000; Young et al., 1985). 5-HT itself and/or its neuromodulatory role is also

strongly implicated in mood disorders, given the many selective serotonin re-uptake

inhibitors (SSRIs) that raise extracellular serotonin are associated with amelioration of

depressive symptoms in individuals suffering from major depressive disorder (Owens

et al., 1994, 1998). Modulators of extracellular 5-HT are important because it is well

established that 5-HT plays a role in physiology and behaviours reported to change

with season including mood, sleep, appetite and energy (Canli et al., 2007).

Collectively, these findings illustrate that the importance of the 5-HTT, in terms of

affect-regulation, is this protein’s influence on extracellular 5-HT levels.

As such, it was our intent evaluate the potential of 5-HTT BPND as a biomarker

for light therapy in healthy volunteers who had not yet developed SAD in order to

develop prevention strategies for new-onset SAD (Boyce et al., 2013). We

hypothesized that administration of light therapy during the fall and winter months

would reduce 5-HTT BPND in the ACC and PFC in healthy volunteers. The ACC and

PFC (and/or subregions of these structures) are often activated in mood induction

studies (reflecting processes that generate sad mood and they also participate in

cognitive functions such as those leading to pessimism that create a sad mood (Liotti

et al., 2001; Liotti et al., 2002; Mayberg et al., 1999; Sharot et al., 2007; Tom et al.,

2007). Other regions included the thalamus, basal ganglia, hippocampus and midbrain

12

were also examined because 5-HTT density is high in these regions (Backstrom et al.,

1989; Cortes et al., 1988; Laruelle et al., 1988).

1.3.3 Study 3: The Effect of Light Therapy on 5-HTT BPND in SAD

Light therapy is an evidence-based first-line treatment for SAD, a highly

impactful disease, consisting of numerous, reoccurring, major depressive episodes

spanning the fall and winter with full remission in the spring and summer (Faedda et

al., 1993; Rosenthal et al., 1984). There have been several advances in the technique

of light therapy, insofar as light intensity of 10,000 lux is superior to 3000 lux, and

treatment in the early morning has greater efficacy relative to evening administration

(Lewy et al., 1998; Terman et al., 1990) . However, only 55 percent of individuals with

SAD experience remission of seasonal depressive symptoms following light therapy

and therefore it is important to develop strategies to further improve this treatment so

as to better ameliorate winter depressive symptoms (Lam et al., 2006).

It is generally recognized that use of PET with specific radioligands is an

effective tool for developing therapeutics. For example, [11C]DASB PET is often

applied to predict optimal dosing of antidepressants with high affinity for the serotonin

transporter while [11C]raclopride PET is often utilized to determine suitable dosing of

antipsychotics with high affinity for the D2 receptor (Kapur et al., 2000; Meyer et al.,

2001; Meyer et al., 2004b). More recently, a newer direction regarding the role of PET

receptor-ligand neuroimaging is its application quantifying markers of disease

pathology, such as amyloid burden in Alzheimer's disease or microglial activation in

major depressive episodes, so as to determine if these markers may be successfully

targeted by therapeutics (Rinne et al., 2010; Setiawan et al., 2015) .

13

The intent of the present study was to develop a PET-based measure of SAD

pathophysiology as a biomarker for light therapy. 5-HTT BPND had previously been

found to be inversely correlated with duration of daily sunshine in health (Kalbitzer et

al., 2010; Praschak-Rieder et al., 2008). As a priori regions, the ACC and PFC were

prioritized because these regions have a key role in mood regulation and cognitive

processing of emotion (Liotti et al., 2002; Mayberg et al., 1999; Ressler et al., 2007;

Sharot et al., 2007; Tom et al., 2007). The ACC participates in production of sad

emotions, where regional activation has been observed during provocation of sad

mood in MDD patients and decreased activity follows recovery from depression (Liotti

et al., 2002; Mayberg et al., 1999; Ressler et al., 2007). The PFC is involved in the

cognitive emotional processing; regional reductions in activity have been found to be

correlated with increased sensitivity to potential loss of reward (i.e. “loss aversion”)

whereas enhanced activity accompanies recall of positive life events (Sharot et al.,

2007; Tom et al., 2007). As such, our primary hypothesis was that, during fall-winter,

5-HTT BPND would be reduced in the ACC and PFC following light therapy. Other

brain regions with high 5-HTT BPND and/or roles in affect regulation were also

examined, including the hippocampus, thalamus, dorsal putamen, ventral striatum,

and midbrain, in order to assess the extent to which the effects of light therapy would

be more global or region-specific as in study two (Backstrom et al., 1989; Cortes et al.,

1988; Laruelle et al., 1988).

14

1.4 Overview of the Serotonin System

1.4.1 The Raphe Nuclei

In the mammalian brain, the raphe nuclei are found in the reticular formation

within the medial portion of the brain stem. These nuclei are divided into (i) the raphe

nuclei of the medulla oblongata (nucleus raphe obscurus, nucleus raphe magnus and

nucleus pallidus), raphe nuclei of the pontine reticular formation (nucleus raphe pontis,

nucleus centralis inferior) and (iii) raphe nuclei of the midbrain reticular formation

(nucleus raphe dorsalis; dorsal raphe nucleus and nucleus centralis superior; median

raphe nucleus) (Lowry et al., 2008). The majority of neurons with the raphe nuclei are

serotonergic, but of these nuclei, it is the dorsal and median raphe nuclei (DRN and

MRN, respectively) of the midbrain that project to ascending brain areas, including

cortical and subcortical regions, in addition to the brainstem (Vertes et al., 2008).

Serotonergic innervation within the central nervous system (CNS) is extensive and the

5-HT neurotransmitter itself has a key role in modulation of affect, cognitive function

and behaviours such as sleep, appetite and energy (Vertes et al., 2008). This latter

topic regarding the influence of 5-HT on mood and behaviour will be covered

extensively in subsequent sections.

There is also evidence that serotonergic neurons in the DRN and MRN play an

important role in regulation of circadian behaviours such as diurnal variation in

temperature and hormone levels, alertness and the sleep-wake cycle. However, the

exact mechanism by which 5-HT modulates circadian rhythmicity in regards to

functional and structural connections within the brain is complex and will require

further research (Deurveilher et al., 2008). Interestingly, there is evidence of a direct

15

retino-raphe projection between a small population of light-sensitive, non-photic retinal

ganglion cells in the eye and serotonergic neurons within the DRN and there are also

pronounced serotonergic reciprocal connections between the DRN and MRN (Ren et

al., 2013; Vertes et al., 2008). The MRN provides the majority of serotonergic input to

the suprachiasmatic nucleus (SCN) of the hypothalamus, the “circadian pacemaker” of

the brain and also receives input from this region, indirectly, via the dorsomedial

nucleus of the hypothalamus (Deurveilher et al., 2008; Vertes et al., 2008). The DRN

projects indirectly to the SCN via the intergenticulate leaflet and receives input from

the SCN via the medial preoptic area (Deurveilher et al., 2008; Vertes et al., 2008). As

previously stated, the DRN and MRN project to both cortical and subcortical regions

within the brain and also areas of the brainstem (Vertes et al., 2008). Thus, this

complex signaling pathway between the retina, serotonergic neurons within raphe

nuclei of the midbrain, SCN and efferent projection regions may be one means by

which photoperiodic information from the environmental is transduced into a biological

signal so that circadian rhythmicity in the CNS and periphery is entrained to the

external light-dark cycle (Figure 1-1).

Figure 1-1: A schematic summary of the reciprocal connections between the suprachiasmatic nucleus (SCN) and

the midbrain dorsal raphe nucleus (DRN) and median raphe nucleus (MRN). The SCN sends efferent projections (solid lines) to the DRN and MRN via putative relays in the medial preoptic area (MPA) and dorsal medial hypothalamus (DMH), respectively: the sub-paraventricular zone (SPVZ) may also serve as an intermediary via its projections to the DMH. In turn, the SCN receives afferent projections (broken lines) directly from serotonergic neurons in the MRN, and indirectly from serotonergic neurons in the DRN via the thalamic intergeniculate leaflet (IGL). The DRN and MRN are also reciprocally connected (solid arrows). [Modified from Deurveilher et al., 2008]

16

1.4.2 Serotonin Biosynthesis, Neurotransmission, Recycling and Degradation

5-HT is an indolamine monoamine neurotransmitter synthesized, in the CNS,

within presynaptic serotonergic neurons with the superior raphe nuclei from its amino

acid precursor L-tryptophan in a three step biosynthesis pathway (Mohammad-Zadeh

et al., 2008). As a first step in this pathway, L-tryptophan is oxidized by tryptophan

hydroxylase 2 (TPH2) to form 5-hydroxy-L-tryptophan (5-HTP) and this is the rate

limiting step of 5-HT biosynthesis (Mohammad-Zadeh et al., 2008). Subsequently, 5-

HTP is decarboxylated by aromatic amino acid decarboxylase (AADC) to form 5-

hydroxytryptamine (serotonin; 5-HT) (Figure 1-2). 5-HT is then packaged into

presynaptic vesicles for eventual release from synaptic terminals and also at

somatodendritic sites (Dankoski et al., 2013).

Figure 1-2: The 5-HT Biosynthesis Pathway Serotonin (5-HT) is synthesized from its amino acid precursor, L-tryptophan in a three step biosynthesis pathway. (i) L-Tryptophan is first oxidized by tryptophan hydroxylase 2 (TPH2) into its metabolic intermediate 5-hydroxy-L-tryptophan (5-HTP). (ii) 5-HTP is then decarboxylated by aromatic amino acid decarboxylase (AADC) to form 5-hydroxytryptamine (5-HT) [Modified from Druce M et al., 2009]

17

After release into the synaptic cleft, this neurotransmitter interacts with both

presynaptic and postsynaptic 5-HT receptors to initiate downstream effects. There are

seven classes and fourteen subtypes of 5-HT receptors, all of which are G-protein

coupled receptors, excepting the 5-HT3R which is a cation-selective ion-gated ligand

channel (Hannon et al., 2008). 5-HT binding to a G-protein coupled 5-HT receptor

activates second messenger cascades that produce either excitatory or inhibitory

effects within the CNS (Hannon et al., 2008). Some 5-HT receptors, such as the 5-

HT1DR, are located predominantly on presynaptic neurons and act as autoreceptors

regulating serotonergic neurotransmission via negative feedback (Hannon et al.,

2008). Other 5-HT receptors such as the 5-HT2AR and 5-HT2cR mediate the

postsynaptic downstream effects of 5-HT such as regulation of mood, sleep, appetite,

locomotion and cognitive function (Hannon et al., 2008). Lastly, 5-HT receptors, such

as the 5-HT1AR or 5-HT1BR, can act as either autoreceptors on presynaptic neurons or

postsynaptic receptors depending upon their regional location with the brain (Hannon

et al., 2008). Regional distribution of 5-HT receptor subtypes also differs within the

brain. For instance, the 5-HT1BR and 5-HT4R are most abundant within subcortical

regions, such as the striatum, thalamus and brainstem, whereas density of the 5-

HT1AR and 5-HT2AR are more heavily concentrated within cortical and limbic regions

such as the hippocampus, amygdala and isocortex (Varnäs et al., 2004).

Serotonergic neurotransmission is terminated via re-uptake of 5-HT by the

serotonin transporter (5-HTT) into the presynaptic neuron upon which is either broken

down by monoamine oxidase A (MAO-A) to produce 5-hydroxyindoleacetic acid (5-

HIAA) or recycled via repackaging into presynaptic vesicles (Mohammad-Zadeh et al.,

2008; Youdim et al., 2004). Thus, there is an inverse relationship between available 5-

18

HTT and clearance of extracellular 5-HT. For example: selective serotonin reuptake

inhibitors (SSRIs), which block the 5-HTT, elevate levels of extracellular 5-HT, 5-HTT

knockout mice have greater extracellular 5-HT levels and mice overexpressing 5-HTT

have low extracellular 5-HT levels (Bel et al., 1992, 1993; Jennings et al., 2006;

Mathews et al., 2004; Shen et al., 2004).

1.4.3 The Serotonin Transporter (5-HTT)

The human 5-HTT is encoded by the SLC6A4 (solute carrier protein family 6

membrane 4) gene which spans approximately 40 kilobases of DNA, is localized on

chromosome 17, centered at 17q11.2 and comprised of 14 exons (Murphy et al.,

2008) (Figure 1-3). The membrane-bound 5-HTT protein is comprised of

approximately 630 amino acids with 12 transmembrane spanning helices, an

extracellular region comprised of three extracellular loops (extracellular loops 2, 4 and

6) and intracellular amino and carboxy-terminal tails (Coleman et al., 2016; Murphy et

al., 2008) (Figures 1-4 and 1-5).

Figure 1-3: Organization of the human serotonin transporter gene (SLC6A4). The human SLC6A4 maps to

chromosome 17q11.2 and is composed of 14 exons that span ∼40 kb. Functional variants that modulate

transcriptional activity include variation in the length of the serotonin-transporter-gene-linked polymorphic region (5‑HTTLPR), single nucleotide polymorphisms, rs25531 and rs25532, located upstream of the transcriptional start site and variable number of tandem repeats (VNTR) at intron 2. [Modified from Murphy et al., 2008]

19

The 5-HTT is a monoamine transporter protein and a member of the

neurotransmitter sodium symporter (NSS) family, which also includes the dopamine

and norepinephrine transporters (DAT and NET, respectively) (Coleman et al., 2016).

The major physiological role of 5-HTT is the transport of 5-HT from the synaptic cleft

into the presynaptic neuron and thus, the 5-HTT has a key role in regulating

magnitude, duration and termination of serotonergic neurotransmission (Mohammad-

Zadeh et al., 2008) . Reuptake of 5-HT by the 5-HTT is coupled with co-transport of

sodium (Na+), chloride (Cl-) and potassium (K+) ions into the cell. Na+ and Cl-

concentrations are higher outside the cell, than intacelluarly, whereas the

concentration of K+ is greater inside relative to outside the cell (Murphy et al., 2004).

This ion concentration gradient is generated by the membrane-bound sodium-

potassium pump (Na+/K+ ATPase) and this ATPase is necessary for transporter-

mediated monoamine reuptake (Murphy et al., 2004).

Figure 1-4: A schematic of the membrane-bound 5-HTT protein. The 5-HTT is comprised of 630 amino acids with 12 transmembrane spanning helices, an extracellular region comprised of three extracellular loops (extracellular loops 2, 4 and 6) and intracellular amino and carboxy-terminal tails [Modified from Murphy et al., 2008]

20

In regards to the mechanism of 5-HT reuptake, a single multifunctional binding

site on the extracellular surface of the 5-HTT allows for movement of Na+, Cl–, K+ ions

and 5-HT across the cell membrane (Murphy et al., 2004). Initially, a 5-HT molecule, a

Na+ and Cl- ion bind simultaneously to this exterior binding site. Subsequent

conformational change impedes access of other molecules/ions to this binding site

and transports both 5-HT, Na+ and Cl- ions to the cytoplasmic side of the membrane

(Murphy et al., 2004). 5-HT, Na+ and Cl- then disassociate from the 5-HTT and one

intracellular K+ ion is transported to the extracellular side of the membrane to restore

the 5-HTT to its active conformation (Murphy et al., 2004). Na+/K+ ATPases maintain

this ion gradient to allow for continued function of the 5-HTT (Murphy et al., 2004).

Figure 1-5: Architecture of Human 5-HTT as visualized by X-ray crystallography. The protein is viewed parallel to the membrane. The (S)-citalopram molecules denoting central and allosteric site are shown as sticks in dark green and cyan, respectively. Sodium ions are shown as spheres in salmon. [Modified from Coleman et al., 2016]

21

Most 5-HTT are located at outer cell membranes, primarily perisynaptically and

along axons (axolemma) (Zhou et al., 1998) (Figures 1-6 and 1-7). Diffusion of 5-HT

away from serotonergic presynaptic terminals to distances of up to 20μm have been

reported (Zhou et al., 1998). Accordingly, perisynaptic 5-HTT may modulate reuptake

of 5-HT near synapses, whereas axonal 5-HTT may facilitate termination of

serotonergic neurotransmission at more distal locations (Zhou et al., 1998). In the

human brain, 5-HTT density varies by region and is highest in the raphe nuclei,

elevated in subcortical regions (i.e. thalamus and basal ganglia) and lower in density

in limbic and cortical regions (i.e. hippocampus, ACC, PFC). The cerebellar cortex

(excepting the vermis), is nearly devoid of 5-HTT thus, in regards to quantification of

the 5-HTT via PET imaging, this brain area is an excellent reference region

(Backstrom et al., 1989; Cortes et al., 1988; Kish et al., 2005; Laruelle et al., 1988).

Figure 1-6: A scanning electron micrograph immunostained for the 5-HTT. A thin section of an asymmetric synapse can be seen along a 5-HTT-positive axon. Small arrows indicate the post-synaptic density and the portion of presynaptic membrane opposed to the synaptic cleft is devoid of 5-HTT. The perisynaptic area adjacent to the presynapstic membrane is distinctly stained (circled in red), indicating the presence of 5-HTT immunograins [Modified from Zhou et al., 1998]

22

Figure 1-7: A scanning electron micrograph immunostained for the 5-HTT of proximal axon bundles near the raphe nucleus. As can be seen, 5-HTT immunograins are located on the along the axolemma (i.e. along the length of the axon) (circled in red) and not within in the axoplasm. [Modified from Zhou et al., 1998]

1.5. Seasonality and the Serotonin System

Of the monoamine neurotransmitter systems (i.e. serotonin, dopamine,

norepinephrine), there is most evidence of seasonal rhythmicity within the serotonin

system. In regards to preclinical investigation, in rodents, a shortened photoperiod (i.e.

to mimic shortened “winter-like” day length) has been found to decrease 5-HT release

and increase both 5-HTT density and clearance of 5-HT in the hypothalamus and

SCN of the hypothalamus (Blier et al., 1989; Rovescalli et al., 1989). In post-mortem

study, Carlsson and colleagues, reported seasonal variation in 5-HT levels in the

human hypothalamus with lower levels in late winter and higher levels in late summer

(Carlsson et al., 1980). Lambert et al. also observed seasonal variation in whole brain

5-HT turnover in a sample of one hundred and one healthy males using jugular vein

catheterization and found that the rate of 5-HT production was correlated with duration

of daily sunshine (Lambert et al., 2002). Using this technique, it is possible to quantify

overflow of 5-HT and its metabolites from the brain into jugular venous effluent, thus

providing an indirect measure of 5-HT turnover in the brain (Lambert et al., 2002).

More recently, in a neuroimaging study involving thirty-six healthy volunteers,

23

Spindelegger et al., used [carbonyl-11C]WAY-100635 PET to examine the relationship

between 5-HT1A receptor binding, an index of 5-HT1A receptor levels, season and light

exposure in both cortical and subcortical limbic regions. A positive correlation was

observed between post-synaptic 5-HT1A receptor binding and duration of daily

sunlight, with greater binding observed in spring-summer relative to fall-winter

(Spindelegger et al., 2012).

As the 5-HTT has a key role in regulating magnitude, duration, and termination

of serotonergic neurotransmission, the majority of neuroimaging studies investigating

seasonal fluctuation in the serotonin system have focused on this transporter protein.

To date, there have been four cross-sectional neuroimaging studies of high to

moderate quality to support evidence of seasonal variation in 5-HTT BPND (Buchert et

al., 2006; Kalbitzer et al., 2010; Praschak-Rieder et al., 2008; Ruhe et al., 2009). In a

study of eighty-eight healthy volunteers in Toronto, Canada, [11C]DASB PET was used

to assess seasonal change in 5-HTT BPND and a marked elevation in 5-HTT BPND was

observed in winter relative to summer in a number of affect-modulating brain regions

including the ACC and PFC, in addition to the basal ganglia, thalamus and midbrain

(Praschak-Rieder et al., 2008) (Figure 1-8).

