intrinsically photosensitive melanopsin retinal ganglion...

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Intrinsically photosensitive

melanopsin retinal ganglion cell

contributions to the

post-illumination pupil response

and circadian rhythm

Emma L. Markwell

B.App.Sc(Optom)

Submitted in fulfilment of the requirements for the degree of Masters of Applied Science

Visual Science and Medical Retina Laboratories School of Optometry and

Institute of Health and Biomedical Innovation Queensland University of Technology (QUT)

Australia

February 2011

Keywords

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Keywords

Keywords: intrinsically photosensitive Retinal Ganglion Cells (ipRGC),

melanopsin, pupil light reflex, post-illumination pupil response, cone photoreceptor,

rod photoreceptor, circadian rhythm, melatonin

Abstract

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Abstract

Intrinsically photosensitive retinal ganglion cells (ipRGCs) in the eye transmit the

environmental light level, projecting to the suprachiasmatic nucleus (SCN) (Berson,

Dunn & Takao, 2002; Hattar, Liao, Takao, Berson & Yau, 2002), the location of the

circadian biological clock, and the olivary pretectal nucleus (OPN) of the

pretectum, the start of the pupil reflex pathway (Hattar, Liao, Takao, Berson & Yau,

2002; Dacey, Liao, Peterson, Robinson, Smith, Pokorny, Yau & Gamlin, 2005).

The SCN synchronizes the circadian rhythm, a cycle of biological processes

coordinated to the solar day, and drives the sleep/wake cycle by controlling the

release of melatonin from the pineal gland (Claustrat, Brun & Chazot, 2005).

Encoded photic input from ipRGCs to the OPN also contributes to the pupil light

reflex (PLR), the constriction and recovery of the pupil in response to light. IpRGCs

control the post-illumination component of the PLR, the partial pupil constriction

maintained for > 30 sec after a stimulus offset (Gamlin, McDougal, Pokorny,

Smith, Yau & Dacey, 2007; Kankipati, Girkin & Gamlin, 2010; Markwell, Feigl &

Zele, 2010). It is unknown if intrinsic ipRGC and cone-mediated inputs to ipRGCs

show circadian variation in their photon-counting activity under constant

illumination. If ipRGCs demonstrate circadian variation of the pupil response under

constant illumination in vivo, when in vitro ipRGC activity does not (Weng, Wong

& Berson, 2009), this would support central control of the ipRGC circadian activity.

A preliminary experiment was conducted to determine the spectral sensitivity of the

ipRGC post-illumination pupil response under the experimental conditions,

confirming the successful isolation of the ipRGC response (Gamlin, et al., 2007) for

the circadian experiment. In this main experiment, we demonstrate that ipRGC

photon-counting activity has a circadian rhythm under constant experimental

conditions, while direct rod and cone contributions to the PLR do not. Intrinsic

ipRGC contributions to the post-illumination pupil response decreased 2:46 h prior

to melatonin onset for our group model, with the peak ipRGC attenuation occurring

1:25 h after melatonin onset. Our results suggest a centrally controlled evening

decrease in ipRGC activity, independent of environmental light, which is

temporally synchronized (demonstrates a temporal phase-advanced relationship) to

the SCN mediated release of melatonin. In the future the ipRGC post-illumination

Abstract

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pupil response could be developed as a fast, non-invasive measure of circadian

rhythm. This study establishes a basis for future investigation of cortical feedback

mechanisms that modulate ipRGC activity.

List of Publications

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List of Publications

Published Papers

Zele, A. J., Feigl, B., Smith, S. S., & Markwell, E. L. (2011) The circadian response

of intrinsically photosensitive retinal ganglion cells. PLoS ONE. DOI:

10.1371/journal.pone.0017860

Markwell, E. L., Feigl, B., & Zele, A. J. (2010). Intrinsically photosensitive

melanopsin retinal ganglion cell contributions to the pupil light reflex and circadian

rhythm (Invited review). Clinical & Experimental Optometry, 93:(3), 137-149.

Conference Abstracts

Markwell, E. L., Feigl, B., Smith, S. S., & Zele, A. J. (2010). Circadian modulation

of the intrinsically photosensitive (melanopsin) retinal ganglion cell driven pupil

light response. Investigative Ophthalmology and Visual Science, 51, ARVO

E-abstract 671.

Markwell, E. L., Feigl, B., Smith, S. S., & Zele, A. J. (2010) Circadian variation in

the response of intrinsically photosensitive retinal ganglion cells. Sleep and

Biological Rhythms, 8:(s1), A22.

Table of Contents

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Table of Contents

KEYWORDS ......................................................................................................................... I

ABSTRACT .......................................................................................................................... II

LIST OF PUBLICATIONS ................................................................................................ IV

TABLE OF CONTENTS ...................................................................................................... V

LIST OF FIGURES ........................................................................................................... VII

LIST OF TABLES .............................................................................................................. IX

ABBREVIATIONS .............................................................................................................. X

SI UNITS ............................................................................................................................ XI

DECLARATION ............................................................................................................... XII

ACKNOWLEDGEMENTS .............................................................................................. XIII

CHAPTER 1. INTRODUCTION. ........................................................................................ 1

CHAPTER 2. LITERATURE REVIEW .............................................................................. 4

2.1 INTRODUCTION .............................................................................................................................................. 4

2.2 HISTOLOGY AND ELECTROPHYSIOLOGY OF ipRGCs ..................................................................... 5

2.3 ipRGCs AND THE PUPIL LIGHT REFLEX ........................................................................................... 14

2.4 CYCLIC VARIATIONS OF THE RETINA ............................................................................................... 19

2.4.1 Circadian variation in the PLR .................................................................................................... 23

2.5 EXPERIMENTAL AIMS AND HYPOTHESES ...................................................................................... 24

CHAPTER 3. EXPERIMENTAL METHODS .................................................................. 26

3.1 PARTICIPANTS ............................................................................................................................................. 26

3.2 PUPILLOMETER APPARATUS ................................................................................................................ 27

3.3 DETERMINATION OF THE OPTIMAL VIEWING DISTANCE FOR THE PUPILLOMETRIC

MEASUREMENTS ......................................................................................................................................... 30

3.3.1 Introduction ........................................................................................................................................ 30

3.3.2 Experimental Methods ................................................................................................................... 30

3.3.3 Results and Discussion ................................................................................................................... 30

3.4 PUPILLOMETER CALIBRATIONS ......................................................................................................... 31

3.4.1 Tungsten Halogen Stimulus Light ............................................................................................. 32

3.4.2 IR LEDs .................................................................................................................................................. 32

3.4.3 Narrow Band Interference Filters ............................................................................................. 33

3.4.4 Neutral Density Filters ................................................................................................................... 34

3.4.5 Luxeon LED.......................................................................................................................................... 34

3.4.6 Photon Calculations ......................................................................................................................... 35

3.5 DATA ANALYSIS OF PUPILLOMETRY RECORDINGS ................................................................... 36

3.5.1 Pupil Diameter Analysis Software ............................................................................................. 36

3.5.2 Analysing the Pupil Light Reflex ................................................................................................ 39

3.6 DETERMINATION OF THE SPECTRAL SENSITIVITY OF THE POST-ILLUMINATION

PUPIL RESPONSE ......................................................................................................................................... 41

Table of Contents

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3.6.1 Introduction ........................................................................................................................................ 41

3.6.2 Experimental Methods ................................................................................................................... 41

3.6.3 Results and Discussion ................................................................................................................... 43

CHAPTER 4. INVESTIGATION OF CIRCADIAN VARIATION OF THE ipRGC PUPIL

RESPONSE ............................................................................................................... 45

4.1 INTRODUCTION ........................................................................................................................................... 45

4.2 METHODS........................................................................................................................................................ 47

4.2.1 Participants ......................................................................................................................................... 47

4.2.2 Apparatus ............................................................................................................................................. 47

4.2.3 Procedures ........................................................................................................................................... 50

4.2.4 Data Analysis ...................................................................................................................................... 53

4.3 RESULTS ......................................................................................................................................................... 58

4.3.1 Cone photoreceptor (outer retina) contributions to the PLR ...................................... 58

4.3.2 Intrinsic and cone-mediated ipRGC contributions to the PLR...................................... 60

4.4 DISCUSSION ................................................................................................................................................... 66

4.4.1 The isolation of the inner and outer retinal responses ................................................... 67

4.4.2 Circadian variation in ipRGC but not cone activity ............................................................ 67

4.4.3 Temporal synchrony of ipRGC and central SCN circadian rhythms .......................... 73

CHAPTER 5. CONCLUSIONS AND FUTURE STUDIES/DIRECTIONS ..................... 75

5.1 CONCLUSIONS ............................................................................................................................................... 75

5.2 FURTHER STUDY ......................................................................................................................................... 76

REFERENCES ................................................................................................................... 79

APPENDICES .................................................................................................................... 96

7.1 PUBLICATION ............................................................................................................................................... 96

7.2 PITTSBURGH SLEEP QUALITY INDEX ............................................................................................. 109

7.3 PITTSBURGH SLEEP DIARY .................................................................................................................. 111

7.4 ACTIGRAPHY OUTPUT ............................................................................................................................ 113

7.5 INDIVIDUAL PUPIL LIGHT REFLEX AND MELATONIN DATA ............................................... 114

7.5.1 Baseline pupil diameter ............................................................................................................... 114

7.5.2 Maximum constriction pupil diameter ................................................................................. 116

7.5.3 Post-illumination pupil response ............................................................................................ 120

7.5.4 Post-illumination pupil response (488 nm) and melatonin ........................................ 124

_ List of Figures

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List of Figures

Figure 2.1. Schematic of the primate retinal layers showing location and synapses of

inner and outer stratifying ipRGCs in primates.. ................................................... 6

Figure 2.2. Intrinsically photosensitive retinal ganglion cell projections to brain

locations and the associated circuits in mice. ......................................................... 9

Figure 2.3. Intracellular voltage recordings of a human ipRGC in vivo. ........................... 12

Figure 2.4. Anatomical drawing showing the direct and consensual pupillary light

reflex pathways and the parasympathetic and sympathetic innervation of

the iris in primates. .......................................................................................................... 15

Figure 2.5. The consensual pupil light reflex (PLR) of a 30 yo female with 6/5 acuity.

.................................................................................................................................................. 16

Figure 2.6. The phase relationship between environmental light, activity, core body

temperature and pineal melatonin secretion. ...................................................... 20

Figure 3.1. Schematic plan view of the pupillometer.. ............................................................. 28

Figure 3.2. Pre-stimulus pupil fluctuations for three fixation accommodative

demands.. ............................................................................................................................. 31

Figure 3.3. The normalised spectral distribution of the 500 W, 240 V tungsten

halogen lamp. ..................................................................................................................... 32

Figure 3.4. The spectral distribution of the IR LEDs. ............................................................... 33

Figure 3.5. The spectral transmission of narrow band interference filters measured

through the pupillometer.. ........................................................................................... 33

Figure 3.6. The normalized spectral distribution of the white Luxeon LED. .................. 34

Figure 3.7. Linear and exponential model of the pupil light reflex for a 10 second,

14.2 log photons.cm-2.s-1, 488 nm stimulus (30 yo female).. .......................... 40

Figure 3.8. The post-illumination pupil response and spectral sensitivity of

intrinsically photosensitive retinal ganglion cells. ............................................. 42

Figure 3.9. Spectral sensitivity of the five human retinal photopigments.. .................... 44

Figure 4.1. Timing of the hourly measurements and protocols for the 24 hour testing

period.. .................................................................................................................................. 51

List of Figures

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Figure 4.2. An example of the skewed baseline cosine function (SBCF) model and

parameters.. ........................................................................................................................ 54

Figure 4.3. Alignment of participants by individual circadian phase. ............................... 55

Figure 4.4. The circadian profiles of the baseline and maximum constriction pupil

components of the pupil light reflex. ....................................................................... 60

Figure 4.5. The individual circadian variation of the post-illumination pupil response

component of the pupil light reflex for two observers.. ................................... 61

Figure 4.6. Temporal synchrony of ipRGC activity with the biological clock.. .............. 65

Figure 4.7. A functional model of the ipRGC, cone-mediated ipRGC and conventional

retinal ganglion cells contributions to the SCN and OPN, and the

hypothesized site/s of action of SCN inhibitory feedback ............................. 72

Figure 7.1. The actigraphic output of a 26 yo M participant over 8 days. .................... 113

Figure 7.2. Individual baseline pupil diameter data and models for the 11 participants

recorded over 20 - 24 hours. .................................................................................... 115

Figure 7.3. Individual maximum constricted pupil diameter data and models for the

11 participants recorded over 20 - 24 hours (488 nm)................................ 117

Figure 7.4. Individual maximum constricted pupil diameter data and models for the

11 participants recorded over 20 - 24 hours (610 nm)................................ 119

Figure 7.5. Individual pupil light reflexes at three circadian times and the post-

illumination pupil response data and models, for the 11 participants... 121

Figure 7.6. Individual post-illumination pupil response (488 nm) and salivary

melatonin data and models for the 11 participants recorded over 20 - 24

hours. ................................................................................................................................. 125

List of Tables

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List of Tables

Table 2.1. Location, distribution and anatomy of primate intrinsically photosensitive

retinal ganglion cells compared with rod and cone photoreceptors. ........... 7

Table 3.1. Ocular screening protocol and inclusion criteria for all participants. ........ 27

Table 3.2. Baseline pupil diameter measured at three fixation distances. .................... 31

Table 3.3. The calibrated optical density measurements of the reflective neutral

density filters. .................................................................................................................... 34

Table 3.4. Photon and candela irradiance of the pupillometer for each narrow band

interference filter. ............................................................................................................ 36

Table 4.1. The habitual sleep and wake times of the 11 participants, recorded for one

week prior to the overnight experiment. ............................................................... 49

Table 4.2. The mean linear model parameters of the baseline pupil diameter

circadian profile. ............................................................................................................... 58

Table 4.3. The mean linear model parameters of the maximum pupil constriction

circadian profile for 488 nm and 610 nm (14.2 log photon.cm-2.s-1, 10 sec)

stimuli. .................................................................................................................................. 59

Table 4.4. Participant (n = 11) post-illumination pupil response amplitudes for the

488 nm and 610 nm stimuli. ........................................................................................ 62

Table 4.5. Participant (n =11) intrinsic ipRGC activity (488 nm), cone-mediated

ipRGC activity (610 nm) and salivary melatonin onset and minimum/peak

times. ..................................................................................................................................... 62

Table 4.6. Participant (n = 11) post-illumination pupil response amplitudes (488

nm); and ipRGC activity onset, ipRGC activity peak and melatonin peak

times, with respect to the melatonin onset time of 14 h ................................. 66

Table 7.1. The mean parameters of the modelled intrinsic ipRGC activity determined

by the post-illumination pupil responses (488 nm stimuli). ...................... 123

Table 7.2. The mean parameters of the modelled cone-mediated ipRGC activity

determined by the post-illumination pupil responses (610 nm stimuli).

............................................................................................................................................... 123

Table 7.3. The mean parameters of the modelled salivary melatonin. ........................ 123

Abbreviations

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Abbreviations

CG ciliary ganglion

COM centre of mass

DLMO dim light melatonin onset

ERG electroretinogram

EW Edinger–Westphal nucleus

FWHM full width-half maximum

GCL ganglion cell layer

INL inner nuclear layer

IPL inner plexiform layer

ipRGC intrinsically photosensitive Retinal Ganglion Cell

IR infra red

LED light emitting diode

LGN lateral geniculate nucleus

ND neutral density

NFL nerve fibre layer

ONL outer nuclear layer

OPL outer plexiform layer

OPN olivary pretectal nucleus

OS outer segment

PghSD Pittsburgh sleep diary

PIPR post-illumination pupil response

PLR pupil light reflex

PSQI Pittsburgh sleep quality index

RPE retinal pigment epithelium

SBCF skewed baseline cosine function

SCG superior cervical ganglion

SCN suprachiasmatic nucleus

UV ultraviolet

SI Units

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SI Units

Angle ° degree

Frequency Hz Hertz

Irradiance W.m-2.s-1 Watt per square metre per second

log photons.cm-2.s-1 log photons per square centimetre per

second

Luminance cd.m-2 candela per square metre

Melatonin level pM picomole per litre

pg.mL-1 picogram per millilitre

(1 pg.ml-1 = 4.31 pM)

Power W watt

Time h hour

Velocity m.s-1 metre per second

mm.s-1 millimetre per second

Voltage V volt

Wavelength nm nanometre

Declaration

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Declaration

The work contained in this thesis has not been previously submitted for a degree or

diploma at this or any other higher education institution. To the best of my

knowledge and belief, the thesis contains no material previously published or

written by another person except where due reference is made.

Student Signature:

Date: 25th February 2011

Acknowledgements

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Acknowledgements

This work was supported by Australian Research Council Discovery Projects ARC-

DP1096354 (AJZ), a QUT Vice Chancellor’s Research Fellowship (BF) and a

Queensland University of Technology Postgraduate Research Award.

I would like to thank my supervisors, Dr Andrew J. Zele, Dr Beatrix Feigl and Dr

Simon Smith for their expertise, advice and the invitation to be part of this

interesting and unique research project.

I am indebted to my participants, all of whom struggled through a long sleepless

night of data collection in the laboratory. Thanks must also go to Rinku Tuli and

Hanna Thrumstom for assistance in testing participants. I enjoyed working with you

both very much, and appreciated your help with those long nights in the laboratory.

My thanks go to Associate Professor Peter Hendicott, Head of the School of

Optometry, for his encouragement, and assistance in facilitating the completion of

this thesis.

I would also like to thank John Stephens, the School of Optometry electronic

technician, for his technical support; Dion Scott, from the University of

Queensland, for his computer programming expertise; Geoff Doyle, from the Prince

Charles Hospital, for his help with actigraphy and the associated software; Diana

Battistutta and Dimitrios Vagenas, from the Research Methods Clinic, for their help

with the statistical analysis; and the Circadian Physiology Group, University of

Adelaide for the melatonin assays.

Finally, I thank my outstanding family and friends for their love, encouragement

and support over the last two years. Thank you for understanding my absences and

pre-occupation during this busy time, and tolerating my sleep deprivation after

overnight data collection. Without you all this thesis would not have been possible.

Chapter 1 Introduction

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Chapter 1.

Introduction

Intrinsically photosensitive Retinal Ganglion Cells (ipRGCs) in the eye project to

the suprachiasmatic nucleus (SCN) (Berson, Dunn & Takao, 2002) which

coordinates a circadian cycle of biological processes (Pickard & Sollars, 2008), and

to the olivary pretectal nucleus (OPN) of the pretectum (Dacey, Liao, Peterson,

Robinson, Smith, Pokorny, Yau & Gamlin, 2005), the start of the pupil reflex

pathway. IpRGCs transmit the environmental light level to the SCN and OPN

(Berson, et al., 2002; Dacey, et al., 2005) and demonstrate long temporal integration

and high efficiency spiking in response to a single photon (Dacey, et al., 2005; Do,

Kang, Xue, Zhong, Liao, Bergles & Yau, 2009). The SCN is the location of the

central circadian clock and controls the release of melatonin from the pineal gland

to drive the sleep/wake cycle (Claustrat, Brun & Chazot, 2005). The inner retina

(ipRGCs) and outer retina (rods and cones) contribute to the pupil light reflex

(PLR) but only ipRGCs control the post-illumination component of the PLR, a

partial pupil constriction maintained for > 30 sec after a stimulus offset (Gamlin,

McDougal, Pokorny, Smith, Yau & Dacey, 2007; Kankipati, Girkin & Gamlin,

2010; Markwell, Feigl & Zele, 2010). Many retinal physiological processes (Boyd

& McLeod, 1964; LaVail, 1976; Anderson, Fisher, Erickson & Tabor, 1980) and

visual functions (Birch, Berson & Sandberg, 1984; Bassi & Powers, 1986;

Sandberg, Pawlyk & Berson, 1986) demonstrate cyclic variation and this includes

ipRGC photopigment mRNA and protein levels (Hannibal, Georg, Hindersson &

Fahrenkrug, 2005; Mathes, Engel, Holthues, Wolloscheck & Spessert, 2007;

González-Menéndez, Contreras, Cernuda-Cernuda & García-Fernández, 2009). It is

unknown if ipRGC activity, determined from the post-illumination pupil response

(PIPR), demonstrates a circadian rhythm.

Research investigating circadian variations in the pupil light reflex has been

inconclusive. The baseline pupil diameter, which is driven primarily by the outer

retina, did not demonstrate circadian variation in one minute recordings, over 24

Introduction Chapter 1

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hours under constant conditions (Loving, Kripke & Glazner, 1996). Wilhelm,

Giedke, Lüdtke, Bittner, Hofmann, and Wilhelm (2001), and Kraemer, Danker-

Hopfe, Dorn, Schmidt, Ehlert, and Herrmann (2000) both demonstrated significant

variation in two hourly baseline pupil diameter recordings (10 - 11 minutes) with

overnight testing. Kraemer, et al., (2000) recorded an evening increase in pupil

diameter which was attributed to a circadian change in the tonic alertness of the

central nervous system. In contrast, Wilhelm, et al., (2001) recorded an evening

pupil diameter decrease, but this decrease did not demonstrate the cyclic variation

of a circadian rhythm. Studies examining circadian variation in pupil constriction

and latency, also driven by the outer retina, are similarly inconclusive (Tiedt, 1963;

Ranzijn & Lack, 1997). These inconclusive results may be attributed to variability

in both pupillometry and circadian experimental design. The studies all used time-

of-day analysis which may mask circadian rhythms due to individual variation in

circadian rhythm phase onset. Instead circadian phase of individual participants

should be aligned using an independent phase marker such as core body

temperature or melatonin levels (Hofstra & de Weerd, 2008). Previous studies have

also used a variety of pupil stimulus wavelengths and irradiances, background

illuminations and durations for pupil recording (Alpern & Campbell, 1962; Barbur,

Harlow & Sahraie, 1992; Loewenfeld, 1999). No research has investigated the

ipRGC driven post-illumination pupil response for circadian variation. (Kraemer, Danker-Hopfe,

Dorn, Schmidt, Ehlert & Herrmann, 2000; Wilhelm, Giedke, Lüdtke, Bittner, Hofmann & Wilhelm, 2001)

This research investigated the cone (outer retina) and ipRGC (inner retina)

contributions to the components of the PLR and their circadian properties. We

investigated the hypothesis that intrinsic ipRGC and cone-mediated ipRGC activity,

determined from the post-illumination pupil response (PIPR), demonstrates

circadian variation under conditions of constant illumination and stimuli. The outer

retina (cone) input to the PLR was also investigated for circadian variability, as

previous research has been inconclusive (Loving, et al., 1996; Kraemer, et al., 2000;

Wilhelm, et al., 2001). Previous research has demonstrated no circadian rhythm in

ipRGC activity in vitro (Weng, Wong & Berson, 2009), and if the ipRGC (inner

retina) post-illumination pupil response displayed circadian variation in vivo this

would demonstrate that ipRGCs are not controlled by a local retinal oscillator, but

instead are under central cortical control. The circadian phase of ipRGC activity can

Chapter 1 Introduction

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be determined relative to the circadian phase of the suprachiasmatic nucleus using

salivary melatonin as the phase marker.

The 24 hour variation in the components of the PLR was compared with the diurnal

variation in salivary melatonin, a direct measure of the central circadian rhythm

(Pandi-Perumal, Smits, Spence, Srinivasan, Cardinali, Lowe & Kayumov, 2007).

Chapter 2 reviews the current research on ipRGCs and leads to the experimental

aims and hypotheses examined in this thesis. Chapter 3 describes the purpose-built

pupillometer, custom software designed to record the pupil light reflex, and a

preliminary experiment conducted to confirm the successful isolation of the cone-

mediated (outer retina) and ipRGC (inner retina) contributions to the pupil light

reflex. Chapter 4 presents the main experiment, conducted to determine the

circadian properties of cone photoreceptor and ipRGC contributions to the PLR.

Chapter 5 discusses future research and implications.

Literature Review Chapter 2

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Chapter 2.

Literature Review

This chapter reviews the current literature on intrinsically photosensitive retinal

ganglion cells and their role in the pupil light reflex and circadian rhythm. This

discussion leads to the experimental aims and hypotheses of the thesis. The chapter

includes sections of text from a published review article included in Appendix 7.1

(Markwell, et al., 2010).

2.1 INTRODUCTION

The discovery of intrinsically photosensitive retinal ganglion cells (ipRGCs)

(Berson, et al., 2002) and their unique photopigment melanopsin (Provencio,

Rodriguez, Jiang, Hayes, Moreira & Rollag, 2000) significantly altered the classical

view of only four types of light sensitive retinal photoreceptors. Substantial

progress has since been made regarding the histological distributions and functional

properties of intrinsically photosensitive ganglion cells in non-primate and primate

eyes. It is established that ipRGCs provide the primary environmental light input to

the suprachiasmatic nucleus (SCN) for photoentrainment of the circadian rhythm

(Ruby, Brennan, Xie, Cao, Franken, Heller & O'Hara, 2002; Gooley, Lu, Fischer &

Saper, 2003; Hattar, Lucas, Mrosovsky, Thompson, Douglas, Hankins, Lem, Biel,

Hofmann, Foster & Yau, 2003). They also contribute to the constriction, recovery

and the post-illumination pupilloconstriction component of the pupil light reflex

(Gamlin, et al., 2007; Kankipati, et al., 2010; Markwell, et al., 2010). In mice

ipRGCs cells contribute to spatial vision, with an acuity of 0.16 cycles.degree-1

recorded in the absence of rods and cones, although this is significantly reduced

from the 0.55 cycles.degree-1 recorded for control mice (Ecker, Dumitrescu, Wong,

Alam, Chen, LeGates, Renna, Prusky, Berson & Hattar, 2010). The role of ipRGCs

in image forming vision is unclear in primates. The temporal properties of ipRGCs

are distinct from rod and cone photoreceptors; the light response of ipRGCs has a

slow onset and sustained depolarization that is maintained for up to 30 seconds after

Chapter 2 Literature Review

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light offset (Dacey, et al., 2005). This post-illumination sustained depolarization is

observed in the pupil reflex after light offset as an unique indicator of ipRGC

function (Gamlin, et al., 2007; Kankipati, et al., 2010; Markwell, et al., 2010) and

has been termed the post-illumination pupil response (PIPR) (Kankipati, et al.,

2010), also called the sustained pupil response. The first part of this review will

discuss the anatomical distribution and electrophysiological properties of

intrinsically photosensitive ganglion cells and compare them with cone and rod

photoreceptors. The review will then examine the role of ipRGC signalling in the

circadian rhythm and the pupil light reflex. The final sections discuss the possibility

of ipRGC activity demonstrating circadian variation, leading to the experimental

aims and hypotheses.

2.2 HISTOLOGY AND ELECTROPHYSIOLOGY OF ipRGCs

Melanopsin is the fifth human retinal photopigment, with the three cone opsins and

the single rod opsin comprising the other four. It was detected in the retinal

ganglion cell layer (GCL) of mice and primates (Provencio, et al., 2000) after its

first discovery in the dermal melanophores of frogs (Provencio, Jiang, De Grip,

Hayes & Rollag, 1998). Several studies have confirmed melanopsin as a retinal

photopigment, in both mammals and humans (Gooley, Lu, Chou, Scammell &

Saper, 2001; Hannibal, Hindersson, Knudsen, Georg & Fahrenkrug, 2002; Hattar,

Liao, Takao, Berson & Yau, 2002; Provencio, Rollag & Castrucci, 2002; Lucas,

Hattar, Takao, Berson, Foster & Yau, 2003; Dacey, et al., 2005). Retinal ganglion

cells encode visual light input as a function of position, wavelength and time, and

project to the visual cortex via the lateral geniculate nucleus (LGN) (Nassi &

Callaway, 2009), as well as projecting to the olivary pretectal nucleus (OPN),

suprachiasmatic nucleus (SCN), the nucleus of the optic tract (NOT), the superior

colliculus (SC), accessory optic system (AOS) and numerous other neural locations

(Hendrickson, Wagoner & Cowan, 1972; Pickard, 1985; Telkes, Distler &

Hoffmann, 2000). Ganglion cells have been classified by soma, dendritic field size

and density (Nassi & Callaway, 2009) into an estimated 20 specialized cell sub-

types (Dacey, Peterson, Robinson & Gamlin, 2003; Dacey, Joo, Peterson & Haun,

2010). Of these, ipRGCs comprise 0.2% of the ~ 1.5 million retinal ganglion cells

in the human retina (Dacey, et al., 2005).