24

Figure 1-8: Reciprocal peaks and troughs of brain 5-HTT BPND and duration of sunshine in the 88 healthy participants. 5-HTT BPND was measured using [11C]DASB PET. 5-HTT and determined in 6 brain regions (anteromedial PFC [A], ACC [B], caudate [C], putamen [D], thalamus [E], and mesencephalon [F]). Circles represent bimonthly moving average means of serotonin transporter binding potential values; error bars, 95% confidence intervals of the mean. The shaded areas represent the average duration of sunshine in Toronto, Ontario, Canada (range, 2.4-9.2 hours a day). [Modified from Praschak-Rieder et al., 2008]

Kalbitzer et al., subsequently replicated this finding in Copenhagen, Denmark,

also applying [11C]DASB PET in a study of fifty-four subjects, reporting seasonal

variation in 5-HTT BPND in the caudate and putamen (Kalbitzer et al., 2010).

Interestingly, in both studies, an inverse relationship was found between duration of

daily sunshine and 5-HTT BPND, suggesting that light exposure may be one external

environmental factor that may facilitate this seasonal change in 5-HTT BPND (Kalbitzer

et al., 2010; Praschak-Rieder et al., 2008) (Table 1-1). Other studies applying different

and lesser quality techniques and radioligands have also shown similar results (i.e. 5-

HTT BPND using [123I]β-CIT SPECT is quantifiable only in the midbrain whereas 5-HTT

BPND cannot be measured in cortical regions using [11C]McN5652 PET) (Brücke et al.,

1993; Ikoma et al., 2002; Parsey et al., 2000). Nonetheless, in Amsterdam,

Netherlands, Ruhé et al., applied [123I]β-CIT SPECT to examine the relationship

between season and 5-HTT BPND in forty-nine healthy and forty-nine depressed

subjects and observed elevated 5-HTT BPND in winter relative to summer in the

25

midbrain (Ruhe et al., 2009). In Hamburg, Germany, Buchert et al., used

[11C]McN5652 PET in thirty-nine healthy participants and also reported the season-

related finding in the midbrain, but not in the thalamus (Buchert et al., 2006).

Collectively, these findings from four independent research groups in four different

countries indicate that 5-HTT BPND is higher across multiple brain regions in winter

relative to summer (Table 1-2).

Table 1-1: Negative correlations between average sunshine duration and day length in Toronto, Ontario, Canada and brain 5-HTT BPND in 88 healthy study participants. [Modified from Praschak-Rieder et al., 2008]

Table 1-2: Seasonal fluctuation in 5-HTT binding as replicated by four independent research groups; aapplicable to only to midbrain regions; b5-HTT binding not measurable in cortex

In conclusion, there is evidence from both preclinical, post-mortem, clinical and

neuroimaging studies in support of seasonal variation within the serotonin system.

However, the majority of these studies focused upon investigation of this

26

monoaminergic system in health. In order to examine the effect of seasonal fluctuation

of the serotonin system in relation to seasonal changes in affect and behaviour, it is

necessary to study a clinical population of individuals who experience marked

seasonal variation in mood, such as those with SAD.

1.6 Seasonal Affective Disorder

1.6.1 Definition

SAD is a subtype of MDD, characterized by a pattern of MDEs that occur

seasonally as part of an existing mood disorder (American Psychiatric Association,

2013). A key criterion is a consistent pattern of onset and remission at characteristic

times of the year, which is not better explained by seasonally linked psychosocial

stressors (American Psychiatric Association, 2013). In addition, this seasonal pattern

of MDE recurrence and remission must persist for a minimum of two consecutive

years, during which there is no occurrence of non-seasonal MDEs (American

Psychiatric Association, 2013). Lastly, the lifetime number of seasonal MDEs must be

greater than the number of non-seasonal MDEs (American Psychiatric Association,

2013). This seasonal pattern of MDEs commonly occurs in fall-winter, with full

remission in spring-summer (i.e. “winter SAD”) (Levitan, 2007; Rosenthal et al., 1984).

Winter MDEs must include low mood or anhedonia, but are also often accompanied

by atypical depressive symptoms such as decreased energy, hypersomnia and

increased appetite or weight gain (Rosenthal et al., 1984).

27

1.6.2 Neuropathophysiology of SAD

There have been several lines of investigation in regards to identifying

biological markers associated with the SAD phenotype. Lavoie and colleagues

examined SAD patients using flash electroretinography and found decreased rod

sensitivity to light in winter relative to summer (Lavoie et al., 2009). Seasonal variation

in nocturnal melatonin secretion has also been reported such that, in SAD as

compared to health, duration the secretion is longer in winter than in summer,

indicating that circadian rhythm dysregulation may play a role in the pathology of this

disorder (Wehr et al., 2001). There is also evidence of circadian rhythm delay in

seasonal depression, such that intrinsic melatonin and temperature rhythms are

misaligned with external light-dark cycle (Lewy et al., 1988). Genetic markers

identified in SAD include altered polymorphic frequencies in the clock genes NPAS

and PER3 and a variable number tandem repeat on exon 3 of the DRD4 gene

(Johansson et al., 2003; Levitan et al., 2006). However, to date, a major body of work

has focused upon the role of monoamine neurotransmitter systems (i.e. serotonin,

dopamine, and norepinephrine) in SAD pathophysiology (Levitan, 2007). Of note,

there has been particular emphasis on the serotonin system given evidence of its

seasonal rhythmicity and the importance of 5-HT in regulating behaviours and

affective states that change with season such as mood, sleep and appetite.

1.6.2.1 Role of Serotonin Physiology in Seasonal Behaviour

It is well established that 5-HT plays a role in physiology and behaviours

related to mood, sleep and appetite. As such, this section will provide an overview of

28

research regarding the importance of 5-HT in regards to the modulation of affective

and behavioural states that change seasonally in SAD.

Results of clinical studies indicate that pharmacological manipulations that

affect function of the serotonin system and/or extracellular 5-HT levels have a

profound impact on mood and also in SAD. Depletion of tryptophan, the amino acid

precursor of 5-HT, has been estimated to reduce 5-HT levels by approximately 80

percent and this decrease in 5-HT is associated with low mood (Young et al., 1985).

Accordingly, reduction of extracellular 5-HT via acute tryptophan depletion (ATD) is

accompanied by the return of depressive symptoms in remitted MDD patients

following antidepressant treatment and relapse into MDE in SAD patients both after

light therapy in winter and during summer remission (Delgado et al., 1990; Neumeister

et al., 1997; Neumeister et al., 1998). Conversely, elevation of extracellular 5-HT

levels via administration of intravenous d-fenfluramine, a medication that causes 5-HT

release, is followed by happier mood and increased energy in healthy individuals, and

a reversal of depressive symptoms in SAD (Meyer et al., 1996; O'Rourke et al., 1989).

Administration of the non-selective 5-HT receptor agonist, m-CPP, to individuals with

SAD during winter, has been associated with reports of increased energy and

euphoria (“activation-euphoria”), and also blunted norepinephrine, cortisol and

prolactin response, suggestive of either a season-dependent downregulation or a

dysfunction of postsynaptic 5-HT receptors (Levitan et al., 1998; Schwartz et al.,

1997). Lastly, both tryptophan supplementation and use of SSRIs, treatments which

raise levels of extracellular 5-HT, are effective in ameliorating symptoms of seasonal

depression during winter (Lam et al., 1997; Lam et al., 2006). Thus, there is

29

substantial evidence to support hypofunction of serotonin system both in regards to

depressed mood and in SAD.

Sleep disturbances are a common in SAD with approximately 80 percent of

patients reporting symptoms of hypersomnia during winter MDEs (Kaplan et al.,

2009). As such, antidepressants that inhibit 5-HT reuptake and increase levels of

extracellular 5-HT can help to alleviate excessive sleepiness during winter in SAD and

these medications are also sometimes associated with the side-effect of transient

insomnia (Aszalos, 2006; Wilson et al., 2005). In addition, there is strong support from

preclinical animal studies of a relationship between the activity of 5-HT releasing

neurons in the DRN and the state of the sleep-wake cycle (Jacobs et al., 1999). To

elaborate, relative to the awake state, firing of 5-HT releasing neurons decreases

during slow wave sleep and is virtually absent during rapid eye movement sleep

(Jacobs et al., 1999; McGinty et al., 1976). Accordingly, during slow wave and rapid

eye movement sleep, respectively, extracellular 5-HT levels have been observed to

decrease by 50 percent and 38 percent, respectively, in the DRN of cats and by 58

percent and 37 percent, respectively, in the frontal cortex of rats (Portas et al., 1998;

Portas et al., 1994). In summary, hypersomnia is a hallmark of the symptomatic phase

of SAD, medications that elevate 5-HT are helpful in alleviating excessive sleepiness,

and extracellular 5-HT levels in both the DRN and efferent projection regions are

altered in a consistent manner during sleep relative to waking, suggestive of a

neuromodulatory role for 5-HT in the sleep-wake cycle.

Hyperphagia and carbohydrate cravings are core features of SAD (Rosenthal

et al., 1984). Appetite control is mediated through serotonergic neuromodulation at the

paraventricular nucleus of the medial hypothalamus (Blundell, 1984; Fletcher et al.,

30

1989) and it has been found that ingestion of high carbohydrate meals increases

uptake of tryptophan from the blood into the brain thereby elevating brain 5-HT levels

(Fernstrom, 1986). Interestingly, individuals with SAD experience seasonal alterations

in the ability to detect “sweet taste” such that this taste sensitivity is blunted in winter

and normalizes during the summer months, a variation that is not observed in healthy

volunteers (Andersen et al., 2014; Arbisi et al., 1996). As such, it has been postulated

that, in SAD, seasonal depressive symptoms of increased appetite and carbohydrate

cravings are attributable to hypofunction of the serotonin system during winter and

that such behaviours may reflect a compensatory mechanism to elevate low levels of

5-HT via excessive carbohydrates (i.e. sugary and starchy foods) (Andersen et al.,

2014; Arbisi et al., 1996).

It is also important to note that there strong evidence of an anorexiogenic role

for 5-HT in regards to appetite regulation; thus, low levels of extracellular 5-HT would

be expected to induce symptoms of hyperphagia and carbohydrate craving, as

commonly seen in the symptomatic phase of SAD, whereas pharmacological

manipulations that elevate extracellular 5-HT would likely reduce appetite (Fernstrom,

1985). Much research regarding 5-HT and appetite stems from both preclinical and

clinical studies of appetite regulation with a primary focus on treatment of obesity, but

as individuals with SAD commonly suffer from bingeing and significant weight gain

during the winter, symptoms which profoundly impact individual health and well-being,

the relationship between 5-HT and food intake is an important topic for discussion

(Rintamaki et al., 2008; Wurtman, 1993). Accordingly, in rodents, injection of 5-HT into

the paraventricular nucleus of the medial hypothalamus has been found to decrease

food intake and administration of d-fenfluramine has been shown to reduce both body

31

weight and food intake (Blundell, 1984; Fletcher et al., 1989). This latter finding

regarding d-fenfluramine has been corroborated clinically in two double-blind studies

of obese volunteers in which chronic use of this medication was significantly reduced

food intake and body weight relative to placebo (Drent et al., 1995; Guy-Grand et al.,

1989). Other supplements and medications that affect the serotonin system or raise

levels of extracellular 5-HT have also been found to suppress appetite (Halford et al.,

2007). For instance, in a double-blind placebo-controlled trial, twenty obese

participants treated for six weeks with 5-HTP experienced a reduction in body weight,

carbohydrate craving and earlier satiety as compared to volunteers receiving placebo

treatment (Cangiano et al., 1992). There also some evidence that treatment with m-

CPP, a medication that causes “activation-euphoria” in SAD during winter, reduces

both appetite and body weight in obese patients and in those with SAD (Levitan et al.,

1998; Sargent et al., 1997; Schwartz et al., 1997). As m-CPP has relatively high

affinity for post-synaptic 5-HT2C receptors that are primarily involved in regulation of

appetite and food intake, these findings suggest that agonism of this receptor subtype

may induce hypophagia and weight loss (Kennett et al., 1997; Sargent et al., 1997).

Lastly, appetite can be reduced by SSRIs, such as fluoxetine, that are commonly used

to treat winter MDEs in SAD and raise levels of extracellular 5-HT (Goldstein et al.,

1995; Lam et al., 1995; Pijl et al., 1991). To conclude, symptoms of hyperphagia,

carbohydrate craving and weight gain, commonly observed in patients with SAD

during winter, may be compensatory behaviours to offset hypofunction of the 5-HT

system and elevate low levels of 5-HT. Accordingly, medications that raise levels of

extracellular 5-HT or mimic the actions of this neurotransmitter are effective in

32

reducing appetite and inducing weight loss in individuals experiencing such

symptoms.

Taken together, these findings suggest that processes that influence

extracellular 5-HT levels across seasons are highly promising mechanisms to explain

seasonal variation in symptoms commonly observed in SAD, such as low mood,

hypersomnia, hyperphagia, carbohydrate craving and weight gain.

1.7 Light Therapy

In 1984, Rosenthal and colleagues published a seminal paper describing SAD

as a syndrome and reporting the efficacy of the light therapy, a chronotherapeutic

treatment, in mitigating the symptoms of seasonal depression (Rosenthal et al., 1984).

Seasonal variation in day-length (i.e. photoperiod) had been observed at latitudes

where SAD was particularly prevalent, and it had been hypothesized that the

symptoms of seasonal depression might be an outcome of shortened photoperiod in

winter (Levitan, 2007; Rosen et al., 1990). Accordingly, it was postulated that

extension of day-length during the winter months might ameliorate seasonal

depressive symptoms and that exposure to daily bright light might be a means by

which to lengthen shortened photoperiod during this season (Levitan, 2007). In a

double-blind, placebo-controlled, cross-over design, Rosenthal et al., randomized

patients with SAD into either bright light (2,500 lux) or dim light conditions (100 lux),

during which participants were asked to sit in front of a fluorescent lamp daily, for

three hours upon waking and before sleeping, for a period of two weeks (Rosenthal et

al., 1984). A robust antidepressant effect of bright light was observed after two weeks

33

of treatment whereas little or no therapeutic effect was found following exposure to the

alternative dim light condition (Rosenthal et al., 1984) (Figure 1-9).

Figure 1-9: Effect of light on mood in seasonal depression. Bright white light had significant antidepressant effects (t=8.45, p<.001, two-tailed paired t-test, Bonferroni intervals). There was no significant effect with dim light. [Modified from Rosenthal et al., 1984]

However, the lengthy duration of such a daily treatment regime required to

achieve therapeutic response necessitated investigating the feasibility of a briefer

more intense period of light exposure as compared to this initial treatment protocol. As

such, Terman and colleagues investigated the efficacy of thirty minutes of light

therapy at an intensity 10,000 lux and found its antidepressant effects to be

comparable to two hours of light therapy at an intensity of 2,500 lux (Terman et al.,

1990). Moreover, light therapy in the early morning was observed to be superior to

34

evening administration, with reported remission rates of 54.3 percent relative to 33.3

percent respectively (Terman et al., 1990; Terman et al., 1998).

Figure 1-10 Remission rates (criterion: post-treatment Structured Interview Guide for the Hamilton Depression Rating Scale – Seasonal Affective Disorder Version score ≤ 8) for four light treatment groups in a cross-over design balanced by parallel group controls ([M1][M2], [E1][E2], [M1][E2] and [E2][M1]). As can be seen, the effects if morning light (47.7% to 65% remission rate) are superior to that of evening light (25.9% to 36.8% remission rate) regardless of sequence within parallel and cross-over groups. [Modified from Terman et al., 1998]

In further support of its therapeutic effects, randomized controlled trials

comparing light therapy to antidepressant treatment found that its efficacy was

comparable to that of SSRIs with overall remission rates of approximately 55 percent

(Lam et al., 2006; Ruhrmann et al., 1998). Interestingly, the positive effects of light

therapy occurred within one to two weeks whereas antidepressant response typically

required two to four weeks of treatment (Lam et al., 2006; Ruhrmann et al., 1998).

At present, light therapy is regarded as a first-line treatment for SAD and

clinical consensus guidelines for treatment of seasonal depression recommend daily

35

use of a fluorescent lamp emitting full-spectrum white light fluorescent white light at a

“dose” of 10,000 lux for thirty minutes each morning (Lam et al., 1999). It is also

recommended that the height of the lamp be adjusted such that individual’s eyes are

level with the centre of the screen and angle of the lamp is tilted approximately 15°

downward. This lamp angle and level of light exposure are considered optimal for

amelioration of seasonal depressive symptoms as clinical trials using this protocol

have reported positive results (Lam et al., 2006; Levitan, 2005; Terman et al., 1998).

However, as 45 percent of SAD cases fail to achieve remission following a course of

light therapy, it is important to develop strategies to further improve this treatment

(Lam et al., 2006).

Despite evidence of the seasonal rhythmicity of the serotonin system, the

importance of 5-HT in regulation of mood and behavioural states that change with

season and evidence of aberrant function of the serotonin system in SAD, there have

been few studies evaluating the effect of deliberate light exposure on the serotonin

system. Some theories as to the mechanism by which light therapy exerts its

antidepressant effects include circadian phase advance, phase shift of endogenous

melatonin rhythm and enhancing resilience against tryptophan depletion (Lam et al.,

1996b; Lewy et al., 1987; Neumeister et al., 1997; Terman et al., 2001). There is

some evidence, albeit from small clinical studies, suggestive of the involvement of

serotonin system in therapeutic response to light in SAD. For example, tryptophan

supplementation has been reported to augment the antidepressant effects of light

therapy, and therapeutic response following combination treatment with light therapy

and citalopram, an SSRI, has been found to be superior to light therapy alone (Lam et

al., 1997; Thorell et al., 1999). Interestingly, in rodents, an inverse relationship

36

between light exposure and 5-HTT density has been observed in the hypothalamus

and SCN of the hypothalamus (Rovescalli et al., 1989). Furthermore, in PET studies

of healthy volunteers, an inverse relationship between duration of daily sunlight and 5-

HTT BPND has also been observed, suggesting this transporter protein may be

sensitive to light exposure and thus a potential brain biomarker by which to assess the

effects of light therapy (Kalbitzer et al., 2010; Praschak-Rieder et al., 2008).

1.8 Summary of Hypotheses

Study 1: The Effect of Season on 5-HTT BPND

Objective: To determine whether magnitude of seasonal variation in 5-HTT BPND, an

index of 5-HTT levels, in the PFC and ACC is greater in SAD as compared to health.

Hyp [1] The magnitude of seasonal fluctuation in 5-HTT BPND in the PFC and

ACC will be significantly greater in SAD relative to health.

Hyp [2] Seasonal variation in 5-HTT BPND in the PFC and ACC will be

associated with degree of seasonal change in mood and behaviour

Study 2: The Effect of Light Therapy on 5-HTT BPND in Health

Objective: To evaluate the effect of light therapy on 5-HTT BPND, an index of 5-HTT

levels, in the ACC and PFC of healthy individuals during fall and winter.

Hyp [3] In fall and winter, 5-HTT BPND will be significantly reduced in the ACC

and PFC after light therapy as compared to placebo treatment in healthy

volunteers.

Study 3: Effect of Light Therapy on 5-HTT BPND in SAD

37

Objective: To evaluate the effects of light therapy on 5-HTT BPND, an index of 5-HTT

levels, in the PFC and ACC of SAD subjects during the winter.

Hyp [4] In SAD, light therapy will be associated with reduced 5-HTT BPND in

the ACC and PFC in winter as compared to baseline (i.e. prior to start of treatment).

Note: In study 3, as we chose to examine the effect of light therapy on 5-HTT

BPND in a sample SAD of patients with fairly severe seasonal depressive symptoms, a

study design including a placebo control group, as in study 2, was not feasible. In

such a clinical situation, in which an evidence based first-line therapeutic is available,

the ethical standard at CAMH is that a placebo should not be given.

In regards to all three studies, the ACC and PFC were prioritized since

structures within these regions have a key role in mood regulation and cognitive

processing of emotion (Liotti et al., 2001; Liotti et al., 2002; Mayberg et al., 1999;

Sharot et al., 2007; Tom et al., 2007). Our secondary hypotheses are that similar

changes in 5-HTT BPND will occur in other brain regions assayed, including the

hippocampus, ventral striatum, dorsal putamen, dorsal caudate, thalamus and

midbrain.