__________________________________________________________________ 6

Figure 2.1. Schematic of the primate retinal layers showing location and synapses of inner and

outer stratifying ipRGCs in primates. (a) Inner stratifying photosensitive ganglion cell bodies

(ipRGCi) are located in the ganglion cell layer (GCL) and their dendrites stratify along the extreme

inner strata (S5) of the inner plexiform layer (IPLi). Outer stratifying photosensitive ganglion cell

bodies (ipRGCo) are located in both the ganglion cell layer (GCL) and the inner nuclear layer (INL)

and their dendrites stratify along the extreme outer strata (S1) of the inner plexiform layer (IPLo).

(b) Cone input is transmitted to ipRGCi via DB6 cone bipolar cells (DB6) (Dacey, et al., 2006; Jusuf,

et al., 2007). Rod input to ipRGCi may be transmitted via rod-cone gap-junctions (GJ) and the DB6

bipolar cells of the cone pathway; rod input along the rod pathway, via ON rod bipolar (RB), AII

amacrine cells (AII) and ON cone (Bon) and OFF cone (Boff) bipolars, is yet to be determined in

primates although synaptic contact has been shown between rod bipolars and ipRGCi in rats

(Ostergaard, et al., 2007). Synaptic contact also occurs between ipRGCo and dopaminergic amacrine

cells (Ad) (Belenky, et al., 2003; Dacey, et al., 2006; Ostergaard, et al., 2007) and bipolar cells (B)

(Jusuf et al. 2007); ipRGCi synapse with unspecified amacrine cells (A) (Belenky, et al., 2003; Jusuf,

et al., 2007; Ostergaard, et al., 2007). Chemical synapses shown as filled circles and electrical

synapses as zigzags. Abbreviations: outer segment (OS), outer nuclear layer (ONL), outer plexiform

layer (OPL) and nerve fibre layer (NFL). Reproduced with permission from Markwell, et al., 2010.


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Table 2.1. Location, distribution and anatomy of primate intrinsically photosensitive retinal

ganglion cells compared with rod and cone photoreceptors. Reproduced with permission from

Markwell, et al., 2010.

ipRGCs Rods L, M and S Cones

Location Inner Retina Outer Retina Outer Retina

Number in Retina ~ 3000 † 92 million ‡ 4.6 million ‡

Peak Cell Density 20 - 25 cells.mm-2 at

2 mm eccentricity from

the fovea †

176 200 cells.mm-2 at ~ 21°

eccentricity ‡

199 000 cells.mm-2 at fovea ‡

(L and M cones)

2600 cells.mm-2 at 0.6°

eccentricity †††† (S cones)

Cells Bodies 40 % INL, 60 % GCL † ONL ONL

Dendrite

Stratification

Extreme outer and

inner IPL †

OPL OPL

Input Intrinsically

photosensitive †

Rod and Cone input †

Intrinsically photosensitive Intrinsically photosensitive

Peak λ Sensitivity 482 nm † 507 nm ¶ 440, 543 and 566 nm at

cornea §§

Photopigment Melanopsin §§§ Rhodopsin ††† Cyanolabe ¶¶¶

Chlorolabe ‡‡‡

and Erythrolabe ‡‡‡

Synapses DB6 Bipolar Cells §

Amacrine Cells §

Rod-Cone gap junctions ††

Rod ON Bipolar cells ¶¶

Cone midget, parasol and

bistratified bipolar cells

Horizontal cells ‡‡

Footnote

† Dacey, et al., (2005) ¶¶ Daw, et al., (1990)

‡ Curcio, et al., (1990) ††† Boll (1877)

§ Jusuf, et al., (2007) ‡‡‡ Rushton (1959)

¶ Crawford (1949) §§§ Provencio, et al., (2000)

†† Schneeweis and Schnapf (1995) ¶¶¶ Marks, et al., (1964)

‡‡ Dacey, et al., (1996) †††† Calkins (2001)

§§ Smith and Pokorny (1975)

Intrinsically photosensitive ganglion cell dendrites branch infrequently along the

inner and outermost edges of the inner plexiform layer (IPL) (Figure 2.1a) to create

an overlapping photoreceptive mesh (Dacey, et al., 2005; Jusuf, Lee, Hannibal &

Grünert, 2007). Although few in number (~ 3000), ipRGCs have the longest

dendrites and largest fields of all known ganglion cells, with diameters of

350 - 1200 µm increasing with retinal eccentricity (Dacey, et al., 2005), as


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compared to midget (~ 4 - 180 µm) (Dacey & Petersen, 1992), small bi-stratified

(~ 30 - 400 µm) (Dacey, 1993) and parasol (~ 20 - 400 µm) (Dacey & Petersen,

1992) ganglion cells. Retinal ganglion cells are absent in the fovea with dendrites

encircling the foveal pit (Dacey, et al., 2005). Like other known ganglion cell types

60 % of ipRGCs have their cell bodies in the ganglion cell layer (GCL) of the inner

retina, however 40 % of ipRGC bodies are located in the inner nuclear layer (INL)

(Dacey, et al., 2005) (Figure 2.1a). A comparison of the anatomy and distribution of

ipRGC, rod and cone photoreceptors is given in Table 2.1.

Intrinsically photosensitive ganglion cells in primates are classified into two

subtypes according to stratification layer (Figure 2.1a). The inner subtype (ipRGCi)

has cell bodies in the GCL and stratifies in the extreme inner IPL (stratum 5),

whereas the outer subtype (ipRGCo) has cell bodies in both the GCL and INL and

stratifies in the extreme outer IPL (stratum 1) (Dacey, et al., 2005; Jusuf, et al.,

2007). The ratio of inner to outer stratifying cells is between 1:1.1 and 1:1.5 in

primates (Dacey, et al., 2005; Dacey, Peterson, Liao & Yau, 2006). Additional inner

stratifying ipRGC subtypes with low melanopsin expression (Ecker, et al., 2010)

and bi-stratifying ipRGCs (Viney, Balint, Hillier, Siegert, Boldogkoi, Enquist,

Meister, Cepko & Roska, 2007; Schmidt, Taniguchi & Kofuji, 2008) have been

identified in mice but not in the primate retina (Dacey, et al., 2005; Jusuf, et al.,

2007).

In addition to their intrinsic response, ipRGCs receive rod and cone input.

Figure 2.1b shows the synapses of inner and outer stratifying ipRGCs with rod and

cone pathways. Inner cells (ipRGCi) contact DB6 bipolar cells in stratum 5 (Dacey,

et al., 2006; Jusuf, et al., 2007) which transmit signals from L, M and S cones (Lee,

Jusuf & Grünert, 2004; Lee & Grünert, 2007). Rod input, which transmits to cones

via gap-junctions (DeVries & Baylor, 1995; Sharpe & Stockman, 1999), may also

pass via the DB6 bipolar of the cone pathway. Inner cells have also been shown to

synapse with amacrine cells (Belenky, Smeraski, Provencio, Sollars & Pickard,

2003; Jusuf, et al., 2007; Ostergaard, Hannibal & Fahrenkrug, 2007) and rod bipolar

cells in rats (Ostergaard, et al., 2007).


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Outer cells (ipRGCo) co-stratify with dopaminergic amacrine cells (Belenky, et al.,

2003; Dacey, et al., 2006; Ostergaard, et al., 2007; Zhang, Wong, Sollars, Berson,

Pickard & McMahon, 2008) and bipolar cells in stratum 1 (Jusuf, et al., 2007). In

mammals ipRGCo and dopaminergic amacrine cells also synapse with bistratified

ON bipolar cells via en passant ribbons (Dumitrescu, Pucci, Wong & Berson, 2009;

Hoshi, Liu, Massey & Mills, 2009). This ipRGCo ON input in the OFF IPL sub

layer has not yet been confirmed in primates. These synapses suggest further

unknown rod and cone pathways to both inner and outer ipRGCs. The receptive

fields of inner and outer stratifying cells overlap, suggesting a difference in roles

that is still to be determined.

Figure 2.2. Intrinsically photosensitive retinal ganglion cell projections to brain locations and

the associated circuits in mice. The ipRGCs and their axons are shown in dark blue and their

principal targets in red. Intrinsically photosensitive ganglion cells project to the suprachiasmatic

nucleus (SCN) for entrainment of the biological circadian rhythm. The SCN regulates the expression

of melatonin from the pineal gland (P), with a sympathetic pathway (orange) synapsing at the

intermediolateral nucleus (IML) and superior cervical ganglion (SCG). Intrinsically photosensitive

ganglion cells also project to the olivary pretectal nucleus (OPN) contributing to both the

sympathetic (not shown) and parasympathetic pupil reflex pathways. The parasympathetic pupil

pathway (light blue) synapses at the Edinger–Westphal nucleus (EW) and the ciliary ganglion (CG)

before reaching the iris muscles (I). The final target of ipRGC projections are two regions of the

lateral geniculate nucleus in the thalamus: the ventral division (LGNv) and the intergeniculate leaflet

(IGL). The LGN processes, integrates and projects to the visual cortex for image formation (pathway

not shown). Reproduced with permission from Berson (2003).


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Intrinsically photosensitive ganglion cells (ipRGCs) give rise to 70 – 90 % of

projections to the suprachiasmatic nucleus (SCN) (Gooley, et al., 2001; Morin,

Blanchard & Provencio, 2003; Sollars, Smeraski, Kaufman, Ogilvie, Provencio &

Pickard, 2003), the location of the circadian biological clock (Inouye & Kawamura,

1979). The majority of SCN projections in mice are from the inner stratifying cell

group (Baver, Pickard, Sollars & Pickard, 2008), but this difference in subgroup

projections has not been demonstrated in primates. If outer and inner stratifying

cells do project to different brain regions this supports a difference in roles for the

cell subgroups. Intrinsically photosensitive retinal ganglion cells also project to the

olivary pretectal nucleus (OPN) of the pretectum, the start of the pupil reflex

pathway (Hattar, et al., 2002; Dacey, et al., 2005; Hattar, Kumar, Park, Tong, Tung,

Yau & Berson, 2006). Intrinsically photosensitive retinal ganglion cells also

synapse in the lateral geniculate nucleus (LGN) of the thalamus (Hattar, et al., 2002;

Dacey, et al., 2005; Hattar, et al., 2006) which relays, integrates and projects visual,

auditory and somoto-sensory information to the cerebral cortex and receives cortical

feedback (Sherman, 2007). In mice ipRGCs additionally project to the superior

colliculus (SC) which contributes to spatial orientation (Hattar, et al., 2006; Ecker,

et al., 2010). The current known ipRGC projections to the SCN, the OPN and the

LGN in mice are displayed in Figure 2.2.

Melanopsin, the ipRGC photopigment, can be fitted with a Vitamin A pigment

nomogram similar to those of rods and cones. The peak sensitivity is 482 nm in

humans (Dacey, et al., 2005; Gamlin, et al., 2007) and 484 nm in rodents (Berson,

et al., 2002; Hattar, et al., 2002), determined both in vitro and in vivo. A second

melanopsin photopigment state with a peak wavelength of 587 nm was recorded in

humans (Mure, Cornut, Rieux, Drouyer, Denis, Gronfier & Cooper, 2009) but has

not been replicated, although such bistable photopigments are known to exist in

invertebrates (Koyanagi, Kubokawa, Tsukamoto, Shichida & Terakita, 2005;

Terakita, Tsukamoto, Koyanagi, Sugahara, Yamashita & Shichida, 2008).

Rod, cone and ipRGC photopigments are isomerised on light absorption, converting

11-cis retinal to all-trans retinal (Lamb & Pugh, 2004; Fu, Zhong, Wang, Luo, Liao,

Maeda, Hattar, Frishman & Yau, 2005; Melyan, Tarttelin, Bellingham, Lucas &


__________________________________________________________________ 11

Hankins, 2005; Qiu, Kumbalasiri, Carlson, Wong, Krishna, Provencio & Berson,

2005; Walker, Brown, Cronin & Robinson, 2008). Rod and cone photopigments

regenerate by binding 11-cis retinal via synthesis in the retinal pigment epithelium

(RPE) to return to the active state. The retinoid processing cycle has been reviewed

in detail elsewhere (Lamb & Pugh, 2004). Recent studies show that Müller cells

also regenerate 11-cis retinal and support the rapid dark adaptation required by the

cones (Mata, Radu, Clemmons & Travis, 2002; Wang & Kefalov, 2009).

Regeneration of the ipRGC photopigment melanopsin is not dependent on the

retinoid processing cycle (Tu, Owens, Anderson, Golczak, Doyle, McCall,

Menaker, Palczewski & Van Gelder, 2006) and may regenerate by a different

mechanism. Some invertebrate opsins and the melanopsin of primitive chordates are

bistable photoisomerases that have an intrinsic light triggered regeneration where

the opsin is isomerised to all-trans retinal with one photon and regenerated to

11-cis retinal with a second photon (Koyanagi, et al., 2005; Terakita, et al., 2008).

Human melanopsin shares a common ancient origin with these bistable invertebrate

opsins (Koyanagi & Terakita, 2008) and early evidence suggests it may also

function as a photoisomerase (Fu, et al., 2005; Melyan, et al., 2005; Panda, Nayak,

Campo, Walker, Hogenesch & Jegla, 2005; Qiu, et al., 2005). The intrinsic photo-

regeneration of melanopsin may be combined with further unknown extrinsic

processes. Müller cells, capable of regenerating 11-cis retinal (Mata, et al., 2002;

Wu, Moiseyev, Chen, Rohrer, Crouch & Ma, 2004), are located adjacent to ipRGCs

and may be a component of the melanopsin pigment cycling mechanism (Lucas,

2006).

Intrinsically photosensitive ganglion cells display both light and dark adaptation,

with response amplitude and latency varying with prior adaptation level (Dacey, et

al., 2005; Wong, Dunn & Berson, 2005; McDougal & Gamlin, 2010). Light

adaptation produces a 0.4 log unit loss of sensitivity with a time constant of ~ 8 sec,

measured using the human pupil light reflex (McDougal & Gamlin, 2010). Dark

adaptation increases the intrinsic sensitivity of rat ipRGCs from

~ 11 log photons.cm-2.s-1 to ~ 9 log photons.cm-2.s-1 with a time constant of

~ 198 minutes (Wong, et al., 2005) and rod input further increases the dynamic

range to ~ 6 log photons.cm-2.s-1 (Dacey, et al., 2005).


__________________________________________________________________ 12

Rods and cones show transient hyperpolarization in response to light (Schneeweis

& Schnapf, 1995) and display photosensitive bleaching and adaptation under

continuous illumination (Hecht, Haig & Chase, 1937). IpRGCs are also intrinsically

photosensitive having a slow onset, sustained depolarization in response to light,

even when detached from the retina (Berson, et al., 2002; Hattar, et al., 2002;

Dacey, et al., 2005). Figure 2.3a shows that the intrinsic response (no rod or cone

input) of a human ipRGC to a 10 second light stimulus (470 nm, 13.3

log photons.cm-2.s-1) has an initial slow onset, with a latency of < 1.78 sec

(McDougal & Gamlin, 2010) followed by sustained depolarization lasting up to 30

seconds after light offset (Dacey, et al., 2005). In comparison, the response of

ipRGCs to rod and cone input (Figures 2.3b and 2.3c), prior to any intrinsic

response, is a rapid onset, transient depolarization with latencies of ~ 150 ms and

~ 30 - 40 ms respectively (Dacey, et al., 2005). Both the intrinsic response

amplitude and time-to-peak of the ipRGC response increase with irradiance

(Berson, et al., 2002; Dacey, et al., 2005); the sustained depolarization (total

number of spikes) is linearly proportional to the retinal irradiance in the photopic

range between 11.5 and 14.7 log photons.cm-2.s-1 (Dacey, et al., 2005; Tu, Zhang,

Demas, Slutsky, Provencio, Holy & Van Gelder, 2005).

Figure 2.3. Intracellular voltage recordings of a human ipRGC in vivo. (a) The slow, sustained

intrinsic photoresponse of the ipRGC in response to a 10 sec, 550 nm, 13.5 log photons.cm-2.s-1 light

pulse under pharmacological blockade of the rod and cone photoreceptors. (b) The rod-mediated

response of the ipRGC to a 10 sec, 550 nm, low scotopic light pulse of 7.6 log photons.cm-2.s-1.

(c) The (L+M) cone ON and S cone OFF isolated responses of the ipRGC. Reproduced with

permission from Dacey, et al., (2005)


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The sustained, linear depolarization of the intrinsic ipRGC response to retinal

irradiance, combined with a latency 100 times slower than with cone input, is

consistent with its intrinsic role for mediating long term steady signalling of

environmental irradiance (Do, et al., 2009). Luxotonic cells in the primate visual

cortex discharge in a sustained, linear response to illumination (Bartlett & Doty,

1974). Because traditional rod and cone image forming pathways encode contrast,

ipRGCs may signal this irradiance input and explain a person’s ability to quantify

brightness in the absence of contrast information, as occurs in a Ganzfeld (Barlow

& Verrillo, 1976). Brightness perception is sustained at short wavelengths near the

ipRGC spectral peak compared to the faster fade-out that occurs with long

wavelength light (Gur, 1989).

Intrinsically photosensitive ganglion cells receive input from rods and cones, via

synapses with amacrine, DB6 and other bipolar cells in the inner plexiform layer

(IPL) (Dacey, et al., 2006; Jusuf, et al., 2007). Rods provide input to ipRGCs along

one of two pathways, depending on the light level (Altimus, Güler, Alam, Arman,

Prusky, Sampath & Hattar, 2010). At high light intensities the rod signal travels via

cones (Altimus, et al., 2010) along gap-junctions between rods and cones

(Figure 2.1b) (DeVries & Baylor, 1995; Sharpe & Stockman, 1999). At low

(scotopic) intensities the rod-ipRGC pathway is via rod bipolar cells in mice

(Altimus, et al., 2010), a pathway yet to be confirmed in primates. Figure 2.3b

shows the rod-mediated sustained ON response of ipRGCs in response to scotopic

stimulation (6 - 7.6 log photons.cm-2.s-1) in the dark-adapted primate retina (Dacey,

et al., 2005). The ipRGC (L+M) cone ON and S cone OFF mediated responses are

shown in Figure 2.3c. The spatially co-extensive S-OFF and (L+M)-ON

components contribute to a colour-opponent receptive field that does not display the

typical surround antagonism common to other retinal ganglion cells (Dacey, et al.,

2005). Intrinsically photosensitive ganglion cells may subserve the S-OFF signal,

which projects to layer 4A of the primary visual cortex (Martin, 2004). Spatial

vision receives a small contribution from ipRGCs in mice (0.16 cycles.degree-1)

(Ecker, et al., 2010), but the role of ipRGCs in primate image formation is not yet

understood .


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2.3 ipRGCs AND THE PUPIL LIGHT REFLEX

The pupil light reflex (PLR) is the constriction and recovery of the pupil in response

to light. In addition to attenuating the retinal illumination, a light responsive pupil

can vary the depth of focus and reduce the visual effects of glare, diffraction and

optical aberrations (McDougal & Gamlin, 2008). A small pupil diameter also

reduces photoreceptor bleaching, allowing faster dark adaptation (Loewenfeld,

1999).

The pupil light reflex is modulated by the autonomic nervous system which

innervates two iris muscles; the sphincter pupillae (parasympathetic innervation), a

smooth muscle ring located around the pupil aperture, and the dilator pupillae

(sympathetic innervation), a thin muscle sheet lying between the iris stroma and the

posterior pigment endothelium, radiating from the sphincter muscle to the ciliary

body (McDougal & Gamlin, 2008). Figure 2.4 overviews the parasympathetic and

sympathetic pupil pathways. Retinal input to the olivary pretectal nucleus (OPN) is

projected to the Edinger-Westphal nucleus (EW) where the parasympathetic

pathway originates. The parasympathetic signal is transmitted via the third cranial

nerve and synapses at the ciliary ganglion (CG) before the postsynaptic short ciliary

nerve innervates the sphincter pupillae muscle (McDougal & Gamlin, 2008). The

sympathetic pathway originates in the intermediolateral columns of the cervical

spinal cord (C8-T1) and synapses at the superior cervical ganglion (SCG) located at

the C2-C3 vertebrae. Post synaptic fibres pass up the neck to the orbit and signals

are primarily transmitted via the long posterior ciliary nerves to the dilator pupillae

muscle in the iris. Other sympathetic fibres may also travel along the short ciliary

nerves (McDougal & Gamlin, 2008). Unlike parasympathetic fibres, sympathetic

fibres do not synapse at the CG (McDougal & Gamlin, 2008).

Pupillary constriction to light occurs when parasympathetic cholinergic stimulation

contracts the sphincter pupillae muscle (Burnstock & Sillito, 1999). At light offset,

pupil dilation occurs via dual pathways; excitation of the α1 adrenergic sympathetic

pathway causes dilation of the dilator pupillae and parasympathetic inhibition of the

EW relaxes the sphincter pupillae (Burnstock & Sillito, 1999). The dual

parasympathetic and sympathetic autonomic innervation creates a balance (tonus) in


__________________________________________________________________ 15

the steady state pupil. Non-photic stimuli can also induce pupil dilation. Noise,

pain, surprise, pleasure and stress cause pupil dilation by increasing the sympathetic

tone of the central autonomic system (Loewenfeld, 1999; Bradley, Miccoli, Escrig

& Lang, 2008; Bär, Schulz, Koschke, Harzendorf, Gayde, Berg, Voss, Yeragani &

Boettger, 2009). Cognitive tasks such as number recall and mental arithmetic also

cause pupil dilation by cortical inhibition of the parasympathetic pathway at the EW

(Hess & Polt, 1964; Granholm, Asarnow, Sarkin & Dykes, 1996; Steinhauer,

Siegle, Condray & Pless, 2004). This dilation increases with the level of demand

(Granholm, et al., 1996; O'Neill & Zimmerman, 2000; Steinhauer, et al., 2004) and

is sustained during continuous cognitive tasks (Beatty, 1982).

Figure 2.4. Anatomical drawing showing the direct and consensual pupillary light reflex

pathways and the parasympathetic and sympathetic innervation of the iris in primates. See text

for details. Reproduced with permission from McDougal and Gamlin (2008).


__________________________________________________________________ 16

Figure 2.5. The consensual pupil light reflex (PLR) of a 30 yo female with 6/5 acuity. The

average initial pupil diameter is indicated by the horizontal black dashed line. Light onset is

indicated by the vertical dashed line, duration by the grey box. Pupil data are represented by the light

and dark grey traces and pupil model data by the blue and red lines. Pupil light reflex components

shown: Baseline pupil diameter (-10 - 0 sec), response latency (0 - 0.3 sec), maximum constriction,

escape (1 - 10 sec) and recovery (10 - 45 sec). (a) Pupil Light Reflex for scotopic

10.1 log photons.cm-2.s-1, 488 nm (blue line) and 610 nm (red line) 10 sec stimuli. (b) Pupil Light

Reflex for photopic (above cone threshold) 12.2 log photons.cm-2.s-1, 488 nm (blue line) and 610 nm

(red line) 10 sec stimuli. (c) Pupil Light Reflex for photopic (above cone and ipRGC threshold)

14.2 log photons.cm-2.s-1, 488 nm (blue line) and 610 nm (red line) 10 sec stimuli. The post-

illumination pupil response of 81 % is shown by the blue dashed line. Data were collected in the

Visual Science and Medical Retina Laboratories, QUT using the pupillometry techniques described

in Chapter 3. Reproduced with permission from Markwell, et al., 2010.


__________________________________________________________________ 17

Figure 2.5 shows the rod, cone and ipRGC contributions to the consensual pupil

light reflex of a healthy, 30 year old observer. The consensual pupil light reflex was

recorded with the techniques described in Chapter 3. Three light stimulus (10 sec)

irradiances were used: 10.1 log photons.cm-2.s-1 (rod only; Figure 2.5a),

12.2 log photons.cm-2.s-1 (rod and cone; Figure 2.5b) and 14.2 log photons.cm-2.s-1

(above the irradiance required for ipRGCs to produce a half-maximal pupil

constriction at 470 nm in primates and humans (Gamlin, et al., 2007);

(Figure 2.5c)). A 488 nm stimulus was used to maximise the intrinsic ipRGC

contribution to the PLR and a 610 nm (control) stimulus was used to minimise it.

The pupil light reflex has four major components; the baseline steady–state pupil

diameter, maximum constriction, escape and recovery. The baseline pupil diameter

is driven by rod, cone and ipRGC signals, with the relative contributions changing

in response to stimulus duration (McDougal & Gamlin, 2010). For light levels

below melanopsin threshold, the steady state pupil diameter is primarily determined

by rod signalling (McDougal & Gamlin, 2010). Above the melanopsin threshold,

ipRGC signals dominate. The cone contribution to the steady state pupil is reduced

after the first 10 seconds and light adaptation limits cone contribution to the

maintenance of steady constant pupil constriction after 30 seconds (McDougal &

Gamlin, 2010).

A light stimulus causes the pupil to rapidly constrict to a minimum diameter

(maximum pupil constriction) which ‘escapes’ to partial constriction during a

prolonged stimulus of several seconds. At light offset the pupil re-dilates returning

to the baseline size. The latency of the initial constriction is the delay in pupil

constriction after light onset. This rapid pupil constriction is driven by rod and cone

input with the latency shortening as light intensity increases up to a minimum

latency of 230 ms (Bergamin & Kardon, 2003; Fotiou, Brozou, Tsiptsios, Fotiou,

Kabitsi, Nakou, Giantselidis & Goula, 2007). The minimum latency is dependent on

the temporal dynamics of cones (30 - 40 ms), rods (~ 150 ms), iris muscle,

innervation pathway and processing (Bergamin & Kardon, 2003; Dacey, et al.,

2005; Fotiou, et al., 2007). Under a low photopic light level (24.6 cd.m-2) a pupil

constriction of 1.6 - 1.9 mm takes ~ 0.73 sec (Bloom, Papakostopoulos, Gogolitsyn,


__________________________________________________________________ 18

Leenderz, Papakostopoulos & Grey, 1993; Piha & Halonen, 1994; Fotiou, et al.,

2007). Intrinsically photosensitive ganglion cells have a latency of 1.78 - 10 sec

(Berson, et al., 2002; Dacey, et al., 2005; McDougal & Gamlin, 2010) and therefore

do not have the temporal dynamics to contribute to the initial constriction.

The maximum pupil constriction varies with stimulus intensity, duration, spectral

composition, retinal size and location (Alpern & Campbell, 1962; Barbur, et al.,

1992; Loewenfeld, 1999). The sensitivity of rods and cones changes with stimulus

wavelength and illumination (Pokorny, Lutze, Cao & Zele, 2006; Cao, Pokorny,

Smith & Zele, 2008), and the Purkinje shift occurs for both the visual system

(Purkinje, 1825) and the pupil light reflex (Loewenfeld, 1999) as light levels change

from photopic to scotopic. Cones are fewer in number (Österberg, 1935) but cover a

broader spectral range compared to rods (Crawford, 1949). Below cone threshold,

greater initial constriction is produced by a short wavelength compared to a long

wavelength stimulus of equal intensity (Kardon, Anderson, Damarjian, Grace,

Stone & Kawasaki, 2009) due to rod sensitivity being higher at shorter wavelengths.

Pupil escape, the re-dilation to a partially constricted state, occurs for light durations

longer than 1 - 2 sec, and is produced by a combination of rod, cone and ipRGC cell

input (McDougal & Gamlin, 2010). This partial constriction may be maintained for

light durations up to 100 seconds (McDougal & Gamlin, 2010).