38

2 MATERIALS AND METHODS

2.1 Rationale For Use Of [11C]DASB Positron Emission Tomography

At present, [11C]DASB (11C-labeled 3-amino-4-[2-dimethylaminomethyl-

phenylsulfanyl]benzonitrile) is the preferred radioligand for neuroimaging of the 5-HTT.

It is a high quality radioligand with several useful properties including selectivity for the

5-HTT, excellent brain uptake, reversible kinetics, negligible sensitivity to endogenous

5-HT, a high ratio of specific to free/non-specific binding allowing for its quantification

in multiple brain regions and its reliability is well established (Ginovart et al., 2001;

Houle et al., 2000; Wilson et al., 2002; Wilson et al., 2000). In addition, binding of

DASB to the 5-HTT, in vitro, has been observed to be saturable only in environments

mimicking extracellular conditions and not intracellularly (Quelch et al., 2012).

Accordingly, this result suggests that changes in 5-HTT BPND observed in vivo using

[11C]DASB PET may largely represent fluctuations in levels of 5-HTT protein on the

cell surface (i.e. extracellular environment) and not within the cell. Furthermore, the

major role of the 5-HTT is its ability to transport extracellular 5-HT from the synaptic

cleft into the presynaptic neuron and this functionality is dependent upon its

embedding in the cell membrane. As such, it would be expected that fluctuations in 5-

HTT BPND observed in vivo using [11C]DASB PET would correlate to alterations in

extracellular 5-HT reuptake.

There are also a number of other radioligands which have been used for 5-HTT

neuroimaging including β[123I]CIT, [11C]McN5652, [123I]ADAM, [11C]MADAM and

[11C]HOMADAM. These radioligands have been applied for several years, and have

modeling, displacement studies, and reliability data in humans. Their properties will be

39

summarized briefly and are outlined in further detail in the table below (Table 2-1)

(Meyer, 2014).

Table 2-1: Comparison of PET and SPECT Radiotracers for the 5-HTT

[Modified from Meyer et al., 2014]

The first 5-HTT radioligands were β[123I]CIT and [11C]McN5652, applied using

SPECT and PET imaging, respectively, although reliable 5-HTT quantification using

both techniques was difficult due to significant methodological constraints (Brücke et

al., 1993; Szabo et al., 1995). Measurement of the 5-HTT using β[123I]CIT SPECT is

feasible only in the midbrain, a region in which density of both the 5-HTT and the DAT

40

are high (Brücke et al., 1993). In addition, the β-CIT ligand has near equal affinity for

both monoamine transporters and this further complicated quantification of the 5-HTT

in the midbrain (Laruelle et al., 1994). In regards to [11C]McN5652, this radioligand has

modest reversibility and a low ratio of specific relative to free and non-specific binding,

both characteristics which prohibited reliable quantification of 5-HTT in cortical regions

(Buck et al., 2000; Ikoma et al., 2002; Parsey et al., 2000).

Later generation radioligands include [11C]DASB, [123I]ADAM, [11C]MADAM and

[11C]HOMADAM (Meyer, 2014). The properties of [11C]DASB were described in the

first paragraph of this section and are further outlined in the table below for

comparison purposes. [123I]ADAM, a SPECT radioligand, has some advantage over

β[123I]CIT in regards to its high affinity for the 5-HTT relative to the DAT and NET, but

its low specific to free and non-specific binding ratio limits its use to midbrain regions

(Catafau et al., 2005; Choi et al., 2000; Erlandsson et al., 2005; Oya et al., 2000). The

[11C]MADAM PET radioligand approaches the quality of [11C]DASB, although

[11C]DASB ligand has been observed to have better brain uptake and test-retest

reliability in comparison to [11C]MADAM (Lundberg et al., 2006). Finally, although

there is evidence that the PET radioligand [11C]HOMADAM displays better reversibility

and a greater specific to free and non-specific binding ratio relative to [11C]DASB, in

vivo investigations of its test-retest reliability and sensitivity to endogenous 5-HT have

not been conducted and such studies are necessary for further development of this

tracer (Ginovart et al., 2001; Nye et al., 2008). Thus, [11C]DASB was applied to

measure 5-HTT BPND in the following series of studies.

Information regarding PET scan radiation dose for [11C]DASB was included in

study consent forms, which also detailed scanning procedure and associated risks.

41

This information was explained to study participants in accordance with CAMH

standard operating procedures, both within the consent forms and in the initial

interview by investigators AET or SJH. Dose of [11C]DASB administered to each

subject was less than 2mSv/scan, well within Health Canada guidelines for a PET

study and less than the amount of radiation received from natural sources over one

year (3mSv). Delay between scans ranged from 2 weeks (study three) to 6 months

(study one). Risk to participants was described as minimal, as such doses and

associated scanning intervals have never been associated with adverse effects.

Additional questions regarding the scanning procedure were addressed by principal

investigator JHM.

2.2 Study 1: The Effect of Season on 5-HTT BPND

2.2.1 Participants

Twenty SAD participants (14 women and 6 men; mean [SD] age: 31.3 [4.8]

years; age range: 24-39) and twenty healthy volunteers (13 women and 7 men; mean

[SD] age: 30.5 [4.2] years; age range: 24-39) were recruited from the Greater Toronto

Area between June 2012 and July 2015. Healthy subjects were age-matched to SAD

subjects within 3 years. Demographics are listed in the table below (Table 2-2)

Table 2-2: Demographic Characteristics

42

Criteria for all included being between the ages of 18 to 40, non-smoking and in

good physical health, no history of alcohol or substance abuse, no antidepressant use

within the past 6 months and no use of prescription medications or herbal

supplements within the past 2 months. In addition, as light exposure has been found

to reduce [11C]DASB 5-HTT BPND during the winter months and [11C]DASB 5-HTT

BPND has been shown to be inversely correlated with duration of daily sunshine, both

use of light therapy within the past 3 months and travel to more southern latitudes

during the study period were exclusionary (Harrison et al., 2015; Praschak-Rieder et

al., 2008). Exclusion criteria for female subjects included use of oral contraceptives,

current pregnancy, postpartum or recent abortion (within one year), and in

perimenopause or menopause. No female subject tested positive for pregnancy at the

time of scanning as indicated by urine dip stick testing for human chorionic

gonadotropin (hCG).

Subjects were asked not to take over the counter medications one week prior

to scanning, to avoid alcohol 4 days prior to scanning and not to consume caffeinated

beverages within 2 days of the PET scan. In addition, lifetime history of Axis I or Axis

II disorders was exclusionary for healthy subjects and comorbid Axis I or Axis II

disorders were exclusionary for SAD subjects. Screening instruments included the

Structured Clinical Interview for DSM-IV-TR (SCID-I/II). Accordingly, all subjects

received both SCID-I/II assessments and a consultation with a study psychiatrist to

verify either (i) the presence of recurrent MDD with a seasonal pattern specifier (SAD

group) or (ii) the absence of lifetime psychiatric illness (healthy volunteers). Urine drug

screening was performed at initial assessment and on each PET scanning day to rule

out recent drug and medication use. Urine toxicology was performed using gas

43

chromatography–mass spectrometry (GC-MS) at the CAMH clinical laboratory (drug

screening sensitive to ethanol, drugs of abuse, all classes of antidepressants,

antipsychotics, anticonvulsants, benzodiazepines, narcotics, NSAIDs, anthelmintics,

statins, β-blockers, muscle relaxants and anti-allergy medications).

Participants also completed the Seasonal Pattern Assessment Questionnaire

(SPAQ) from which a summed global seasonality score was calculated to determine

degree of seasonality (i.e. seasonal change in sleep, mood, energy, appetite, weight

and social activity) (Rosenthal et al., 1987). The SPAQ was administered in

accordance with the season in which subjects were scanned. All participants were

scanned first in the season during which they were assessed (i.e. winter or summer)

and the distribution of subjects scanned initially in summer or winter, did not differ

between groups (summer: 10 healthy, 8 SAD; winter: 10 healthy, 12 SAD; χ2(1)=0.40,

p=0.53). Subjects were defined categorically as healthy (with no seasonality) for

SPAQ scores below 12, moderate SAD for scores between 12 and 16, and severe

SAD for scores equal to or greater than 16 (Bartko et al., 1989; Terman, 1988). All

healthy participants had SPAQ scores of less than 7 (mean [SD]: 2.1 [1.7], range 0-6,

Table 2.1). On each scan day the Structured Interview Guide for the Hamilton

Depression Rating Scale with Seasonal Affective Disorder Supplement (SIGH-SAD)

was also administered. For each participant, written informed consent was obtained

after the procedures were fully explained. The study and recruitment procedures were

approved by the Research Ethics Board for Human Subjects at the Centre for

Addiction and Mental Health (CAMH), University of Toronto.

44

2.2.2 Image Acquisition and Analysis

All scans occurred at the CAMH Research Imaging Centre. All participants

underwent two [11C]DASB PET and MRI scans: one in spring-summer and the other in

fall-winter, in randomized order, to measure the seasonal percent change in 5-HTT

BPND. Scan dates of healthy controls were matched to SAD participants within two to

four weeks. To minimize any potential effects of circadian rhythm all scans were

scheduled in the morning and took place at either 9:30am or 11:30am. All participants

were non-smoking and on each PET scan day underwent laboratory tests (plasma

sampling for cotinine, calcium and thyroid hormones, complete blood cell count) to

verify non-smoking status and ensure physical health.

Synthesis of [11C]DASB has been described previously (Ginovart et al., 2001;

Wilson et al., 2000). Briefly, [11C]-CH3I was trapped in a high-performance liquid

chromatography sample loop coated with a solution of the N-normethyl precursor (1

mg) in dimethylformamide (80 µl). After five minutes at ambient temperature, the

contents of the sample loop were injected onto a reverse-phase high-performance

liquid chromatography column, and the fraction containing the product was collected,

evaporated to dryness, formulated in saline, and filtered through a 0.2-µ filter. Prior to

each scan, an intravenous bolus of 10 mCi (370 MBq) of [11C]DASB was injected. The

[11C]DASB was of high radiochemical purity (98.10% ± 5.16%) and high specific

activity (65.62 ± 26.36 GBq/μmol) at the time of injection. PET images were obtained

using a high-resolution PET/CT Siemens-Biograph HiRez XVI scanner (81 axial

sections of 2mm; Siemens Molecular Imaging, Knoxville, TN, USA). The emission

scan was reconstructed in 15 frames of 1 minute, followed by 15 frames of 5 minutes,

45

totaling to a scan duration of 90 minutes in length. The images were corrected for

attenuation using a germanium 68–labeled transmission scan and reconstructed using

2D filtered back projection algorithms with a ramp filter. Subsequent to the initial PET

scan, each participant also underwent a magnetic resonance imaging scan (GE 3.0-T

scanner, fast spin echo – XL sequence, proton density–weighted image, x, y, z voxel

dimensions; 0.37, 0.37, and 0.90 mm, GE Medical Systems, Milwaukee, WI, USA).

Regions of interest (ROIs) on the MRI were determined using a semi-

automated method in which regions of a template MRI are transformed onto the

individual MRI based on a series of transformations and deformations that matched

the template image to the individual co-registered MRI, as well as segmentation of the

individual MRI to select gray matter voxels as previously described (Meyer et al.,

2009; Rusjan et al., 2006). ROIs on the MRI were subsequently located on the PET

image using the rigid body transformations from co-registration of the MRI to PET

image via a mutual information algorithm. ROIs included the prefrontal cortex, anterior

cingulate cortex, ventral striatum, dorsal caudate, dorsal putamen, thalamus,

hippocampus, midbrain and cerebellar cortex. The location of the ROIs were verified

by visual assessment of their display on the integral [11C]DASB PET image. The

cerebellar cortex reference region was defined as the posterior half of the cerebellar

cortex, excluding the vermis and cerebellar white matter. Reference tissue methods

have been validated for [11C]DASB to calculate 5-HTT BPND (Ginovart et al., 2001;

Ichise et al., 2003). We applied the non-invasive Logan method, which has a modest

underestimate but the advantage of having the lowest coefficient of variation (i.e.

standard deviation/mean) of calculated BPND values (Logan et al., 1996). An additional

analysis was conducted using the simplified reference tissue method 2 (SRTM2)

46

which has a negligible underestimate but a higher coefficient of variation (Wu et al.,

2002). Test-retest variability of 5-HTT BPND values using the non-invasive Logan

method have been reported have a mean regional change of 0% with a standard

deviation of ±4.75% in the prefrontal cortex, ±3.7% in the anterior cingulate cortex,

±1.6% in the bilateral caudate, ±2.6% in the bilateral putamen, ± 2.5% in the thalamus

and ±0.3% in the midbrain/superior raphe nuclei, with similar results obtained using

the SRTM2 method (Praschak-Rieder et al., 2005).

2.2.3 Statistical Analysis

Seasonal percent change in 5-HTT BPND (% Δ 5-HTT BPND) was calculated in

each region for each subject [(winter 5-HTT BPND - summer 5-HTT BPND)/summer 5-

HTT BPND)]. As one value pertaining to a particularly severe SAD case was outside of

the normal distribution, non-parametric tests were used for all statistics. The % Δ 5-

HTT BPND in the PFC and ACC was compared between SAD and healthy groups

using the Mann-Whitney U test. To assess the relationship of % Δ 5-HTT BPND to

severity, the primary method was to categorize SAD into two groups, moderate and

severe, applying a cut-off of greater than 16 on the SPAQ as previously described

(Bartko et al., 1989; Terman, 1988). To determine whether a difference was present

amongst healthy, moderate SAD and severe SAD groups, the Kruskal–Wallis H test

was applied to assess % Δ 5-HTT BPND in the PFC and ACC, and then the Mann-

Whitney U test was used to compare healthy with severe SAD. As a secondary

approach, all analyses were applied in the other regions of interest including the

dorsal putamen, thalamus, dorsal caudate, midbrain, ventral striatum and

hippocampus.

47

As an additional analysis, the effect of severity of SAD upon seasonal change

in 5-HTT BPND was investigated. Seasonal change in 5-HTT BPND (Δ 5-HTT BPND)

was calculated for each participant (winter 5-HTT BPND - summer 5-HTT BPND). To

determine whether a difference was present amongst healthy, moderate SAD and

severe SAD groups, the Kruskal–Wallis H test was applied to assess Δ 5-HTT BPND in

the PFC and ACC and other examined regions, followed by the Mann-Whitney U to

compare healthy with severe and moderate SAD groups. The Kruskal–Wallis H test

was also used to determine if 5-HTT BPND values differed across groups in winter and

in summer.

Further assessments of the relationship to severity of symptoms, were to

determine the Spearman correlation coefficients between % Δ 5-HTT BPND in the PFC

and ACC and seasonal depressive symptoms, as measured by the SPAQ. These

correlations were also determined for other regions of interest. Lastly, as an

exploratory analysis, Spearman correlation coefficients were used to assess the

relationship between Δ 5-HTT BPND and seasonal depressive symptoms in all brain

regions assayed.

2.3. Study 2: The Effect of Light Therapy on 5-HTT BPND in Health

2.3.1 Participants

Twenty-one healthy volunteers (11 women and 10 men; mean [SD] age 25.8

[5.8] years; age range 19-39) were recruited through fliers posted in community

locations within the Toronto area. We elected to study healthy volunteers, rather than

SAD patients because we were interested in investigating the potential of 5-HTT BPND

as a biomarker for light exposure, so as to develop prevention strategies for those

48

who had not yet developed SAD. This was plausible because previous observations of

correlations between 5-HTT BPND and season were in healthy samples (Kalbitzer et

al., 2010; Praschak-Rieder et al., 2008). For each participant, written informed

consent was obtained after the procedures were fully explained. The study and

recruitment procedures were approved by the Research Ethics Board for Human

Subjects at the Centre for Addiction and Mental Health, University of Toronto.

Participants were recruited as non-smoking healthy volunteers, with no history of

major medical or psychiatric illness, no history of alcohol or substance abuse and no

recent use of prescription or over the counter medications, including herbal

supplements. Female subjects were free from oral contraceptives. Subjects were

screened using the Structured Clinical Interview for DSM-IV–Non-Patient-Edition to

rule out psychiatric disorders (current or in remission), current suicidal ideation, history

of self-harm, anger dyscontrol or impulsive behavior. Urinalysis and urine drug

screening were also performed to rule out recent herbal, drug or medication use (i.e.

within 5 half-lives).

Two subjects did not complete the study. One subject withdrew due to

discomfort in the scanner. A second subject had a positive urine drug screen for a

recreational drug and was subsequently withdrawn. Thus, 19 participants (10 women

and 9 men) completed the study.

2.3.2 Study Design

Volunteers participated in a single-blind, placebo-controlled, within-subject,

counterbalanced, crossover design. All subjects completed two experimental

conditions separated by a four week washout period. In Condition 1, participants

49

received five sessions of daily light therapy and in Condition 2, participants received

five sessions of placebo treatment. In each treatment condition, sessions were 30

minutes in length and took place between 7:00am and 7:30am for five consecutive

mornings. Subjects were randomly assigned in regards to the order in which they

received each condition and assignment of conditions was balanced in both subject

number and order.

Condition 1 (Light Therapy): Subjects were seated approximately 30 cm in front

of a Day-Light Classic light box (Uplift Technologies Inc., Dartmouth, NS, Canada),

which emitted broad-spectrum fluorescent white light at 10,000 lux for 30 minutes; the

standard treatment dose for SAD (Terman et al., 1990; Terman et al., 2005). The

height of the box was adjusted so that the subject’s eyes were level with the centre of

the screen and the box was tilted at an angle of approximately 15° downward. During

the sessions, subjects were asked to read during so that gaze was cast downward

and head position and posture were monitored by a study investigator.

Condition 2 (Placebo): In a room separate from that in which subjects had

undergone the light therapy condition, subjects were tethered to an inactive negative

ionization system (Sphere One Inc., Chattanooga, TN) with a Velcro wrist strap and

seated approximately 30 cm in front of the ionizer. Posture and position were

monitored by a study investigator. As the negative ionizer was turned off with only the

fan running to simulate activity, this was not expected to produce an effect on either

mood or 5-HTT BPND. In addition, to control for expectancy bias, this intervention was

described as another active condition. Subjects were instructed to read for the

duration of the 30 minute session.

50

Mood-related symptoms were assessed at screening and on day five of each

condition using the Structured Interview Guide for the Hamilton Depression Rating

Scale with Atypical Depression Supplement (SIGH-ADS), Beck Depression Inventory

(BDI) and the 10cm Visual Analogue Scale (VAS) for mood (i.e. happy–depressed),

energy (i.e. most–least), and anxiety (i.e. relaxed–tense). Participants also wore an

Actiwatch (Philips Electronics, Murrysville, PA), a waterproof light-weight wrist-watch,

for 24 hours/day during both light therapy and placebo treatment to collect information

about how such variables might differ across conditions. At the end of each condition

subjects also underwent a [11C]DASB PET scan to measure 5-HTT BPND.

2.3.3 Image Acquisition and Analysis

Participants underwent a [11C]DASB PET scan at the end of each treatment

condition, spaced a minimum of four weeks apart. All scans were scheduled for the

morning and took place at either 9:30am or 11:30am. Lighting conditions in the scan

room were kept constant during scanning and participants were instructed to keep

their eyes closed for the scan duration. Participants were required not to consume any

caffeine or alcohol 48 hours prior to scanning. On the day of scanning, subjects also

underwent laboratory tests (complete blood cell count, plasma sampling for calcium,

cotinine and thyroid hormones) to ensure physical health and non-smoking status, and

a urine drug screen to rule out recent herbal, drug or medication use (within 5 half-

lives). All subjects were medication-free prior to scanning, save for three participants

who tested positive for over the counter medications, none of which seemed likely to

influence the serotonin transporter (ibuprofen, aspirin, pseudoephedrine). In addition,

51

removal of these subjects from analysis did not affect the significance of the results

and therefore, these data were included in the final analyses.

Synthesis and measurement of 5-HTT BPND with [11C]DASB reported in this

study are the same as those used in previous studies (Ginovart et al., 2001; Houle et

al., 2000; Meyer et al., 2001; Meyer et al., 2004b; Praschak-Rieder et al., 2008;

Praschak-Rieder et al., 2005). Briefly, PET images were acquired using a High

Resolution Research Tomograph (HRRT) PET camera (in-plane resolution; full-width

half-maximum, 3.1mm; 207 axial sections of 1.2mm; Siemens Molecular Imaging,

Knoxville, TN, USA). Prior to each scan, an intravenous bolus of 10 mCi (370 MBq) of

[11C]DASB was injected. The [11C]DASB was of high radiochemical purity (99.35% ±

0.57%) and high specific activity (84.78 ± 31.14 GBq/μmol) at the time of injection.

The emission scan was reconstructed in 15 frames of 1 minute, followed by 15 frames

of 5 minutes, totaling to a scan duration of 90 minutes in length (Ginovart et al., 2001;

Ichise et al., 2003). The images were corrected for attenuation using a cesium 137–

labeled transmission scan and reconstructed by filtered back-projection (Hann filter) at

Nyquist cut-off frequency. Additional details regarding the scanning acquisition have

been previously published (Meyer et al., 2009). Each participant also underwent

magnetic resonance imaging (GE Signa 1.5-T scanner, spin-echo sequence proton

density–weighted image, x, y, z voxel dimensions; 0.78, 0.78, and 3mm, GE Medical

Systems, Milwaukee, WI USA) for the region of interest (ROI) delineation. ROIs were

delineated on these magnetic resonance images using a semi-automated method

based on linear and nonlinear transformations of an ROI template in standard space

to each individual magnetic resonance image (MRI), followed by a refinement process

based upon the gray matter probability (Rusjan et al., 2006). The MRI was co-

52

registered to the summated [11C]DASB PET image using a mutual information

algorithm and the resulting transformation was applied to sample the ROIs from the

PET image (Studholme et al., 1999). The location of the ROI was verified by visual

assessment on the summated [11C]DASB PET image. The posterior half of the

cerebellar cortex under exclusion of vermis and cerebellar white matter served as the

reference region. The borders of the reference tissue were at least 1 full width at half

maximum (5.5 mm) from the venous sinuses and occipital cortex. At a distance of 1

full width at half maximum, spillover from the occipital cortex (which has specific

binding) or the venous sinuses is negligible.

5-HTT BPND values were determined using the non-invasive Logan method

(PMOD Technologies Ltd, Zurich, Switzerland) (Logan et al., 1996). This method

provides valid and reproducible [11C]DASB PET measurements of 5-HTT BPND values

with low between-subject variance in 5-HTT BPND for most brain regions (Meyer et al.,

2004a; Meyer et al., 2004b; Praschak-Rieder et al., 2007; Praschak-Rieder et al.,

2005). A secondary analysis was conducted using SRTM2 (Wu et al., 2002).

2.3.4 Statistical Analysis

Participants were divided into fall (September 22 to December 20) and winter

(December 21 to March 19) groups corresponding to the season in which they were

scanned. The mean scan date [SD] of the fall group was, November 17, 2009 [18.6

days] and the mean scan date of the winter group was February 13, 2010 [16.1 days].

For each region, within each group, the Shapiro-Wilk test was applied to

assess the normality of the distribution of the difference in 5-HTT BPND between light

53

and placebo conditions. PET data was also plotted as a histogram for each group and

visually inspected to ensure normality and concordance with the Shapiro-Wilk test. In

the fall group, the difference in 5-HTT BPND between light and placebo was normally

distributed across all brain regions assayed. In the winter group, PET data was

normally distributed across all brain regions with the exception of the thalamus

(Shapiro-Wilk test, W=0.82, p=0.03).

The primary analyses were repeated-measures multivariate analyses of

variance (MANOVA) applied to each group to assess the effect of treatment (light

therapy versus placebo) on 5-HTT BPND (the repeated dependent variable) in the ACC

and PFC within each season. Regional univariate repeated-measures ANOVAs were

applied only if there was a significant omnibus effect in the repeated-measures

MANOVA. To correct for 4 multiple comparisons (2 seasons, fall and winter; 2 a priori

brain regions, ACC and PFC), significance for each ANOVA was set at a p-value of

0.0125. As a secondary analysis, for each group, a repeated-measures MANOVA was

applied to assess for a global effect of treatment (light therapy versus placebo) on 5-

HTT BPND in all brain regions assayed, with the exclusion of data from the thalamus

pertaining to the winter group, which was not normally distributed.

In addition, for each group, exploratory two-tailed paired t-tests were also

performed to examine the effects of treatment in all brain regions, with the exception

of winter data from the thalamus, for which the non-parametric Wilcoxon Signed

Ranks test was applied. For these exploratory analyses, to correct for 14 multiple

comparisons (2 seasons, fall and winter; 7 brain regions), significance was set at a p-

value of 0.0036. For each group (with the exclusion of winter data from the thalamus),

effect size (Cohen’s d) was calculated for all regions, defined as mean difference

54

across conditions (light versus placebo) divided by the standard deviation of the

difference across conditions. For winter data from the thalamus, effect size was

calculated by dividing the test statistic from the non-parametric Wilcoxon Signed

Ranks Test by the square root of the number of observations.

As an additional exploratory measure, Pearson correlation coefficients were

also applied to determine whether there was a relationship between reduction in 5-

HTT BPND in the ACC, a region heavily involved in mood regulation, following light

therapy relative to placebo, and improvement on scales of mood-related symptoms

(BDI, VAS Mood, Anxiety, Energy and SIGH-ADS) (Ressler et al., 2007). All analyses

were performed using the Statistical Package for Social Sciences version 20 (IBM

SPSS Statistics, Chicago, IL).

2.4 Study 3: Effect of Light Therapy on 5-HTT BPND in SAD

2.4.1 Participants

For each participant, written informed consent was obtained after the

procedures were fully explained. The study and recruitment procedures were

approved by the Research Ethics Board for Human Subjects at the Centre for

Addiction and Mental Health, University of Toronto. Eleven SAD participants (9

women and 2 men; mean [SD] age: 32.8 [2.6] years; age range: 26-39) were recruited

from the Greater Toronto Area between December 2013 and December 2015.

Participant demographics are included in Table 2-3.

Criteria included being age 18 to 40, non-smoking and in good physical health

with no history of alcohol or substance abuse, no antidepressant use within the past 6

55

months and no use of prescription medications or herbal supplements within the past

2 months. Comorbid Axis I or Axis II disorders were exclusionary. In addition, both use

of light therapy within the past 3 months and travel to more southern latitudes during

the study period were also exclusionary as potentially confounding variables (Harrison

et al., 2015; Praschak-Rieder et al., 2008). Exclusion criteria for female subjects

included use of oral contraceptives, current pregnancy, postpartum or recent abortion

(within one year), and in perimenopause or menopause. Seven of eleven participants

had previous trials of antidepressant medications (SSRIs, SNRIs or bupropion) for

treatment of winter MDEs, although no participant had used psychotropic medication

in the past six months. One participant had previously bought a light box but had

never used it for a consistent period and had not used it within the past six months.

Screening instruments included the Structured Clinical Interview for DSM-IV-TR

(SCID-I/II) and Seasonal Pattern Assessment Questionnaire (SPAQ) from which a

summed global seasonality score was calculated to determine degree of seasonality

(i.e. seasonal change in sleep, mood, energy, appetite, weight and social activity

(Rosenthal et al., 1987).

Table 2-3: Demographic Characteristics

56

All SAD subjects received a consultation with a study psychiatrist to verify

diagnosis and attended clinical follow-up appointments two weeks after beginning light

therapy and at the end of treatment (week six). Initial consultations and follow-up

appointments included assessment for emergence of manic symptoms (which did not

occur in this sample), but a scale based measurement specifically for manic

symptoms (i.e. Young Mania Rating Scale) was not applied (Sit et al., 2007). Urine

drug screening was performed at initial assessment and on each PET scanning day to

rule out recent drug and medication use.

2.4.2 Treatment Protocol

Volunteers participated in an open-label, within-subject investigation during

which they received a total of six weeks of daily morning light therapy. Enrollment

began in late fall, starting from November 1 and all participants completed the light

therapy protocol by February 15, so as reduce the possibility of spontaneous spring

remission (Lam et al., 2006). The study was conducted over three consecutive winters

(2013/2014-2015/2016). Participants were asked to use a Day-Light Classic light box

(Uplift Technologies Inc., Dartmouth, NS, Canada) each morning for a 30 minute

period between 7:00am and 8:00am. All subjects underwent two [11C]DASB PET and

MRI scans: one set of scans before beginning light therapy and another after two

weeks of treatment to measure the effect of light therapy on 5-HTT BPND. This

duration between scans was chosen because usually, most of the clinical response of

seasonal MDEs to light therapy occurs within two weeks and it was our intent to

evaluate neurochemical changes concomitant with this dramatic reduction in

57

depressive symptoms (Lam et al., 2006; Ruhrmann et al., 1998). At the end of

treatment, subjects also came in for a final clinical assessment.

Participants were trained in how to use the light box and provided with an

instruction sheet detailing its use. Training and instructions were consistent with

clinical consensus guidelines for the use of light therapy for treatment of SAD (Lam et

al., 1999). Briefly, subjects were asked to sit approximately 30cm in front the light box

which emitted broad-spectrum fluorescent white light at 10,000 lux for 30 minutes; the

standard treatment dose for SAD (Terman et al., 1990; Terman et al., 2005). The

height of the box was adjusted so that the subject’s eyes were level with the centre of

the screen and the box was tilted at an angle of approximately 15° downward. During

the sessions, participants were instructed not to stare directly at the light and instead,

asked to read so that gaze and head position were cast downward.

Mood-related symptoms were assessed at screening with the Structured

Clinical Interview for DSM-IV, and in addition, on each PET scan day and when

participants returned for their final assessment following six weeks of treatment,

assessment tools included the Hamilton Depression Rating Scale with the Seasonal

Affective Disorder Supplement (SIGH-SAD), Beck Depression Inventory (BDI),

Dysfunctional Attitudes Scale (DAS) and the 10cm Visual Analogue Scale (VAS) for

mood (i.e. happy–depressed), energy (i.e. most–least), and anxiety (i.e. relaxed–

tense) (Williams J et al., 1994). Applying the SIGH-SAD, treatment response was

defined as a reduction of at least 50% compared to baseline with a score of less than

or equal to 14 and remission was defined as a score less than or equal to 8 (Terman

et al., 1998).

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2.4.3 Image Acquisition and Analysis

Participants underwent two [11C]DASB PET and MRI scans, one set of scans

before beginning light therapy and another after two weeks of treatment to measure

the effect of light therapy on 5-HTT BPND. Participants were asked not to take over-

the-counter medications one week prior to scanning, to avoid alcohol 4 days prior to

scanning and not to consume caffeinated beverages within 2 days of the PET scan. In

addition, to minimize any potential effects of circadian rhythm all scans were

scheduled in the morning and took place at either 9:30am or 11:30am. All participants

were non-smoking and on each PET scan day underwent laboratory tests (plasma

sampling for cotinine, calcium and thyroid hormones, complete blood cell count) to

verify non-smoking status and ensure physical health.

Synthesis of [11C]DASB has been described previously (Ginovart et al., 2001;

Wilson et al., 2000). Briefly, [11C]-CH3I was trapped in a high-performance liquid

chromatography sample loop coated with a solution of the N-normethyl precursor (1

mg) in dimethylformamide (80 µl). After 5 minutes at ambient temperature, the

contents of the sample loop were injected onto a reverse-phase high-performance

liquid chromatography column, and the fraction containing the product was collected,

evaporated to dryness, formulated in saline, and filtered through a 0.2-µ filter. Prior to

each scan, an intravenous bolus of 10 mCi (370 MBq) of [11C]DASB was injected. The

[11C]DASB was of high radiochemical purity (98.60% ± 0.61%) and high specific

activity (58.10 ± 33.17 GBq/μmol) at the time of injection. PET images were obtained

using a high-resolution PET/CT Siemens-Biograph HiRez XVI scanner (81 axial

sections of 2mm; coverage from medulla through to superior frontal cortex; Siemens

59

Molecular Imaging, Knoxville, TN, USA). The emission scan was reconstructed in 15

frames of 1 minute, followed by 15 frames of 5 minutes, totaling to a scan duration of

90 minutes in length. The images were corrected for attenuation using a germanium

68–labeled transmission scan and reconstructed using 2D filtered back projection

algorithms with a ramp filter. Subsequent to the initial PET scan, each participant also

underwent a magnetic resonance imaging scan (GE 3.0-T scanner, fast spin echo –

XL sequence, proton density–weighted image, x, y, z voxel dimensions; 0.37, 0.37,

and 0.90 mm, GE Medical Systems, Milwaukee, WI, USA) for the region of interest

(ROI) delineation.

To minimize head movement during the PET scan, a custom fitted

thermoplastic mask was made for each subject and used with a head fixation system.

After reconstruction denoised dynamical images were visually inspected for potential

head movement. Motion was determined by defining the outline of the cortex in the

transverse and coronal planes of the integral PET image and then by transposing this

outline onto sequential frames which were visually inspected for shift using Analyze

version 9.0 (AnalyzeDirect, Inc., KS, USA). When motion was visible, it was corrected

using frame realignment with respect a reference frame characterized by a high

signal-to-noise ratio (Mawlawi et al., 2001). Roto-translation transformations were

calculated on denoised frames images using the Automatic Image Registration

algorithm (Woods et al., 1992). When motion correction was applied the reference

frame was the first 5 minute frame.

ROIs were delineated on the magnetic resonance images using a semi-

automated method in which regions of a template MRI are transformed onto the

individual MRI based on a series of transformations and deformations that matched

60

the template image to the individual co-registered MRI, as well as segmentation of the

individual MRI to select gray matter voxels as previously described (Meyer et al.,

2009; Rusjan et al., 2006). ROIs on the MRI were subsequently located on the PET

image using the rigid body transformations from co-registration of the MRI to PET

image via a mutual information algorithm. All ROIs were bilateral and included the

anterior cingulate cortex, prefrontal cortex, ventral striatum, dorsal putamen, thalamus,

hippocampus, midbrain and cerebellar cortex. The location of the ROI was verified by

visual assessment on the summated [11C]DASB PET images. The posterior half of the

cerebellar cortex excluding the vermis and cerebellar white matter served as the

reference region.

Reference tissue methods have been validated for [11C]DASB to calculate 5-

HTT BPND (Ginovart et al., 2001; Ichise et al., 2003). We applied the non-invasive

Logan method, which has a modest underestimate but the advantage of having the

lowest coefficient of variation (i.e. standard deviation/mean) of calculated 5-HTT BPND

values (Logan et al., 1996). An additional analysis was conducted using the simplified

reference tissue method 2 (SRTM2) which has a negligible underestimate but a higher

coefficient of variation (Wu et al., 2002).

2.4.4 Statistical Analysis

The primary analysis for the region of interest method was a repeated-

measures multivariate analysis of variance (rm-MANOVA) to assess the effect of light

therapy on 5-HTT BPND (the repeated dependent variable) in the ACC and PFC. As a

secondary important analysis, a rm-MANOVA was also applied to evaluate the effect

of light therapy across all examined brain regions. Regional univariate repeated-

61

measures ANOVAs were applied only if there was a significant omnibus effect in the

rm-MANOVA. Effect size (Cohen’s d), was calculated for all regions as mean

difference across conditions (light vs. baseline) divided by the standard deviation of

the difference across conditions. Percentage change in 5-HTT BPND was calculated

for each region as: [(Light-Baseline)/Baseline].

As exploratory measure, Pearson correlation coefficients were calculated to

determine whether there was a relationship between reduction in 5-HTT BPND,

following light therapy, in the ACC and PFC, regions heavily involved in mood

regulation (26), and improvement on scales of mood-related symptoms (BDI, VAS

Mood, Anxiety, Energy and SIGH-SAD), as well as SPAQ scores.

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3 RESULTS

3.1 Study 1: The Effect of Season on 5-HTT BPND

3.1.1 Rationale

Although information regarding the neuropathophysiology of SAD is

accumulating, a critical gap is the lack of direct brain investigations of this illness. As

such, there is a clear need to identify brain biomarkers associated with SAD to better

understand its underlying neurobiology and develop strategies for prevention and

treatment. There is strong evidence that 5-HTT BPND displays seasonal fluctuation

with greater binding in the winter than summer in healthy participants (Buchert et al.,

2006; Kalbitzer et al., 2010; Praschak-Rieder et al., 2008; Ruhe et al., 2009), but there

have been no longitudinal studies examining seasonal change in 5-HTT BPND, in SAD

or in health. Thus, we scanned healthy participants and individuals with SAD, using

[11C]DASB PET, in summer and in winter, to measure seasonal variation in 5-HTT

BPND in the PFC and ACC and compare the magnitude of seasonal change in this

biomarker in these brain regions between groups. Furthermore, as SAD is a

dimensional illness with a continuous distribution within health (such that 25% of

healthy individuals experience mild seasonal symptoms) through to SAD of moderate

to high severity, we also assessed the relationship between degree of seasonal

change in mood and behaviour and magnitude of seasonal fluctuation in 5-HTT BPND

(Bartko et al., 1989; Kasper et al., 1989; Rohan et al., 2011; Terman, 1988).

63

3.1.2 Effect of SAD and Severity Category on % Δ 5-HTT BPND

Seasonal % Δ 5-HTT BPND was greater in SAD as compared to health in the

PFC and ACC (Mann-Whitney U, U=126.5 and 114.0, p=0.046 and 0.02, respectively,

Table 3-1). However, the strongest finding was a main effect of group (healthy,

moderate SAD, and severe SAD) upon seasonal fluctuation in 5-HTT BPND in the PFC

and ACC (Kruskal–Wallis H test, χ2(2)=8.82, p=0.01 and χ2(2)=9.62, p=0.008,

respectively, Figure 3-1 and Table 3-2). Similar findings were present across other

regions of interest (χ2(2)=7.01-10.45, p=0.005-0.03, Figure 3-1 and Table 3-2),

excepting the hippocampus in which a trend-level effect was observed (χ2(2)=5.26,

p=0.07; Figure 3-1 and Table 3-2).

Table 3-1: Group differences in seasonal percent change in 5-HTT BPND

These findings were primarily explained by greater seasonal % Δ 5-HTT BPND

in the PFC and ACC of severe SAD cases relative to healthy volunteers, (Mann-

64

Whitney U, U= 42.5 and 37.0, p=0.005 and 0.003, respectively, Figure 3-1 and Table

3-2), an effect also observed in other regions of interest (U=40.0-62.0, p=0.004-0.048;

Figure 3-1 and Table 3-2), excepting the midbrain for which seasonal % Δ 5-HTT

BPND was significantly greater across all SAD participants relative to healthy

volunteers (U=96.0, p=0.005; Figure 3-1 and Table 3-1). In contrast, seasonal % Δ 5-

HTT BPND in moderate SAD subjects was similar to healthy individuals with no

difference in any region of interest (Mann-Whitney U, U=51.0-106.0, p=0.07-0.96;

Figure 3-1 and Table 3-2). Seasonal % Δ 5-HTT BPND was consistent within

individuals across brain regions (Cronbach's alpha, α=0.89)

Table 3-2: Group differences in seasonal percent change in 5-HTT BPND in participants undergoing [11C]DASB PET in spring-summer and fall-winter.

65

Seasonal % Δ 5-HTT BPND measured applying the SRTM2 was similar to that

of the non-invasive Logan method (Pearson correlation coefficient, r=0.93-0.98,

p<0.0001, across regions) and yielded similarly consistent main results.

Figure 3-1: Seasonal percent change in serotonin transporter binding potential (% Δ 5-HTT BPND) as measured in 8 brain regions of interest (ROIs). Open (healthy, n=20) and red (moderate SAD, n=9) and blue (severe SAD, n=11) triangles represent individual subject % Δ 5-HTT BPND. Black bars represent mean % Δ 5-HTT BPND for each group. % Δ 5-HTT BPND was significantly greater in the prefrontal and anterior cingulate cortices (severe SAD vs health; Mann-Whitney U, U= 42.5 and 37.0, p=0.005 and 0.003, respectively; greater magnitude in severe SAD of 35.10% and 14.23%, respectively) with similar findings observed in other regions (U= 40.0 to 62.0, p=0.004 to 0.048; greater magnitude in severe SAD of 13.16% to 17.49%). To compare groups, the Kruskal-Wallis H test was also applied at each ROI. ap-value ≤ 0.005; bp-value ≤ 0.01; cp-value ≤ 0.05. Seasonal % Δ 5-HTT BPND was consistent

within individuals across brain regions (Cronbach's alpha, α=0.89).