The ipRGC signal is responsible for the post-illumination pupil response (PIPR)

which occurs in the recovery phase of the pupil light reflex. The PIPR is the

difference between the pupil diameter prior to stimulus onset and after light offset

(Gamlin, et al., 2007; Kankipati, et al., 2010; Markwell, et al., 2010). The PIPR,

also referred to as the sustained pupil response, is characterised by 8 - 10 seconds of

re-dilation after light offset before stabilizing at ~ 1.5 mm less than the baseline

pupil diameter, and is maintained for at least 30 seconds (stimulus: 60º, 470 nm,

13 log photons.cm-2.s-1, 10 sec) (Kankipati, et al., 2010). The PIPR depends on the

intensity and wavelength of the stimulus (Gamlin, et al., 2007) and the baseline

pupil diameter (Kankipati, et al., 2010), but the effect of age, race and gender on

ipRGC function was not determined in a small sample of 37 participants

(26 - 80 yr) (Kankipati, et al., 2010). Further investigations are required to


__________________________________________________________________ 19

determine how age affects ipRGC function. Short wavelength light produces the

greatest PIPR, with a half-maximal pupilloconstriction of 1.5 mm occurring for a

470 nm, 13.6 log photons.cm-2.s-1 stimulus as demonstrated in primates under

pharmacological blockade of the rod and cone photoreceptors (Gamlin, et al.,

2007). An increased irradiance is required to produce an equivalent PIPR at longer

wavelengths (Gamlin, et al., 2007).

The post-illumination pupil response for a single observer is demonstrated in

Figure 2.5c (blue line, dark grey trace) with a PIPR of 1.12 mm (81 % of baseline

pupil diameter) in response to a 7.15º, 488 nm, 14.2 log photons.cm-2.s-1 stimulus.

In contrast, a long wavelength stimulus of the same irradiance (red line)

demonstrates a small PIPR occurring due to cone-mediated ipRGC activity,

returning to within 0.29 mm (~ 95.5 %) of the baseline pupil diameter within 8 - 10

sec after light offset (red line, Figure 2.5c). Cone signals must transmit via the

ipRGCs rather than conventional ganglion cells for the cone-mediated PIPR.

Prolonged pupil constriction after light offset has been confirmed as the unique

result of ipRGC activity (Gamlin et al., 2007).

Figure 2.5b shows that the PIPR is not evident at the lower irradiance

(12.2 log photons.cm-2.s-1) for either the 488 or 610 nm stimuli. The PIPR observed

in Figure 2.5c displays similar temporal properties to the sustained depolarization

seen in vitro, in the macaque and human retina (Figure 2.3a; (Berson, et al., 2002;

Dacey, et al., 2005). It has been confirmed in primates that the ipRGC signal is

responsible for the PIPR after pharmacologically blocking rod and cone signals

(Gamlin, et al., 2007).

2.4 CYCLIC VARIATIONS OF THE RETINA

The circadian rhythm is a cycle of biochemical, physiological and behavioural

processes coordinated by the suprachiasmatic nucleus (SCN) of the anterior

hypothalamus (Pickard & Sollars, 2008). The SCN has an intrinsic rhythm of

23.81 - 24.31 hours (Czeisler, Duffy, Shanahan, Brown, Mitchell, Rimmer, Ronda,

Silva, Allan, Emens, Dijk & Kronauer, 1999; Wyatt, Ritz-De Cecco, Czeisler &

Dijk, 1999; Wright, Hughes, Kronauer, Dijk & Czeisler, 2001) and is synchronized


__________________________________________________________________ 20

to the solar day in a process called photoentrainment. Light is the primary zeitgeber

(time setter), with ipRGCs transmitting encoded environmental light levels (Ruby,

et al., 2002; Hattar, et al., 2003) to the SCN which regulates core body temperature

(Kräuchi, 2002) and the release of melatonin from the pineal gland to drive the

sleep/wake cycle (Benloucif, Burgess, Klerman, Lewy, Middleton, Murphy, Parry

& Revell, 2008).

Figure 2.6. The phase relationship between environmental light, activity, core body

temperature and pineal melatonin secretion. (a) Diurnal variation in environmental light as the

earth rotates on its axis. (b) Activity is greatest in the daytime phase. (c) Pineal melatonin secretion

is regulated by the suprachiasmatic nucleus which is entrained to the light-dark cycle by ipRGCs.

Melatonin levels are lowest during the day, and begin to rise 2 – 3 h prior to habitual sleep time,

shown here modelled with a skewed baseline cosine function (Equation 4.1). (d) Core body

temperature is regulated by the SCN and is lowest in the early morning and peaks in the late

afternoon. Core body temperature can be modelled with a simple cosine function (Benloucif, et al.,

2007). Adapted from Figure 1 in Challet (2007).


__________________________________________________________________ 21

Figure 2.6d displays the circadian variation of core body temperature, which is

lowest in the early morning (~ 5 am) and peaks in the late afternoon (~ 5 pm)

(Hofstra & de Weerd, 2008). Melatonin release begins 2 - 3 hours prior to habitual

sleep time and peaks during the night, before dropping back to daytime levels

within a few hours of waking (Pandi-Perumal, et al., 2007; Benloucif, et al., 2008)

(Figure 2.6c). IpRGC input to the SCN also regulates phase shifting (Ruby, et al.,

2002; Hattar, et al., 2003), where the circadian rhythm is advanced or delayed by

exposure to light, with the degree of phase shifting dependent on the duration,

intensity and wavelength of the light (Skene & Arendt, 2006). Phase shifts are not

linearly responsive to light, instead varying in both magnitude and direction

depending on the circadian phase position of the stimulus (Czeisler, Kronauer,

Allan, Duffy, Jewett, Brown & Ronda, 1989; Khalsa, Jewett, Cajochen & Czeisler,

2003). A light stimulus prior to the minimum core body temperature induces a

phase delay, while a light after causes a phase advance (Khalsa, et al., 2003).

The intrinsic ipRGC photoresponse dominates phase shifting and circadian

entrainment for high irradiance and longer duration stimuli (Gooley, Rajaratnam,

Brainard, Kronauer, Czeisler & Lockley, 2010; Lall, Revell, Momiji, Al Enezi,

Altimus, Güler, Aguilar, Cameron, Allender, Hankins & Lucas, 2010). A 460 nm,

12.64 - 12.82 log photons.cm-2.s-1 full-field ganzfeld stimulus, maintained for

6.5 hours, produces a half-maximal melatonin suppression response maintained for

the duration of the light exposure in humans (Gooley, et al., 2010). Rod and cone

input to ipRGCs also contribute to circadian photoentrainment (Altimus, et al.,

2010; Dollet, Albrecht, Cooper & Dkhissi-Benyahya, 2010; Lall, et al., 2010). The

cone input to ipRGCs is limited by light adaptation, with the greatest cone-mediated

ipRGC driven phase shifts occurring for temporally discontinuous stimuli (a series

of 15 one minute light pulses over 43 min) in mice (Lall, et al., 2010). Humans also

demonstrate a decrease in cone-mediated ipRGC phase-shifting with an increase in

stimulus duration (Gooley, et al., 2010). Photoentrainment for light levels below the

intrinsic ipRGC threshold relies primarily on rod input to ipRGCs (Altimus, et al.,

2010; Lall, et al., 2010). The intrinsic ipRGC response, with some additional rod-

input, dominates photoentrainment and phase-shifting for all light levels above the

ipRGC threshold (~ 12 log photons.cm-2.s-1, 500 nm, in mice) (Lall, et al., 2010).


__________________________________________________________________ 22

Visual functions and performance show circadian variation in sensitivity to

optimize vision for photopic (cone only) and scotopic (rod only) conditions. Visual

luminance sensitivity increases during the night with the greatest scotopic

sensitivity occurring between midnight and 2:30 am in normally entrained

participants (Bassi & Powers, 1986; O'Keefe & Baker, 1987). The cone pathway

shows faster latency (ERG b- and d-wave) in the light phase of the circadian cycle

compared to the dark phase (Hankins, Jones & Ruddock, 1998; Hankins, Jones,

Jenkins & Morland, 2001) and the rod ERG b-wave amplitude decreases early in

the light cycle (Birch, et al., 1984) in humans. Thus rod and cone function vary

diurnally in response to environmental light. The circadian expression of

melanopsin mRNA peaks near dark onset while immunopositive ipRGC cell

numbers peak just before light onset in mice entrained by artificial light in an

animal house (Sakamoto, Liu & Tosini, 2004; Hannibal, et al., 2005; Mathes, et al.,

2007; González-Menéndez, et al., 2009). The circadian rhythms of mRNA and

melanopsin-expressing cell numbers are lost in a continuous dark environment

(Mathes, et al., 2007; González-Menéndez, et al., 2009) demonstrating that

melanopsin production may be driven either directly by environmental light or

involve feedback from the SCN. These circadian variations in the ipRGC

photopigment, melanopsin, suggest the possibility of circadian changes in the

signalling activity of ipRGCs. The rhythm of ipRGC function is unknown and this

research was designed to investigate ipRGC activity for diurnal variation.

Although the SCN is the master synchronizer, peripheral tissues such as the retina,

heart, liver, lungs, pituitary and skeletal muscle show self-sustained circadian

oscillation of clock genes and protein expression when isolated from the SCN

(Yamazaki, Numano, Abe, Hida, Takahashi, Ueda, Block, Sakaki, Menaker & Tei,

2000; Yoo, Yamazaki, Lowrey, Shimomura, Ko, Buhr, Siepka, Hong, Oh, Yoo,

Menaker & Takahashi, 2004; Ruan, Zhang, Zhou, Yamazaki & McMahon, 2006).

The precise location of the retinal oscillator is unknown; clock gene expression has

been demonstrated in rod and cone photoreceptors and the inner nuclear, inner

plexiform and ganglion cells layers, in horizontal, bipolar, amacrine and ganglion

cells (Namihira, Honma, Abe, Masubuchi, Ikeda & Honmaca, 2001; Witkovsky,

Veisenberger, LeSauter, Yan, Johnson, Zhang, McMahon & Silver, 2003; Ruan, et


__________________________________________________________________ 23

al., 2006; Tosini, Davidson, Fukuhara, Kasamatsu & Castanon-Cervantes, 2007).

The retinal oscillator regulates the circadian rhythm of several retinal processes and

functions independently of the SCN in vitro (Tosini, Pozdeyev, Sakamoto &

Iuvone, 2008). For example, the retinal clock controls circadian shedding of rod

outer segments, which peak 1.5 hours into the light cycle (LaVail, 1976; LaVail,

1980; Terman, Remé & Terman, 1993). The retinal clock also regulates the nightly

synthesis of melatonin in the outer photoreceptors, the inner nuclear layer and the

ganglion cell layer (reviewed by (Iuvone, Tosini, Pozdeyev, Haque, Klein &

Chaurasia, 2005)) which can also be photoentrained in vitro in retinal tissue (Tosini

& Menaker, 1996; Tosini & Menaker, 1998). Many other retinal processes such as

dopamine synthesis and cone outer segment shedding also show a cyclic variation

but it is not yet known if these are also regulated by a local retinal clock (Young,

1978; Nir, Haque & Iuvone, 2000; Doyle, Grace, McIvor & Menaker, 2002). The

effects of the central circadian rhythm and the local retinal rhythm on ipRGC

function are not yet fully understood.

2.4.1 Circadian variation in the PLR

Evidence of circadian rhythm in the PLR parameters, baseline pupil, maximum

constriction, escape and recovery, is inconclusive. One study recorded an evening

increase in baseline pupil diameter (Kraemer, et al., 2000), another demonstrated an

evening decrease in diameter (Wilhelm, et al., 2001), while a third demonstrated no

significant changes over 27 hours (Loving, et al., 1996). Evidence of circadian

variations in pupil constriction and latency, driven by the rod and cone

photoreceptors (outer retina) are similarly inconclusive (Tiedt, 1963; Fosnaugh,

Bunker & Pickworth, 1992; Ranzijn & Lack, 1997). No research has examined the

post-illumination pupil response over 24 hours for circadian changes. Sleepiness

can also cause a decrease in pupil diameter and an increase in pupil fluctuations

(Lowenstein & Loewenfeld, 1964; Ranzijn & Lack, 1997; Wilhelm, Wilhelm,

Lüdtke, Streicher & Adler, 1998; Kraemer, et al., 2000) and studies must be

designed to reduce fatigue to unmask any PLR circadian variation. The inconsistent

results of previous circadian PLR studies may be the result of differences in both

circadian and pupillometry methodology. For circadian variation to be unmasked

the external circadian cues of activity, sleep, posture, caffeine, ambient temperature


__________________________________________________________________ 24

and caloric intake must be controlled in a constant routine protocol (Duffy & Dijk,

2002). No previous study of PLR circadian variation has aligned the circadian phase

of individual participants using an independent phase marker such as core body

temperature or melatonin levels (Kräuchi, 2002; Claustrat, et al., 2005), instead

using time-of-day to analyse results. Time-of day analysis can mask circadian

variation by not considering the variability in circadian phase which may exist

between participants. Variation in the pupillometry techniques used include

differences in the pupil stimulus wavelength and irradiance, the background

illumination and the duration of pupil recording, all of which vary the PLR (Alpern

& Campbell, 1962; Barbur, et al., 1992; Loewenfeld, 1999).

2.5 EXPERIMENTAL AIMS AND HYPOTHESES

Intrinsically photosensitive retinal ganglion cells provide the primary photic input to

the SCN, which demonstrates a temporal variation in oscillation (Jagota, de la

Iglesia & Schwartz, 2000), and controls circadian photoentrainment and phase

shifting (Pickard & Sollars, 2008). The spectral sensitivity of the retinal circadian

phototransduction system, determined by melatonin suppression, varies with time of

day (Figueiro, Bullough, Parsons & Rea, 2005). Circadian variation in ipRGC

sensitivity may contribute to this circadian change in the spectral sensitivity of

melatonin suppression, especially since ipRGCs display circadian rhythmicity in

melanopsin mRNA and protein expression (Sakamoto, et al., 2004; Hannibal, et al.,

2005; Mathes, et al., 2007; González-Menéndez, et al., 2009) and it has previously

been demonstrated that the absolute visual sensitivity changes with time of day

(Bassi & Powers, 1986; Tassi & Pins, 1997). A moderate evening increase of in

vitro ipRGC spiking for a constant stimuli, but no significant circadian rhythm, was

recently demonstrated in rats (Weng, et al., 2009). The central finding of Weng, et

al., (2009) is the absence of cell-autonomous circadian modulation in in vitro rat

ipRGC sensitivity, but this does not preclude in vivo SCN controlled ipRGC

circadian variation. The post-illumination pupil response (PIPR), as a direct

measure of ipRGC activity, may be used to determine if a circadian variation in

ipRGC sensitivity occurs in vivo in humans. If established, the presence of circadian

variation in ipRGC sensitivity under constant illumination conditions would provide

a greater understanding of the extrinsic circadian control of retinal function and the


__________________________________________________________________ 25

relationship between environmental light signals and central biological clock

function.

AIM 1: To isolate the cone-mediated (outer retinal) and intrinsic ipRGC (inner

retinal) contributions to the pupil light reflex (PLR).

HYPOTHESIS 1: Direct cone, cone-mediated ipRGC and intrinsic ipRGC

contributions to the PLR can be isolated with the custom-built experimental

apparatus under our laboratory conditions, using stimuli with appropriately chosen

wavelengths and irradiance.

AIM 2a: To determine if the direct cone photoreceptor, intrinsic ipRGC and/or

cone-mediated input to the ipRGCs demonstrate circadian variation over a 24 hour

period in their contributions to the pupil light reflex.

HYPOTHESIS 2a: The intrinsic ipRGC and the cone-mediated photoreceptor

contributions to the ipRGC driven post-illumination pupil response will

demonstrate a circadian rhythm but direct cone inputs to the maximum pupil

constriction will not.

AIM 2b: To determine the temporal synchrony, where synchrony is defined as

demonstrating a temporal relationship, of cone and/or ipRGC diurnal variation to

the central circadian rhythm as measured using melatonin.

HYPOTHESIS 2b: IpRGC activity as measured via the post-illumination pupil

response will be temporally synchronized with the central circadian rhythm as

measured via salivary melatonin.

Experimental Methods Chapter 3

__________________________________________________________________ 26

Chapter 3.

Experimental Methods

This chapter describes the design and calibration of a pupillometer, custom

designed Matlab software and the analysis and modelling of the pupil light reflex.

An experiment was conducted to determine the spectral sensitivity of the post-

illumination pupil response, confirming the successful isolation of ipRGC activity

under the experimental conditions. These experimental methods are then used for

the main experiment described in Chapter 4.

3.1 PARTICIPANTS

Participants with normal colour vision, visual acuity (≥ 6/6) and no medical, ocular

or sleep disorders were recruited in accordance with the Institutional Ethics

Requirements and the tenets of the Declaration of Helsinki

(Ethics No: 0800000546). Written informed consent was obtained after the purpose

and possible risks of the experiment were explained.

A comprehensive visual screening was conducted by an optometrist and included

visual acuity, subjective refraction, contrast sensitivity, colour vision, stereo acuity,

intraocular pressure, eye colour and an internal ocular examination. Lenses were

graded for cortical, nuclear and posterior subcapsular cataract (Age-Related Eye

Disease Study Research Group, 2001). Table 3.1 shows the ocular screening

protocol and inclusion criteria for participants. Participants were all non-smokers

and moderate caffeine consumers (< 4 beverages/day). All participants were

uncorrected during the experiment and viewed the 4° black fixation cross with the

left eye. The right eye was dilated with cyclopentolate 1 % and subjective

accommodation was assessed hourly using an optometer (Hartinger, Rodenstock).

Cyclopentolate 1% was chosen for its longer duration mydriatic action compared to

1% Tropicamide, which required less re-instillation over the 24 hour testing period.

Chapter 3 Experimental Methods

__________________________________________________________________ 27

Table 3.1. Ocular screening protocol and inclusion criteria for all participants.

Technique / Equipment Criteria

Visual Acuity Bailey Lovie chart ≥ 6/6

Colour Vision

HRR Pseudoisochromatic Plate Test

and Lanthony Desaturated Panel D-15

No abnormality

Stereo Acuity Stereo Fly Test < 60”

Contrast Sensitivity Pelli-Robson chart ≥ 1.75

IOP I-care tonometer ≤ 21 mmHg

Ophthalmoscopy Ophthalmoscopy and Fundus

photography

No retinal or optic nerve

disease

Lens Health

Nikon photo slit lamp

No cataract, Grade ≤ 1

(AREDS, 2001)

3.2 PUPILLOMETER APPARATUS

The pupillometer presented a stimulus light in Maxwellian view to the right eye and

recorded the consensual pupil response of the left eye. The apparatus, shown in

Figure 3.1 was mounted on an optical breadboard (ThorLabs, 750 x 750 x 60 mm)

supported on a rigid breadboard support frame. The 500 W, 240 V tungsten halogen

lamp stimulus light (Section 3.4.1) was shielded by PVC pipe (37 cm x Ф 15.2 cm)

and cooled by a rotary fan (10 cm2, 50 Hz / 240 V). The stimulus passed through

four achromatic lens doublets Ф 50 mm, FL 100, FL 200, FL 100, FL 200 (Techspec,

Edmund Optics) and two Ф 25 mm diaphragms, and was reflected by two 45° silver

surfaced mirrors (50 mm2) to subtend a 7.15o visual angle at the right eye. The

positions of the narrow band interference filters (Edmund Optics, 50.8 mm2)

(Section 3.4.3) and reflective neutral density filters (Ealing Catalog Inc, Rocklin,

CA, USA) are shown in Figure 3.1 (shown at IF, ND). Calibrations of the optical

components are described in Section 3.4.

The left eye fixated a 4° black cross on a 6.3° x 8.9° white rear projection screen at

1.15 m (12.7 x 17.8 cm, Da-Lite, DA-100) backlit to 116 cd.m-2 by a Luxeon V

LED (Lambertian Star, 700 mA) through a hot mirror (45° incident angle, 50 mm2,

Edmund Optics) positioned at a 45° angle in front of the eye. The left eye was

illuminated by a ring of six infrared LEDs (Section 3.4.2) positioned 4 - 7 cm from


__________________________________________________________________ 28

Figure 3.1. Schematic plan view of the pupillometer. Scale 1:5.56. Irradiance from a

500 W, 240 V tungsten halogen lamp (TH) passes through four achromatic lens doublets Ф 50 mm

with focal lengths of 100 and 200 mm (FL100, FL200), two Ф 25 mm iris diaphragms (D), two 4°

black fixation crosses printed on UV filters (UV) and two 45° silver surfaced mirrors (50 mm2) (M)

and is presented in Maxwellian view to the right eye location (RE). Narrow band interference

(50.8 mm2) (IF) and reflective neutral density filters (50.8 mm2) (ND) attenuate irradiance. The left

eye (LE) views a 6.3° x 8.9° white rear projection screen (S) with a 4° black fixation cross (F)

through a 45° incident angle hot mirror (50 mm2) (HM) while illuminated by a ring of six infrared

LEDs (IR). The left eye is recorded with an infrared Pixelink camera with telecentric lens (C).


__________________________________________________________________ 29

the camera. The right eye viewed two 4° black crosses printed on UV filters

(Gelatin, Lee Filters), incorporated in the pupillometer at positions shown in

Figure 3.1. The participant’s head was positioned so the two crosses, visible only to

the right eye, were fused. To align the two eyes, the cross visible to the left eye was

slid horizontally until it fused with the crosses viewed by the right eye; when

aligned the participant perceived one fused black cross. Temple bars, head restraint

and chin rest stabilized the participant’s head in position in the pupillometer.

This method of recording the pupil light reflex of the contralateral eye to a direct

light stimulus was previously used to measure the post-illumination pupil response

(Gamlin, et al., 2007; Kankipati, et al., 2010). The irradiance exposure was

maximised and quantified by the use of mydriatic eyedrops in the stimulated eye.

Monocular eye drops can reach the retina and aqueous humour of the contralateral

eye (Koevary, 2003; Patsiopoulos, Lam, Lake & Koevary, 2003) but evidence of a

mydriatic effect in the contralateral, untreated eye is inconclusive (Lahdes,

Huupponen & Kaila, 1994; Patsiopoulos, et al., 2003). In this study any possible

contralateral effect of cyclopentolate would have a constant effect on the recorded

PLR throughout the testing period.

A Pixelink camera (IEEE-1394, PL-A741 FireWire) with a telecentric lens

(Computar 2/3" 55 mm Telecentric Lens and 2X Extender C-Mount) was positioned

to view the left eye reflected by the hot mirror, while the participant simultaneously

fixated on the cross directly ahead. The camera was set to an ISO Speed of 400 mb,

image size of 640 x 480 pixels and a frame rate of 62 frames.sec-1. The program

automatically controlled exposure optimising the shutter speed and gain. The

researcher viewed a live feed on a computer screen shielded by a 0.9 ND filter (Lee

Filters) to attenuate the screen luminance during pupillometry. Custom designed

Matlab software controlled the temporal properties of the stimulus, recording time

and camera settings. The software, in conjunction with Active DCam camera

software (A&B software, New London, USA), started and terminated the camera

recording.


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3.3 DETERMINATION OF THE OPTIMAL VIEWING DISTANCE

FOR THE PUPILLOMETRIC MEASUREMENTS

3.3.1 Introduction

During the pupillometric recordings, the participants are required to maintain

accommodation and convergence for up to one minute. This pilot study was

conducted to determine the optimum fixation distance to minimise pupil

fluctuations due to accommodation and convergence. An increase in

accommodative stimulus decreases pupil diameter, increases convergence but does

not increase pupil fluctuation for alternating near and far accommodative demand

(Hunter, Milton, Lüdtke, Wilhelm & Wilhelm, 2000). No previous research has

investigated the effect on pupil fluctuations of maintained rather than varied

accommodative demand.

3.3.2 Experimental Methods

The 4° fixation cross was positioned at three distances of 0.57 m, 1.15 m and

1.43 m, producing accommodative demands of 1.75 D, 0.87 D and 0.70 D

respectively. The fixation screen luminance was constant (116 cd.m-2). One 30 year

old participant, with a right pupil dilated with 1 % cyclopentolate, viewed the

fixation cross with the left eye and pupil diameter was continuously recorded for 30

seconds. Five recordings were averaged for each fixation distance.

3.3.3 Results and Discussion

Figure 3.2 shows the baseline pupil diameter decreased for an accommodative

demand of 1.75 D (blue trace), compared to 0.70 D and 0.87 D (black and red

traces). The decrease in pupil diameter with decreasing fixation distance can be

explained by an increase in convergence for closer fixation targets (Hunter, et al.,

2000). Pupil fluctuations also increased with accommodative demand (blue trace,

Figure 3.2) with greatest fluctuations from the average pupil diameter occurring for

the 1.75 D target (Table 3.2). The results for this participant indicate that

accommodation may contribute to pupil fluctuations, with standard deviations of

0.25 mm, 0.26 mm and 0.52 mm respectively for 0.7 D, 0.87 D and 1.75 D


__________________________________________________________________ 31

accommodative demands (Table 3.2), a result which has been previously

unreported. The fixation demand of 1.15 m (0.87 D) was selected for the

pupillometer design because the pupil diameter was larger than at 0.57 m and the

1.43 m fixation distance made only 0.09 mm difference in maximum fluctuations or

the pupil diameter variability. Table 3.2 gives the baseline pupil diameters (mean ±

SD) and maximum deviations from mean diameter (mean ± SD) for the three

accommodative demands.

Table 3.2. Baseline pupil diameter measured at three fixation distances.

Fixation Target

Distance (m)

Accommodative

Demand (D)

Mean Baseline Pupil

Diameter (mm)

Maximum deviation from

mean pupil diameter (mm)

1.43 0.70 6.92 ± 0.25 0.28 ± 0.11

1.15 0.87 6.66 ± 0.26 0.37 ± 0.15

0.57 1.75 5.88 ± 0.52 0.66 ± 0.24

Figure 3.2. Pre-stimulus pupil fluctuations for three fixation accommodative demands. The

traces show baseline pupil diameter (mm) with accommodative demands 0.7 D (1.43 m; black),

0.87 D (1.15 m; red) and 1.75 D (0.57 m; blue). Pupil diameter decreased as convergence increased.

3.4 PUPILLOMETER CALIBRATIONS

This section discusses the calibrations of components of the pupillometer described

in Section 3.2 and calculations of photon irradiance. These components include the

tungsten halogen stimulus light, infrared LEDs, narrow band interference filters,

neutral density filters and the Luxeon LED.


__________________________________________________________________ 32

3.4.1 Tungsten Halogen Stimulus Light

Figure 3.3 shows the spectral distribution of the tungsten halogen lamp (500 W,

240 V), measured at the eye position in the pupillometer. Twenty irradiance

measurements, each integrated over 10 seconds, were taken using a fibre optic

spectrometer with a cosine receptor (StellarNet, Florida, USA). The mean

irradiance of the tungsten halogen lamp was 373.31 ± 3.18 W.m-2.s-1.

Figure 3.3. The normalised spectral distribution of the 500 W, 240 V tungsten halogen lamp.

3.4.2 IR LEDs

A ring (diameter 4 cm) of six infra red LEDs (Thorlabs) illuminated the left eye for

camera recording of the pupil light reflex. Three calibration measurements recorded

every 15 – 30 minutes over a two hour period showed a stable peak wavelength of

863.17 ± 0.75 nm (Figure 3.4) and an irradiance of 70.83 ± 4.44 W.m-2.s-1. Values

above 900 nm were outside the spectroradiometer range.

Due to variations in pupillary distance and eye socket anatomy between

participants, the LEDs were repositioned to produce even illumination for the

camera image for each participant. Depending on the location of the LEDs with

respect to the eye (4 - 7cm), the recorded irradiance varied between 70.83 and

77.7 W.m-2.s-1. The recorded irradiance did not follow the Inverse Square Law due

to the angling of the multiple LEDs. The IR illumination was constant during all

measurements for a single observer.


__________________________________________________________________ 33

Figure 3.4. The spectral distribution of the IR LEDs.

3.4.3 Narrow Band Interference Filters

Interference filters (Edmund Optics, 50.8 mm2) were used to produce narrow band

illumination. These filters have a manufacturer specified central wavelength

tolerance of 2 nm and a full width-half maximum (FWHM) of 10 ± 2 nm with

measured values shown in Figure 3.5. Pilot measurements showed a FWHM

increase of 2.38 ± 0.38 nm with a filter tilt of 20°. Care was taken in all experiments

to position the filters to prevent the effect of tilt.

Figure 3.5. The spectral transmission of narrow band interference filters measured through

the pupillometer. Filter λmax and (FWHM): 431.8 ± 0.7 nm (10.5); 450.4 ± 0.2 nm (11.7);

469.4 ± 0.3 nm (12.2); 487.2 nm (11.8); 509.1 nm (12.1); 532.9 (11.6); 549.8 (11.90); 567.7 (12.0);

589.6 (11.1); 610.2 ± 0.42 (9.8). The dashed line represents the approximate position of where the

spectroradiometer approached the lower limits of its dynamic range for the interference filter and

light source combination.


__________________________________________________________________ 34

3.4.4 Neutral Density Filters

Table 3.3 shows the measured optical density of the reflective neutral density filters

(50.8 mm2, Ealing Catalog Inc, Rocklin, CA, USA) used to attenuate the irradiance

of the pupillometer stimulus light.

Table 3.3. The calibrated optical density measurements of the reflective neutral density filters.

Theoretical Optical Density Measured Optical Density Log10 (Io/It)

0.1 0.113

0.3 0.301

0.5 0.487

0.6 0.620

1.0 1.004

2.0 1.732

3.0 2.856

3.4.5 Luxeon LED

Figure 3.6 shows the spectral distribution of a white Luxeon LED (CIE x, y: 0.340,

0.372; Dominant λ: 563 nm) positioned 26 cm behind a rear projection screen

(Section 3.2) to illuminate the fixation target viewed by the left eye. The LED was

powered by a 0.35 A, 10.3 V voltage regulated power supply. The photopic

luminance of the fixation screen was 116 cd.m-2.

Figure 3.6. The normalized spectral distribution of the white Luxeon LED.


__________________________________________________________________ 35

3.4.6 Photon Calculations

The pupillometer (Figure 3.1) was designed to produce a minimum irradiance of

13.6 log photons.cm-2.s-1 at 488 nm at the plane of the eye, the irradiance required

to produce a half-maximal post-illumination pupil response (PIPR) at a wavelength

of 493 nm (Gamlin, et al., 2007). Photon irradiance was calculated from irradiance

measurements taken at the eye position using a fibre optic spectrometer with a

cosine receptor (StellarNet, Florida, USA). Three measurements were taken for

each narrow band interference filter on three separate days, randomised for filter

sequence. Irradiance values (W.m-2) were converted to photon irradiance

(photons.m-2.s-1) according to the following calculation.

The relationship between photon flux (Φ) and radiant power (φ) is expressed by

Φ = φ * λ/hc (Equation 3.1)

where λ is wavelength in metres, h is Planks constant (6.626 x 10-34) and c is the

speed of light (2.998 x 108 m.s-1). For a point source, photon flux (photons.s-1) and

radiant power (W) are related to irradiance (W.m-2) and photon irradiance

(photons.m-2.s-1) by

φ = ER * A and

Φ = EP * A

where ER is irradiance, EP is photon irradiance and A is the area of the receiver. By

substitution, irradiance (W.m-2) is converted to photon irradiance (photons.m-2.s-1)

where

ER = EP * λ/hc hc (Equation 3.2)

Pupillometer photon irradiance output was calculated for all the narrow band

interference filters (Section 3.4.3) and the results are presented in Table 3.4. Neutral

density filters (Section 3.4.4) were used to attenuate photon irradiance to the

required level.

Luminance (cd.m-2) values were calculated from irradiance (W.m-2) according to

the following calculation

HI = HJ K L(M) K 683 (Equation 3.3)


__________________________________________________________________ 36

where ER is irradiance, EL is luminance and V(λ) is the spectral luminous efficiency

function.

Table 3.4. Photon and candela irradiance of the pupillometer for each narrow band

interference filter.

Interference Filter (nm) Pupillometer Irradiance at the Eye

log photons.cm-2.s-1 cd.m-2

430 13.37 ± 0.05 0.86

450 13.70 ± 0.10 5.78

470 13.99 ± 0.05 26.87

488 14.22 ± 0.04 90.03

510 14.35 ± 0.06 309.68

532 14.46 ± 0.05 682.72

550 14.59 ± 0.07 920.10

568 14.77 ± 0.05 1361.37

589 14.78 ± 0.07 988.77

610 14.91 ± 0.12 1048.43

Note: Irradiance (W.m-2) values measured using a fibre optic spectrometer with a cosine receptor at

the cornea, using the pupillometer described in Section 3.2. Values were converted to log

photons.cm-2.s-1 and cd.m-2 using Equations 3.2 and 3.3.

3.5 DATA ANALYSIS OF PUPILLOMETRY RECORDINGS

The infrared pupillometer recordings were analysed for pupil diameter using custom

designed Matlab analysis software and the pupil light reflex data were fitted with a

simple linear and exponential model so that individual components could be

analysed statistically.

3.5.1 Pupil Diameter Analysis Software

The pupil diameter was determined post-hoc using custom designed Matlab analysis

software of the greyscale video images. The pupil margin was extracted from the

iris by reducing the image size by a factor of four (to reduce computer processing

time) and sharpening the pupil boundary to improve edge detection. The

complement image was next determined by reversing the black and white 256 grey

scale image.


__________________________________________________________________ 37

A centre of mass (COM) was located in the complement image to give an initial

reference point within the pupil area according to

N. O. P. Q B RST9UVKTWRRV:RST XYWZZ TWRR (Equation 3.4)

and N. O. P. [ B RST9\VKTWRRV:

RST XYWZZ TWRR (Equation 3.5)

where i B 1:N (where N is the total number of pixels in the image).

Before proceeding, the software confirmed that the COM value lay within an

assigned central maximum area chosen to minimise eyelid interference. A COM

value outside this area could be caused by artefacts such as the detection of

eyelashes and these frames were excluded from the analysis.

Next a “seed point” location was detected using a weighting scheme. The weight of

each pixel was calculated by multiplying the distance from the COM with the grey

scale value according to

^_`abcdeUfZ B g`hc ci NOPdeUfZ K ajk[hlkm_ nkmo_deUfZ , (Equation 3.6)

The seed point was the pixel with the smallest calculated weight. Pixels outside a

minimum central area, chosen by experimentation, had their weight increased by a

factor of three,

^_`abcdeUfZ B g`hc ci NOPdeUfZ K ajk[hlkm_ nkmo_deUfZ K p (Equation 3.7)

to prevent the seed point being located in the peripheral eyelashes which was

another dark region within the image.

The seed point was then the starting location for the flood fill algorithm which

segmented the pupil area from the remainder of the image. Starting from the seed

point, all connected pixels with a gray scale value within a specified range of the

seed point value were detected. The flood fill area expanded until no further

connected pixels met the gray scale criteria.. All flood fill points were assigned as

black and allocated a value of zero to approximate the pupil area. The outer margin

of this approximate pupil area was determined using a convex hull algorithm which

connected all the edge pixels.


__________________________________________________________________ 38

To estimate pupil area an ellipse was fitted to the pupil margin using least square

regression. The conic equation of an ellipse is

Hmm`qh_ = k K Qr s t K Q K [ s l K [r s g K Q s _ K [ s u (Equation 3.8)

where a, b, c, d, e and f are constants and (x, y) are the coordinates of a single point

on the ellipse. The orientation of the ellipse (Φ) was removed by substituting

Q = vQ s w[

and

[ = (xwQ s v[)

to get the conic representation for a non-tilted ellipse

Hmm`qh_ = k(vQ s w[)r s t(vQ s w[)(xwQ s v[) s l(xwQ s v[)r s g(vQ s w[) s

_(xwQ s v[) s u (Equation 3.9)

where m = cos(Φ) and n = sin (Φ) are constants and Φ is the ellipse rotation.

Since b = 0, the orientation (Φ) was calculated by

2kvw s 9vr x wr:t x 2lvw B 0 (Equation 3.10)

and y B z

r K kckw 9 {|}W: (Equation 3.3)

The representation of the non-tilted ellipse is now

Hmm`qh_ B ~U}��W �

rs ~\}��

{ �r, (Equation 3.4)

where x and y are the major and minor axes (in pixels), (XO,YO) is the centre of the

ellipse and Φ is the ellipse orientation. Values of x and y axes were then multiplied

by four to correct for the earlier image rescaling. Pixels were converted into

millimetres using the pixel:mm ratio determined by imaging a 10 x 10 mm

calibration grid located at the plane of the eye. The pupil diameter was taken as the

mean value of the longest and shortest axes of the ellipse and these values were

exported to a data file for analysis.

Each video had 3300 - 3500 frames recorded at 62 frames per second, but 50 - 100

frames were excluded during software analysis of each video file. Reasons for

frame drop included blink artefacts, a COM outside the central maximum area, the

ellipse axes not within 40 - 250 pixels of each other, insufficient pupil edge pixels

to fit an ellipse, or if a parabola or hyperbola was fitted instead of an ellipse.


__________________________________________________________________ 39

Overall, only a small proportion of frames (1 - 3 %) were dropped and each

accounted for only 16 ms of the 55 second measuring time. Therefore the 62 Hz

frame rate of the camera provided sufficient resolution to record all the dynamics of

the pupil light reflex.

3.5.2 Analysing the Pupil Light Reflex

The pupil light reflex was divided into the four components of baseline pupil

diameter (pre-stimulus), constriction velocity (maximum constriction), maintained

constriction and post-illumination pupil response, for statistical analysis

(Figure 3.7). The PLR was fitted with a simple linear and exponential model, by

floating all parameters and minimizing the sum of squares differences between the

data and the model parameters with an Excel solver Add-in. Diurnal variation in the

experimental parameters of baseline pupil diameter (mm) (pre-stimulus),

constriction velocity (mm.s-1), maximum amplitude of constriction (mm), rate of

pupil escape (mm.s-1), re-dilation velocity (mm.s-1) and PIPR plateau (mm) were

examined experimentally.

The first component, the baseline pupil diameter, is the diameter until stimulus

onset. This was fitted with a straight line with a slope of zero, to give a mean

baseline diameter value of

[ =∑ dSdeZ �eWTf�f��

��ST{f� XY �f�TR , (Equation 3.5)

where t is the time of light onset.

The second component is the pupil constriction velocity, from light onset to the

maximum pupil constriction. This was fitted with a straight line

[ B vQ s l, (Equation 3.6) where [ is pupil diameter in mm, Q is time in seconds, l is a constant and v is the

constriction velocity in mm.s-1. The amplitude of total constriction was calculated

as the difference between the baseline pupil diameter and the maximum pupil

constriction.


__________________________________________________________________ 40

The third component of the pupil reflex, the maintained constriction, is the time

between the maximum pupil constriction and light offset. This component was

fitted with a straight line

[ = vQ s l, (Equation 3.7) where [ is pupil diameter in mm, Q is time in seconds, l is a constriction constant

and v is the rate of pupil escape in mm.s-1.

Figure 3.7. Linear and exponential model of the pupil light reflex for a 10 second,

14.2 log photons.cm-2

.s-1

, 488 nm stimulus (30 yo female). Light duration is indicated by the blue

bar. The PLR data (grey trace) is fitted with linear and exponential functions (black line). The

modelled PLR components (blue lines) are: (a) The baseline pupil diameter fitted with the linear

function y B x s 6.20, (b) the amplitude of constriction fitted with the linear function

y B -4.09 * x s 47.21, (c) the stimulus constriction fitted with the linear function

y B -0.002 * x s 2.63 and (d) the post-illumination pupil response fitted with an exponential

function y B -1.16*106 * exp9-0.67 * x: s 4.42.

The fourth component is the pupil reflex after light offset, where the pupil partially

re-dilates and plateaus in a post-illumination pupil response which is dependent on


__________________________________________________________________ 41

the wavelength and intensity of the light (Gamlin, et al., 2007). This component was

fitted with an exponential function

[ = � K _Qq(� K Q) s J, (Equation 3.8)

where � is a constant, � is the re-dilation velocity in mm.s-1and J is the plateau

pupil diameter (mm) of the PIPR.

3.6 DETERMINATION OF THE SPECTRAL SENSITIVITY OF THE

POST-ILLUMINATION PUPIL RESPONSE

3.6.1 Introduction

The ipRGC melanopsin photopigment has a peak spectral sensitivity of 482 nm,

recorded in vitro and in vivo in primates, when fitted with a Vitamin A1 pigment

nomogram (Dacey, et al., 2005; Gamlin, et al., 2007). It has been confirmed in

primates that the ipRGC signal is responsible for the PIPR after pharmacologically

blocking rod and cone signals (Gamlin, et al., 2007). Using the pupillometer and

experimental conditions described earlier in this chapter, the following experiment

aimed to confirm isolation of the ipRGC signal via the post-illumination pupil

response by measuring its spectral sensitivity.

3.6.2 Experimental Methods

Two participants (25 yo F and 30 yo F) with normal visual acuity (≥ 6/6) and who

met the participant inclusion criteria in Table 3.1 provided written informed

consent. The pupillometer (Section 3.2) was used to record the pupil light reflex

while participants viewed the 116 cd.m-2 backlit rear projection screen. The PLR

was recorded for 55 seconds consisting of 10 seconds pre-stimulus (adaptation to

fixation screen), 10 seconds with the stimulus light and 35 seconds after light offset.

The stimulus light (7.15° diameter field, 10 sec duration) was presented in

Maxwellian view to the (cyclopleged) right eye at seven selected narrowband

wavelength lights (450 – 568 nm) with a range of energy levels (13.4 - 14.7 log

photons.cm-2.s-1) using neutral density filters (Section 3.4.4) and narrow band

interference filters (Section 3.4.3) to determine a criterion PIPR. Components of the

consensual PLR were modelled (Section 3.5.2) to determine the post-illumination

pupil response.


__________________________________________________________________ 42

Figure 3.8. The post-illumination pupil response and spectral sensitivity of intrinsically

photosensitive retinal ganglion cells. (a) Retinal irradiance-PIPR response plot for 493 nm. Data

reproduced from Gamlin, et al., (2007) using GraphClick (Arizona Software, v2.9.2). (b) PIPR of

participant 1 to 488 nm (blue trace) and 610 nm (red trace), 14.2 log photons.cm-2.s-1 stimuli.

Horizontal black dashed line indicates baseline pupil diameter of 6.10 mm and blue dashed line

indicates PIPR of 4.97 mm. Vertical dashed line indicates stimulus light onset. (c) Criterion pupil

responses (mm) for participant 1 at each wavelength (nm) (average of up to three measurements).

Retinal irradiance (log photons.cm-2.s-1) at each wavelength is indicated above each data point. Mean

criterion response was 0.57 ± 0.08 mm (red dashed line) (d) Criterion pupil responses (mm) for

participant 2 (single measurement for each λ). Mean criterion response was 1.26 ± 0.15 mm (red

dashed line). (e) Log relative sensitivity of the PIPR modelled using a vitamin A1 pigment


__________________________________________________________________ 43

nomogram with a λmax = 482 nm (black line). Data are shown for two participants (filled circles,

participant 1; open circles, participant 2). The A1 pigment nomogram model from Gamlin, et al.,

(2007) is indicated by the dotted red line. (f) Log sensitivity difference between model in Figure 3.8e

and the model of Gamlin, et al., (2007). Black circles show the wavelengths of the interference

filters.

For each stimulus wavelength (450 – 568 nm) the corneal irradiance was adjusted to

achieve an equivalent post-illumination pupil response (Gamlin, et al., 2007)

according to the principle of univariance (Naka & Rushton, 1966). The corneal

irradiance was matched to values used by Gamlin, et al., (2007) to produce a

criterion PIPR of 1.15 mm (Gamlin, et al., 2007). Figure 3.8a shows the irradiance-

PIPR response function for 493 nm fitted with a Hill equation (Gamlin, et al.,

2007). Note the half-maximal PIPR pupilloconstriction of 1.7 mm occurs for a

retinal irradiance of 13.6 log photons.cm-2.s-1 for 493 nm (Figure 3.8a). The

irradiance-PIPR response plot was used to adjust the PIPR values where these

deviated from the criterion response. The adjusted irradiance values were

normalised and fitted with Vitamin A1 pigment nomogram (Dartnall, 1953) used to

describe the spectral sensitivity of retinal A1 opsins as defined by

Iia � B ∑ k��mia ~��K��z��

�� , (Equation 3.17)

where kz x k� are constants, M is the wavelength of the corneal irradiance (nm)

and MTWU is the peak wavelength of spectral sensitivity function (nm).

3.6.3 Results and Discussion

Figure 3.8b demonstrates a PIPR of 1.13 mm (19 % pupilloconstriction relative to

baseline) to a 488 nm light of 14.2 log photons.cm-2.s-1 (90.03 cd.m-2) for participant

1. The PIPR was evident for short (488 nm) but not long (610 nm) wavelength light

stimuli (compare the red and blue trace), consistent with published reports (Gamlin,

et al., 2007). Figure 3.8c and 3.8d display the criterion PIPR pupil responses of

0.57 ± 0.08 mm and 1.26 ± 0.15 mm respectively for the two participants, achieved

by varying corneal irradiance. Figure 3.8e illustrates the normalized irradiance data

for the two participants, following correction for deviations from the criterion

response for the two observers and those reported by Gamlin, et al., (2007), using


__________________________________________________________________ 44

the irradiance-PIPR response from Figure 3.8a, and fitted with Vitamin A1 pigment

nomogram with a peak sensitivity of 482 nm.

The measured ipRGC spectral sensitivity model (black line) closely matches the

model of Gamlin, et al., (2007) (red line, Figure 3.8e). Figure 3.8f shows the log

sensitivity difference between the measured A1 pigment nomogram model in Figure

3.8e (black line) and the model of Gamlin, et al., (2007), with a maximum

difference of 0.073 log units at 600 nm. The best fitting nomogram (λmax = 482 nm)

was consistent with published reports (Dacey, et al., 2005; Gamlin, et al., 2007) and

confirms the successful isolation of the ipRGC post-illumination pupil response

with our pupillometer for use in the main experiment described in Chapter 4. Figure

3.9 demonstrates the position of the ipRGC spectral sensitivity function relative to

the rod and cone photoreceptors.

Figure 3.9. Spectral sensitivity of the five human retinal photopigments. S-cone (λmax = 440 nm),

ipRGC (λmax = 482 nm), Rod (λmax = 507 nm), M-cone (λmax = 543 nm) and L-cone (λmax = 566 nm).

Cone spectral data from Smith and Pokorny (1975); Rod spectral data from Crawford (1949); ipRGC

spectral data from Figure 3.8e.

Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response

__________________________________________________________________ 45

Chapter 4.

Investigation of circadian variation

of the ipRGC Pupil Response

4.1 INTRODUCTION

Previous research investigating circadian variation in the components of the pupil

light reflex (PLR) has been inconclusive. Differences in the circadian and

pupillography experimental methodologies, and the differing PLR components

recorded, have resulted in inconsistent results. As pupil diameter can be influenced

by both autonomic stimuli such as pain, surprise and stress (Burnstock & Sillito,

1999; Loewenfeld, 1999; Bradley, et al., 2008; Bär, et al., 2009) and cortical

inhibition at the Edinger-Westphal nucleus due to cognitive tasks (Hess & Polt,

1964; Granholm, et al., 1996), these factors also need to be considered when

comparing studies.

The baseline pupil diameter receives input from rod, cone and ipRGC

photoreceptors, with the relative proportion of contributions varying with

irradiance, wavelength and duration (McDougal & Gamlin, 2010). Circadian

variation in the baseline pupil diameter has been demonstrated in two studies

investigating pupil variations associated with alertness and time-of-day (Kraemer, et

al., 2000; Wilhelm, et al., 2001). Pupils were recorded for 10 – 11 minutes in

darkness every two hours, after 2 minutes of prior dark adaptation (Kraemer, et al.,

2000; Wilhelm, et al., 2001). These scotopic experimental conditions resulted in a

baseline pupil diameter dominated by rod activity. In contrast, Loving, et al., (1996)

was unable to demonstrate any circadian variation in baseline pupil diameter over

27 hours. Loving, et al., (1996) recorded pupils under < 5 lux mesopic, red

(unreported λ) illumination with no adaptation period, conditions where cone input

would dominate the baseline pupil diameter. The conflict between these studies of

Investigation of circadian variation of the ipRGC Pupil Response Chapter 4

__________________________________________________________________ 46

baseline pupil diameter may be the result of differing (or no) diurnal variation in rod

and cone contributions to the pupil diameter.

Studies examining circadian variation in the PLR components of pupil constriction

and latency are similarly inconclusive (Tiedt, 1963; Ranzijn & Lack, 1997). Tiedt

(1963) demonstrated significant circadian variation in the PLR, with an increased

constriction response during the day and a decreased response at night. This result

has not been replicated, and more recent research demonstrated no circadian

variation in PLR maximum constriction (Ranzijn & Lack, 1997). The post-

illumination pupil response is a direct measure of ipRGC activity (Gamlin, et al.,

2007) but this component of the PLR has not previously been examined for

circadian variation.

Variability in pupillometry and circadian experimental methodology accounts for

the inconclusive results of previous studies. Previous studies have used a variety of

pupil stimulus wavelengths and irradiances, background illuminations and durations

for pupil recording, all factors which influence the PLR (Alpern & Campbell, 1962;

Barbur, et al., 1992; Loewenfeld, 1999). The pupillometry method used in the

following experiment was designed to control these factors to isolate the inner and

outer photoreceptor contributions to the pupil light reflex. Previous studies also did

not align the circadian phase of individual participants using an independent phase

marker such as core body temperature or melatonin levels (Kräuchi, 2002;

Claustrat, et al., 2005). Instead results were analysed using time-of-day which, as

individuals may exhibit variability in circadian phase for the same time-of-day, may

mask circadian variation (Tiedt, 1963; Loving, et al., 1996; Ranzijn & Lack, 1997;

Kraemer, et al., 2000; Wilhelm, et al., 2001). The circadian experiment described in

this chapter uses salivary melatonin as a phase marker to align participants,

enabling a more accurate estimate of PLR circadian rhythms than previous research.

The research described in this chapter investigates if ipRGC activity, as measured

via the post-illumination pupil response, demonstrates a circadian rhythm. A recent

in vitro rat study has demonstrated no significant circadian rhythm in ipRGC

activity, excluding light responsive cell-autonomous circadian modulation (Weng,


__________________________________________________________________ 47

et al., 2009). By isolating both the intrinsic and the cone-mediated ipRGC activity

with the post-illumination response in vivo, it was anticipated the results of this

study would provide information regarding SCN central control of any

photoreceptor circadian rhythms recorded. It was hypothesized that any ipRGC

circadian activity would also be synchronized with the central circadian rhythm as


4.2 METHODS

This section describes the participants, circadian testing protocols and circadian

data analysis of the experiment.

4.2.1 Participants

Eleven participants (4M, 7F; age range: 18 – 31; mean ± SD: 25.67 ± 4.21) with

normal visual acuity (≥ 6/6) and who met the participant inclusion criteria in Table

3.1 provided written informed consent. All participants (Refraction, mean ± SD;

RE: -0.36 ± 0.93 /-0.23 ± 0.24 D, LE: -0.36 ± 0.92 /-0.25 ± 0.32 D) were

uncorrected during the experiment and viewed the 4° black fixation cross with the

left eye while the right eye was dilated with cyclopentolate 1 %.

4.2.2 Apparatus

The pupillometer used in the following experiment has been described in Section

3.2. The light stimuli used were 488 nm, 14.23 ± 0.04 log photons.cm-2.s-1

(90.03 cd.m-2) and 610 nm, 14.29 ± 0.12 log photons.cm-2.s-1 (217.90 cd.m-2). The

methods used to measure and convert irradiance values have previously been

described in Section 3.4.6.

4.2.2.1 Pittsburgh Sleep Quality Index (PSQI)

The Pittsburgh Sleep Quality Index (PSQI) is a clinical tool to assess subjective

sleep quality during the prior month (Buysse, Reynolds, Monk, Berman & Kupfer,

1989) (Appendix 7.2). It can be used as a clinical screening tool for detection of

sleep disturbances and to monitor any progression. Nineteen questions measure

seven components of sleep quality (subjective sleep quality, sleep latency, sleep


__________________________________________________________________ 48

duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication

and daytime dysfunction) to calculate a global score of 0 - 21. A score > 5 indicates

a “poor” sleeper, with a significant probability of a sleep disturbance (Buysse, et al.,

1989). All participants were screened with this questionnaire (PSQI mean ± SD;

3.3 ± 1.3) and determined to have normal sleep quality.

4.2.2.2 Pittsburgh Sleep Diary (PghSD)

Appendix 7.3 shows the Pittsburgh Sleep Diary (PghSD), a clinical tool designed to

investigate subjective sleep, waking and daytime patterns (Monk, Reynolds,

Kupfer, Buysse, Coble, Hayes, Machen, Petrie & Ritenour, 1994). The diary was

filled out by participants twice daily and comprises two sections, one completed at

bedtime and the other at wake time. The bedtime component records the daytime

activities of meals, exercise and naps as well as alcohol, caffeine, nicotine and

medication intake. The wake time component documents sleep onset and duration,

method of final waking, night time disturbances and sleep quality. In the context of

this experiment the PghSD was used to determine habitual self-reported sleep

patterns and wake time, and participants were determined to have regular sleep

patterns before inclusion in the study.

The habitual sleep pattern of participants was recorded for one week prior to

participation in the experiment using the Pittsburgh Sleep Diary. Participants

recorded a subjective mean wake time of 7:55 am ± 0:54 and sleep time of

11:59 pm ± 0:28 (Table 4.1) and were determined to have regular sleep patterns.

4.2.2.3 Actigraph

Actigraphy is the measurement of a participant’s sleep/wake cycle from motion and

light exposure detection. The AW-L actiwatch (Mini Mitter, now trading as Phillips

Respironics, Bend, Oregon 97701 USA) record motor activity and light data (range:

0.4 – 150000 lux) every minute for up to 15 days and is worn on the wrist. Data

were downloaded at the end of the collection period using the Actiware 5.2 software

(Philips Respironics, Bend, Oregon 97701 USA).


__________________________________________________________________ 49

Actigraphy is a valid tool for differentiating sleep and wake in healthy adults

(Ancoli-Israel, Cole, Alessi, Chambers, Moorcroft & Pollak, 2003). Unlike

polysomnography sleep assessment, which uses wired sensors to record

physiological activity during sleep in a laboratory (EEG, EMG, EOG, ECG),

actigraphy is minimally invasive and allows participants to remain in their home

sleeping environment. Additionally actigraphy can be assessed across several days

to record a more stable estimate of the habitual sleep cycle. The eleven participants

wore an actigraph for one week prior to testing to assess their habitual sleep pattern.

Participants recorded a mean actigraphic wake time of 8:00 am ± 1:14 and sleep

time of 11:54 pm ± 0:48 (Table 4.1).

The actigraphic output of a 26 yo M participant is included as Figure 7.1, Appendix

7.4. Light exposure (yellow trace) and motion (black trace) were recorded, and

sleep periods (aqua bars) were identified by the Actiware 5.2 software using the

Actiware Sleep Interval Detection Algorithm. The experimental period (final 24

hours in the figure) contains no sleep periods because the test protocol required

participants to remain awake.

Table 4.1. The habitual sleep and wake times of the 11 participants, recorded for one week

prior to the overnight experiment. Values were determined from a motion and light sensor

Actigraph worn on the wrist and the Pittsburgh Sleep Diary (PghSD).

Mean SD Min Max

Wake Time: PghSD 7:55 am 0:54 6:07 am 9:18 am

Actigraph 8:00 am 1:14 5:27 am 9:59 am

Sleep Time: PghSD 11:59 pm 0:28 11:24 pm 12:54 am

Actigraph 11:54 pm 0:48 10:44 pm 1:31 am

4.2.2.4 Participant circadian rhythm inclusion criteria

Because the presence of a circadian disorder could be associated with abnormal

retinal circadian rhythms (Sack, Auckley, Auger, Carskadon, Wright, Vitiello &

Zhdanova, 2007), only participants with robust, normal circadian rhythms were

included in this study. All eleven participants demonstrated normal sleep quality

(PSQI score ≤ 5, Section 4.2.2.1) and a regular sleep-wake cycle assessed with both


__________________________________________________________________ 50

actigraphy (Section 4.2.2.3) and the Pittsburgh Sleep Diary (Section 4.2.2.2)

(Table 4.1). Participants who were smokers, shift-workers, excessive consumers of

caffeine (> 3 cups/day), who used sleeping medications or had crossed more than

one time zone in the prior month were not included.