3.1.3 Effect of SAD and Severity Category on Δ 5-HTT BPND

The effects of SAD and severity on Δ 5-HTT BPND were consistent with those

on % Δ 5-HTT BPND (figure 3-2). A main effect of group was observed on Δ 5-HTT

BPND in the PFC and ACC (Kruskal–Wallis H test, χ2(2)=8.32, p=0.016 and χ2

(2)=9.00,

p=0.01, respectively) and across other regions of interest (χ2(2)= 6.45-10.56, p=0.005-

0.04, Figure 3-2). These findings were similarly driven by increased Δ 5-HTT BPND of

66

severe SAD cases relative to healthy volunteers (Mann-Whitney U, U=38.5-52.5,

p=0.003-0.018) with no difference in Δ 5-HTT BPND values upon comparison of

moderate SAD and healthy groups (U=60.0-108.0, p=0.16-0.91, Figure 3-2).

As compared to healthy and moderate SAD groups, mean regional 5-HTT BPND

values of severe SAD cases were greater in fall-winter by 0-7.89% and lower in

spring-summer by 11.47-20.83%, however, these differences were not significant

(Kruskal-Wallis H test, fall-winter: χ2(2)=0.01-1.59, p=0.45-0.99; spring-summer:

χ2(2)=1.61-5.11, p=0.08-0.45, Table 3-3).

Figure 3-2: Seasonal change in serotonin transporter binding potential (Δ 5-HTT BPND) as measured in 8 brain regions of interest (ROIs). Open (healthy, n=20) and red (moderate SAD, n=9) and blue (severe SAD, n=11) triangles represent individual subject Δ 5-HTT BPND. Black bars represent mean Δ 5-HTT BPND for each group. Δ 5-HTT BPND was significantly greater in the prefrontal and anterior cingulate cortices (severe SAD vs health; Mann-Whitney U, U= 45.0 and 38.5, p=0.007 and 0.003, respectively) with similar findings observed in other regions (U= 41.0 to 52.5, p=0.004 to 0.018). In the severe SAD group, an increase in 5-HTT BPND values in winter relative to summer was observed in approximately 90% of subjects whereas direction of change was evenly distributed in moderate SAD and healthy groups. To compare groups, the Kruskal-Wallis H test was also applied at each region of interest ap-value ≤ 0.005; bp-value ≤ 0.01; cp-value ≤ 0.05.

67

Table 3-3: Group differences in 5-HTT BPND values in participants undergoing [11C]DASB PET in spring-summer and fall-winter

3.1.4 Relationship of Symptoms to % Δ [11C]DASB 5-HTT BPND

In SAD participants, a positive correlation was also observed between

magnitude of seasonal % Δ 5-HTT BPND and SAD severity, as measured by the

SPAQ GSS, which was significant in the PFC (Spearman’s rank correlation

coefficient, ϼ=0.52, p=0.018) and trend-level in the ACC (Spearman’s rank correlation

coefficient, ϼ=0.42, p=0.07, Table 3-4). Similar relationships were observed in other

examined brain regions (ϼ=0.44-0.55, p=0.012-0.055), with the exception of the

midbrain, in which no significant correlation was observed (ϼ=0.25, p=0.29; Table 3-

4). However, in healthy participants, for which the range in SPAQ GSS scores was

narrow, no significant correlations were observed between seasonal % Δ 5-HTT BPND

and SPAQ GSS in any brain region (ϼ=-0.18-0.36, p=0.12-0.92; Table 3-4).

Table 5: Group Differences in Brain 5-HTT BPND Values in Participants Undergoing [11

C]DASB PET in Spring-Summer and Fall-Winter

Brain Region Healtha

Moderate SADb

Severe SADc

p -valued

Healtha

Moderate SADb

Severe SADc

p -valued

Prefrontal Cortex 0.24 (0.08) 0.26 (0.07) 0.19 (0.09) 0.19 0.22 (0.07) 0.24 (0.05) 0.23 (0.09) 0.82

Anterior Cingulate Cortex 0.41 (0.09) 0.43 (0.10) 0.37 (0.12) 0.44 0.38 (0.07) 0.42 (0.05) 0.41 (0.11) 0.45

Dorsal Putamen 1.21 (0.15) 1.22 (0.13) 1.08 (0.22) 0.08 1.18 (0.14) 1.17 (0.10) 1.20 (0.19) 0.99

Thalamus 1.56 (0.24) 1.61 (0.15) 1.38 (0.34) 0.24 1.54 (0.22) 1.55 (0.17) 1.58 (0.28) 0.98

Dorsal Caudate 1.11 (0.21) 1.17 (0.20) 0.95 (0.23) 0.08 1.06 (0.16) 1.12 (0.20) 1.06 (0.16) 0.85

Ventral Striatum 1.17 (0.16) 1.21 (0.13) 1.04 (0.22) 0.09 1.14 (0.13) 1.15 (0.12) 1.15 (0.17) 0.99

Midbrain 1.10 (0.22) 0.99 (0.16) 0.97 (0.30) 0.45 1.10 (0.32) 1.10 (0.20) 1.12 (0.22) 0.62

Hippocampus 0.55 (0.15) 0.58 (0.15) 0.47 (0.12) 0.21 0.54 (0.14) 0.57 (0.13) 0.54 (0.12) 0.94

an=20 (Health) ,

bn=9 (Moderate SAD) ,

cn=11 (Severe SAD) ,

dKruskal-Wallis H Test

[11

C]DASB 5-HTT BPND, Mean (SD)

Spring-Summer Fall-Winter

Table 3-4: Correlations between seasonal percent change in 5-HTT BPND and seasonal pattern assessment questionnaire global seasonality score

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Correlations between SPAQ scores and Δ 5-HTT BPND in SAD and healthy

groups were comparable to those of seasonal % Δ 5-HTT BPND in all examined brain

regions (Figure 3-3, Table 3-5).

Figure 3-3: In SAD participants, a positive correlation was observed between magnitude of seasonal Δ 5-HTT BPND and SAD severity, as measured by the SPAQ GSS which was significant in the PFC (Spearman’s rank correlation coefficient, ϼ=0.52, p=0.02) and trend-level in the ACC (Spearman’s rank correlation coefficient, ϼ=0.41, p=0.07). Correlations between SPAQ scores and Δ

[11C]DASB 5-HTT BPND were comparable to those of seasonal % Δ [11C]DASB 5-HTT BPND.

Table 3-5: Correlations between seasonal change in 5-HTT BPND value and Seasonal Pattern Assessment Questionnaire Global Seasonality Score

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In SAD, post-hoc exploratory analyses comparing a breakdown of the GSS by

mood, energy and appetite clusters found that the strongest correlations between

seasonal change in mood symptoms and seasonal % Δ 5-HTT BPND occurred in the

PFC (ϼ=0.53, p=0.015), dorsal putamen (ϼ=0.66, p=0.002), dorsal caudate (ϼ=0.62,

p=0.004), ventral striatum (ϼ=0.63, p=0.003) and thalamus (ϼ=0.59, p=0.006). As for

seasonal changes in energy levels, the strongest correlations were also observed in

the PFC (ϼ=0.52, p=0.019), dorsal putamen (ϼ=0.70, p=0.001), dorsal caudate

(ϼ=0.68, p=0.001), ventral striatum (ϼ=0.71, p<0.0001) and thalamus (ϼ=0.66,

p=0.001). Similar relationships were found between changes in appetitive behaviors

and seasonal % Δ 5-HTT BPND with the strongest correlations observed in the PFC

(ϼ=0.50, p=0.024), thalamus (ϼ=0.54, p=0.014), dorsal putamen (ϼ=0.48, p=0.03),

hippocampus (ϼ=0.48, p=0.033) and ventral striatum (ϼ=0.42, p=0.069). Results are

presented in Table 3-6.

Table 3-6: Correlations between seasonal percent change in 5-HTT BPND and domains of the seasonal pattern assessment questionnaire

Table 7: Correlations Between Seasonal Change in 5-HTT BPND and Domains of the Seasonal Pattern Assessment Questionnaire

ϼa p -value ϼa p -value ϼa p -value ϼa p -value ϼa p -value ϼa p -value

Prefrontal Cortex 0.53 0.015 0.52 0.019 0.50 0.024 0.23 0.33 0.10 0.67 -0.33 0.15

Anterior Cingulate Cortex 0.39 0.09 0.51 0.02 0.34 0.15 0.10 0.67 0.12 0.62 -0.27 0.25

Dorsal Putamen 0.66 0.002 0.70 0.001 0.48 0.03 0.23 0.34 0.23 0.34 -0.43 0.06

Thalamus 0.59 0.006 0.66 0.001 0.54 0.014 0.28 0.23 0.46 0.04 -0.33 0.16

Dorsal Caudate 0.62 0.004 0.68 0.001 0.40 0.08 0.44 0.05 0.48 0.03 -0.07 0.77

Ventral Striatum 0.63 0.003 0.71 <0.0001 0.42 0.069 0.27 0.24 0.27 0.24 -0.37 0.11

Midbrain -0.032 0.90 0.04 0.87 0.28 0.23 -0.30 0.20 -0.36 0.13 -0.11 0.64

Hippocampus 0.28 0.24 0.40 0.078 0.48 0.03 0.22 0.34 -0.01 0.97 -0.43 0.06aSpearman's Rank Correlation Coefficiant

Brain Region

Health

Mood Energy Appetite

SAD

Mood Energy Appetite

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3.2 Study 2: The Effect of Light Therapy on 5-HTT BPND in Health

3.2.1. Rationale

Seasonal change in mood and behavior is a significant problem as

approximately twenty-five percent of healthy individuals report impaired functioning in

winter due to low mood, increased appetite and decreased energy (Chotai et al.,

2004; Kasper et al., 1989; Okawa et al., 1996; Perry et al., 2001; Rosen et al., 1990).

In addition, only 55% of individual suffering from seasonal depressive symptoms fully

remit after light therapy, a first-line treatment for SAD (Lam et al., 2006). As such,

there is an urgent need for research to optimize this treatment for prevention of SAD.

In two previous studies of seasonal variation in 5-HTT BPND using [11C]DASB PET, an

inverse correlation between duration of daily sunlight and 5-HTT BPND was observed

in health, suggesting that this might be a promising biomarker for light therapy

(Kalbitzer et al., 2010; Praschak-Rieder et al., 2008). However, there has been no

investigation of the effects of deliberate bright light exposure upon 5-HTT BPND in

human brain. Thus, we used [11C]DASB PET to examine the effects of light therapy on

5-HTT BPND in the ACC and PFC during the fall and winter, in healthy volunteers, in

order to investigate the potential of 5-HTT BPND as a biomarker for light exposure to

develop prevention strategies for new-onset SAD

3.2.2 5-HTT BPND Decreases in the ACC Following Light Therapy in Winter

In winter group, there was a main effect of treatment on 5-HTT BPND in the ACC

and PFC (repeated-measures MANOVA, F(2,8)=19.54, p=0.001). Subsequent

univariate pairwise ANOVAs showed this treatment effect to be significant only in the

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ACC (F(1,9)=18.04, p=0.002) after correction for multiple comparisons where a

decrease in 5-HTT BPND of 12% was observed following light therapy relative to

placebo, but not in the PFC (magnitude -1.1%, F(1,9)=0.23, p=0.64). As a secondary

analysis, the repeated-measures MANOVA was re-run with all ROIs, excluding the

thalamus, and an effect of treatment was observed (F(6,4)=14.53, p=0.01); however,

since this was an exploratory analysis among a number of such analyses, greater

than five, this was viewed as a non-significant finding. Subsequent exploratory t-tests

revealed some level of reduction in 5-HTT BPND in the ventral striatum after light

therapy compared to placebo (magnitude -9.9%, paired t test, t9=2.85, p=0.02) and

hippocampus (magnitude -10%, paired t test, t9=1.73, p=0.12), but the effect in the

ventral striatum did not remain significant after correction for multiple comparisons

(Figure 3-4 and Table 3-7). In addition, in the winter group, upon application of the

two-tailed non-parametric Wilcoxon Signed Ranks Test, we did not observed a

significant effect of treatment (light therapy versus placebo) on 5-HTT BPND in the

thalamus (Z= -1.79, p=0.08). In the fall group, there was no significant effect of

treatment on 5-HTT BPND observed in the ACC and PFC (repeated-measures

MANOVA, F(2,7)=0.93, p=0.44) or, upon additional comparison, in any other examined

region (Table 3-7).

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Table 3-7: Group differences in brain 5-HTT BPND values in healthy subjects undergoing [11C]DASB PET in winter (n=10) and fall (n=9) following light therapy and placebo conditions

(inactive negative ionization)

Figure 3-4: Serotonin transporter binding potential (5-HTT BPND) measured across conditions in 6 brain regions of interest (ROIs) during the winter (n=10). Open (placebo) and closed (light therapy) triangles represent individual subject 5-HTT BPND values for each condition and red bars represent the group mean. ACC refers to anterior cingulate cortex. 5-HTT BPND was significantly decreased in the ACC of healthy individuals following light therapy relative to placebo. Trend-level reductions in 5-HTT BPND were also observed in the ventral striatum and hippocampus. Error bars were omitted for clarity.

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Similar results were obtained when analyzing 5-HTT BPND values obtained

from applying SRTM2. 5-HTT BPND values were highly correlated across the two

methods in each region, within both light therapy and placebo conditions (Pearson

correlation coefficient, r=0.90-0.99, p=<0.001 and r=0.92-0.99, p=<0.001,

respectively).

An exploratory analysis was also conducted to determine whether there was a

relationship between reduction in 5-HTT BPND in the ACC following light therapy

relative to placebo and improvement of mood-related symptoms. As small changes in

mood-related symptoms would be expected of healthy subjects prior to and after light

therapy, behavioural data was pooled across fall and winter groups (n=19) to increase

statistical power. Accordingly, a modest positive, trend-level correlation was observed

between reduction in 5-HTT BPND in the ACC following light therapy relative to

placebo and improvement of scores on the BDI (Pearson correlation coefficient,

r=0.45, p=0.06). However, no significant or trend level correlations were observed

between the other scales of mood symptoms (VAS Mood, Anxiety, Energy, or SIGH-

ADS) and change in 5-HTT BPND in the ACC or PFC.

We also collected sleep data via actigraphy (Actiwatch Spectrum, Philips

Respironics, Pennsylvania, USA) because we were interested in investigating the

relationship between changes in sleep measures and in 5-HTT BPND following light

therapy. However, we did not observe a significant correlation between change in

ACC 5-HTT BPND and any sleep measure (time of awakening, bed-time, number of

awakenings, sleep duration, sleep efficacy, sleep latency, r=0.20-0.04, p=0.40-0.87)

across conditions and upon further analysis, did not find group differences in changes

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in these parameters (Table 3-8). Most likely the reason for this was that the sample

had fairly normative levels of these measures.

3.3 Study 3: The Effect of Light Therapy on 5-HTT BPND in SAD

3.3.1 Rationale

The efficacy of light therapy, a first-line treatment for SAD, is comparable that

of antidepressant use (Lam et al., 1995; Lam et al., 2006; Moscovitch et al., 2004). In

addition, the positive effects of light therapy tend to occur within one to two weeks

whereas response to antidepressants typically begins after two to four weeks. This

treatment is also associated with a lower frequency of adverse effects relative to

antidepressant use (Lam et al., 2006; Ruhrmann et al., 1998). However, only 55% of

individual suffering from SAD fully remit following light therapy, indicating a need to

improve this treatment (Lam et al., 2006). In the first study, we observed a seasonal

fluctuation in 5-HTT BPND across examined regions, including the PFC and ACC in

SAD, predominantly in severe SAD cases, with an elevation in winter relative to

Table 3-8: Group differences in sleep parameters in healthy subjects undergoing [11C]DASB PET in winter (n=10) and fall (n=9) following light therapy and placebo conditions

(inactive negative ionization)

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summer. In the second study, we observed a region-specific decrease in 5-HTT BPND

in the ACC of healthy individuals, a brain region involved in affect-regulation following

light therapy (Ressler et al., 2007). In this study, we scanned individuals with severe

SAD, in which we had previously observed increased magnitude of seasonal change

in 5-HTT BPND before and after light therapy to determine (i) if light therapy could

reduce the winter elevation in 5-HTT BPND to ameliorate seasonal depressive

symptoms and (ii) whether the findings of study two of, in health, were generalizable

to individuals with full-syndrome severe SAD.

3.3.2 Global Reduction in 5-HTT BPND Following Light Therapy in SAD

The primary finding was a main effect of treatment on 5-HTT BPND in the ACC

and PFC (repeated-measures MANOVA, F(2,9)=6.82, p=0.016). Subsequent univariate

pairwise ANOVAs showed this effect to be significant in both the ACC (F(1,10)=15.11,

p=0.003) and PFC (F(1,10)=8.33, p=0.016), with decreases in 5-HTT BPND of 11.94%

and 9.13%, respectively, following light therapy (Figure 3-5, Table 3-9). Similarly, a

multivariate effect of treatment was also observed when the repeated-measures

MANOVA included 5-HTT BPND as the dependent variable from all examined brain

regions (repeated-measures MANOVA, F(4,7)=8.54, p=0.028). Significant reductions in

5-HTT BPND were found in the hippocampus, ventral striatum, dorsal putamen,

thalamus and midbrain (F(1,10)= 36.94 to 8.02, p<0.0001 to 0.018; magnitude -16.74 to

-8.83; Figure 3-5, Table 3-9). Effect sizes in the ACC and PFC were 1.17 and 1.03,

respectively, with similar values observed in the hippocampus, ventral striatum, dorsal

putamen, thalamus and midbrain (Cohen’s d: 0.85-1.83, Table 3.9). The change in 5-

HTT BPND following light therapy measured applying the SRTM2 was similar to that of

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the non-invasive Logan method (Pearson correlation coefficient, r=0.93-0.98,

p<0.0001, across regions) and yielded similarly consistent main results.

Table 3-9: Change in 5-HTT BPND values in SAD participants before and after light therapy

Figure 3-5: Serotonin transporter binding potential (5-HTT BPND) measured across conditions in 7 brain regions of interest (ROIs) in SAD participants before and after two weeks of daily morning light therapy. Closed (baseline) and open (light therapy) triangles represent individual subject 5-HTT BPND values for each condition with the connecting lines depicting respective change in 5-HTT BPND for each subject. Red bars represent the group mean. ACC refers to anterior cingulate cortex. A significant global reduction in 5-HTT BPND was observed following light therapy (repeated-measures MANOVA, F(4,7)=8.54, p=0.028). Region-specific significant reductions were present in the ACC (F(1,10)=15.11, p=0.003, magnitude of decrease, 11.94%), PFC (F(1,10)=8.33, p=0.016, magnitude of decrease, 9.13%,) and also in the hippocampus, ventral striatum, dorsal putamen, thalamus and midbrain (F(1,10)=8.02 to 36.94, p<0.0001 to 0.018; magnitude of decrease, 8.83% to 16.74%). Effect sizes in the ACC and PFC were 1.17 and 1.03, respectively, with similar values observed in the hippocampus, ventral striatum, dorsal putamen, thalamus and midbrain (Cohen’s d: 0.85-1.83). Error bars were omitted for clarity.

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There was some tendency for a relationship between degree of seasonal

variation in mood and behaviour as measured by the SPAQ and reduction in 5-HTT

BPND in the ACC and PFC following treatment (Pearson correlation coefficient, r=-

0.49, p=0.13 and r=-0.52, p=0.10, respectively). After two weeks of light therapy,

clinical response and remission were observed in 54.5% and 27.3% of SAD cases,

respectively, and after six weeks of light therapy, clinical response and remission

occurred in 81.8% and 54.5% of SAD cases, respectively. During clinical assessment,

there was no evidence of manic symptoms in any participant, consistent with mood

and energy ratings on the SIGH-SAD and VAS scales. There was no significant

relationship between reduction in 5-HTT BPND in the ACC and PFC following light

therapy and improvement in scores on scales of mood symptoms (SIGH-SAD, VAS

Mood and Energy or BDI).

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4 GENERAL DISCUSSION, CONCLUSION AND RECOMMENDATIONS

4.1 General Discussion of Results

4.1.1 Study 1: The Effect of Season on 5-HTT BPND

This is the first study to investigate seasonal change in 5-HTT BPND in SAD.

While greater seasonal variation of 5-HTT BPND in SAD as compared to health was

observed across most brain regions sampled, including the PFC and ACC, the

strongest finding was a more pronounced seasonal fluctuation of 5-HTT BPND in

severe SAD across all brain regions relative to moderate SAD and asymptomatic

healthy groups. These results indicate that 5-HTT BPND, as measured with PET, is

detecting a brain phenotype of SAD and suggests new opportunities for applying this

neuroimaging method in biomarker based approaches to develop new strategies for

both prevention and treatment.

Greater seasonal fluctuation of 5-HTT BPND across all examined brain regions,

including the PFC and ACC, has important implications for SAD pathophysiology,

particularly in regards to severe SAD. [11C]DASB has a strong preferential binding to

5-HTT on the cell surface where functional 5-HTT are located and the binding of

[11C]DASB is insensitive to competition by endogenous serotonin as demonstrated by

the lack of effect of physiologically tolerable serotonergic manipulations in humans,

such as acute tryptophan depletion (Praschak-Rieder et al., 2005; Quelch et al., 2012;

Talbot et al., 2005). As such, the changes in 5-HTT BPND observed in vivo using

[11C]DASB PET are best interpreted to reflect greater availability of the 5-HTT to clear

5-HT from extracellular space in the winter, thereby lowering levels of extracellular 5-

HT. This is a key issue, given that overexpression of 5-HTT in the PFC is associated

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with decreased stimulation induced release of 5-HT from serotonergic neurons and

differential expression of the 5-HTT is associated with magnitude of response to

anxiogenic stimuli (Jennings et al., 2010; Lesch et al., 1996; Mouri et al., 2012). In

addition, while greater seasonal variation in 5-HTT BPND was observed in severe SAD

relative to health, 5-HTT BPND values did not differ across groups in summer and

winter seasons, suggesting that, in SAD, change across seasons is more relevant

than the 5-HTT BPND levels, themselves. Taken together, these findings suggest that

across the shift from summer to winter, seasonal change in 5-HTT levels and/or

affinity may alter the dynamics of extracellular 5-HT release within the PFC, ACC and

subcortical structures thereby dysregulating systems adversely affected in severe

SAD, such as mood, energy and appetite.

Identifying a new brain biomarker in SAD is critical for therapeutic advances

because brain biomarkers are an essential guide for developing treatments of

complex neuropsychiatric illnesses with multiple underlying biological phenotypes.

While it is well accepted that novel therapeutics require target engagement, it is a

newer direction in therapeutic development to assess the effects of treatment on the

target biomarker itself. Knowledge that the biomarker has been engaged then allows

for assessment of whether an adequate number of phenotypes have been targeted in

clinical trials with symptom burden as the primary outcome. In the present

investigation, the biomarker identified provides opportunities to create novel

prevention methods for SAD: it is clear that the environmental combination of

seasonal variables including light, temperature, and humidity, influence 5-HTT BPND,

especially in those with severe SAD. Future studies could identify combinations of

specific environmental factors and their exposure thresholds that induce seasonal

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change in 5-HTT BPND so that by staying below such thresholds or adding other

preventative interventions, such as light therapy, the winter elevation in 5-HTT BPND

could be avoided. Avoidance of environmental qualities and exposure thresholds that

induce seasonal fluctuations in 5-HTT BPND could then be incorporated into larger

scale clinical studies as prevention strategies in high risk communities, such as those

at more Northern latitudes where the prevalence of SAD exceeds six percent

(Magnusson, 2000).

The present study found no seasonal differences in 5-HTT BPND in healthy

volunteers with minimal seasonality, as measured by the SPAQ, or in SAD

participants with mild-moderate symptom severity, whereas several previous studies

found seasonal changes in 5-HTT BPND in healthy subjects who were not selected on

the basis of seasonal fluctuation in mood and, presumably, had a wide range of

seasonal variation in symptoms (Buchert et al., 2006; Kalbitzer et al., 2010; Praschak-

Rieder et al., 2008; Ruhe et al., 2009). It is important to note that the sample

populations of the present study are different from those of earlier studies and likely

account for this discrepancy. For example, it may be seasonal variation in 5-HTT BPND

would be observed in healthy individuals with a greater degree of change in seasonal

symptoms. In regards to why 5-HTT BPND did not change with season in mild-

moderate SAD, it is plausible that multiple pathophysiological mechanisms contribute

to SAD and that, of these, a single individual pathology, such as seasonal fluctuation

in 5-HTT BPND, is more likely to be present and of greater magnitude in those

suffering from a more severe form of the illness.

There are some limitations of this study typical of SAD investigations and

human brain studies of neuropsychiatric disease. First, it was not always possible to

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scan SAD participants when their winter MDE was at its most severe. As such,

completion of winter scanning prior to the symptomatic nadir may have

underestimated the strength of the relationship between severity of SAD and seasonal

% Δ 5-HTT BPND. Second, as our approach was to determine if the severity of SAD

was related to seasonal change in 5-HTT BPND, we deliberately chose healthy

volunteers with negligible severity of seasonal symptoms for comparison to SAD

subjects with more extreme symptoms, and this may have reduced our ability to

detect seasonal differences in healthy subjects. Since 25 percent of healthy people

experience seasonal variation in mood symptoms, an additional interesting future

direction would be to assess % Δ 5-HTT BPND in a group of healthy subjects with

substantial seasonality to further characterize this brain phenotype (Kasper et al.,

1989). Third, exposure to different seasonal environmental influences (i.e. light,

temperature, humidity) are highly inter-correlated and this does not allow for

differentiation of their individual effects in regards to the contribution of each factor to

seasonal change in 5-HTT BPND. Lastly, while has strongly preferential binding to 5-

HTT on outer cell membranes, 5-HTT BPND, is a measure of both 5-HTT density and

its affinity for [11C]DASB (Quelch et al., 2012). Thus, it is not possible to differentiate

between changes in 5-HTT density and affinity, although it would be expected that

when the affinity of the serotonin transporter is altered there is a similar functional

effect on the dynamics of extracellular 5-HT concentrations.

In summary, this is first investigation to compare seasonal variation in 5-HTT

BPND in SAD participants, across a spectrum of illness severity, to a group of healthy

volunteers, asymptomatic for seasonal changes in mood and behaviour. The primary

finding is that, across brain regions sampled, including the PFC and ACC, 5-HTT

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BPND was significantly elevated in winter as compared to summer in SAD, particularly

in severe SAD. Given that [11C]DASB binds preferentially to the 5-HTT on the cell

surface, this has important pathophysiological implications for the dynamics of

serotonin release and is best interpreted as reflecting a key phenotype of SAD

(Quelch et al., 2012). As a brain biomarker, greater seasonal percent change in 5-HTT

BPND is an important breakthrough because it can be applied to develop interventions

to reduce environmental impact on this target and create very specific prevention

strategies for SAD.

4.1.2 Study 2: The Effect of Light Therapy on 5-HTT BPND in Health

This is the first investigation to compare the effect of light therapy to placebo

upon 5-HTT BPND in the human brain. The primary finding is that, during the winter,

administration of light therapy significantly decreased 5-HTT BPND in the ACC of

healthy humans and this reduction remained significant after correction for multiple

comparisons. In the fall group, no significant change was observed in any examined

brain region. These results have important implications as they suggest a novel

mechanism by which light exposure may exert an antidepressant effect and also

provide a basis upon which to better develop light therapy for prevention of SAD.

At present, there is no consensus by which light therapy facilitates amelioration

of depressive symptoms such as low mood. However, the finding that light therapy, a

first-line treatment for SAD, affects 5-HTT BPND in the ACC is in accordance with

literature supporting the role of this brain region in regulation of mood and

antidepressant response. The ACC participates in production of sad emotions, where

regional activations occur during transient sadness and a reduction in activity follows

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recovery from depression (Mayberg et al., 1999). In addition, activity in the ACC has

been observed to decrease in response to antidepressant drug treatment, cognitive

behavioural therapy, transcranial magnetic stimulation, and deep brain stimulation

(DBS) (Kreuzer et al., 2015; Mayberg et al., 1999; Mayberg et al., 2005; Ressler et al.,

2007; Siegle et al., 2006). Interestingly, 5-HT function in the ACC may be important for

treatment response since, in rodent models of depressive behaviour, 5-HT depletion

abolishes the antidepressant effects of DBS in the ACC (Hamani et al., 2010; Hamani

et al., 2012).

One explanation for reduced 5-HTT BPND in the ACC following light therapy is

that, during the winter, light exposure may increase signaling between the retina,

dorsal raphe nucleus (DRN) of the midbrain and ACC to influence 5-HTT BPND in the

ACC. To elaborate upon this putative mechanism, it has been reported that retinal

sensitivity, as measured by electroretinography, changes after light therapy in

individuals with SAD and also varies seasonally in both subsyndromal and full-

syndrome SAD (Hebert et al., 2002; Lavoie et al., 2009). In addition, in a recent

preclinical study, it was shown that, following light exposure, a distinct population of

non-visual DRN-projecting retinal ganglion cells (RGCs) were able to modulate

affective behavior via increased input to 5-HT neurons in the DRN (Ren et al., 2013).

Furthermore, in rodents, sleep deprivation, an anti-depressant treatment that

increases neuronal firing in the DRN, has been found to down-regulate levels of

monoamine transporters such as the 5-HTT and NET in efferent limbic structures

(Hipolide et al., 2005; Mogilnicka et al., 1980; Ursin, 2002). The DRN has projections

to the ACC and 5-HTT BPND in the ACC varies inversely with duration of daily

sunshine (Porrino et al., 1982; Praschak-Rieder et al., 2008). In future study,

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preclinical investigation would be helpful in regards to identifying specific cellular

mechanisms underlying this light-induced change in 5-HTT BPND.

As approximately twenty-five percent of healthy individuals experience

seasonal changes in mood and energy that impair functioning and only 55 percent of

individuals suffering from seasonal depressive symptoms fully remit after light therapy,

there is an urgent need for research to optimize this treatment for prevention of SAD

(Chotai et al., 2004; Kasper et al., 1989; Lam et al., 2006; Okawa et al., 1996; Rosen

et al., 1990). [11C]DASB PET is currently applied to guide new antidepressant drug

development for medications that bind to the 5-HTT (Meyer, 2012; Meyer et al.,

2004a; Meyer et al., 2001; Meyer et al., 2004b). Our finding of a decrease in 5-HTT

BPND in the ACC of healthy individuals and an associated increase in mood after light

therapy, represents the first central biomarker associated with therapeutic response to

light exposure. This reduction in 5-HTT BPND in the ACC, a brain region heavily

involved in mood regulation, following light therapy, may provide a means to improve

the efficacy of this treatment (Ressler et al., 2007). For example: [11C]DASB PET

could be similarly applied to determine what aspects (i.e. light colour, duration,

intensity, time of day, etc.) of light exposure best reduce 5-HTT BPND in the ACC to

optimize this treatment.

One limitation of the present study is that it may have been underpowered to

detect changes in 5-HTT BPND in regions other than the ACC, such as the ventral

striatum and hippocampus which had similar magnitudes of change but greater

variability of such change. Second, while our cut-off between winter and fall was

based on standardized definitions of season, it is possible that the true cut-off at which

5-HTT BPND in the ACC decreases after light therapy might be earlier, as a cut-off of

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November 27 would have yielded similar results (magnitude -12.81%, repeated-

measures ANOVA, F(1,11)=7.34, p=0.02). Third, we chose to examine the effect of light

therapy in healthy volunteers, thus, the extent to which these findings are

generalizable to a clinical population of individuals with SAD is unclear. Another

challenge in studying healthy volunteers is that they would be expected to score low

on measures of depressed mood and energy both prior to and after light therapy. This

floor effect may have limited our ability to detect significant correlations between 5-

HTT BPND and symptom-related scales.

We studied healthy individuals in the fall and winter since the primary aim of

this study was to evaluate the potential of 5-HTT BPND as a biomarker for light

exposure to develop prevention strategies for new-onset SAD. Nevertheless, these

results suggest several future directions: one future direction should examine the

effect of light therapy on 5-HTT BPND in individuals with sub-syndromal or full-

syndrome SAD to determine whether there would be a greater level of response.

Another interesting future direction would be to assess the effect of light therapy on 5-

HTT BPND in the spring and summer to further characterize the seasonal variation in

response.

In summary, this is the first study to examine the effects of light therapy upon 5-

HTT BPND in the living human brain. The main finding is that 5-HTT BPND in the ACC

was significantly reduced after light therapy relative to placebo treatment in healthy

individuals during the winter months. These results provide evidence of a relationship

between 5-HTT binding in the ACC and light therapy, and identify, for the first time, a

central biomarker associated with the therapeutic intervention of light therapy. As

such, this central biomarker represents a new approach by which, in the future,

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different candidate light therapy strategies could be screened with the best performing

method upon reducing 5-HTT BPND in the ACC being advanced to clinical trial for

preventing SAD.

4.1.3 Study 3: The Effect of Light Therapy on 5-HTT BPND in SAD

This is the first study of the effects of light therapy on 5-HTT BPND in SAD. The

primary finding was a main effect of treatment on 5-HTT BPND across all brain regions,

including a significant reduction in 5-HTT BPND in the ACC and PFC. Similarly,

significant decreases in 5-HTT BPND were also observed in other brain regions

assayed including the hippocampus, ventral striatum, dorsal putamen, thalamus and

midbrain. These findings provide important direction for discerning the most salient

environmental factor in the etiology of the seasonal change in 5-HTT BPND in SAD.

These results also demonstrate that light therapy reaches an key therapeutic target in

the treatment of SAD and provide a basis for further development of this treatment

through application of [11C]DASB PET.

The findings of the present study have implications for understanding the

etiology of the seasonal change in 5-HTT BPND across summer and winter seasons.

We previously found that SAD subjects, particularly severe SAD cases with SPAQ

global seasonality scores of greater than 16, had significantly greater seasonal

variation in 5-HTT BPND relative to healthy controls across all brain regions assayed

(Tyrer et al., 2016). In the present study, all participants had SPAQ global seasonality

scores of 16 or greater (Tyrer et al., 2016). However, in the previous study of seasonal

variation, identification of the environmental cause inducing this seasonal change in 5-

HTT BPND was not resolvable since the main environmental factors that vary with

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season, such as light, temperature and humidity, are highly inter-correlated and

cannot be differentiated with respect to their relationship to seasonal fluctuation in this

biomarker. In the present study, both the magnitude of the effect on 5-HTT BPND

following two weeks of light therapy and the global reduction in this brain biomarker

was in close accordance with the seasonal change in 5-HTT BPND across winter to

summer seasons previously observed in SAD (Mc Mahon et al., 2016; Tyrer et al.,

2016). As such, our findings suggest that changes in light exposure represent a

sufficient environmental condition to account for the seasonal variation in 5-HTT BPND

in SAD.

Our results of significant reductions in 5-HTT BPND in the ACC and PFC

following light therapy, a first-line treatment for SAD, are in accordance with literature

supporting the role of these regions in affect control (Liotti et al., 2002; Mayberg et al.,

1999; Ressler et al., 2007; Sharot et al., 2007; Tom et al., 2007). However, it is

notable that, following treatment, decreases in 5-HTT BPND were observed across all

other examined regions including the hippocampus, ventral striatum, dorsal putamen,

thalamus and midbrain, suggesting that the effects of light therapy on this biomarker

may be more global than region-specific. Interestingly, dysregulated functioning,

neurochemistry and/or structure has been implicated in some of these other regions in

MDD. For instance, in the hippocampus, increased CA1 pyramidal cell neuron density

and reduced astrocyte activation have been found to be associated with illness

duration in recurrent/chronic MDD (Cobb et al., 2016; Cobb et al., 2013). The ventral

striatum plays a role in both reward-processing and in the symptom of anhedonia, and

this region has been a target of DBS for treatment of MDD with positive results in

some open trials (Grubert et al., 2011). Lastly, the superior raphe nuclei within the

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midbrain have a strong influence on circadian rhythm, including the sleep wake cycle

(Vertes et al., 2008). Hence it is plausible that changes in 5-HTT BPND in these other

regions could also contribute to antidepressant response following light therapy.

Hence it is plausible that changes in 5-HTT BPND in these other regions could also

contribute to effects of light therapy.

Determination of the mechanism underlying the substantial global reduction in

5-HTT BPND following light therapy will require further study. A possible explanation for

the global reduction in brain 5-HTT BPND following light therapy is that light induces

signalling directly between the retina and the DRN and also, indirectly, through the

suprachiasmatic nucleus and then the dorsomedial nucleus of the hypothalamus to

the median raphe nuclei MRN (Ren et al., 2013; Shen et al., 1994; Vertes et al., 2008)

. There are also strong reciprocal connections between the DRN and MRN (Vertes et

al., 2008). This signalling to the raphe nuclei of midbrain may modulate firing of

efferent serotonergic neurons within these structures to decrease 5-HTT levels in

subcortical and cortical brain regions. A direct link between alterations in firing of the

DRN and MRN and reduction of 5-HTT levels in efferent regions has not been fully

established, but in rodents, sleep deprivation, a chronotherapeutic treatment, has

been shown to increase neuronal firing in the DRN and down-regulate 5-HTT levels in

efferent limbic structures (Hipolide et al., 2005; Mogilnicka et al., 1980; Ursin, 2002).

Identification of specific cellular mechanisms underlying this light-induced change in 5-

HTT BPND would be a valuable direction for future study

While results of phase 3 studies are the overriding determinant of therapeutic

usefulness, to address the phenotypic heterogeneity of neuropsychiatric illnesses and

verify target engagement, a new direction for advancing personalized medicine in

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clinical trials is to apply PET receptor-ligand neuroimaging in phase 0 studies. This is

intended as a solution to the concern that the reason clinical trials fail at a high

frequency for treatment of neuropsychiatric illnesses is due to a mismatch between

the therapeutic and the target as a result of illness heterogeneity or because the

treatments themselves do not affect the target. Other insightful information can be

obtained from such trials in similar circumstances. For example, the novel therapeutic

Bapineuzumab, a humanized monoclonal antibody against the β-amyloid (Aβ) N-

terminus, has been found to reduce accumulation of amyloid more prominently in

mild-moderate relative to severe Alzheimer’s disease, suggesting that the therapeutic

might be best developed for an earlier phase of the illness than initially intended (Liu

et al., 2015). Although it is the clinical response which ultimately matters, similar

strategies of pure target engagement have a longstanding history of use in MDD; for

instance, threshold occupancies of 80% for the 5-HTT and greater than 14% for the

DAT are considered optimal in new antidepressant development (Meyer et al., 2002;

Meyer et al., 2001; Meyer et al., 2004b). Accordingly, the present investigation was

designed as a phase 0 study to develop a PET-based biomarker of light therapy. As

such, in future study, the magnitude of reduction in 5-HTT BPND, as measured by

[11C]DASB PET, could be used to identify aspects of light therapy that could be

modified so as to improve this biological response. It is anticipated that treatments

with a greater biological response (i.e. decrease in 5-HTT BPND) would achieve a

greater level of clinical response in large scale clinical trials.