4.2.3 Procedures

4.2.3.1 Circadian Experiment Testing Procedure

Testing occurred in the Visual Science and Medical Retina Laboratories at the

Institute of Health and Biomedical Innovation, QUT. The eleven participants wore

an actigraph (Section 4.2.2.3) and kept a Pittsburgh Sleep Diary (PghSD,

Section 4.2.2.2) for one week prior to testing and were screened for sleep disorders

(PSQI, Section 4.2.2.1). On the day of testing, participants arrived at the laboratory

at 8 am for a comprehensive visual screening (Section 3.1) and alignment of the

pupillometer (Section 3.2), prior to the commencement of testing at 9 am. Caffeine

was prohibited from 6 am. To maximise the right pupil diameter (> 6.5 mm) and

control the retinal illumination to the right eye, the participant’s right pupil was

cyclopleged with 1 % cyclopentolate. Cyclopentolate 1 %, with a longer duration of

action, was chosen instead of 1 % Tropicamide, to minimise eyedrop instillation

over the 24 hours. Subjective accommodation was assessed using an optometer

(Hartinger, Rodenstock) and cyclopentolate was re-instilled as required during the

24 hour test. During the experiment, participants remained in the laboratory for

20 - 24 hours, until the participant felt too tired to continue. A constant routine

protocol was maintained in the laboratory for the entire testing period. Figure 4.1

displays the timing of the hourly measurements and protocols, repeated each hour

of the testing period.

Circadian variation of the pupil light reflex was determined by four consensual

pupil recordings of 55 seconds (10 seconds pre-stimulus, 10 seconds stimulus and

35 seconds post-stimulus) repeated every hour for 20 - 24 hours using the

pupillometer described in Section 3.2. Wavelengths of 488 nm and 610 nm were

alternated for the 10 second, 14.2 log photons cm-2.s-1, 7.15° stimuli (2 x 488 nm;

2 x 610 nm). The infrared camera recordings were analysed with custom developed

software (Section 3.5.1) and the dynamic cone, cone-mediated input to ipRGCs and


__________________________________________________________________ 51

intrinsic ipRGC PLR parameters (baseline pupil diameter, maximum constriction,

maintained constriction and post-illumination pupil response) were determined

(Section 3.5.2) every hour for up to 24 h.

Figure 4.1. Timing of the hourly measurements and protocols for the 24 hour testing period.

The timing of events each hour is shown for an hourly time scale (vertical left). Data collection each

hour comprised of four pupil recordings and one saliva collection.

To determine if circadian PLR variation was independent of external illumination,

both the laboratory illumination (< 10 lux) and pupillometer stimuli were kept

constant during the 24 h test. In this experiment the SCN circadian rhythm was

assessed from hourly salivary melatonin levels (Section 4.2.3.2). A 24 hour constant

routine protocol was used to control the external circadian cues of activity, sleep,

posture, caffeine, ambient temperature and caloric intake to reveal the underlying

endogenous circadian rhythm (Duffy & Dijk, 2002). Participants remained awake in

the laboratory (< 10 lux, 23 - 25°C) watching DVDs (screen < 10 lux) in an upright


__________________________________________________________________ 52

seated position with limited physical activity monitored by the actigraph. Hourly

snacks (< 500 kJ.hr-1) were provided and caffeine was prohibited. Toilet breaks

were offered immediately after each session of hourly recordings, with sunglasses

worn outside the laboratory to minimise light exposure.

4.2.3.2 DLMO Melatonin Assay

To determine the circadian phase of the suprachiasmatic nucleus (SCN), the

melatonin circadian rhythm was determined from hourly saliva samples collected

after completion of each hourly pupil recording (Section 4.2.3.1). Core body

temperature and melatonin expression from the pineal gland both demonstrate

circadian rhythms regulated by the SCN (Kräuchi, 2002; Claustrat, et al., 2005)

(Figure 2.6). Core body temperature is lowest in the early morning (~ 5 am) and

peaks in the late afternoon (~ 5 pm) (Hofstra & de Weerd, 2008). In normal

participants, melatonin levels remain at a low baseline level during the day,

beginning to rise 2 - 3 hours before habitual sleep time, and peaking during the

night before dropping back to daytime levels within a few hours of waking (Pandi-

Perumal, et al., 2007; Benloucif, et al., 2008). Melatonin is less easily masked than

core body temperature (Benloucif, Guico, Reid, Wolfe, L'Hermite-Balériaux & Zee,

2005) and can be measured in blood plasma, saliva and as a melatonin metabolite in

urine (Pandi-Perumal, et al., 2007). Salivary melatonin is established as a valid

marker of circadian phase compared to plasma melatonin (Voultsios, Kennaway &

Dawson, 1997) and saliva collection has the additional benefits of being less

invasive than blood collection and of allowing more frequent sampling than urine

collection.

As environmental light suppresses melatonin production (Lewy, Wehr, Goodwin,

Newsome & Markey, 1980), saliva collection is conducted in dim light conditions

(< 10 lux). The evening onset of melatonin secretion under dim light conditions is

termed dim light melatonin onset (DLMO) and is a robust and accurate phase

marker (Pandi-Perumal, et al., 2007). The optimal assessment of circadian phase

requires hourly melatonin measurements for a full 24 hour circadian cycle under

dim light conditions. While DLMO is robust, the following external factors may

mask the endogenous melatonin rhythm: physical activity (Monteleone, Maj, Fusco,


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Orazzo & Kemali, 1990), sleep (Zeitzer, Duffy, Lockley, Dijk & Czeisler, 2007),

posture (Deacon & Arendt, 1994), caffeine (Wright, Badia, Myers, Plenzler &

Hakel, 1997; Shilo, Sabbah, Hadari, Kovatz, Weinberg, Dolev, Dagan &

Shenkman, 2002), medications (Murphy, Myers & Badia, 1996; Stoschitzky,

Sakotnik, Lercher, Zweiker, Maier, Liebmann & Lindner, 1999) and evening

caloric intake (Kräuchi, Cajochen, Werth & Wirz-Justice, 2002). To determine the

endogenous melatonin profile a constant routine protocol is used to minimise the

influence of these exogenous factors (Duffy & Dijk, 2002).

Saliva samples were collected hourly (Figure 4.1) by participants gently chewing on

a cotton swab (Salivettes; Sarstedt, Nümbrecht, Germany) for 2 minutes. Dim light

melatonin onset (DLMO) saliva collection protocols were followed (Pandi-Perumal,

et al., 2007), with subjects rinsing their mouths 15 minutes prior to each

measurement, sitting in < 10 lux illumination, refraining from strenuous physical

exertion and not brushing their teeth. Saliva samples were centrifuged (3 mins,

3000 rpm; Hettich Universal 320 centrifuge) and stored at -80 °C within 24 hours of

collection, before being shipped on dry ice to the Circadian Physiology Group at the

University of Adelaide Medical School for analysis. Melatonin levels were

determined by radioimmunoassay (sensitivity < 4.3 pM ) using the methods

described by Voultsios, Kennaway and Dawson (Voultsios, et al., 1997) using

Bϋhlmann Laboratories assay reagents (Schönenbuch, Switzerland).

4.2.4 Data Analysis

4.2.4.1 Alignment of Participant Circadian Phase

In this experiment the circadian phase of each participant was calculated from

salivary melatonin levels. Dim light melatonin onset (DLMO) can be calculated

using a threshold melatonin level (8.6 - 43.1 pM or 2 SD above baseline levels) or

visually estimating the point of rise above the baseline level (Benloucif, et al.,

2008). However the most accurate assessment of circadian phase requires 24 hours

of measurements modelled with a baseline cosine function to determine DLMO,

peak melatonin and offset times (Van Someren & Nagtegaal, 2007). In this study

the calculation of circadian phase was optimized by modelling the individual

participants’ 24 hour melatonin data as a function of time with the skewed baseline


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cosine function (SBCF) (Figure 4.2) described by Van Someren and Nagtegaal,

(2007) where:

[ = t s (�

291 x l:: 9lih �c x y s n lih9c x y:� x l s �lih �c x y s nlih9c x y:� x l�:

(Equation 4.1)

and t is baseline salivary melatonin, � is amplitude height above baseline, l is the

width parameter, y is phase and n is skewness. Time 9c: is in radians representing

0 – 24 hours.

The time above baseline is calculated from the width parameter l as:

�`v_ 9biojh: B 24 K kjllih9l: /� (Equation 4.2)

The modelled melatonin peak is defined as the time when the modelled melatonin

value is equal to the sum of baseline melatonin 9t: and the amplitude 9�:,

P_mkciw`w �_k� �`v_ `h c 9jkg`kwh: �b_w [ B t s � (Equation 4.3)

Figure 4.2. An example of the skewed baseline cosine function (SBCF) model and parameters.

Time (t) is shown in radians for 0 - 2π (bottom horizontal axis) and hours for 0 – 24 h (top

horizontal axis). Parameters displayed are: baseline level (b), amplitude above baseline (H), time of

peak amplitude (Φ), width parameter (c) and skew (υ). A function with no skew (υ = 0, solid black

line) and a skewed function (υ = -0.5, red dashed line) are displayed.


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The dim light melatonin onset time (DLMO) of each participant was calculated as

the time (c) when the individual modelled melatonin first increased by 0.01 pM

above the baseline value. The melatonin offset time was calculated as the time (c)

after the melatonin peak when the individual modelled melatonin was 0.01 pM

above the baseline value such that:

P_mkciw`w Owh_c �`v_ `h c 9jkg`kwh: �b_w [ B t s 0.01 kwg c

� P_mkciw`w �_k� �`v_

(Equation 4.4)

P_mkciw`w Ouuh_c �`v_ `h c 9jkg`kwh: �b_w [ B t s 0.01 kwg c

� P_mkciw`w �_k� �`v_

(Equation 4.5)

Once individual DLMO, peak melatonin and offset times were calculated, DLMO

time was used to align the circadian phase of all participants. The clock time of

DLMO varies between individuals, so group analysis required all the individual

DLMO times to be aligned (Figure 4.3). In this study DLMO was assigned the

arbitrary value of 14 hours in a 0 – 24 hour circadian cycle, with clock time shifted

to circadian time with:

N`jlkg`kw c`v_ 9b: v`w: B lmil� c`v_ s IPO x 14 (Equation 4.6)

Figure 4.3. Alignment of participants by individual circadian phase. The data sets of two

participants (blue and yellow bars) with the clock time (below each bar) and individual salivary

melatonin onset (bold, red vertical lines) displayed. (a) Participant data sets aligned for the clock

time scale. (b) Participant data sets aligned for circadian phase, determined by individual melatonin

onset time. Melatonin onset time assigned the value of 14 h in a 0 – 24 circadian cycle.


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4.2.4.2 Modelling of the Pupil Light Reflex Components

The baseline pupil diameter, the maximum constriction diameter and the post-

illumination pupil response (PIPR) were each plotted against circadian time and

modelled twice, with both a linear function and a skewed baseline cosine function.

The parameters of both functions were changed to minimise the sum of squares

differences between the data and the model parameters, and the best fitting model

was used describe the PLR parameter. The first model was the linear function:

[ = c K k s g (Equation 4.7)

where k is the slope (%.radian-1), g is a constant and time (c) is in radians

representing 0 - 24 hours.

The second model was the skewed baseline cosine function (SBCF) (Figure 4.2)

used to model salivary melatonin (Equation 4.1) where t is baseline pupil diameter

instead of salivary melatonin. The model inflection points were defined as a 0.01 %

threshold increase or decrease in the modelled pupil diameter. As for melatonin, the

pupil onset, peak and offset times were calculated using the equations:

�`v_ iu �_k� �oq`m `kv_c_j `h c (jkg`kwh) �b_w [ = t s � (Equation 4.8)

�oq`m `kv_c_j Owh_c �`v_ `h c (jkg`kwh) �b_w [ = t s 0.01 kwg c �

�oq`m �_k� �`v_

(Equation 4.9)

�oq`m `kv_c_j Ouuh_c �`v_ `h c (jkg`kwh) �b_w [ = t s 0.01 kwg c

� �oq`m �_k� �`v_

(Equation 4.10)

For each PLR parameter (baseline pupil diameter, the maximum constriction

diameter and the post-illumination pupil response) participants were grouped by

rounding circadian time to the nearest 1-hour bin and a group model was derived

from the mean data of participants fitted with a single linear or SBCF model. In

addition, for each participant, an individual model was derived to confirm the

robustness of the group linear model and to observe individual physiological

variation. All individual participant models for all PLR parameters are included in

Appendix 7.5.


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4.2.4.3 Statistical Analysis of Pupil Light Reflex Components

While the individual participant PLR models could be fitted with resolutions of one

minute, participants could not be analysed or modelled as a group without rounding

circadian time to the nearest 1-hour time bin. This introduced an error margin of

less than one hour. The rounded circadian time values covered a range of 0 –

27 hours. The circadian times of 25, 26 and 27 hours were excluded from analysis

due to small participant numbers (≤ 2) at these data points.

Three components of the pupil light reflex, the baseline pupil diameter, the

maximum constriction diameter and the post-illumination pupil response (PIPR),

were investigated in this experiment. Statistical analysis used the Linear Mixed

Model (random effects) univariate ANOVA to determine if each PLR component

varied significantly with circadian time. The Linear mixed model is designed for

analysis of unbalanced repeated measures, and can accept missing values without

excluding whole sections of data. The hypothesis of the Linear Mixed Model is that

the dependent variable (baseline, maximum constriction or PIPR) is not

significantly different when the fixed factor (time or repeat) is varied. Participant

number is included in the analysis as a random factor because, although each

measurement is independent, some observations come from the same individual. In

this experiment the dependent variables investigated were each PLR component and

the independent factors were:

Independent Fixed Factors: Circadian Time (1 – 24), Repeat (Run 1 or Run 2)

Independent Random Factor: Participant number (#1 – 11)

Interaction Term: Circadian Time * Repeat (Run 1 or Run 2)

The questions this statistical design addressed were:

Does the PLR component vary significantly with Circadian Time or Repeat?

Do the Run 1 and Run 2 pupil diameter (of the PLR component) values

significantly differ over Circadian Time?


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4.3 RESULTS

4.3.1 Cone photoreceptor (outer retina) contributions to the PLR

4.3.1.1 Baseline Pupil Diameter

Figure 4.4a displays the circadian profile of the baseline pupil diameter derived

from the mean data of eleven participants fitted with a single linear function. Table

4.2 shows the parameters of the baseline pupil diameter (n = 11) group model,

obtained by rounding circadian time to the nearest 1-hour bin. Table 4.2 also shows

the mean ± SD of individually derived linear models for all 11 participants. These

individual participant linear functions were derived to confirm the robustness of the

group linear model. Baseline pupil diameter did not vary significantly with

circadian time (p = 0.668; mixed model univariate ANOVA), confirmed by a slope

of 0.01 %.h-1 (n = 11). When modelled individually, the baseline pupil diameter

varies significantly between participants (Table 4.2), consistent with past reports

(Loewenfeld, 1999) (p < 0.001; mixed model univariate ANOVA), as anticipated,

due to physiological inter-individual variation. The individual baseline pupil data

and linear models over the 24 hour period are shown in Figure 7.2, Appendix 7.5.1.

Table 4.2. The mean linear model parameters of the baseline pupil diameter circadian profile.

Pupil diameter shown as % of average baseline diameter (n = 11).

Group Model Individual Models

Mean SD Min Max

Slope (%.h-1

) 0.01 -0.02 0.39 -0.71 0.65

Constant (%) 99.70 100.41 4.63 92.85 108.43

NOTE: Group model derived from the mean data of participants fitted with a single linear function.

Individual models derived from the individual participants linear functions.

4.3.1.2 Maximum Pupil constriction

To determine if the cone activity (outer retina) showed circadian variation, the

maximum pupil constriction was derived from the PLR recordings for 24 hours.

This PLR component occurs ~ 0.24 – 1.5 s after light onset, receiving no input from

slow latency (> 1.78 sec) ipRGCs (McDougal & Gamlin, 2010). Figure 4.4b and

4.4c display the 24 hour maximum constriction derived from the mean data of

eleven participants, grouped by rounding circadian time to the nearest 1-hour bin,


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fitted with a single linear model. Table 4.3 shows the linear group model (n = 11)

for 488 nm and 610 nm (14.2 log photon.cm-2.s-1, 10 sec) stimuli. An individual

model was also derived for each of the 11 participants (for both 488 and 610 nm

stimuli), shown in Figures 7.3 and 7.4 (Appendix 7.5.2). Table 4.3 shows the

mean ± SD of these individually derived linear models.

Maximum pupil constriction significantly decreased with time (p < 0.001; mixed

model univariate ANOVA), decreasing (pupil diameter increasing) by 0.19 %.h-1

and 0.26 %.h-1 for the 488 and 610nm stimuli respectively in the 24 hour period.

Individual maximum pupil constriction decreased (and pupil diameter increased) in

8/11 of participants for the 488 nm light and in 10/11 for the 610 nm stimulus. As

mean maximum constriction continued to decrease rather than returning to the 0 h

level at 24 h, this is not evidence of a circadian rhythm. Wavelength also

significantly affected the maximum constriction, with 2.73 % greater constriction

produced by the 610 nm compared to the 488 nm stimulus (p < 0.001; mixed model

univariate ANOVA). A significant difference between the first and second pupil

measurement each hour was detected for maximum constriction for both the

488 and 610 nm stimuli (p ≤ 0.001; mixed model univariate ANOVA). When

modelled individually, the maximum constriction diameter varied significantly

between participants (Table 4.3), for both the 488 and 610 nm stimuli (p < 0.001;

mixed model univariate ANOVA).

Table 4.3. The mean linear model parameters of the maximum pupil constriction circadian

profile for 488 nm and 610 nm (14.2 log photon.cm-2

.s-1

, 10 sec) stimuli. Pupil diameter shown as

% of average baseline diameter (n = 11).


Mean SD Min Max

488 nm stimulus

Slope (%.h-1

) 0.19 0.10 0.29 -0.35 0.61

Constant (%) 44.11 46.04 7.82 32.57 59.46

610 nm stimulus

Slope (%.h-1

) 0.26 0.17 0.27 -0.24 0.52

Constant (%) 41.38 43.32 7.22 32.15 56.20

NOTE: Group model derived from the mean data of participants fitted with a single linear function.

Individual models derived from the individual participants linear functions.


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Figure 4.4. The circadian profiles of the baseline and maximum constriction pupil components

of the pupil light reflex. Symbols show the mean ± SD of 11 participants. Lines are the best-fitting

linear models. Pupil diameter is represented as a % of the mean baseline diameter. Inset is the pupil

light reflex of one individual (19 yo, Female) at two circadian times referenced to DLMO at 14 h.

(a) Baseline pupil diameter recorded hourly while viewing a uniform photopic screen (116 cd.m-2)

(Black unfilled circles, mean ± SD). Linear model y = 0.01*x + 99.70 (R2 = 0.004) (Black line).

(b) Maximum pupil constriction recorded for an hourly 488 nm, 14.2 log photon.cm-2.s-1, 10 sec

stimulus (Blue filled squares, mean ± SD). Linear model y = 0.19*x + 44.11 (R2 = 0.47) (Blue line)

(c) Maximum pupil constriction recorded for an hourly 610 nm, 14.2 log photon.cm-2.s-1, 10 sec

stimulus (Red filled squares, mean ± SD). Linear model y = 0.26*x + 41.38 (R2 = 0.64) (Red line)

4.3.2 Intrinsic and cone-mediated ipRGC contributions to the PLR

To determine if the intrinsic (inner retina) and cone-mediated (outer retina) ipRGC

activity showed circadian variation, the PIPR component of the PLR was recorded

for 488 nm and 610 nm stimuli for 24 hours. Figure 4.5a and 4.5b illustrate the

change in the PIPR (15 – 45 seconds) for a 488 nm stimulus, with circadian phase

for two observers (19 yo F and 18 yo M). The first observer (Figure 4.5a)

demonstrated a post-illumination constriction diameter of 75.0 % (5.83 mm) of the

mean baseline pupil diameter at 3.5 h increasing to 92.5 % (7.18 mm) at 15.4 h.


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Figure 4.5. The individual circadian variation of the post-illumination pupil response

component of the pupil light reflex for two observers. A 488 nm 10 second,

14.2 log photons.cm-2.s-1, 7.15º stimulus used for two observers (19 yo F, 18 yo M). (a) Post-

illumination pupil responses at three circadian times were 75.0 % (3.5 h), 83.2 % (9.4 h) and 92.5 %

(15.4 h) of the mean baseline pupil diameter of 7.77 mm, for a 19 yo F. (b) Post-illumination pupil

responses of 71.4 % (5.4 h), 77.0 % (10.4 h) and 99.6 % (15.4 h) of the mean baseline pupil diameter

of 6.68 mm, for an 18 yo M. (c) The post-illumination component of the pupil light reflex fitted with

a skewed baseline cosine function (data show mean ± SD, filled circles; model, line;) (R2 = 0.79) for

a 19 yo F. Inset: Post-illumination pupil response (blue lines) from Figure 4.5a at 3.5 and 15.4 h. (d)

The post-illumination component of the pupil light reflex fitted with a skewed baseline cosine

function (data show mean ± SD, filled circles; model, line;) (R2 = 0.71) for an 18 yo M. Inset: Post-

illumination pupil response (blue lines) from Figure 4.5b at 5.4 and 15.4 h.

When the PIPR of this participant was modelled with a skewed baseline cosine

function, baseline PIPR was 72.9 % of the baseline diameter and increased to

93.7 % at the peak time of 17:19 h (Figure 4.5c). This diurnal variation in the

intrinsic ipRGC post-illumination pupil response was demonstrated in all 11

participants with a mean baseline PIPR diameter of 82.5 ± 7.8 % and a peak

amplitude 11.7 ± 5.7 % above the baseline PIPR value (Table 4.4). Figure 7.5 in

Appendix 7.5.3 displays three pupil light reflexes and the PIPR skewed baseline


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Table 4.4. Participant (n = 11) post-illumination pupil response amplitudes for the 488 nm and

610 nm stimuli.


Mean SD Min Max

488 nm stimulus

Baseline PIPR % 83.5 82.5 7.8 71.5 96.0

Minimum PIPR % 90.0 94.4 6.6 80.7 103.7

PIPR Difference % 6.5 11.7 5.7 2.2 20.9

610 nm stimulus

Baseline PIPR % 86.5 86.2 7.2 73.6 96.6

Minimum PIPR % 93.3 98.0 4.9 86.9 107.9

PIPR Difference % 6.8 11.9 5.7 2.6 24.4

NOTE: Group model derived from the mean data of participants fitted with a single skewed baseline

cosine function. Individual models derived from the individual participants skewed baseline cosine

function

Table 4.5. Participant (n =11) intrinsic ipRGC activity (488 nm), cone-mediated ipRGC

activity (610 nm) and salivary melatonin onset and minimum/peak times.

Group

Model Individual Models (h:min)*

(h:min)* Mean SD Min Max

Intrinsic ipRGC activity decrease** 11:14 11:04 2:28 5:52 13:49

Intrinsic ipRGC activity minimum 15:25 14:39 1:29 11:31 17:19

Cone-mediated ipRGC activity

decrease**

9:40 11:41 1:41 8:05 14:10

Cone-mediated ipRGC activity

minimum

15:42 14:53 1:37 12:08 17:23

Melatonin onset *** 14:00 14:00 0:00 14:00 14:00

Melatonin peak 18:56 18:39 1:28 17:08 21:54

NOTE: Group model derived from the mean data of participants fitted with a single skewed baseline

cosine function. Individual models derived from the individual participants skewed baseline cosine

functions.

* Clock time was replaced with circadian time by aligning the dim-light melatonin onset (DLMO) of

each individual participant to 14:00 h.

** Threshold decrease in ipRGC activity (model inflection point) defined as 0.01 % rise above

baseline PIPR model value.

***Threshold increase salivary melatonin defined as 0.01 pM rise above baseline melatonin model

value.


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cosine function model for each participant (n = 11). The mean parameters of the

individually modelled intrinsic and cone-mediated ipRGC post-illumination pupil

responses are shown in Columns 2 – 5, Tables 7.1 and 7.2 (Appendix 7.5.3)

Intrinsic ipRGC activity (488 nm stimulus) and cone-mediated ipRGC activity

(610 nm stimulus) were each derived from the mean data of participants fitted with

a single skewed baseline cosine function. The post-illumination pupil response

results for the 488 and 610 nm stimuli were grouped by rounding circadian time to

the nearest 1-hour bin. Figure 4.6a and b display the circadian variation in both the

intrinsic (Figure 4.6a, 488 nm) and cone-mediated (Figure 4.6b, 610 nm) ipRGC

activity. For the grouped participants analysis, the intrinsic ipRGC activity began to

decrease at 11:14 h and reached minimum ipRGC activity (maximum PIPR

diameter) at 15:25 h (Figure 4.6a) (Table 4.5). The cone-mediated ipRGC activity

also demonstrated a circadian variation, decreasing from 9:40 h to a minimum

activity level at 15:42 h (Figure 4.6b) (Table 4.5). The mean parameters of the

group models (488 and 610 nm) are presented in Column 1, Table 7.1 and 7.2

(Appendix 7.5).

IpRGC activity onset and minimum times were also derived from individual

participants skewed baseline cosine functions to confirm the robustness of the

results. Table 4.5 displays the mean ± SD of the individually derived models for all

11 participants (n = 11). For the individually derived models the intrinsic ipRGC

response was greatest (smallest PIPR pupil diameter) at circadian times prior to

11:04 ± 2:28 h. After this onset time, intrinsic ipRGC activity decreased until

minimum activity was reached at 14:39 ± 1.29 h, demonstrated by a minimum PIPR

diameter (Table 4.5). The cone-mediated ipRGC response was greatest at circadian

times before 11:41 ± 1.41 h and reached a minimum at 14:53 ± 1:37 h (Table 4.5).

In order to determine the temporal relationship of diurnal ipRGC activity and the

biological clock, the central SCN circadian phase of the eleven participants was

determined by salivary melatonin assay data (Section 4.2.3.2) and modelled with

skewed baseline cosine functions. The melatonin onset of the eleven participants

was set to the circadian time 14:00 h to align for individual circadian phase (see


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Section 4.2.4.1). Table 4.5 gives the group (n = 11) salivary melatonin onset, peak

and offset times modelled with a single skewed baseline cosine function, and the

mean ± SD of the individually derived models for all 11 participants. With

melatonin onset set to 14:00 h (clock time; 9:27 pm ± 1:21), peak melatonin

occurred at 18:39 h (clock time; 2:06 am ± 1:01) and melatonin offset time at

25:44 h (clock time; 9:10 am ± 1:24). All individual melatonin models

(R2 mean ± SD; 0.96 ± 0.4) are included in Figure 7.6 and the mean parameters are

displayed in Table 7.3 (Appendix 7.5.4).

The circadian phase of both the intrinsic and cone-mediated ipRGC activity is

temporally synchronized to, and in phase advance of the central SCN rhythm

measured by salivary melatonin. The decrease in intrinsic ipRGC activity occurred

2:46 h prior to the peak salivary melatonin secretion at 19:10 h, with minimum

ipRGC activity (maximum PIPR diameter) occurring 1:25 h after melatonin onset.

Figure 4.6c illustrates the 2:46 h phase difference in intrinsic ipRGC activity (blue

line) and the central circadian modulation of melatonin (dashed black line). Cone-

mediated ipRGC activity also demonstrated a phase 4:20 h in advance of the central

circadian rhythm (red and black lines, Figure 4.6c). Figure 7.6, Appendix 7.5.4

shows the temporal synchrony of individual melatonin and post-illumination pupil

response (488 stimuli) data and models over the 24 hour period.

The post-illumination pupil response demonstrates significant variation under

conditions of constant illumination and stimulus irradiance for both 488 nm and

610 nm stimuli (p < 0.001; mixed model univariate ANOVA). Post-hoc analysis

indicate the PIPR is significantly different at 15 h compared to other circadian times

for 488 nm (p < 0.05 for the circadian hours: 2 – 13, 17 - 24 h; Tukey post hoc

analysis) and 610 nm (p < 0.05 for the circadian hours: 2 – 12, 17, 18, 20 and 24 h;

Tukey post hoc analysis). As for the other components of the PLR (Section 4.3.1),

the PIPR also showed significant variation between the individual participants

(p < 0.001; mixed model univariate ANOVA). A significant difference between the

first and second PIPR measurement each hour was detected for the 488 nm stimulus

(p ≤ 0.001; mixed model univariate ANOVA) but not the 610 nm stimulus

(p = 0.173; mixed model univariate ANOVA).


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Figure 4.6. Temporal synchrony of ipRGC activity with the biological clock. Data show

mean ± SD (symbols) for 11 participants aligned for circadian phase, modelled with skewed baseline

cosine functions (solid lines). Model onsets and peaks indicated by arrows. (a) The post-illumination

pupil response to 488 nm stimuli (data, filled blue circles; model, blue line) (R2 = 0.65). PIPR

diameter began to increase at 11:14 h and peaked at 15:25 h. (b) The post-illumination pupil

responses to 610 nm stimuli (data, filled red circles; model, red line) (R2 = 0.80). PIPR diameter

began to increase at 9:40 h and peaked at 15:42 h. (c) The salivary melatonin (model, dashed black

line) (R2 = 0.96) and the PIPR models from Figure 4.6a (blue line) and 4.6b (red line). Melatonin

began to increase at 14:00 h, 2:46 hours after PIPR (488 nm) began to increase and peaked at

18:56 h. The PIPR and melatonin group model parameters are displayed in Column 1, Table 7.1, 7.2

and 7.3 (Appendix 7.5).

Table 4.6 displays the individual variation in the post-illumination pupil response

(488 nm) models derived for each participant, for the 24 h testing period. The

baseline PIPR, minimum PIPR, ipRGC activity decrease and ipRGC activity

minimum all display physiological inter-individual variation. The individual

minimum circadian variation of the PIPR displayed is 6.75 % while the maximum is

20.87 %. The onset time of ipRGC activity decrease also varies between 5:52 h and

13:49 for individual participants.


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Table 4.6. Participant (n = 11) post-illumination pupil response amplitudes (488 nm); and

ipRGC activity onset, ipRGC activity peak and melatonin peak times, with respect to the

melatonin onset time of 14 h **. Values are derived from individual participants skewed baseline

cosine functions.

Participant Baseline PIPR %

Minimum PIPR %

PIPR Difference %

ipRGC activity decrease * (h:min) **

ipRGC activity minimum (h:min) **

Melatonin peak (h:min) **

30, F 81.88 91.51 9.63 -0:11 +1:02 +4:41

31, F 91.63 99.35 7.71 -2:17 +0:13 +5:32

19, F 72.87 93.74 20.87 -4:34 +3:19 +4:05

27, F 96.04 98.20 2.16 -2:08 -0:37 +3:14

27, F 84.11 99.42 15.31 -1:39 +1:20 +7:54

30, F 79.43 86.18 6.75 -4:35 -2:29 +3:22

24, M 84.24 97.50 13.26 -1:01 +0:29 +5:05

21, F 73.58 80.67 7.10 -1:48 +1:33 +6:18

18, M 71.53 91.22 19.69 -5:33 +1:18 +3:58

26, M 84.47 96.51 12.04 -0:25 +1:17 +3:57

24, M 89.42 103.69 14.27 -8:08 -0:22 +3:08

NOTE: Values derived from best-fitting skewed baseline cosine functions (Equation 4.1) to

individual participants PIPR and melatonin data (individual graphs shown in Figure 7.6).

* Threshold decrease in ipRGC activity (model inflection point) defined as 0.01 % rise above

baseline PIPR model value.

** Times are circadian with melatonin onset set to 14:00 h.

4.4 DISCUSSION

Using a custom developed pupillometer and analysis protocols, the cone

photoreceptor, cone-mediated and intrinsic ipRGC contributions to the pupil light

reflex were successfully isolated (Figure 4.4 and 4.6). It was determined that cone

contributions to the PLR do not demonstrate circadian variation over the 24 h

period (Figure 4.4). In contrast, both the cone-mediated inputs to the ipRGCs and

the intrinsic ipRGC activity displayed statistically significant circadian variation

(p < 0.001), demonstrating that ipRGCs mediate this circadian rhythm (Figure 4.6).

It was established that the group average ipRGC circadian response is 2:46 h in

phase advance of the central SCN rhythm, determined from the onset of melatonin


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secretion, and minimum ipRGC activity occurs 1:25 h after melatonin onset

(Table 4.5, Column 1).

4.4.1 The isolation of the inner and outer retinal responses

The first aim of this study was to isolate cone-mediated (outer retinal) and ipRGC

(inner retinal) responses using the pupil light reflex. The post-illumination pupil

response is a direct measure of the ipRGC response (Gamlin, et al., 2007). Here the

PIPR was used to determine the ipRGC photopigment melanopsin Vitamin A

pigment nomogram and confirm it as a direct measure of ipRGC activity

(Figure 3.8). The recorded melanopsin spectral sensitivity (λmax = 482 nm) of this

study is in agreement with previous in vivo and in vitro measurements in humans

(Dacey, et al., 2005; Gamlin, et al., 2007), successfully confirming the PIPR as a

direct measure of ipRGC activity under our experimental conditions. The cone-

mediated (610 nm) and intrinsic (488 nm) ipRGC responses were isolated with the

PIPR, and maximum pupil constriction was used to measure the direct cone (outer

retinal) activity.

4.4.2 Circadian variation in ipRGC but not cone activity

4.4.2.1 Circadian variation in ipRGC activity

The second aim of this study was to determine if the cone, intrinsic ipRGC and

cone-mediated inputs to ipRGCs display circadian variation over a 24 hour period.

The major finding of this research was that both intrinsic and cone-mediated ipRGC

activity demonstrated significant circadian variation, under conditions of constant

illumination (Figure 4.6). As exogenous factors such as light (Lewy, Cutler & Sack,

1999), physical activity (Monteleone, et al., 1990), sleep (Zeitzer, et al., 2007),

posture (Deacon & Arendt, 1994), caffeine (Wright, et al., 1997; Shilo, et al., 2002),

medications (Murphy, et al., 1996; Stoschitzky, et al., 1999) and evening caloric

intake (Kräuchi, et al., 2002) can mask endogenous circadian rhythm, the 24 hour

constant routine protocol controlled for these factors. The intrinsic ipRGC activity

displayed a decrease from 11:14 h; with minimum activity occurring at 15:25 h for

the group model. Cone-mediated ipRGC activity demonstrated a circadian phase

which is synchronized to the intrinsic ipRGC activity, decreasing from 9:40 h and

with minimum activity at 15:42 h for the group model. This confirmed our


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hypothesis that ipRGC activity as measured via the post-illumination pupil response

demonstrates a circadian rhythm.

The onset times in this research are dependent on the model used to fit the circadian

variation of the PIPR data. The skewed baseline cosine function used in this study

was chosen as the best fitting model. Future research may consider other models

when fitting the circadian variation of the PIPR, and this may alter the onset time.

The intrinsic ipRGC activity demonstrated a significant difference between the first

and second pupil measurement each hour, while the cone-mediated ipRGC activity

did not. As cone input to ipRGCs is transient compared to the intrinsic ipRGC

photoresponse (Dacey, et al., 2005) this may be the result of these different

temporal response dynamics. The first hourly measurement was always recorded for

a 488 nm stimulus (intrinsic ipRGC activity), followed by the randomized

presentation of a second 488 nm and two 610 nm stimuli (cone-mediated ipRGC

activity) (Figure 4.1). The second baseline pupil diameter measurement was always

reduced. Kankipati, et al., (2010) has demonstrated a smaller baseline pupil results

in a smaller PIPR. This would also explain the significant difference between the

first and second PIPR measurement each hour, detected for the 488 nm but not the

610 nm stimuli.

This significant circadian variation in ipRGC activity is in contrast to the only

previous study of ipRGC circadian activity, recorded in rats (Weng, et al., 2009).

Electrophysiological recordings of in vitro rat ipRGCs demonstrated a non-

significant evening increase in the light-induced ipRGC firing rate (Weng, et al.,

2009), when ipRGCs were isolated from the SCN for one hour prior. This increase

in the ipRGC firing rate correlates to the nocturnal rats’ active night-time phase. In

diurnal humans the greatest ipRGC spiking would be expected in the day, consistent

with the greater daytime ipRGC activity recorded in this study. If ipRGC activity is

under central SCN rather than local retinal control this would explain why the

Weng, et al., (2009) in vitro study did not demonstrate significant circadian

variation, compared to our in vivo finding of a circadian rhythm. Additionally, we

demonstrated circadian variation in the cone-mediated ipRGC activity, which was


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not examined by Weng who pharmacologically blocked rod and cone input to

ipRGCs.

4.4.2.2 No circadian variation in direct cone activity

Direct cone activity, determined from maximum constriction and baseline pupil

diameter, did not demonstrate circadian variation over the 24 hour period under

constant conditions (Figure 4.4). Baseline pupil diameter is a measure of rod, cone

and ipRGC input; however due to saturation, rods had a very minor contribution to

the pupil diameter when viewing the photopic illuminated fixation screen.

McDougal and Gamlin, (2010) recently showed a photic stimulus duration > 10

seconds is required for ipRGCs to contribute to the spectral sensitivity of the half-

maximal baseline pupil diameter. Under photopic illumination, cone input drives

the baseline pupil diameter until ~ 10 seconds when ipRGCs begin to dominate

(McDougal & Gamlin, 2010). A larger ipRGC contribution would be expected for a

stimulus duration longer than 10 seconds or after light offset. In this research the 10

second baseline pupil diameter was recorded 1 – 2 seconds after the photopic screen

was illuminated, and was primarily a measure of cone activity with a small rod and

ipRGC contribution.

The relative contribution of rods, cones and ipRGCs to the baseline pupil diameter

depends on the illumination of the viewing conditions (McDougal & Gamlin,

2010). For the 116 cd.m-2 background luminance of our pupillometer (above the

ipRGC threshold) ipRGCs have a 3:1 relative contribution to the baseline pupil

compared to cones (Tsujimura, Ukai, Ohama, Nuruki & Yunokuchi, 2010). In this

experiment the baseline pupil diameter was recorded every hour, while participants

viewed a 4° black cross on a uniform photopic screen backlit to 116 cd.m-2 for 10

seconds, after prior adaptation to 10 lux room illumination. For photopic conditions,

the contribution of cone photoreceptors to the baseline pupil diameter was likely to

be greatest during the first 10 seconds recorded with this paradigm (McDougal &

Gamlin, 2010).

In contrast to our result, a significant circadian variation of recorded baseline pupil

diameter was reported previously (Kraemer, et al., 2000; Wilhelm, et al., 2001) for


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prolonged (10 - 11 minute) recordings. These recordings were conducted in

scotopic conditions after prior dark adaptation where pupil diameter and circadian

photoentrainment (Altimus, et al., 2010) are predominantly driven by rods. As our

experiment used a photopic fixation screen any circadian variation in rod activity

would not significantly impact on our baseline pupil recording. In concert with our

results, Loving, et al., (1996) also failed to demonstrate a circadian rhythm in the

baseline pupil diameter. The study of Loving, et al., (1996) was conducted under

< 5 lux red illumination where a cone contribution to the baseline pupil is likely.

Taken together with our findings, the evidence demonstrates a possible circadian

variation baseline pupil activity when dominated by rod activity (Kraemer, et al.,

2000; Wilhelm, et al., 2001) which drives photoentrainment for stimuli below the

ipRGC threshold (Altimus, et al., 2010), but not when cones provide the major

input to baseline diameter (Loving, et al., 1996).

Further evidence that cone activity does not display circadian variation is derived

from our maximum constriction results. Cone photoreceptors drive the rapid pupil

constriction to a maximum pupil diameter (< 1.5 sec) in response to light, while the

ipRGCs of the inner retina do not contribute due to a slower latency of > 1.78 sec

(Dacey, et al., 2005; McDougal & Gamlin, 2010). The mean constriction amplitude

was 2.73 % greater for the 610 nm compared to the 488 nm stimulus, a result which

is consistent with the spectral sensitivity of the pupil reflex (Alpern & Campbell,

1962). This research demonstrated no circadian variation in the maximum

constriction, as previously demonstrated by Ranzijn and Lack, (1997). The results

did display a small, non-circadian increase in maximum constriction diameter of

0.19 %.h-1 and 0.26 %.h-1, for the 488 and 610 nm stimulus respectively, over 24

hours.

Our study demonstrates the pupil light reflex produced by an identical stimulus

becomes less extensive over a 24 hour period. Very few investigations have

examined the pupil constriction diameter of the PLR for circadian variation, with

previous research focussed instead on the effect of fatigue with repeated measures.

Lowenstein and Loewenfeld (Lowenstein & Loewenfeld, 1951, 1952a, 1952b,

1964) reported reduced PLR constriction amplitudes for a series of repeated


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measures in fatigued participants which corresponds to the reduced constriction

displayed by our participants.

We hypothesize that our experimental increase in maximum constriction diameter is

due to cumulative task demand. During this study participants became progressively

more sleep deprived, and required increased concentration to maintain fixation. The

result was a 4.6 % (488 nm) and 6.2 % (610 nm) increase in maximum constriction

diameter for the 24 hours period. Research by Steinhauer, et al., (2004) provides

confirmation that increased task difficulty causes pupil dilation, due to cortical

inhibition of the parasympathetic pathway at the Edinger-Westphal nucleus.

In summary, this research isolated and studied the direct cone, cone-mediated

ipRGC and intrinsic ipRGC activity over the 24 hour circadian period under

controlled illumination. The results demonstrate there is no circadian variation in

the outer retina (cone) driven baseline pupil diameter or maximum constriction

diameter components of the PLR. In contrast both the intrinsic and cone-mediated

ipRGC driven post-illumination response display a diurnal variation in activity,

confirming the second hypothesis that ipRGC activity demonstrates a circadian

rhythm but cone contribution to the pupil light reflex does not.

4.4.2.3 Control of ipRGC circadian variation

The SCN is the master pacemaker of the body and synchronizes the intrinsic

rhythms of peripheral oscillators throughout the body, such as the liver, lungs,

kidneys and retina (Yamazaki, et al., 2000; Yoo, et al., 2004; Pickard & Sollars,

2008). Many retinal functions display circadian rhythms (Bassi & Powers, 1986;

Sandberg, et al., 1986) and some, such as retinal melatonin synthesis and clock gene

expression, are entrained by light in vitro (Cahill & Besharse, 1993; Tosini &

Menaker, 1996; Ruan, Allen, Yamazaki & McMahon, 2008) requiring no cortical

input. This raised the possibility that ipRGC activity might be controlled by a local

retinal oscillator, but the results of our study are inconsistent with this proposal.


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Figure 4.7. A functional model of the ipRGC, cone-mediated ipRGC and conventional retinal

ganglion cells contributions to the SCN and OPN (black dashed arrows), and the hypothesized

site/s of action of SCN inhibitory feedback (red arrows). Maximum pupil constriction is driven by

direct cone input transmitted to the OPN via conventional RGCs. The PIPR and circadian

photoentrainment are driven by ipRGC and cone-mediated ipRGC input transmitted to the OPN and

SCN.

Instead we infer that the central pacemaker controls the mechanism by which

ipRGC photic signalling is inhibited. Figure 4.7 displays the hypothesized site/s of

action of possible central feedback mechanisms. The SCN could provide inhibitory

feedback directly to the ipRGCs to reduce firing rate, via retinal clock gene

expression. Alternatively, SCN controlled neurotransmitters could act on post-

retinal pathways to attenuate ipRGC signal transmission. The central feedback

gating mechanism would need to specifically target ipRGCs in the inner retina, and

not the outer retinal cone photoreceptors that do not demonstrate a circadian

variation. IpRGCs differ from outer retinal photoreceptors in photopigment, retinal


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location and post-retinal pathways (see Table 2.1) and an ipRGC gating mechanism

should act on an ipRGC-specific process. Possible mechanisms for the specific

gating of ipRGC activity include neuromodulation of melanopsin expression, or

inhibition of ipRGC-dominated signals at synapses of the post-retinal pathway to

the SCN, with one possible site at the post-retinal pathway-SCN synapse. It is

hypothesised that no circadian variation occurs in the outer retinal cone contribution

to the pupil because cones signal primarily to the visual cortex for image formation,

with less contribution to the SCN for circadian photoentrainment and to the pupil

pathway.

The central SCN controlled mechanism driving the phase-advanced, circadian

ipRGC activity may operate via negative feedback loops, which oscillate protein

concentrations or gene expression levels (Pigolotti, Krishna & Jensen, 2007). The

presence of circadian oscillations in ipRGC photopigment mRNA and protein levels

(González-Menéndez, et al., 2009) adds support to this idea. The hypothesis of this

thesis, displayed in Figure 4.7, is that unknown neuroendocrine agents act directly

on ipRGCs or the ipRGC activity transmission pathway to the SCN. Such a SCN

feedback mechanism may utilise neuroendocrine agents acting directly on ipRGCs

to decrease ipRGC firing rate. This is supported by the non-significant but small

evening increase in the light-induced ipRGC firing rate demonstrated in rats (Weng,

et al., 2009). As rats are nocturnal while humans are diurnal, a decrease in the

human ipRGC firing rate would be consistent with the research of Weng, et al.,

(2009). Alternatively a SCN feedback loop could utilise unknown neuromodulators

to inhibit ipRGC signals at synapses along the post-retinal pathway to the SCN,

such as the post-retinal pathway-SCN synapse. A neural mechanism is less likely

than an endocrine mechanism, as at present there is no known neural pathway from

the SCN to the retina. This study establishes the time course of cortical feedback

mechanisms that modulate ipRGC activity, but the feedback mechanism remains

unknown.

4.4.3 Temporal synchrony of ipRGC and central SCN circadian rhythms

The third aim of this research was to determine if ipRGC activity was synchronized

with the central circadian rhythm, by demonstrating a temporal relationship in


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which ipRGC activity onset always preceded melatonin onset. Our research

confirmed the ipRGC circadian phase is synchronized to, and is in phase advance

of, the central SCN rhythm by 2:46 h, as determined from the onset of melatonin

secretion. The minimum ipRGC activity follows melatonin onset 1:25 h later. Since

the ipRGC rhythm is under SCN control, the previous 24 hours of light exposure

will drive the ipRGC circadian phase in advance of melatonin secretion. A decrease

in the ipRGC signal to the SCN, independent of environmental light, could assist in

driving the sleep/wake cycle. The attenuation of ipRGC activity would reduce the

overall photic input to the SCN independent of the environmental light levels. As

light acutely suppresses melatonin (Lewy, et al., 1980), it is hypothesised that a yet

to be determined net decrease in photic input to the SCN may be needed to trigger

melatonin release or to phase shift the SCN. The attenuated ipRGC signals to the

SCN may also assist in driving melatonin release under conditions of seasonal

variation. The SCN can phase shift in response to changes in the light-dark cycle,

and its flexibility may be assisted by circadian variation in the photic input from

ipRGCs.

Conclusions and Future Studies/Directions

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Chapter 5.


5.1 CONCLUSIONS

This thesis addressed important pending questions of if intrinsically photosensitive

retinal ganglion cell activity displayed circadian variation and if there was temporal

synchrony of diurnal variation in ipRGC activity with the SCN. It also broached the

issue of whether this rhythm was controlled by the local retinal oscillator or the

central SCN clock.

AIM 1: To isolate the cone-mediated (outer retinal) and intrinsic ipRGC (inner

retinal) contributions to the pupil light reflex (PLR).

HYPOTHESIS 1: Direct cone, cone-mediated ipRGC and intrinsic ipRGC

contributions to the PLR will be measured with the custom-built experimental

apparatus under our laboratory conditions, using stimuli with appropriately chosen

wavelengths and irradiance.

To examine these research questions a purpose built Maxwellian view pupillometer

with custom designed analysis software was developed. Figure 3.8 and 3.9 display

our ipRGC spectral sensitivity results, derived from the post-illumination pupil

response, which are consistent with published reports (Dacey, et al., 2005; Gamlin,

et al., 2007) and confirm the isolation of ipRGC activity. The direct cone, cone-

mediated ipRGC (inner retina) and intrinsic ipRGC (outer retina) contributions to

the PLR were all successfully isolated, utilising the PLR components of baseline

diameter, maximum constriction diameter and the post-illumination pupil response.

AIM 2a: To determine if the direct cone photoreceptor, intrinsic ipRGC and/or

cone-mediated input to the ipRGCs demonstrate circadian variation over a 24 hour

period in their contributions to the pupil light reflex.

HYPOTHESIS 2a: The intrinsic ipRGC and the cone-mediated photoreceptor

contributions to the ipRGC driven post-illumination pupil response will


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demonstrate a circadian rhythm but direct cone inputs to the maximum pupil

constriction will not.

The intrinsic ipRGC activity and the cone-mediated ipRGC activity, as measured

via the post-illumination pupil response, demonstrated circadian rhythms (Figure

4.5 and 4.6), unlike the direct cone activity measured via baseline and maximum

constriction pupil diameters (Figure 4.4). Weng, et al., (2009) demonstrated no

significant in vitro circadian variation in ipRGC activity; whereas our in vivo results

do show a circadian rhythm in intrinsic and cone-mediated ipRGC activity. This

supports our hypothesis that ipRGCs are not controlled by a local retinal oscillator,

but instead are under central cortical control.

AIM 2b: To determine the temporal synchrony of cone and/or ipRGC diurnal

variation to the central circadian rhythm as measured using melatonin.

HYPOTHESIS 2b: IpRGC activity as measured via the post-illumination pupil

response will be temporally synchronized with the central circadian rhythm as


The circadian variation of ipRGC activity was temporally synchronized to the

central SCN circadian phase, measured via melatonin secretion. The greatest

attenuation of intrinsic ipRGC activity occurred 1:25 h after the onset of melatonin

secretion, with ipRGC activity beginning to reduce 2:46 h prior to melatonin onset

(Figure 4.6c). The ipRGC circadian variation could result from inhibitory feedback

from the SCN which may decrease the ipRGC spike frequency or inhibit

transmission at a synapse along the post-retinal pathway to the SCN, with one

possible site at the post-retinal pathway-SCN synapse (Figure 4.7). The post-

illumination pupil response has the potential to be developed as a non-invasive

measure of circadian function.

5.2 FURTHER STUDY

This research has demonstrated in human participants that ipRGC signals

demonstrate a circadian rhythm, independent of extrinsic environmental light levels

and this rhythm is in temporal phase advance of central SCN function. There are a


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number of issues that this present study raises that can be considered in future

experiments. These issues include forced desynchrony research, unknown cortical

feedback mechanisms, “blue-enriched” irradiance for night-time environments and

clinical applications of the post-illumination pupil response.

Confirmation of the central SCN control of the ipRGC circadian phase could be

demonstrated using a forced desynchrony sleep laboratory protocol. In a controlled

environment the light/dark cycle is changed to a period outside the range of

entrainment for several weeks, causing desynchrony between the sleep-wake cycle

and the free running intrinsic SCN circadian rhythm. If the ipRGC circadian rhythm

is under strict intrinsic SCN control it should remain temporally synchronized to the

free running SCN rhythm, but not to the sleep/wake times. Future research may also

determine the feedback mechanism of SCN controlled circadian changes in ipRGC

activity.

The use of “blue-enriched” (460 nm) light has been demonstrated to improve

alertness and performance (Lockley, Evans, Scheer, Brainard, Czeisler &

Aeschbach, 2006; Viola, James, Schlangen & Dijk, 2008). Our results demonstrate

that an increased irradiance (~ 482 nm) is required in the evening to produce an

equivalent post-illumination pupil response as occurs in the daytime (Figure 4.5). It

is unknown if increased irradiance (~ 482 nm) would also be required in the

evening to produce an equivalent alerting effect as in the daytime, and this has

implications for determining the “blue-enriched” irradiance for night-time

environments.

The current methods for measuring circadian rhythm include blood, urine or saliva

testing, which are invasive, costly and time-intensive. Shift work is becoming

increasingly prevalent with 16 % of all Australian employees (1.4 million) doing

shiftwork (Australian Bureau of Statistics, November, 2009) and potentially

suffering from disrupted circadian rhythms. In addition the aetiologies of many

sleep disorders are unknown (Sack, et al., 2007). Further investigation of the

temporal phase synchrony of melatonin onset and ipRGC activity could increase the

resolution of melatonin onset times derived from the post-illumination pupil


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response. We propose the post-illumination pupil response as a non-invasive tool to

determine the circadian phase of the body clock and to predict the onset of

melatonin secretion.

To develop the PIPR as a clinical tool, the physiological variability of ipRGCs

needs to be studied within the larger population. A recent preliminary study

recorded an average PIPR of 1.5 mm (SEM 0.1) with a range of 0.5 - 2.3 mm

(~ 7 - 41 % of baseline pupil diameter) for 37 participants (60°, 10 sec, 470 nm,

13 log photons.cm-2.s-1 stimulus at the retina), indicating some physiological

variation exists (Kankipati, et al., 2010) which was confirmed in our study.

The post-illumination pupil response may also be developed for retinal disease

detection and monitoring of progression. The aetiologies of some outer and inner

retinal diseases are unknown and the PIPR may be used to differentiate between

inner and outer retinal damage, as discussed by Markwell, et al., (2010). Damage to

ipRGCs may cause a reduction in ipRGC photic input to the SCN, although the

SCN may continue to entrain with the less powerful circadian cues of activity and

meals. Young patients with optic nerve disease demonstrate increased wake-time

instability and daytime napping, compared to equivalently vision impaired patients

with healthy optic nerves (Wee & Van Gelder, 2004). Evidence of increased

prevalence of sleep-disordered breathing and obstructive sleep apnoea (OSA) in

primary open angle glaucoma patients is inconclusive (Onen, Mouriaux,

Berramdane, Dascotte, Kulik & Rouland, 2000; Geyer, Cohen, Segev, Rath,

Melamud, Peled & Lavie, 2003; Bendel, Kaplan, Heckman, Fredrickson & Lin,

2008). The assessment of ipRGC activity in patients with inner retinal diseases is

needed to discover how ipRGC loss impacts on the circadian rhythm and the

sleep/wake cycle. : (Boll, 1877; Crawford, 1949; Rushton, 1959 ; Marks, Dobelle & MacNichol, 1964; Smith &

Pokorny, 1975; Curcio, Sloan, Kalina & Hendrickson, 1990; Daw, Jensen & Brunken, 1990; Schneeweis & Schnapf,

1995; Dacey, Lee, Stafford, Pokorny & Smith, 1996; Provencio, et al., 2000; Calkins, 2001; Dacey, et al., 2005; Dacey,

et al., 2006) (Wilhelm, et al., 2001) and Kraemer (Kraemer, et al., 2000) (Berson, 2003; McDougal & Gamlin, 2008)

(Steinhauer, et al., 2004) (Ranzijn & Lack, 1997). (Markwell, et al., 2010).(Smith & Pokorny, 1975; Challet, 2007)

References

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Chapter 6. References

Age-Related Eye Disease Study Research Group. (2001). The age-related eye disease study (AREDS) system for classifying cataracts from photographs: AREDS report no. 4. American Journal of Ophthalmology, 131:(2), 167-175.

Alpern, M., & Campbell, F. W. (1962). The spectral sensitivity of the consensual

light reflex. The Journal of Physiology, 164, 478-507. Altimus, C. M., Güler, A. D., Alam, N. M., Arman, A. C., Prusky, G. T., Sampath,

A. P., & Hattar, S. (2010). Rod photoreceptors drive circadian photoentrainment across a wide range of light intensities. Nature

Neuroscience, 13:(9), 1107-1112. Ancoli-Israel, S., Cole, R., Alessi, C., Chambers, M., Moorcroft, W., & Pollak, C.

P. (2003). The role of actigraphy in the study of sleep and circadian rhythms. Sleep, 26:(3), 342-392.

Anderson, D. H., Fisher, S. K., Erickson, P. A., & Tabor, G. A. (1980). Rod and

cone disc shedding in the rhesus monkey retina: a quantitative study. Experimental Eye Research, 30:(5), 559-574.

Australian Bureau of Statistics. (November, 2009). Working time arrangements,

Australia (cat no. 6342.0). from http://www.ausstats.abs.gov.au/ Bär, K.-J., Schulz, S., Koschke, M., Harzendorf, C., Gayde, S., Berg, W., Voss, A.,

Yeragani, V. K., & Boettger, M. K. (2009). Correlations between the autonomic modulation of heart rate, blood pressure and the pupillary light reflex in healthy subjects. Journal of the Neurological Sciences, 279:(1-2), 9-13.

Barbur, J. L., Harlow, A. J., & Sahraie, A. (1992). Pupillary responses to stimulus

structure, colour and movement. Ophthalmic and Physiological Optics,

12:(2), 137-141. Barlow, R. B., Jr, & Verrillo, R. T. (1976). Brightness sensation in a ganzfeld.