As we chose to examine the effect of light therapy on 5-HTT BPND in a sample

SAD patients with fairly severe seasonal depressive symptoms, a study design

including a placebo control group was not feasible. In this clinical situation, in which an

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evidence based first-line therapeutic is available the ethical standard at our site is that

a placebo should not be given. As such, it is difficult to differentiate the effects of light

therapy on 5-HTT BPND from putative effects of placebo on this biomarker. However, it

should be noted that therapeutic interventions for treatment for MDD, such as

mirtazapine, a tricyclic antidepressant with an efficacy equal to that of SSRIs, which

do not selectively target the 5-HTT, but presumably include placebo effects, do not

induce change in 5-HTT BPND as observed in the present study (Cipriani et al., 2009;

Lundberg et al., 2012). In addition, there is reason to believe 5-HTT BPND is

reasonably resilient to effects of social interaction since simple administration of a

protocol in a laboratory environment, such as ATD, does not affect 5-HTT BPND.

(Praschak-Rieder et al., 2005; Talbot et al., 2005). Lastly, although we did not include

a control group of healthy volunteers, we have previously published two sets of

reliability data using this technique demonstrating no systematic change in 5-HTT

BPND in health, thus it is unlikely that our results can be attributed to repeated-

measurement alone (Meyer et al., 2001; Praschak-Rieder et al., 2005). As such, we

favour the interpretation that the decrease in 5-HTT BPND is best attributed to effect of

the light therapy.

There are a few limitations of this study. First, we acknowledge it would have

been desirable to conduct this study with a larger sample. However, it is notable that

decreases in 5-HTT BPND following light therapy were observed in all 11 subjects in

the ACC and in 10 of 11 subjects in the PFC, with similar reductions in other regions,

so it is highly unlikely that these decreases could be attributable to chance alone.

Second, given our sample size, we may have been underpowered to detect significant

correlations between therapeutic response to light therapy and reduction in 5-HTT

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BPND. However, this investigation was designed as a “proof of concept” phase 0 study

to develop a PET-based biomarker for light therapy and we note that sample sizes of

over one hundred patients are typically required to distinguish dose effects of

therapeutics, such as SSRIs in MDD (Fabre et al., 1995; Feighner et al., 1999). Third,

the present study demonstrates the influence of light therapy on one therapeutic

target, but other targets may also be important in regards to predicting treatment

response. For example, Kohno et al., using PET, recently found increased uptake of

[18F]-fluorodeoxyglucose in the right olfactory bulb of healthy humans after light

therapy (Kohno et al., 2016). Fourth, while [11C]DASB has strong preferential binding

to 5-HTT on outer cell membranes, 5-HTT BPND, is a measure of both 5-HTT density

and its affinity for [11C]DASB (Quelch et al., 2012). Thus, it is not possible to

differentiate between changes in these measures; however, it would be anticipated

that alterations in both parameters would have similar effects upon extracellular 5-HT

concentrations. Lastly, as we chose to investigate the effects of light therapy on 5-HTT

BPND in SAD, it is unclear as to whether the effects of this treatment are specific to

SAD, generalizable to other psychiatric populations or may also occur in healthy

subjects. As such, a key direction of future study would be to examine the effect of this

treatment on 5-HTT BPND in psychiatric illnesses for which light therapy has been

shown to have efficacy, such as in bipolar disorder or in unipolar MDD, and to

compare the effects in healthy non-seasonal healthy controls (Lam et al., 2016; Tseng

et al., 2016).

In summary, this is the first investigation of the effects of light therapy on 5-HTT

BPND during winter in SAD. The primary findings were significant reductions in 5-HTT

BPND following light therapy in the ACC, PFC, as well as the hippocampus, ventral

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striatum, dorsal putamen, thalamus and midbrain. Since [11C]DASB preferentially

binds to functional 5-HTT on the cell surface, this implies that one mechanism by

which light therapy may exert antidepressant effects is via an elevation in extracellular

serotonin levels (Quelch et al., 2012). These results also identify, for the first time, a

central brain marker that has potential for use as a predictor to identify optimal

modifications of light therapy in SAD, via use of [11C]DASB PET as an imaging-

calibration technique, which could then be brought forward for assessment in clinical

trials. Given that the magnitude of the effect of light therapy on 5-HTT BPND in SAD

and the global reduction in this brain biomarker following light therapy is in close

accordance with the effect of season on 5-HTT BPND, these results strongly suggest

that seasonal change in light is the environmental factor mediating the seasonal

variation in 5-HTT BPND in SAD.

4.2 Summary of Findings and Conclusions

The importance of the 5-HTT, in regards to affect-regulation, is its influence on

extracellular 5-HT levels. There is an inverse relationship between available 5-HTT

and clearance of extracellular 5-HT. In support of this relationship, SSRIs that block

the 5-HTT raise extracellular 5-HT, 5-HTT knockout mice have greater extracellular 5-

HT and mice with overexpression of 5-HTT have low extracellular 5-HT (Bel et al.,

1992, 1993; Jennings et al., 2006; Mathews et al., 2004). In addition, many SSRIs that

raise extracellular 5-HT are associated with amelioration of depressive symptoms in

individuals suffering from MDD and such modulators of extracellular 5-HT are

important because it is well established that 5-HT plays a role in physiology and

behaviours reported to change with season including mood, energy and appetite

(Owens et al., 1994, 1998). There is also strong support from neuroimaging research

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of seasonal variation in 5-HTT BPND, an index of 5-HTT levels, in healthy volunteers

and 5-HTT BPND has been found to be inversely correlated with exposure to light

(Buchert et al., 2006; Kalbitzer et al., 2010; Praschak-Rieder et al., 2008; Ruhe et al.,

2009). However, until this series of studies, there had been no longitudinal

investigation of the effect of season on 5-HTT BPND in SAD as compared to health. In

addition, there had been no studies evaluating the effect of deliberate bright light

exposure upon 5-HTT BPND in human brain, although light therapy is a front-line

treatment for SAD and there is limited knowledge of the mechanism by which it exerts

its antidepressant effects. Thus, the primary aim of this thesis was to examine the

effects of both season and light exposure, upon 5-HTT BPND in SAD and health.

The purpose of study one was to determine whether the magnitude of seasonal

variation in 5-HTT BPND was greater in SAD as compared to health in the PFC and

ACC, brain regions involved in affect-regulation (Ressler et al., 2007). SAD

participants and healthy volunteers underwent [11C]DASB positron emission

tomography scans in summer and winter to measure seasonal change in 5-HTT BPND.

In accordance with our hypotheses, seasonal variation in 5-HTT BPND was greater in

SAD relative to health, across all examined brain regions, including in the PFC and

ACC, and this fluctuation was primarily due to differences between severe SAD cases

and healthy volunteers (Tyrer et al., 2016). We also observed significant and trend-

level positive correlations between seasonal fluctuation in regional 5-HTT BPND and

degree of seasonality in our SAD participants (Tyrer et al., 2016). Our interpretation of

these findings is that the seasonal change in 5-HTT BPND reflects greater availability

of the 5-HTT to clear 5-HT from extracellular space in winter, thereby lowering levels

of extracellular 5-HT to dysregulate systems adversely affected in SAD, such as

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mood, energy and appetite, and predispose such individuals to experiencing recurrent

seasonal MDEs.

There are a number of highly inter-correlated environmental factors that vary

with season, such as light, temperature, and humidity, which might promote seasonal

change in 5-HTT BPND. If such variables and their exposure thresholds inducing this

seasonal change were to be identified, it might be possible for individuals predisposed

to developing recurrent winter MDEs via avoidance of the winter elevation in 5-HTT

BPND by staying below such thresholds or adding other preventative interventions,

such as light therapy. In both previous studies of seasonal variation in 5-HTT BPND

using [11C]DASB PET, including one from our laboratory, an inverse correlation was

observed between 5-HTT BPND and duration of daily sunshine in health, suggesting

that changes in exposure to light might affect 5-HTT BPND levels (Kalbitzer et al.,

2010; Praschak-Rieder et al., 2008). Accordingly, the objective of study two was to

investigate the effect light therapy, a standard intervention for SAD treatment, on 5-

HTT BPND during fall and winter in health. We elected to first study healthy volunteers,

rather than SAD patients, because we were interested in investigating the potential of

5-HTT BPND as a biomarker for light exposure to develop prevention strategies for

new-onset SAD, with the intent to further examine this effect in full-syndrome SAD in

study three. Our hypothesis was that, in fall and winter, 5-HTT BPND would be

significantly reduced in the ACC and PFC after light therapy. The primary finding was

a region-specific decrease in 5-HTT BPND in the ACC of healthy volunteers following

light therapy in winter, with no significant change observed in any examined brain

region in individuals scanned during the fall (Harrison et al., 2015).

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The results of study two suggest that, in healthy participants who have not yet

developed SAD, the response to light therapy may be confined to the ACC, a brain

region heavily involved in regulation of affect and antidepressant response (Ressler et

al., 2007). The ACC participates in production of sad emotions, where regional

activations have been observed during transient sadness in healthy volunteers and

decreases in activity follow recovery from depression (Mayberg et al., 1999). Activity

in this region has also been observed to decrease following various antidepressant

treatments (i.e. use of SSRIs, cognitive behavioural therapy, transcranial magnetic

stimulation, and DBS) (Ressler et al., 2007). Furthermore, appropriate 5-HT function

in the ACC may be important for treatment response, as in rodent models of

depressive behaviour, 5-HT depleting lesions abolish the antidepressant effects of

DBS (Hamani et al., 2010; Hamani et al., 2012). Finally, twenty-five percent of healthy

individuals experience daily impairment during the winter due to marked seasonal

changes in mood, energy and appetite (Kasper et al., 1989). Thus, our observation of

a region-specific decrease in 5-HTT BPND following light therapy in winter suggests

that the effects of light exposure on this biomarker are most pronounced during a

period in which even healthy individuals experience some level of seasonal

depressive symptoms.

In study one, we observed a greater magnitude seasonal variation in 5-HTT

BPND in SAD, primarily in severe SAD cases, as compared to health across multiple

brain regions suggesting that it might be a biomarker associated with SAD pathology

(Tyrer et al., 2016). In study two, we found a region-specific decrease in 5-HTT BPND

in the ACC following light therapy during winter in healthy volunteers, suggesting that

5-HTT BPND might have potential as a biomarker in regards to evaluating the effects of

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light therapy (Harrison et al., 2015). As a final step, it was our intent to examine light

therapy’s effect on 5-HTT BPND in severe SAD, in order to determine whether this

PET-based measure of SAD pathophysiology could be useful in assessing treatment

response to light in this clinical population and ameliorate seasonal change in 5-HTT

BPND. As such, we scanned SAD participants before and after light therapy during the

winter, with the hypothesis that 5-HTT BPND would be reduced across brain regions,

including the ACC and PFC, following this treatment. Interestingly, we discovered a

marked and consistent decrease in 5-HTT BPND across all examined regions,

including the ACC and PFC, following light therapy, with a greater spatial distribution

of effect and more robust statistical significance relative to study two. 5-HTT BPND has

been previously observed to be elevated in winter relative to summer across multiple

brain regions in SAD (Mc Mahon et al., 2016; Tyrer et al., 2016). Although we did not

observe an association between magnitude of decrease in 5-HTT BPND and clinical

remission or response following treatment, our conceptualization of light therapy is

that this treatment may facilitate it therapeutic effects via interaction with multiple

biological targets and it is our view that 5-HTT is one such target. As such, the lack of

relationship between treatment response and reduction in this biomarker would be

expected if light therapy affects multiple biological targets each of which, collectively,

contribute to its antidepressant effects. Accordingly, our findings suggest that one

means by which light therapy may exert its antidepressant effects during winter in

SAD is to ameliorate the seasonal change in 5-HTT BPND, thereby decreasing the

availability of the 5-HTT to clear 5-HT from synapse and increasing levels of

extracellular 5-HT.

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In summary, we have identified a biomarker that is affected by season and

associated with SAD pathology, which is also involved in treatment response to light.

As such, the results of this series of studies have two important implications. First, the

findings of studies one and three provide a basis for understanding the etiology of the

seasonal variation in 5-HTT BPND in SAD. To elaborate, in SAD, the magnitude of

seasonal change in 5-HTT BPND observed across regions moving from winter to

summer in study one was comparable to the global reduction in 5-HTT BPND found

following light therapy during winter in study three. Taken together, these results

suggest that seasonal change in light exposure may be a sufficient environmental

factor to mediate the seasonal variation in 5-HTT BPND in SAD, an explanation

supported by findings from both preclinical and clinical neuroimaging studies, in which

5-HTT binding has been observed to be inversely correlated with exposure to light

(Kalbitzer et al., 2010; Praschak-Rieder et al., 2008; Rovescalli et al., 1989). Second,

the results of studies two and three demonstrate that light therapy reaches an

important therapeutic target relevant to both prevention and treatment of SAD and

these findings provide an opportunity to further develop this therapeutic through

application of [11C]DASB PET. Twenty-five percent of healthy individuals experience

marked seasonal changes in mood, energy and appetite which impair function and

full-syndrome SAD is a common problem with an annual prevalence rate of 1 to 6

percent. However, only 55 percent of individual suffering from seasonal depressive

symptoms fully remit after light therapy, indicating a need for improvement. Our finding

of a decrease in 5-HTT BPND in both in healthy individuals who have not yet

developed SAD and in those with full-syndrome SAD, represents the first central

biomarker associated with treatment response to light therapy. Accordingly, in future

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study, the magnitude of reduction in 5-HTT BPND, as measured by [11C]DASB PET,

could be used as a predictor to identify aspects of light therapy (i.e. light colour,

duration, intensity, time of day, etc.) that could be modified so as to so as to optimize

SAD prevention and treatment.

4.3 Recommendations for Future Studies

4.3.1 Differentiating the Effects of Mood vs. Seasonality on 5-HTT BPND in SAD

There is evidence that the SAD represents a complex phenotype influenced by

multiple interacting, but independent, vulnerability factors including both the presence

seasonality (i.e. seasonal change in mood and behaviour) and a predisposition to

development of MDEs, among other factors (Levitan, 2007). Although there is strong

relationship between individuals who report a high degree seasonality, as measured

by the SPAQ GSS and incidence of SAD, the former measure is generally regarded

as a dimensional construct (Bartko et al., 1989; Terman, 1988). As such, seasonality

describes seasonal changes in mood and behaviour that may or may not cause

clinical impairment, as is necessary for diagnosis of SAD and there is some overlap in

degree of seasonality between those suffering from subsyndromal SAD and full-

syndrome SAD (American Psychiatric Association, 2013; Terman, 1988).

In study one, we investigated the seasonal change in 5-HTT BPND in SAD by

scanning participants in winter during their symptomatic phase and in summer

following remission from their seasonal MDEs. As such, all SAD subjects reported

significantly greater seasonality, relative to asymptomatic healthy controls and all met

criteria for moderate to severe MDEs during the winter months (Tyrer et al., 2016). In

this first study we found greater seasonal variation 5-HTT BPND in SAD relative to

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health, primarily due to increased magnitude of seasonal change in 5-HTT BPND in

severe SAD relative to moderate SAD cases (as measured by the SPAQ GSS) (Tyrer

et al., 2016). The third study included SAD participants with a high degree of

seasonality (SPAQ GSS ≥ 16), and moderate to severe MDEs, subjects who had

previously displayed an increased magnitude of seasonal change in 5-HTT BPND in

study one. In this subsequent study, we found a significant decrease 5-HTT BPND

across all brain examined brain regions after light therapy during winter. As both

studies included SAD participants with a high degree of seasonality and moderate to

severe MDEs, it was not possible to conclusively establish whether the effects of

season and light therapy on 5-HTT BPND in SAD were primarily attributable to degree

of seasonality or to scanning an MDE and again while in remission. Determination of

key components modulating the season and light-induced change in 5-HTT BPND is

critical in order to better identify individuals with SAD in which this biological

abnormality is present and whom may achieve greater benefit from interventions that

preferentially target the 5-HTT.

As a first step, separation of the components of the mood and seasonality in

SAD as contributing factors to change in 5-HTT BPND, as observed in studies one and

three would require individual assessment the impact of each factor (i.e. seasonality

or mood) on 5-HTT BPND, while keeping the other variable constant, as not to

confound results. There are two means by which to do this; the first would involve

using [11C]DASB PET to scan participants with SAD before and after acute

manipulation of mood during summer remission (i.e. lower mood during a

characteristic period of remission, without the confound of change of season) to

assess change in 5-HTT BPND. A second way by which to this would require scanning

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participants with unipolar MDEs that report low seasonality but are similar in severity

and degree of atypical depressive symptoms to those with SAD, within the same

summer and winter scanning windows as those previously used in study one (i.e. alter

season without the confound of change in mood, as individuals would, presumably, be

depressed at during both time-points) (Tyrer et al., 2016). The following two

subsections will review the methods by which the contributions of the effects of

seasonality and mood could be assessed, followed by brief outline of study design

4.3.1.1 Acute Manipulation of Mood in SAD during Summer Remission

In regards to the first point, common procedures to facilitate acute and transient

lowering of mood include mood induction paradigms and acute tryptophan depletion.

There are numerous mood induction procedures (MIPs), all of which engender sad

mood in individuals by different means. Interestingly, to the best of our knowledge,

manipulation of mood via MIPs has never been examined in SAD. Common MIPs

include Velten mood induction (reading aloud self-referential statements progressing

from neutral to negative mood), music mood induction (participants listen to sad or

neutral musical pieces so as to invoke the mood expressed by the music) and film-clip

induction (subjects are presented a short film and explicitly asked to imagine

themselves in the situation in order to generate the desired mood) (Gilet, 2008;

Velten, 1968). Of these, the Velten MIP is used most widely by researchers studying

changes in affective states, given its efficacy an altering subjective emotional states

and it is often applied combination with other approaches such as music mood

induction in order to increase efficacy (Dowlati et al., 2014; Frost et al., 1982; Gilet,

2008; Velten, 1968). Thus, a combination MIP, preferentially using the Velten and

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music MIPs simultaneously, could be used to induce neutral (i.e. control condition)

and sad mood, in SAD participants during summer remission in a randomized order

with [11C]DASB PET scanning before and after each MIP (Dowlati et al., 2014; Gilet,

2008). If the effects of season and light on 5-HTT BPND were primarily attributable to

changes in mood as opposed to seasonality, it would be expected that an increase in

5-HTT BPND would be observed following induction of sad mood in summer, with no

change after induction of neutral mood. A change in 5-HTT BPND would not be

expected if seasonality was the primary factor mediating change in this biomarker.

In regards to acute tryptophan depletion (ATD), Neumeister and colleagues

examined the effects of this procedure during characteristic summer remission in

individuals with SAD (Neumeister et al., 1998). In comparison with sham depletion,

ATD caused transient relapse into MDE in 73 percent of the remitted SAD sample,

suggestive of a “trait-like” vulnerability toward depressive relapse following

perturbation of the serotonergic function in SAD (Neumeister et al., 1998). In a follow-

up study Neumeister et al., found that all but one of the SAD participants who had

relapsed after ATD in summer, experienced a recurrent seasonal depressive episode

during the subsequent winter (Neumeister et al., 1999). Furthermore, ATD has been

found to facilitate relapse in remitted SAD patients following light therapy (Lam et al.,

1996). These results suggest that individuals with SAD, sensitive to acute

serotonergic manipulations, may be at high risk for development of recurrent winter

MDEs. It is notable that there is evidence of a similar “trait-like” vulnerability toward

serotonergic function in unipolar MDD since ATD has also been shown to facilitate

depressive relapse in unmedicated remitted MDD participants(Neumeister et al.,

2004). Taken together, these results suggest that vulnerability to serotonergic

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dysfunction, as unmasked by ATD, may be an underlying trait that is present in those

predisposed to development of MDEs.

There are only two neuroimaging studies that have applied receptor-ligand

neuroimaging techniques to investigate the effect of ATD relative to sham depletion on

5-HTT BPND (Praschak-Rieder et al., 2008; Talbot et al., 2005). Both investigations

used [11C]DASB PET, were conducted using healthy volunteers and found negligible

differences in 5-HTT BPND across brain regions between conditions. Thus, there have

been no PET or SPECT investigations studying the effects of ATD on mood and 5-

HTT BPND in SAD. As such, to differentiate the contributions of the effects of

seasonality and mood on 5-HTT BPND, an alternative approach to an MIP would be to

use [11C]DASB to scan individuals with SAD, during summer, before and after sham

and ATD. [11C]DASB is insensitive to displacement by endogenous 5-HT, use of an

ATD procedure (i.e. a serotonergic manipulation) would not be a confounding factor in

regards to quantification of 5-HTT BPND. For example, if 5-HTT BPND was found to be

unaffected by ATD, yet mood was altered, this would suggest that the effect of season

and light on this biomarker do not result from alterations in mood as induced by ATD.