Vision Research, 16:(11), 1291-1297. Bartlett, J. R., & Doty, R. W., Sr. (1974). Response of units in striate cortex of

squirrel monkeys to visual and electrical stimuli. Journal of

Neurophysiology, 37:(4), 621-641. Bassi, C. J., & Powers, M. K. (1986). Daily fluctuations in the detectability of dim

lights by humans. Physiology and Behavior, 38:(6), 871-877.

References

__________________________________________________________________ 80

Baver, S. B., Pickard, G. E., Sollars, P. J., & Pickard, G. E. (2008). Two types of melanopsin retinal ganglion cell differentially innervate the hypothalamic suprachiasmatic nucleus and the olivary pretectal nucleus. The European

Journal of Neuroscience, 27:(7), 1763-1770. Beatty, J. (1982). Phasic not tonic pupillary responses vary with auditory vigilance

performance. Psychophysiology, 19:(2), 167-172. Belenky, M. A., Smeraski, C. A., Provencio, I., Sollars, P. J., & Pickard, G. E.

(2003). Melanopsin retinal ganglion cells receive bipolar and amacrine cell synapses. The Journal of Comparative Neurology, 460:(3), 380-393.

Bendel, R. E., Kaplan, J., Heckman, M., Fredrickson, P. A., & Lin, S.-C. (2008).

Prevalence of glaucoma in patients with obstructive sleep apnoea - a cross-sectional case-series. Eye, 22:(9), 1105-1109.

Benloucif, S., Burgess, H. J., Klerman, E. B., Lewy, A. J., Middleton, B., Murphy,

P. J., Parry, B. L., & Revell, V. L. (2008). Measuring melatonin in humans. Journal of Clinical Sleep Medicine, 4:(1), 66-69.

Benloucif, S., Guico, M. J., Reid, K. J., Wolfe, L. F., L'Hermite-Balériaux, M., &

Zee, P. C. (2005). Stability of melatonin and temperature as circadian phase markers and their relation to sleep times in humans. Journal of Biological

Rhythms, 20:(2), 178-188. Bergamin, O., & Kardon, R. H. (2003). Latency of the pupil light reflex: Sample

rate, stimulus intensity, and variation in normal subjects. Investigative

Ophthalmology and Visual Science, 44:(4), 1546-1554. Berson, D. M. (2003). Strange vision: Ganglion cells as circadian photoreceptors.

Trends in Neurosciences, 26:(6), 314-320. Berson, D. M., Dunn, F. A., & Takao, M. (2002). Phototransduction by retinal

ganglion cells that set the circadian clock. Science, 295:(5557), 1070-1073. Birch, D. G., Berson, E. L., & Sandberg, M. A. (1984). Diurnal rhythm in the

human rod ERG. Investigative Ophthalmology and Visual Science, 25:(2), 236-238.

Bloom, P. A., Papakostopoulos, D., Gogolitsyn, Y., Leenderz, J. A.,

Papakostopoulos, S., & Grey, R. H. B. (1993). Clinical and infrared pupillometry in central retinal vein occlusion. The British Journal of

Ophthalmology, 77:(2), 75-80. Boll, F. (1877). Zur anatomie und physiologie der retina. Archiv für Anatomie und

Physiologie, Physiol Abt, 4-35. Boyd, T. A., & McLeod, L. E. (1964). Circadian rhythms of plasma corticoid levels,

intraocular pressure and aqueous outflow facility in normal and

References

__________________________________________________________________ 81

glaucomatous eyes. Annals of the New York Academy of Sciences, 117, 597-613.

Bradley, M. M., Miccoli, L., Escrig, M. A., & Lang, P. J. (2008). The pupil as a

measure of emotional arousal and autonomic activation. Psychophysiology,

45:(4), 602-607. Burnstock, G., & Sillito, A. M. (1999). Nervous control of the eye. In Burnstock, G.

(Ed.), The Autonomic Nervous System (Vol. 13). Amsterdam: Harwood Academic.

Buysse, D. J., Reynolds, C. F., III., Monk, T. H., Berman, S. R., & Kupfer, D. J.

(1989). The Pittsburgh sleep quality index: A new instrument for psychiatric practice and research. Psychiatry Research, 28:(2), 193-213.

Cahill, G. M., & Besharse, J. C. (1993). Circadian clock functions localized in

Xenopus retinal photoreceptors. Neuron, 10:(4), 573-577. Calkins, D. J. (2001). Seeing with S cones. Progress in Retinal and Eye Research,

20:(3), 255-287. Cao, D., Pokorny, J., Smith, V. C., & Zele, A. J. (2008). Rod contributions to color

perception: Linear with rod contrast. Vision Research, 48:(26), 2586-2592. Challet, E. (2007). Minireview: Entrainment of the suprachiasmatic clockwork in

diurnal and nocturnal mammals. Endocrinology, 148:(12), 5648-5655. Claustrat, B., Brun, J., & Chazot, G. (2005). The basic physiology and

pathophysiology of melatonin. Sleep Medicine Reviews, 9:(1), 11-24. Crawford, B. H. (1949). The scotopic visibility function. Proceedings of the

Physical Society Section B, 62, 321-334. Curcio, C. A., Sloan, K. R., Kalina, R. E., & Hendrickson, A. E. (1990). Human

photoreceptor topography. The Journal of Comparative Neurology, 292:(4), 497-523.

Czeisler, C. A., Duffy, J. F., Shanahan, T. L., Brown, E. N., Mitchell, J. F., Rimmer,

D. W., Ronda, J. M., Silva, E. J., Allan, J. S., Emens, J. S., Dijk, D.-J., & Kronauer, R. E. (1999). Stability, precision, and near-24-hour period of the human circadian pacemaker. Science, 284:(5423), 2177-2181.

Czeisler, C. A., Kronauer, R. E., Allan, J. S., Duffy, J. F., Jewett, M. E., Brown, E.

N., & Ronda, J. M. (1989). Bright light induction of strong (type 0) resetting of the human circadian pacemaker. Science, 244:(4910), 1328-1333.

Dacey, D. M. (1993). Morphology of a small-field bistratified ganglion cell type in

the macaque and human retina. Visual Neuroscience, 10:(6), 1081-1098.

References

__________________________________________________________________ 82

Dacey, D. M., Joo, H. R., Peterson, B. B., & Haun, T. J. (2010). Morphology, mosaics and targets of diverse ganglion cell populations in macaque monkey retina: Approaching a complete account. Investigative Ophthalmology and

Visual Science, 51:(5), ARVO E-abstract 889. Dacey, D. M., Lee, B. B., Stafford, D. K., Pokorny, J., & Smith, V. C. (1996).

Horizontal cells of the primate retina: Cone specificity without spectral opponency. Science, 271:(5249), 656-659.

Dacey, D. M., Liao, H. W., Peterson, B. B., Robinson, F. R., Smith, V. C., Pokorny,

J., Yau, K. W., & Gamlin, P. D. (2005). Melanopsin-expressing ganglion cells in primate retina signal colour and irradiance and project to the LGN. Nature, 433:(7027), 749-754.

Dacey, D. M., & Petersen, M. R. (1992). Dendritic field size and morphology of

midget and parasol ganglion cells of the human retina. Proceedings of the

National Academy of Sciences of the United States of America, 89:(20), 9666-9670.

Dacey, D. M., Peterson, B. B., Liao, H.-W., & Yau, K.-W. (2006). Two types of

melanopsin-containing ganglion cells in the primate retina: Links to dopaminergic amacrine and DB6 cone bipolar cells. Investigative

Ophthalmology and Visual Science, 47, ARVO E-Abstract 3111. Dacey, D. M., Peterson, B. B., Robinson, F. R., & Gamlin, P. D. (2003). Fireworks

in the primate retina: in vitro photodynamics reveals diverse LGN-projecting ganglion cell types. Neuron, 37:(1), 15-27.

Dartnall, H. J. A. (1953). The interpretation of spectral sensitivity curves. British

Medical Bulletin, 9:(1), 24-30. Daw, N. W., Jensen, R. J., & Brunken, W. J. (1990). Rod pathways in mammalian

retinae. Trends in Neurosciences, 13:(3), 110-115. Deacon, S., & Arendt, J. (1994). Posture influences melatonin concentrations in

plasma and saliva in humans. Neuroscience Letters, 167:(1-2), 191-194. DeVries, S. H., & Baylor, D. A. (1995). An alternative pathway for signal flow

from rod photoreceptors to ganglion cells in mammalian retina. Proceedings

of the National Academy of Sciences of the United States of America,

92:(23), 10658-10662. Do, M. T. H., Kang, S. H., Xue, T., Zhong, H., Liao, H.-W., Bergles, D. E., & Yau,

K.-W. (2009). Photon capture and signalling by melanopsin retinal ganglion cells. Nature, 457:(7227), 281-287.

Dollet, A., Albrecht, U., Cooper, H. M., & Dkhissi-Benyahya, O. (2010). Cones are

required for normal temporal responses to light of phase shifts and clock gene expression. Chronobiology International, 27:(4), 768-781.

References

__________________________________________________________________ 83

Doyle, S. E., Grace, M. S., McIvor, W., & Menaker, M. (2002). Circadian rhythms of dopamine in mouse retina: The role of melatonin. Visual Neuroscience,

19:(5), 593-601. Duffy, J. F., & Dijk, D.-J. (2002). Getting through to circadian oscillators: Why use

constant routines? Journal of Biological Rhythms, 17:(1), 4-13. Dumitrescu, O. N., Pucci, F. G., Wong, K. Y., & Berson, D. M. (2009). Ectopic

retinal ON bipolar cell synapses in the OFF inner plexiform layer: Contacts with dopaminergic amacrine cells and melanopsin ganglion cells. The

Journal of Comparative Neurology, 517:(2), 226-244. Ecker, J. L., Dumitrescu, O. N., Wong, K. Y., Alam, N. M., Chen, S.-K., LeGates,

T., Renna, J. M., Prusky, G. T., Berson, D. M., & Hattar, S. (2010). Melanopsin-expressing retinal ganglion-cell photoreceptors: Cellular diversity and role in pattern vision. Neuron, 67:(1), 49-60.

Figueiro, M. G., Bullough, J. D., Parsons, R. H., & Rea, M. S. (2005). Preliminary

evidence for a change in spectral sensitivity of the circadian system at night. Journal of Circadian Rhythms, 3, 14-14.

Fosnaugh, J. S., Bunker, E. B., & Pickworth, W. B. (1992). Daily variation and

effects of ambient light and circadian factors on the human light reflex. Methods and Findings In Experimental and Clinical Pharmacology, 14:(7), 545-553.

Fotiou, D. F., Brozou, C. G., Tsiptsios, D. J., Fotiou, A., Kabitsi, A., Nakou, M.,

Giantselidis, C., & Goula, A. (2007). Effect of age on pupillary light reflex: Evaluation of pupil mobility for clinical practice and research. Electromyography and Clinical Neurophysiology, 47:(1), 11-22.

Fu, Y., Zhong, H., Wang, M.-H. H., Luo, D.-G., Liao, H.-W., Maeda, H., Hattar, S.,

Frishman, L. J., & Yau, K.-W. (2005). Intrinsically photosensitive retinal ganglion cells detect light with a vitamin A-based photopigment, melanopsin. Proceedings of the National Academy of Sciences of the United

States of America, 102:(29), 10339-10344. Gamlin, P. D. R., McDougal, D. H., Pokorny, J., Smith, V. C., Yau, K.-W., &

Dacey, D. M. (2007). Human and macaque pupil responses driven by melanopsin-containing retinal ganglion cells. Vision Research, 47:(7), 946-954.

Geyer, O., Cohen, N., Segev, E., Rath, E. Z., Melamud, L., Peled, R., & Lavie, P.

(2003). The prevalence of glaucoma in patients with sleep apnea syndrome: Same as in the general population. American Journal of Ophthalmology,

136:(6), 1093-1096. González-Menéndez, I., Contreras, F., Cernuda-Cernuda, R., & García-Fernández,

J. M. (2009). Daily rhythm of melanopsin-expressing cells in the mouse retina. Frontiers in Cellular Neuroscience, 3:(3).

References

__________________________________________________________________ 84

Gooley, J. J., Lu, J., Chou, T. C., Scammell, T. E., & Saper, C. B. (2001). Melanopsin in cells of origin of the retinohypothalamic tract. Nature

Neuroscience, 4:(12), 1165-1165. Gooley, J. J., Lu, J., Fischer, D., & Saper, C. B. (2003). A broad role for melanopsin

in nonvisual photoreception. The Journal of Neuroscience, 23:(18), 7093-7106.

Gooley, J. J., Rajaratnam, S. M. W., Brainard, G. C., Kronauer, R. E., Czeisler, C.

A., & Lockley, S. W. (2010). Spectral responses of the human circadian system depend on the irradiance and duration of exposure to light. Science

Translational Medicine, 2:(31), 31ra33. Granholm, E., Asarnow, R. F., Sarkin, A. J., & Dykes, K. L. (1996). Pupillary

responses index cognitive resource limitations. Psychophysiology, 33:(4), 457-461.

Gur, M. (1989). Color and brightness fade-out in the ganzfeld is wavelength

dependent. Vision Research, 29:(10), 1335-1341. Hankins, M. W., Jones, R. J. M., & Ruddock, K. H. (1998). Diurnal variation in the

b-wave implicit time of the human electroretinogram. Visual Neuroscience,

15:(1), 55-67. Hankins, M. W., Jones, S. R., Jenkins, A., & Morland, A. B. (2001). Diurnal

daylight phase affects the temporal properties of both the b-wave and d-wave of the human electroretinogram. Brain Research, 889:(1-2), 339-343.

Hannibal, J., Georg, B., Hindersson, P., & Fahrenkrug, J. (2005). Light and

darkness regulate melanopsin in the retinal ganglion cells of the albino Wistar rat. Journal of Molecular Neuroscience, 27:(2), 147-155.

Hannibal, J., Hindersson, P., Knudsen, S. M., Georg, B., & Fahrenkrug, J. (2002).

The photopigment melanopsin is exclusively present in pituitary adenylate cyclase-activating polypeptide-containing retinal ganglion cells of the retinohypothalamic tract. The Journal of Neuroscience, 22:(RC191), 1-7.

Hattar, S., Kumar, M., Park, A., Tong, P., Tung, J., Yau, K.-W., & Berson, D. M.

(2006). Central projections of melanopsin-expressing retinal ganglion cells in the mouse. The Journal of Comparative Neurology, 497:(3), 326-349.

Hattar, S., Liao, H.-W., Takao, M., Berson, D. M., & Yau, K.-W. (2002).

Melanopsin-containing retinal ganglion cells: Architecture, projections, and intrinsic photosensitivity. Science, 295:(5557), 1065-1070.

Hattar, S., Lucas, R. J., Mrosovsky, N., Thompson, S., Douglas, R. H., Hankins, M.

W., Lem, J., Biel, M., Hofmann, F., Foster, R. G., & Yau, K.-W. (2003). Melanopsin and rod-cone photoreceptive systems account for all major accessory visual functions in mice. Nature, 424:(6944), 76-81.

References

__________________________________________________________________ 85

Hecht, S., Haig, C., & Chase, A. M. (1937). The influence of light adaptation on subsequent dark adaptation of the eye. The Journal of General Physiology,

20, 831-850. Hendrickson, A. E., Wagoner, N., & Cowan, W. M. (1972). An autoradiographic

and electron microscopic study of retino-hypothalamic connections. Zeitschrift für Zellforschung und Mikroskopische Anatomie, 135:(1), 1-26.

Hess, E. H., & Polt, J. M. (1964). Pupil size in relation to mental activity during

simple problem-solving. Science, 143:(3611), 1190-1192. Hofstra, W. A., & de Weerd, A. W. (2008). How to assess circadian rhythm in

humans: A review of literature. Epilepsy and Behavior, 13:(3), 438-444. Hoshi, H., Liu, W.-L., Massey, S. C., & Mills, S. L. (2009). ON inputs to the OFF

layer: Bipolar cells that break the stratification rules of the retina. The

Journal of Neuroscience, 29:(28), 8875-8883. Hunter, J. D., Milton, J. G., Lüdtke, H., Wilhelm, B., & Wilhelm, H. (2000).

Spontaneous fluctuations in pupil size are not triggered by lens accommodation. Vision Research, 40:(5), 567-573.

Inouye, S. T., & Kawamura, H. (1979). Persistence of circadian rhythmicity in a

mammalian hypothalamic "island" containing the suprachiasmatic nucleus. Proceedings of the National Academy of Sciences of the United States of

America, 76:(11), 5962-5966. Iuvone, P. M., Tosini, G., Pozdeyev, N., Haque, R., Klein, D. C., & Chaurasia, S. S.

(2005). Circadian clocks, clock networks, arylalkylamine N-acetyltransferase, and melatonin in the retina. Progress in Retinal and Eye

Research, 24:(4), 433-456. Jagota, A., de la Iglesia, H. O., & Schwartz, W. J. (2000). Morning and evening

circadian oscillations in the suprachiasmatic nucleus in vitro. Nature

Neuroscience, 3:(4), 372-376. Jusuf, P. R., Lee, S. C. S., Hannibal, J., & Grünert, U. (2007). Characterization and

synaptic connectivity of melanopsin-containing ganglion cells in the primate retina. The European Journal of Neuroscience, 26:(10), 2906-2921.

Kankipati, L., Girkin, C. A., & Gamlin, P. D. (2010). Post-illumination pupil

response in subjects without ocular disease. Investigative Ophthalmology

and Visual Science, 51:(5), 2764-2769. Kardon, R., Anderson, S. C., Damarjian, T. G., Grace, E. M., Stone, E., &

Kawasaki, A. (2009). Chromatic pupil responses: Preferential activation of the melanopsin-mediated versus outer photoreceptor-mediated pupil light reflex. Ophthalmology, 116:(8), 1564-1573.

References

__________________________________________________________________ 86

Khalsa, S. B. S., Jewett, M. E., Cajochen, C., & Czeisler, C. A. (2003). A phase response curve to single bright light pulses in human subjects. The Journal

of Physiology, 549, 945-952. Koevary, S. B. (2003). Pharmacokinetics of topical ocular drug delivery: potential

uses for the treatment of diseases of the posterior segment and beyond. Current Drug Metabolism, 4:(3), 213-222.

Koyanagi, M., Kubokawa, K., Tsukamoto, H., Shichida, Y., & Terakita, A. (2005).

Cephalochordate melanopsin: Evolutionary linkage between invertebrate visual cells and vertebrate photosensitive retinal ganglion cells. Current

Biology, 15:(11), 1065-1069. Koyanagi, M., & Terakita, A. (2008). Gq-coupled rhodopsin subfamily composed

of invertebrate visual pigment and melanopsin. Photochemistry and

Photobiology, 84:(4), 1024-1030. Kraemer, S., Danker-Hopfe, H., Dorn, H., Schmidt, A., Ehlert, I., & Herrmann, W.

M. (2000). Time-of-day variations of indicators of attention: Performance, physiologic parameters, and self-assessment of sleepiness. Biological

Psychiatry, 48:(11), 1069-1080. Kräuchi, K. (2002). How is the circadian rhythm of core body temperature

regulated? Clinical Autonomic Research, 12:(3), 147-149. Kräuchi, K., Cajochen, C., Werth, E., & Wirz-Justice, A. (2002). Alteration of

internal circadian phase relationships after morning versus evening carbohydrate-rich meals in humans. Journal of Biological Rhythms, 17:(4), 364-376.

Lahdes, K. K., Huupponen, R. K., & Kaila, T. J. (1994). Ocular effects and

systemic absorption of cyclopentolate eyedrops after canthal and conventional application. Acta Ophthalmologica, 72, 698-702.

Lall, G. S., Revell, V. L., Momiji, H., Al Enezi, J., Altimus, C. M., Güler, A. D.,

Aguilar, C., Cameron, M. A., Allender, S., Hankins, M. W., & Lucas, R. J. (2010). Distinct contributions of rod, cone, and melanopsin photoreceptors to encoding irradiance. Neuron, 66:(3), 417-428.

Lamb, T. D., & Pugh, E. N., Jr. (2004). Dark adaptation and the retinoid cycle of

vision. Progress in Retinal and Eye Research, 23:(3), 307-380. LaVail, M. M. (1976). Rod outer segment disk shedding in rat retina: Relationship

to cyclic lighting. Science, 194:(4269), 1071-1074. LaVail, M. M. (1980). Circadian nature of rod outer segment disc shedding in the

rat. Investigative Ophthalmology and Visual Science, 19:(4), 407-411.

References

__________________________________________________________________ 87

Lee, S. C. S., & Grünert, U. (2007). Connections of diffuse bipolar cells in primate retina are biased against S-cones. The Journal of Comparative Neurology,

502:(1), 126-140. Lee, S. C. S., Jusuf, P. R., & Grünert, U. (2004). S-cone connections of the diffuse

bipolar cell type DB6 in macaque monkey retina. The Journal of

Comparative Neurology, 474:(3), 353-363. Lewy, A. J., Cutler, N. L., & Sack, R. L. (1999). The endogenous melatonin profile

as a marker for circadian phase position. Journal of Biological Rhythms,

14:(3), 227-236. Lewy, A. J., Wehr, T. A., Goodwin, F. K., Newsome, D. A., & Markey, S. P.

(1980). Light suppresses melatonin secretion in humans. Science,

210:(4475), 1267-1269. Lockley, S. W., Evans, E. E., Scheer, F. A. J. L., Brainard, G. C., Czeisler, C. A., &

Aeschbach, D. (2006). Short-wavelength sensitivity for the direct effects of light on alertness, vigilance, and the waking electroencephalogram in humans. Sleep, 29:(2), 161-168.

Loewenfeld, I. E. (1999). The Pupil: Anatomy, physiology and clinical applications

(Vol. 1). Boston: Butterworth-Heinemann. Loving, R. T., Kripke, D. F., & Glazner, L. K. (1996). Circadian rhythms in the

human pupil and eyelid. American Journal of Physiology. Regulatory,

Integrative and Comparative Physiology, 271:(2), R320-324. Lowenstein, O., & Loewenfeld, I. E. (1951). Types of central autonomic

innervation and fatigue: Pupillographic studies. A. M. A. Archives of

Neurology and Psychiatry, 66:(5), 580-599. Lowenstein, O., & Loewenfeld, I. E. (1952a). Disintegration of central autonomic

regulation during fatigue and its reintegration by psychosensory controlling mechanisms. I. Disintegration; pupillographic studies. The Journal of

Nervous and Mental Disease, 115:(1), 1-21. Lowenstein, O., & Loewenfeld, I. E. (1952b). Disintegration of central autonomic

regulation during fatigue and its reintegration by psychosensory controlling mechanisms. II. Reintegration; pupillographic studies. The Journal of

Nervous and Mental Disease, 115:(2), 121-145. Lowenstein, O., & Loewenfeld, I. E. (1964). The sleep-waking cycle and pupillary

activity. Annals of the New York Academy of Sciences, 117, 142-156. Lucas, R. J. (2006). Chromophore regeneration: Melanopsin does its own thing.

Proceedings of the National Academy of Sciences of the United States of

America, 103:(27), 10153–10154.

References

__________________________________________________________________ 88

Lucas, R. J., Hattar, S., Takao, M., Berson, D. M., Foster, R. G., & Yau, K. W. (2003). Diminished pupillary light reflex at high irradiances in melanopsin-knockout mice. Science, 299:(5604), 245-247.

Marks, W. B., Dobelle, W. H., & MacNichol, E. F., Jr. (1964). Visual pigments of

single primate cones. Science, 143:(3611), 1181-1183. Markwell, E. L., Feigl, B., & Zele, A. J. (2010). Intrinsically photoreceptive

melanopsin retinal ganglion cell contributions to the pupil light reflex and circadian rhythm. Clinical and Experimental Optometry, 93:(3), 137-149.

Martin, P. R. (2004). Colour through the thalamus. Clinical & Experimental

Optometry, 87:(4-5), 249-257. Mata, N. L., Radu, R. A., Clemmons, R. S., & Travis, G. H. (2002). Isomerization

and oxidation of vitamin A in cone-dominant retinas: A novel pathway for visual-pigment regeneration in daylight. Neuron, 36:(1), 69-80.

Mathes, A., Engel, L., Holthues, H., Wolloscheck, T., & Spessert, R. (2007). Daily

profile in melanopsin transcripts depends on seasonal lighting conditions in the rat retina. Journal of Neuroendocrinology, 19:(12), 952-957.

McDougal, D. H., & Gamlin, P. D. (2010). The influence of intrinsically

photosensitive retinal ganglion cells on the spectral sensitivity and response dynamics of the human pupillary light reflex. Vision Research, 50:(1), 72-87.

McDougal, D. H., & Gamlin, P. D. R. (2008). Pupillary control pathways. In

Masland, R. H. & Albright, T. (Eds.), The Senses: A Comprehensive

Reference (Vol. 1, pp. 521-536). Oxford: Academic Press. Melyan, Z., Tarttelin, E. E., Bellingham, J., Lucas, R. J., & Hankins, M. W. (2005).

Addition of human melanopsin renders mammalian cells photoresponsive. Nature, 433:(7027), 741-745.

Monk, T. H., Reynolds, C. F., III., Kupfer, D. J., Buysse, D. J., Coble, P. A., Hayes,

A. J., Machen, M. A., Petrie, S. R., & Ritenour, A. M. (1994). The Pittsburgh sleep diary. Journal of Sleep Research, 3, 111-120.

Monteleone, P., Maj, M., Fusco, M., Orazzo, C., & Kemali, D. (1990). Physical

exercise at night blunts the nocturnal increase of plasma melatonin levels in healthy humans. Life Sciences, 47:(22), 1989-1995.

Morin, L. P., Blanchard, J. H., & Provencio, I. (2003). Retinal ganglion cell

projections to the hamster suprachiasmatic nucleus, intergeniculate leaflet, and visual midbrain: Bifurcation and melanopsin immunoreactivity. The

Journal of Comparative Neurology, 465:(3), 401-416.

References

__________________________________________________________________ 89

Mure, L. S., Cornut, P.-L., Rieux, C., Drouyer, E., Denis, P., Gronfier, C., & Cooper, H. M. (2009). Melanopsin bistability: A fly's eye technology in the human retina. PLoS ONE, 4:(6), e5991.

Murphy, P. J., Myers, B. L., & Badia, P. (1996). Nonsteroidal anti-inflammatory

drugs alter body temperature and suppress melatonin in humans. Physiology

and Behavior, 59:(1), 133-139. Naka, K. I., & Rushton, W. A. H. (1966). S-potentials from colour units in the

retina of fish (Cyprinidae). The Journal of Physiology, 185:(3), 536-555. Namihira, M., Honma, S., Abe, H., Masubuchi, S., Ikeda, M., & Honmaca, K.

(2001). Circadian pattern, light responsiveness and localization of rPer1 and rPer2 gene expression in the rat retina. Neuroreport, 12:(3), 471-475.

Nassi, J. J., & Callaway, E. M. (2009). Parallel processing strategies of the primate

visual system. Nature Reviews. Neuroscience, 10:(5), 360-372. Nir, I., Haque, R., & Iuvone, P. M. (2000). Diurnal metabolism of dopamine in the

mouse retina. Brain Research, 870:(1-2), 118-125. O'Keefe, L. P., & Baker, H. D. (1987). Diurnal changes in human psychophysical

luminance sensitivity. Physiology and Behavior, 41:(3), 193-200. O'Neill, W. D., & Zimmerman, S. (2000). Neurological interpretations and the

information in the cognitive pupillary response. Methods of Information in

Medicine, 39:(2), 122-124. Onen, S. H., Mouriaux, F., Berramdane, L., Dascotte, J.-C., Kulik, J.-F., &

Rouland, J.-F. (2000). High prevalence of sleep-disordered breathing in patients with primary open-angle glaucoma. Acta Ophthalmologica

Scandinavica, 78:(6), 638-641. Österberg, G. (1935). Topography of the layer of rods and cones in the human

retina. Acta Ophthalmologica. Supplement, 6, 11-97. Ostergaard, J., Hannibal, J., & Fahrenkrug, J. (2007). Synaptic contact between

melanopsin-containing retinal ganglion cells and rod bipolar cells. Investigative Ophthalmology and Visual Science, 48:(8), 3812-3820.

Panda, S., Nayak, S. K., Campo, B., Walker, J. R., Hogenesch, J. B., & Jegla, T.