Interestingly, Lam et al. have demonstrated that tryptophan depletion affects mood in

SAD responders to light therapy, but measurement of 5-HTT BPND using [11C]DASB

PET was not a component of this study (Lam et al., 1996). The results of such as

study would also be important, as this would be the first investigation of the effect of

ATD on 5-HTT BPND in unmedicated SAD subjects in remission from depression.

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4.3.1.2 Assessment of 5-HTT BPND in Non-Seasonal MDD during Winter and Summer

Acute manipulations of mood, as proposed in the experiments outlined, above

are useful as they are allow for assessment of the effects of alterations in mood in the

population of interest (i.e. SAD), during the summer season, which is a period of

characteristic remission and can be completed in a relatively short time period.

However, mood induction paradigms are acute and transient in nature, spanning

minutes (i.e. MIPs), to days (i.e. ATD), and the season and light-induced changes in

5-HTT BPND upon mood as observed in studies one and three occurred over months

and weeks, respectively. As such, it is possible that a brief period of depressed mood

invoked by mood induction, as outlined above, might not be sufficient in duration to

elevate 5-HTT BPND in those with SAD.

An alternative method by which to differentiate the components of the mood

and seasonality in SAD as contributing factors to change in 5-HTT BPND would be to

apply [11C]DASB PET to scan individuals with unipolar MDD, low in seasonality, but of

similar severity and degree of atypical symptoms to those of the severe SAD group,

longitudinally, during summer and winter (participants would be experiencing MDEs at

both time-points). In this population, changes in 5-HTT BPND across summer and

winter seasons could then be compared to that of healthy and SAD subjects (Tyrer et

al., 2016). If 5-HTT BPND was found to be elevated during both seasons relative to

non-seasonal healthy controls and levels of 5-HTT BPND were roughly equivalent to

those observed in SAD during winter, this would be evidence of depressed mood as a

primary factor mediating the season and light-induced changes observed in SAD.

Furthermore, as light therapy has shown efficacy as a therapeutic for non-seasonal

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unipolar MDD and has been found to reduce 5-HTT BPND in SAD, as observed in

study three, an additional promising future direction would be to extend the

investigation regarding the effects of light therapy on 5-HTT BPND to those with non-

seasonal unipolar depression (Lam et al., 2016).

4.3.2 Generalizability of Findings to “Seasonal Bulimia Nervosa”

It has become clear that some biological abnormalities are not specific to a

single disorder, but present across a range of neuropsychiatric illnesses that share

common behavioural features (i.e. dysphoric mood, anhedonia, etc.). For example,

levels of MAO-A, an enzyme that degrades dopamine, norepinephrine, and 5-HT, as

measured by [11C]harmine PET, have been found to be elevated in MDD, postpartum

depression, alcoholism, perimenopause and borderline personality disorder (Kolla et

al., 2016; Matthews et al., 2014; Meyer et al., 2009; Rekkas et al., 2014; Sacher et al.,

2015). Similarly, the density of translocator protein (TSPO), a marker of

neuroinflammation, as measured by [18F]FEPPA PET has been observed to be

elevated both in both MDD and Alzheimer’s disease. (Setiawan et al., 2015; Suridjan

et al., 2015). As such, it has become increasingly common to study behavioural

phenotypes and/or biomarkers that may be shared across a neuropsychiatric

disorders to better understand the underlying neurochemistry of such illnesses, clarify

overlap and boundaries between disorders and identify optimal therapeutic targets to

improve treatment response (Morris et al., 2012).

Seasonal patterns of behaviour are present in psychiatric disorders other than

SAD such as bulimia nervosa (BN) (Lam et al., 1996a; Levitan et al., 1994; Yamatsuji

et al., 2003). BN is an eating disorder commonly characterized by dysfunctional eating

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behaviours such as periods of extreme overeating (i.e. bingeing) followed by

compensatory behaviours such as self-induced vomiting, purging (i.e. laxative and/or

diuretic use), over-exercise or fasting (American Psychiatric Association, 2013). It is

notable that there is a high rate of psychiatric comorbidity between SAD and this

eating disorder, as it has been reported that 21 to 32 percent of patients with BN also

meet criteria for SAD, with a concomitant decrease in mood and marked increase in

carbohydrate craving, hyperphagia and compensatory purging behaviours during

winter relative to summer (Lam et al., 1996a; Lam et al., 1991). Accordingly, the

prevalence of SAD in BN is 4 to 5 times greater than the 1 to 6 percent prevalence

rate reported in the general population at similar latitudes. In addition, many

individuals with BN who do not meet full criteria for SAD still display seasonal patterns

in dysfunctional eating behaviours and mood of greater magnitude than of healthy

controls (Gruber et al., 1996; Lam et al., 1996a; Lam et al., 1991). Interestingly, in one

study evaluating “seasonal BN” patients, 31 percent had SPAQ global seasonality

scores equal to or greater than 16, equivalent to those of our severe SAD patients in

which we observed seasonal change in 5-HTT BPND in study one and decrease in 5-

HTT BPND following light therapy in study three (Lam et al., 1991; Tyrer et al., 2016).

Furthermore, a correlation has also been observed between frequency of bingeing

and purging behaviours in BN in photoperiod, with a peak in dysfunctional eating

behaviours during winter, and a symptomatic nadir in summer (Blouin et al., 1992).

Similar to SAD, BN is often a chronic illness with poor outcome following

treatment as only 45 percent of individuals attain full recovery, 27 percent achieve

partial remission, but remain symptomatic while 23 percent experience a protracted

course of illness spanning many years (Steinhausen et al., 2009). The efficacy of

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fluoxetine, the only approved pharmacotherapy for BN, in reducing the behavioural

symptoms of this disorder is comparable to its antidepressant efficacy in SAD, as only

56 and 67 percent of BN patients experience significant reductions in vomiting and

binge-eating, respectively, after 8 weeks of treatment, while only 55 percent of SAD

patients remit from winter depression following 8 weeks of treatment with this SSRI

(Fluoxetine Bulimia Nervosa Collaborative Study Group., 1992). Interestingly, light

therapy also been found to ameliorate both low mood and bingeing and purging

behaviours in patients with seasonal BN. For example: in an open-label 4 week trial of

light therapy of bulimic patients with a comorbid diagnosis of moderate to severe SAD,

clinical response was observed in 56 percent of participants as indicated by a 50

percent reduction in SIGH-SAD scores and a reduction in bingeing and purging

behaviour of 50 percent was observed in 55 and 45 percent of patients, respectively

(Lam et al., 2001). Similar studies of effects of light therapy on reducing dysfunctional

eating behaviours in “seasonal” BN patients have shown similar results (Lam et al.,

1994).

Just as there is evidence of hypofunction of the serotonin system in SAD, so

too is there similarly strong evidence of serotonergic dysfunction in BN. Significantly

greater levels of depression, sadness and desire to binge have been found in women

with BN relative to healthy volunteers following ATD, suggesting increased

vulnerability to serotonergic manipulations and altered modulation of the serotonin

system (Kaye et al., 2000). 5-HIAA levels, a metabolite of 5-HT, have been found to

be reduced in the cerebral spinal fluid of symptomatic BN patients and this reduction

is inversely correlated with frequency of binge-eating episodes (Jimerson et al., 1992).

Brewerton and colleagues administered oral m-CPP, a non-selective 5-HT receptor

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agonist, to BN patients and found seasonal variation in prolactin response in BN with

no difference in healthy volunteers and also lower peak cortisol and prolactin

responses in BN compared to health (Brewerton et al., 1992). Seasonal variation in

prolactin levels was also seen following administration of L-tryptophan in BN and

notably, blunted prolactin and cortisol responses were observed patients with both BN

and MDD, relative to health (Brewerton et al., 1992). In a double-blind, randomized,

placebo-controlled study, Levitan et al., replicated this finding of blunted cortisol and

prolactin response following intravenous m-CPP challenge in bulimic patients and also

observed subjective responses on mood ratings such as decreased anxiety, increased

feeling of calmness and altered self-awareness, similar to mood-altering effects

reported using the same paradigm in symptomatic individuals with SAD (Levitan et al.,

1998; Levitan et al., 1997). Hormonal response following infusion of intravenous 5-

HTP, a metabolic intermediate of 5-HT biosynthesis, has also been investigated with

blunted prolactin levels observed in BN patients relative to healthy volunteers, further

indicating hypofunction of the serotonin system (Goldbloom et al., 1996). In support of

this observation, administration of d-fenfluramine, a 5-HT releasing agent, has been

associated with similarly blunted prolactin response in this clinical group, whereas this

neuroendocrine response is not observed in those who have recovered from BN

(Jimerson et al., 1997; Monteleone et al., 1998; Wolfe et al., 2000). Collectively, these

findings provide strong support of hypofunction of the serotonin system in BN.

However, despite evidence of a seasonal dimension to BN, the large body of

research supporting hypofunction of the serotonin system in this disorder, a high rate

of comorbidity between SAD and BN, and poor treatment response to SSRIs, a first-

line evidence-based treatment for this disorder, there have been relatively few PET

108

studies of investigating 5-HTT BPND in BN and no study investigating seasonal change

in 5-HTT BPND in “seasonal BN” or in BN following light therapy. To date, most studies

have included relatively small samples of recovered, or symptomatic non-seasonal BN

patients (i.e. ≤10 participants), applying inferior neuroimaging techniques and

importantly, have not controlled for seasonal effects on 5-HTT BPND, thus findings

regarding 5-HTT BPND in BN have been inconclusive. For example: Taucher and

colleagues used [123I]β-CIT SPECT and found reduced 5-HTT BPND in the thalamus

and hypothalamus of 10 BN patients relative to 10 healthy controls, although the

authors acknowledged potential confounds such as the near equal affinity of the tracer

for the 5-HTT and DAT, the small sample size and history of anorexia nervosa (AN) in

20 percent of the BN group (Tauscher et al., 2001). Bailer et al., used [11C]McN5652

PET to assess 5-HTT BPND in 9 women recovered from BN and found higher binding

in the antero-ventral striatum relative to 7 women who had recovered from bulimia-

type AN, but also noted the small sample size and limitations of the neuroimaging

technique which precluded measurement of 5-HTT BPND in the neocortex (Bailer et

al., 2007). The only study to apply [11C]DASB PET found higher 5-HTT BPND in the

anterior cingulate cortex and superior temporal gyrus in recovered BN patients relative

to healthy volunteers, but this investigation was also confounded by small sample size

and an unequal division of scan dates by season, thus it was not possible to assess

the effect of season on 5-HTT BPND (Pichika et al., 2012).

Given evidence of a seasonality present in this eating disorder subtype, the

high rate of comorbidity between SAD and BN, findings of serotonergic dysfunction in

this disorder and the efficacy of both light therapy and SSRI treatment in ameliorating

binge-purge behaviour in BN, it would be valuable to conduct a [11C]DASB PET study

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with an identical design to that of study one, to determine whether individuals with BN

that are high in seasonality (i.e. as measured by the SPAQ with a GSS score ≥ 16,

without comorbid SAD), also display seasonal variation in 5-HTT BPND. The

magnitude of this seasonal fluctuation in “seasonal BN” patients could then be

compared to that previously observed in SAD and health (Mc Mahon et al., 2016;

Tyrer et al., 2016). Such a study would provide insight into the neurobiology

underlying the symptomatic peak in dysphoric mood and dysfunctional eating

behaviours that individuals with “seasonal BN” report during the winter months. In

addition, as both individuals with SAD and “seasonal” BN report low mood and

dysregulating eating during winter, this would suggest the presence of a common

neurobiological abnormality associated with seasonal fluctuations in these symptoms

that are shared across separate neuropsychiatric disorders. As a logical next step, the

effects of light therapy on 5-HTT BPND could also be investigated during winter in a

small sample of ”seasonal BN” patients, to determine if a similar reduction symptoms

and in this biomarker would be observed, as was found in SAD in study three.

4.3.3 Neurobiology Underlying the Effect of Season and Light on 5-HTT BPND

In this series of studies we observed greater magnitude of seasonal variation in

5-HTT BPND, an index of 5-HTT levels, in SAD relative to health with an elevation in

winter relative to summer. We also found that, during winter, light therapy could

ameliorate this seasonal change in 5-HTT BPND in region-specific manner in health,

with a significant reduction in this biomarker in the ACC and more globally in SAD,

with significant decreases observed across all examined brain regions. However,

preclinical investigation of the neurobiology underlying the effect of season and light

110

on the 5-HTT is beyond the scope of this thesis. This is a key issue for future study

because at present there is no consensus as to a putative serotonergic mechanism

underlying SAD development and maintenance, and there is limited understanding of

the means by which light therapy exerts its antidepressant effects; such an

investigation is needed to better understand the interplay between season, light and

seasonal changes in mood and behaviour, and their effects on the 5-HTT on a cellular

level. Possible mechanisms underlying the effect of season and light on the 5-HTT

include alterations in gene expression of the 5-HTT or changes in trafficking (i.e.

upregulation or downregulation) of the 5-HTT protein to the cell surface. As [11C]DASB

binds preferentially to the 5-HTT on the cell surface, we favour the view that changes

in 5-HTT BPND may reflect an alterations in regulation of the 5-HTT to the plasma

membrane and that the study of molecular and cellular mechanisms by which this may

occur is a promising direction for future study.

Endogenous regulation of 5-HTT expression and activity is complex and occurs

at multiple levels (i.e. transcriptional, translational, post-translational) (Blakely et al.,

1998). Nonetheless, in order to explain the effect of season and light on the 5-HTT, an

environmental stressor must be identified that, when present, is able to activate

cellular machinery in serotonergic neurons within the brain to alter 5-HTT levels.

Interestingly, there is evidence that regulation of stress-activated p38 mitogen-

activated protein kinase (p38MAPK) is under photoneural control, such that activity of

this kinase peaks during darkness and is inhibited by exposure to bright light (Chik et

al., 2004). In addition, serotonergic neurons in the DRN express p38MAPK which,

when phosphorylated, increases both trafficking of the 5-HTT to the cell surface and

5-HT clearance. Accordingly, this kinase has a critical role in mediating 5-HTT density

111

at the plasma membrane of serotonergic neurons and also in regulation of

extracellular 5-HT levels (Samuvel et al., 2005; Zhu et al., 2005).

Importantly, there is also evidence that activity of p38MAPK is critical for

appropriate behavioural response to stress (Bruchas et al., 2011). It is notable that, in

rodent models of depressive-like behaviour, genetic deletion or pharmacological

inhibition of this kinase in serotonergic neurons of the DRN reduces social avoidance

following a social defeat paradigm, stress-induced reward seeking behaviour and

immobility time during the forced swim test, relative to animals with intact p38MAPK

(Bruchas et al., 2011). On a cellular level, exposure to these stressors has been found

activate this kinase, increase 5-HTT density on the plasma membrane of serotonergic

neurons and decrease extracellular 5-HT levels, a result that is not observed upon

genetic knock-out or pharmacological inhibition of p38MAPK (Bruchas et al., 2011).

Collectively, these findings indicate that, in animal models, exposure to stress initiates

a cascade of cellular events in which activation of p38aMAPK induces a

hyposerotonergic state leading to depressive-like behaviours in rodents.

It is notable that, DASB has been shown to bind preferentially to 5-HTT on

outer cell membranes suggesting that the season and light-induced changes in 5-HTT

BPND observed in this series of [11C]DASB PET studies reflect altered 5-HTT density

at the plasma membrane. Consequently, alterations in activity of p38MAPK may be

critical in mediating these changes in cell surface 5-HTT levels. To elaborate,

individuals with SAD are sensitive to changes in photoperiod and as such, in winter,

shortened photoperiod and/or lack of light exposure may act as environmental

stressors facilitating activation of stress-induced p38MAPK, subsequent upregulation

in trafficking of the 5-HTT to the cell surface, extracellular 5-HT loss and development

112

of winter MDEs (Wehr et al., 2001). In the absence of such a stressor, for instance,

during the summer months or following bright light exposure in winter (i.e. light

therapy) this increase in trafficking of the 5-HTT t the cell surface and concomitant

lowering of mood would not be expected to occur.

In future study, in order investigate the possible role of p38MAPK in mediating

the effects of season and light on the 5-HTT and in SAD, the selection of an

appropriate animal model is critical. To date, such studies have been challenging, as a

most rodent species used in preclinical research are nocturnal and thus have opposite

rest-activity cycles to humans and other diurnal species (Barak et al., 2013). In

addition, most tests of depressive-like behaviour in animal models have been

designed for commonly used nocturnal laboratory rodents (i.e. rats and mice), and

therefore modification of these paradigms is often necessary (Barak et al., 2013).

However, there has been significant progress in regards to establishing both face,

construct and predictive validity of certain diurnal rodent species.

Specifically, the diurnal Mongolian gerbil has been investigated as a highly

promising animal model of SAD. Notably, in these animals, afferents have been found

to project directly from a select population of light-sensitive non-visual retinal ganglion

cells (RGCs) to serotonergic neurons in the DRN and light-deprivation has been

shown to induce depressive-like behavior (Fite et al., 1999; Lau et al., 2011; Luan et

al., 2011). Ren and colleagues found that this distinct population of non-visual DRN-

projecting RGCs was critical in modulating affective behavior in response to light via

increased input to serotonergic neurons in the DRN and also that the level of neural

activity in these RGCs was highly correlated with both 5-HT levels in the DRN and

reduction in depressive-like behaviour (Ren et al., 2013). Serotonergic neurons within

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the DRN projects to both subcortical and cortical areas and increased neuronal firing

in this region has been found to down-regulate levels of monoamine transporters,

including the 5-HTT (Hipolide et al., 2005; Mogilnicka et al., 1980; Porrino et al.,

1982). Thus, it is possible that light exposure may exert its antidepressant effects via

increased signaling along this retina-raphe pathway, thereby regulating 5-HTT levels

in efferent brain regions.

As such, a possible direction of future study would be to expose groups of

Mongolian gerbils to different photoperiodic conditions (i.e. 12:12 LD, 16:8 LD 8:16

LD, constant light, constant darkness). Subsequent to exposure to these

photoperiodic conditions, western blotting would be useful in regards to quantifying

levels of phosphorylated (i.e. active) p38MAPK in different brain regions. To measure

levels of 5-HTT on the cell-surface, a membrane impenetrant biotinylation procedure

could be used followed by affinity chromatography to isolate this protein and western

blotting for 5-HTT quantification. In terms of analysis of these results, one could then

examine the relationship between phosphorylated p38MAPK and both total and cell

surface 5-HTT across photoperiodic conditions. If exposure to darkness facilitates

trafficking of the 5-HTT to the cell surface via interaction with p38MAPK, it would be

expected that levels of this kinase and cell-surface 5-HTT would elevated in conditions

of shortened photoperiod and constant darkness, whereas p38MAPK and cell-surface

5-HTT levels would be reduced following exposure to constant bright light or

lengthened photoperiod. This could be extended into studies evaluating the

dependence of this process on the presence or absence of p38MAPK to further

establish causality.

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LIST OF PUBLICATIONS

Tyrer AE, Levitan RD, Houle S, Wilson AA, Nobrega JN, Meyer JH. Increased Seasonal Variation in Serotonin Transporter Binding in Seasonal Affective Disorder. Neuropsychopharmacology 2016; doi: 10.1038/npp.2016.54.

Harrison SJ & Tyrer AE, Levitan RD, Xu X, Houle S, Wilson AA, Nobrega JN, Rusjan PM, Meyer JH. Light Therapy and Serotonin Transporter Binding in the Anterior Cingulate and Prefrontal Cortex. Acta Psychiatrica Scandinavica 2015; 132: 379-388.

(co-first author)

Tyrer AE, Levitan RD, Xu X, Houle S, Wilson AA, Nobrega JN, Rusjan PM, Meyer JH. Serotonin transporter binding is reduced in seasonal affective disorder following light therapy. Acta Psychiatrica Scandinavica (in press)