(2005). Illumination of the melanopsin signaling pathway. Science,

307:(5709), 600-604. Pandi-Perumal, S. R., Smits, M., Spence, W., Srinivasan, V., Cardinali, D. P.,

Lowe, A. D., & Kayumov, L. (2007). Dim light melatonin onset (DLMO): A tool for the analysis of circadian phase in human sleep and chronobiological disorders. Progress in Neuro-psychopharmacology and

Biological Psychiatry, 31:(1), 1-11.

References

__________________________________________________________________ 90

Patsiopoulos, G., Lam, V., Lake, S., & Koevary, S. B. (2003). Insulin and tropicamide accumulate in the contralateral, untreated eye of rats following ipsilateral topical administration by a mechanism that does not involve systematic uptake. Optometry, 74:(4), 226-232.

Pickard, G. E. (1985). Bifurcating axons of retinal ganglion cells terminate in the

hypothalamic suprachiasmatic nucleus and the intergeniculate leaflet of the thalamus. Neuroscience Letters, 55:(2), 211-217.

Pickard, G. E., & Sollars, P. J. (2008). The suprachiasmatic nucleus. In Masland, R.

H. & Albright, T. (Eds.), The Senses: A Comprehensive Reference (Vol. 1, pp. 537-555). Oxford: Academic Press.

Pigolotti, S., Krishna, S., & Jensen, M. H. (2007). Oscillation patterns in negative

feedback loops. Proceedings of the National Academy of Sciences of the

United States of America, 104:(16), 6533-6537. Piha, S. J., & Halonen, J.-P. (1994). Infrared pupillometry in the assessment of

autonomic function. Diabetes Research and Clinical Practice, 26:(1), 61-66. Pokorny, J., Lutze, M., Cao, D., & Zele, A. J. (2006). The color of night: Surface

color perception under dim illuminations. Visual Neuroscience, 23, 525-530. Provencio, I., Jiang, G., De Grip, W. J., Hayes, W. P., & Rollag, M. D. (1998).

Melanopsin: An opsin in melanophores, brain, and eye. Proceedings of the


Provencio, I., Rodriguez, I. R., Jiang, G., Hayes, W. P., Moreira, E. F., & Rollag,

M. D. (2000). A novel human opsin in the inner retina. The Journal of

Neuroscience, 20:(2), 600-605. Provencio, I., Rollag, M. D., & Castrucci, A. M. (2002). Photoreceptive net in the

mammalian retina. Nature, 415, 493. Purkinje, J. (1825). Beobachtungen und Versuche zur Physiologie der Sinne Neue

Beiträge zur Kenntniss des Sehens in Subjectiver Hinsicht (pp. 192). Berlin: Reimer.

Qiu, X., Kumbalasiri, T., Carlson, S. M., Wong, K. Y., Krishna, V., Provencio, I., &

Berson, D. M. (2005). Induction of photosensitivity by heterologous expression of melanopsin. Nature, 433:(7027), 745-749.

Ranzijn, R., & Lack, L. (1997). The pupillary light reflex cannot be used to measure

sleepiness. Psychophysiology, 34:(1), 17-22. Ruan, G.-X., Allen, G. C., Yamazaki, S., & McMahon, D. G. (2008). An

autonomous circadian clock in the inner mouse retina regulated by dopamine and GABA. PLoS Biology, 6:(10), e249-e249.

References

__________________________________________________________________ 91

Ruan, G.-X., Zhang, D.-Q., Zhou, T., Yamazaki, S., & McMahon, D. G. (2006). Circadian organization of the mammalian retina. Proceedings of the


Ruby, N. F., Brennan, T. J., Xie, X., Cao, V., Franken, P., Heller, H. C., & O'Hara,

B. F. (2002). Role of melanopsin in circadian responses to light. Science,

298:(5601), 2211-2213. Rushton, W. A. H. (1959 ). Visual pigments in the intact human eye. Proceedings of

the National Academy of Sciences of the United States of America, 45:(1), 114–115.

Sack, R. L., Auckley, D., Auger, R. R., Carskadon, M. A., Wright, K. P., Jr.,

Vitiello, M. V., & Zhdanova, I. V. (2007). Circadian rhythm sleep disorders: Part II, advanced sleep phase disorder, delayed sleep phase disorder, free-running disorder, and irregular sleep-wake rhythm. Sleep, 30:(11), 1484-1501.

Sakamoto, K., Liu, C., & Tosini, G. (2004). Classical photoreceptors regulate

melanopsin mRNA levels in the rat retina. The Journal of Neuroscience,

24:(43), 9693-9697. Sandberg, M. A., Pawlyk, B. S., & Berson, E. L. (1986). Electroretinogram (ERG)

sensitivity and phagosome frequency in the normal pigmented rat. Experimental Eye Research, 43:(5), 781-789.

Schmidt, T. M., Taniguchi, K., & Kofuji, P. (2008). Intrinsic and extrinsic light

responses in melanopsin-expressing ganglion cells during mouse development. Journal of Neurophysiology, 100:(1), 371-384.

Schneeweis, D. M., & Schnapf, J. L. (1995). Photovoltage of rods and cones in the

macaque retina. Science, 268:(5213), 1053-1056. Sharpe, L. T., & Stockman, A. (1999). Rod pathways: The importance of seeing

nothing. Trends in Neurosciences, 22:(11), 497-504. Sherman, S. M. (2007). The thalamus is more than just a relay. Current Opinion in

Neurobiology, 17:(4), 417-422. Shilo, L., Sabbah, H., Hadari, R., Kovatz, S., Weinberg, U., Dolev, S., Dagan, Y., &

Shenkman, L. (2002). The effects of coffee consumption on sleep and melatonin secretion. Sleep Medicine, 3:(3), 271-273.

Skene, D. J., & Arendt, J. (2006). Human circadian rhythms: Physiological and

therapeutic relevance of light and melatonin. Annals of Clinical

Biochemistry, 43:(5), 344-353. Smith, V. C., & Pokorny, J. (1975). Spectral sensitivity of the foveal cone

photopigments between 400 and 500 nm. Vision Research, 15:(2), 161-171.

References

__________________________________________________________________ 92

Sollars, P. J., Smeraski, C. A., Kaufman, J. D., Ogilvie, M. D., Provencio, I., & Pickard, G. E. (2003). Melanopsin and non-melanopsin expressing retinal ganglion cells innervate the hypothalamic suprachiasmatic nucleus. Visual

Neuroscience, 20:(6), 601-610. Steinhauer, S. R., Siegle, G. J., Condray, R., & Pless, M. (2004). Sympathetic and

parasympathetic innervation of pupillary dilation during sustained processing. International Journal of Psychophysiology, 52:(1), 77-86.

Stoschitzky, K., Sakotnik, A., Lercher, P., Zweiker, R., Maier, R., Liebmann, P., &

Lindner, W. (1999). Influence of beta-blockers on melatonin release. European Journal of Clinical Pharmacology, 55:(2), 111-115.

Tassi, P., & Pins, D. (1997). Diurnal rhythmicity for visual sensitivity in humans?

Chronobiology International, 14:(1), 35-48. Telkes, I., Distler, C., & Hoffmann, K. P. (2000). Retinal ganglion cells projecting

to the nucleus of the optic tract and the dorsal terminal nucleus of the accessory optic system in macaque monkeys. The European Journal of

Neuroscience, 12:(7), 2367-2375. Terakita, A., Tsukamoto, H., Koyanagi, M., Sugahara, M., Yamashita, T., &

Shichida, Y. (2008). Expression and comparative characterization of Gq-coupled invertebrate visual pigments and melanopsin. Journal of

Neurochemistry, 105:(3), 883-890. Terman, J. S., Remé, C. E., & Terman, M. (1993). Rod outer segment disk shedding

in rats with lesions of the suprachiasmatic nucleus. Brain Research, 605:(2), 256-264.

Tiedt, N. (1963). The 24-hour rhythm of the kinetics of the light reflex in the human

pupil. Pflügers Archiv : European Journal of Physiology, 277, 458-472. Tosini, G., Davidson, A. J., Fukuhara, C., Kasamatsu, M., & Castanon-Cervantes,

O. (2007). Localization of a circadian clock in mammalian photoreceptors. The FASEB Journal 21:(14), 3866-3871.

Tosini, G., & Menaker, M. (1996). Circadian rhythms in cultured mammalian

retina. Science, 272:(5260), 419-421. Tosini, G., & Menaker, M. (1998). The clock in the mouse retina: Melatonin

synthesis and photoreceptor degeneration. Brain Research, 789:(2), 221-228.

Tosini, G., Pozdeyev, N., Sakamoto, K., & Iuvone, P. M. (2008). The circadian

clock system in the mammalian retina. Bioessays, 30:(7), 624-633. Tsujimura, S., Ukai, K., Ohama, D., Nuruki, A., & Yunokuchi, K. (2010).

Contribution of human melanopsin retinal ganglion cells to steady-state

References

__________________________________________________________________ 93

pupil responses. Proceedings of the Royal Society of London. Series B,

Biological Sciences, 277:(1693), 2485-2492. Tu, D. C., Owens, L. A., Anderson, L., Golczak, M., Doyle, S. E., McCall, M.,

Menaker, M., Palczewski, K., & Van Gelder, R. N. (2006). Inner retinal photoreception independent of the visual retinoid cycle. Proceedings of the


Tu, D. C., Zhang, D., Demas, J., Slutsky, E. B., Provencio, I., Holy, T. E., & Van

Gelder, R. N. (2005). Physiologic diversity and development of intrinsically photosensitive retinal ganglion cells. Neuron, 48:(6), 987-999.

Van Someren, E. J. W., & Nagtegaal, E. (2007). Improving melatonin circadian

phase estimates. Sleep Medicine, 8:(6), 590-601. Viney, T. J., Balint, K., Hillier, D., Siegert, S., Boldogkoi, Z., Enquist, L. W.,

Meister, M., Cepko, C. L., & Roska, B. (2007). Local retinal circuits of melanopsin-containing ganglion cells identified by transsynaptic viral tracing. Current Biology, 17:(11), 981-988.

Viola, A. U., James, L. M., Schlangen, L. J. M., & Dijk, D.-J. (2008). Blue-enriched

white light in the workplace improves self-reported alertness, performance and sleep quality. Scandinavian Journal of Work, Environment and Health,

34:(4), 297-306. Voultsios, A., Kennaway, D. J., & Dawson, D. (1997). Salivary melatonin as a

circadian phase marker: Validation and comparison to plasma melatonin. Journal of Biological Rhythms, 12:(5), 457-466.

Walker, M. T., Brown, R. L., Cronin, T. W., & Robinson, P. R. (2008).

Photochemistry of retinal chromophore in mouse melanopsin. Proceedings

of the National Academy of Sciences of the United States of America,

105:(26), 8861-8865. Wang, J.-S., & Kefalov, V. J. (2009). An alternative pathway mediates the mouse

and human cone visual cycle. Current Biology, 19:(19), 1665-1669. Wee, R., & Van Gelder, R. N. (2004). Sleep disturbances in young subjects with

visual dysfunction. Ophthalmology, 111:(2), 297-302. Weng, S., Wong, K. Y., & Berson, D. M. (2009). Circadian modulation of

melanopsin-driven light response in rat ganglion-cell photoreceptors. Journal of Biological Rhythms, 24:(5), 391-402.

Wilhelm, B., Giedke, H., Lüdtke, H., Bittner, E., Hofmann, A., & Wilhelm, H.

(2001). Daytime variations in central nervous system activation measured by a pupillographic sleepiness test. Journal of Sleep Research, 10:(1), 1-7.

References

__________________________________________________________________ 94

Wilhelm, B., Wilhelm, H., Lüdtke, H., Streicher, P., & Adler, M. (1998). Pupillographic assessment of sleepiness in sleep-deprived healthy subjects. Sleep, 21:(3), 258-265.

Witkovsky, P., Veisenberger, E., LeSauter, J., Yan, L., Johnson, M., Zhang, D.-Q.,

McMahon, D., & Silver, R. (2003). Cellular location and circadian rhythm of expression of the biological clock gene Period 1 in the mouse retina. The

Journal of Neuroscience, 23:(20), 7670-7676. Wong, K. Y., Dunn, F. A., & Berson, D. M. (2005). Photoreceptor adaptation in

intrinsically photosensitive retinal ganglion cells. Neuron, 48:(6), 1001-1010.

Wright, K. P., Jr., Badia, P., Myers, B. L., Plenzler, S. C., & Hakel, M. (1997).

Caffeine and light effects on nighttime melatonin and temperature levels in sleep-deprived humans. Brain Research, 747:(1), 78-84.

Wright, K. P., Jr., Hughes, R. J., Kronauer, R. E., Dijk, D.-J., & Czeisler, C. A.

(2001). Intrinsic near-24-h pacemaker period determines limits of circadian entrainment to a weak synchronizer in humans. Proceedings of the National

Academy of Sciences of the United States of America, 98:(24), 14027-14032. Wu, B. X., Moiseyev, G., Chen, Y., Rohrer, B., Crouch, R. K., & Ma, J.-x. (2004).

Identification of RDH10, an all-trans retinol dehydrogenase, in retinal muller cells. Investigative Ophthalmology and Visual Science, 45:(11), 3857-3862.

Wyatt, J. K., Ritz-De Cecco, A., Czeisler, C. A., & Dijk, D.-J. (1999). Circadian

temperature and melatonin rhythms, sleep, and neurobehavioral function in humans living on a 20-h day. The American Journal of Physiology, 277:(4 Pt 2), R1152-1163.

Yamazaki, S., Numano, R., Abe, M., Hida, A., Takahashi, R., Ueda, M., Block, G.

D., Sakaki, Y., Menaker, M., & Tei, H. (2000). Resetting central and peripheral circadian oscillators in transgenic rats. Science, 288:(5466), 682-685.

Yoo, S.-H., Yamazaki, S., Lowrey, P. L., Shimomura, K., Ko, C. H., Buhr, E. D.,

Siepka, S. M., Hong, H.-K., Oh, W. J., Yoo, O. J., Menaker, M., & Takahashi, J. S. (2004). Period2::Luciferase real-time reporting of circadian dynamics reveals persistent circadian oscillations in mouse peripheral tissues. Proceedings of the National Academy of Sciences of the United

States of America, 101:(15), 5339-5346. Young, R. W. (1978). The daily rhythm of shedding and degradation of rod and

cone outer segment membranes in the chick retina. Investigative

Ophthalmology and Visual Science, 17:(2), 105-116.

References

__________________________________________________________________ 95

Zeitzer, J. M., Duffy, J. F., Lockley, S. W., Dijk, D.-J., & Czeisler, C. A. (2007). Plasma melatonin rhythms in young and older humans during sleep, sleep deprivation, and wake. Sleep, 30:(11), 1437-1443.

Zhang, D.-Q., Wong, K. Y., Sollars, P. J., Berson, D. M., Pickard, G. E., &

McMahon, D. G. (2008). Intraretinal signaling by ganglion cell photoreceptors to dopaminergic amacrine neurons. Proceedings of the


Appendices

__________________________________________________________________ 96

Chapter 7. Appendices

7.1 PUBLICATION

This appendix contains a published review article (Markwell, et al., 2010) which

summarises the recent literature on intrinsically photosensitive retinal ganglion

cells, their role in the pupil light reflex and circadian rhythm, and introduces a

clinical framework for using the pupillary light reflex to evaluate inner retinal

(ipRGC) and outer retinal (rod and cone photoreceptor) function in the detection of

retinal eye disease. Sections of text from this publication were included in

Chapter 2.

CLINICAL AND EXPERIMENTAL

OPTOMETRY

INVITED REVIEW

Intrinsically photosensitive melanopsin retinal ganglion cell contributions to the pupillary light reflex and circadian rhythm

Clin Exp Optom 2010; 93: 3: 137-149

Emma L Markwell BAppSci (Optom)

Beatrix Feigl MD PhD

Andrew J Zele PhD Visual Science and Medical Retina Laboratory, Institute of Health and

Biomedical Innovation and School of

Optometry, Queensland University of Technology, Brisbane, Queensland,

Australia E-mail: [email protected]

Submitted: 13 November 2010

Revised: 17 February 2010

Accepted for publication: 1 March 2010

DOI:10.111l/j.1444-0938.2010.00479.x

Recently discovered intrinsically photosensitive melanopsin retinal ganglion cells con

tribute to the maintenance of pupil diameter, recovery and post-illumination components of the pupillary light reflex and provide the primary environmental light input to

the suprachiasmatic nucleus for photoentrainment of the circadian rhythm. This review summarises recent progress in understanding intrinsically photosensitive ganglion cell

histology and physiological properties in the context of their contribution to the pupil

lary and circadian functions and introduces a clinical framework for using the pupillary light reflex to evaluate inner retinal (intrinsically photosensitive melanopsin ganglion

cell) and outer retinal (rod and cone photoreceptor) function in the detection of retinal

eye disease.

Key words: circadian rhythm, melanopsin, pupillary reflex, retinal disease, retinal ganglion cells, retinal photoreceptors

halla

Due to copyright restrictions, this article is not available here. Please consult the hardcopy thesis available from QUT Library

Appendices

__________________________________________________________________ 109

7.2 PITTSBURGH SLEEP QUALITY INDEX

ID # _________________ Date __________________

Instructions:

The following questions relate to your usual sleep habits during the past month only. Your

answers should indicate the most accurate reply for the majority of days and nights in the past

month.

Please answer all questions.

1. During the past month, when have you usually gone to bed at night?

BED TIME _______________

2. During the past month, how long (in minutes) has it usually taken you to fall asleep each night?

NUMBER OF MINUTES _______________

3. During the past month, when have you usually gotten up in the morning?

GETTING UP TIME _______________

4. During the past month, how many hours of actual sleep did you get at night? (This may be

different than the number of hours you spend in bed.)

HOURS OF SLEEP PER NIGHT _______________

For each of the remaining questions, check the one best response. Please answer all questions.

5. During the past month, how often have you had trouble sleeping because you…

a) Cannot get to sleep within 30 minutes

Not during the Less than Once or Three or more

past month ____ once a week ____ twice a week ____ times a week ____

b) Wake up in the middle of the night or early morning



c) Have to get up to use the bathroom



d) Cannot breathe comfortably



e) Cough or snore loudly



f) Feel too cold



Appendices

__________________________________________________________________ 110

During the past month, how often have you had trouble sleeping because you…

g) Feel too hot



h) Had bad dreams



i) Have pain



j) Other reason(s), please describe

_____________________________________________________________________

_____________________________________________________________________

How often during the past month have you had trouble sleeping because of this?



6. During the past month, how would you rate your sleep quality overall?

Very good __________

Fairly good __________

Fairly bad __________

Very bad __________

7. During the past month, how often have you taken medicine to help you sleep (prescribed or

“over the counter”)?



8. During the past month, how often have you had trouble staying awake while driving, eating

meals, or engaging in social activity?



9. During the past month, how much of a problem has it been for you to keep up enough

enthusiasm to get things done?

No problem at all ____________

Only a very slight problem ____________

Somewhat of a problem ____________

A very big problem ____________

© 1989, University of Pittsburgh. All rights reserved. Developed by Buysse,D.J., Reynolds,C.F., Monk,T.H. Berman,S.R., and Kupfer,D.J. of the University of Pittsburgh using National Institute of Mental Health Funding.

Buysse DJ, Reynolds CF, Monk TH, Berman SR, Kupfer DJ: Psychiatry Research, 28:193-213, 1989.

This form may only be used for non-commercial education and research purposes. If you would like to use this instrument for commercial purposes or for commercially sponsored research, please contact the Office of Technology Management at the University of Pittsburgh at 412-648-2206 for licensing information.

Appendices

__________________________________________________________________ 111

7.3 PITTSBURGH SLEEP DIARY

SLEEP DIARY BEDTIME KEEP BY BED Study ID# _______

Please fill out this part of the diary last thing at the night.

day __________ date __________

Today when did you have: breakfast __________

(if none, leave blank) lunch __________

dinner __________

How many of the following did you have in each time period?

(if none, leave blank)

Before

or with

breakfast

after breakfast

before/with

lunch

after lunch

before/with

dinner

after

dinner

caffeinated drinks ___________ ___________ ___________ ___________

alcoholic drinks ___________ ___________ ___________ ___________

cigarettes ___________ ___________ ___________ ___________

cigars/pipes/plugs

(of chewing tobacco) ___________ ___________ ___________ ___________

Which drugs and medications did you take today?

(prescribed & over the counter)

name time dose

______________________ ______________________ ______________________

______________________ ______________________ ______________________

______________________ ______________________ ______________________

______________________ ______________________ ______________________

What exercise did you take today? (if none, check here _______)

start: _______ end: _______ type: _________

start: _______ end: _______ type: _________

How many daytime naps did you take today? (if none, write 0) _________

start: _________ end: _________ start: _________ end: ________

Copyright 1991. University of Pittsburgh. All rights reserved. Developed by Monk,T.H., Reynolds,C.F., Kupfer,D.J. and Buysse,D.J., of the University of Pittsburgh using National Institute of Mental Health Funding. This form may only be used for non-commercial education and research purposes. If you would like to use this instrument for commercial purposes or for commercially sponsored research, please contact the Office of Technology Management at the University of Pittsburgh at 412-648-2206 for licensing information.

Appendices

__________________________________________________________________ 112

SLEEP DIARY WAKETIME KEEP BY BED

Please fill out this part of the diary first thing in the morning.

day __________ date __________

went to bed last night at _____________________

attempted to fall asleep _____________________

minutes until fell asleep _____________________

finally woke at _____________________

Awakened by (check one) alarm clock/radio

someone whom I asked to wake me

noises

just woke

After falling asleep, woke up this many times during the night (circle)

0 1 2 3 4 5 or more

total number of minutes awake ___________

- woke to use bathroom (circle # times

0 1 2 3 4 5 or more

- awakened by noises/child/bedpartner (circle # times)

0 1 2 3 4 5 or more

- awakened due to discomfort or physical complaint (circle # times)

0 1 2 3 4 5 or more

- just woke (circle # times)

0 1 2 3 4 5 or more

Ratings (place a mark somewhere along the line):

Sleep Quality:

very bad __________________________________________ very good

Mood on Final Wakening:

very tense __________________________________________ very calm

Alertness on Final Wakening

very sleepy __________________________________________ very alert

Copyright 1991. University of Pittsburgh. All rights reserved. Developed by Monk,T.H., Reynolds,C.F., Kupfer,D.J. and Buysse,D.J., of the University of Pittsburgh using National Institute of Mental Health Funding. This form may only be used for non-commercial education and research purposes. If you would like to use this instrument for commercial purposes or for commercially sponsored research, please contact the Office of Technology Management at the University of Pittsburgh at 412-648-2206 for licensing information.

________________________________

7.4 ACTIGRAPH

Figure 7.1. The actigraphic output of a 26 yo

trace) and motion (black trace) were recorded, and sleep periods (aqua bars) were identified by the

Actiware software. The final 24 hours contain no sleep period, as the testing protocol required

participants to remain awake.

________________________________________________________________

ACTIGRAPHY OUTPUT

. The actigraphic output of a 26 yo M participant over 8 days. Light exposure (yellow



nts to remain awake.

Appendices

__________________________________ 113

Light exposure (yellow



Appendices

__________________________________________________________________ 114

7.5 INDIVIDUAL PUPIL LIGHT REFLEX AND MELATONIN DATA

7.5.1 Baseline pupil diameter

Appendices

__________________________________________________________________ 115

Figure 7.2. Individual baseline pupil diameter data and models for the 11 participants

recorded over 20 - 24 hours. Each panel shows the response of one of the 11 participants. Pupil

light reflex (data, circles; linear model, line) recorded while participants viewed a 4° black cross on a

uniform photopic screen backlit to 116 cd.m-2. Pupil diameter (open circles) was determined using

custom-designed software and modelled with a linear function (black line). Pupil diameter is shown

in mm on the left axis and as a % of mean baseline pupil diameter on the right axes.

Appendices

__________________________________________________________________ 116

7.5.2 Maximum constriction pupil diameter (488 nm)

Appendices

__________________________________________________________________ 117

Figure 7.3. Individual maximum constricted pupil diameter data and models for the 11

participants recorded over 20 - 24 hours (488 nm). Each panel shows the response of one of the

11 participants. Pupil light reflex recorded for 11 participants over 20 – 24 hours, (data, circles;

linear model, line) for the 488 nm, 10 sec, 14.2 log photons.cm-2.s-1 test stimulus. Pupil diameter

(blue filled circles) was determined using custom-designed software (mean of 2 measurements) and

modelled with a linear function (blue line). Pupil diameter is shown in mm on the left axis and as a

% of mean baseline pupil diameter on the right axes.

Appendices

__________________________________________________________________ 118

Maximum constriction pupil diameter (610 nm)

Appendices

__________________________________________________________________ 119

Figure 7.4. Individual maximum constricted pupil diameter data and models for the 11

participants recorded over 20 - 24 hours (610 nm). Each panel shows the response of one of the

11 participants. Pupil light reflex recorded for 11 participants over 20 – 24 hours, (data, circles;

linear model, line) for the 610 nm, 10 sec, 14.2 log photons.cm-2.s-1 test stimulus. Pupil diameter (red

filled circles) was determined using custom-designed software (mean of 2 measurements) and

modelled with a linear function (red line). Pupil diameter is shown in mm on the left axis and as a %

of mean baseline pupil diameter on the right axes.

Appendices

__________________________________________________________________ 120

7.5.3 Post-illumination pupil response

Appendices

__________________________________________________________________ 121

Appendices

__________________________________________________________________ 122

Figure 7.5. Individual pupil light reflexes at three circadian times and the post-illumination

pupil response data and models, for the 11 participants. Each panel shows the response of one of

the 11 participants. Left panels: Pupil light reflex (thin traces) for 11 individual participants at three

circadian times and the best-fitting linear and exponential functions (thick lines). Right Panels:

Circadian variation in the post-illumination pupil response diameter (mean of two measurements

± SD) as a function of time of day fitted with a skewed baseline cosine function (Equation 4.1)

(black line). Insets show post-illumination pupil responses from left panels. Pupil diameter is shown

as a % of mean baseline pupil diameter on the left axes and in mm on the right axes.

Appendices

__________________________________________________________________ 123

Table 7.1.The mean parameters of the modelled intrinsic ipRGC activity determined by the

post-illumination pupil responses (488 nm stimuli). Pupil diameter is shown as % of average

baseline diameter. (n = 11)


Mean SD Min Max

Baseline (b) 83.50 82.66 7.91 71.53 96.04

Peak Height (H) 6.55 11.71 5.72 2.16 20.87

Width (c) 0.05 0.35 0.62 -0.76 0.86

Phase (Φ) 4.37 3.98 0.56 3.00 4.64

Skewness (υ) 0.35 0.20 0.76 -1.01 1.23

R2 0.65 0.36 0.26 0.07 0.79

Table 7.2. The mean parameters of the modelled cone-mediated ipRGC activity determined by

the post-illumination pupil responses (610 nm stimuli). Pupil diameter is shown as % of average

baseline diameter. (n = 11)


Mean SD Min Max

Baseline (b) 86.51 86.18 7.15 73.60 96.60

Peak Height (H) 6.78 11.85 5.75 2.62 24.42

Width (c) -0.62 0.22 0.64 -0.79 0.94

Phase (Φ) 4.55 4.54 0.35 3.95 5.01

Skewness (υ) 0.49 0.88 0.37 0.13 1.33

R2 0.80 0.41 0.30 0.10 0.97

Table 7.3. The mean parameters of the modelled salivary melatonin. (n = 11)


Mean SD Min Max

Baseline (b) ^ 4.62 4.55 ^ 0.67 4.30 ^ 6.54

Peak Height (H) 65.53 71.51 32.67 33.99 118.23

Width (c) -0.02 0.02 0.13 -0.19 0.27

Phase (Φ) 5.25 5.22 0.11 5.03 5.41

Skewness (υ) 0.30 0.42 0.45 -0.44 1.04

R2 0.96 0.96 0.04 0.88 1.00

^ Sensitivity of salivary melatonin assay was 4.3 picomoles per litre (pM)

NOTE FOR ALL: Group model derived from the mean data of participants fitted with a SBCF

(Equation 4.1). Individual models derived from the individual participant functions.

Appendices

__________________________________________________________________ 124

7.5.4 Post-illumination pupil response (488 nm) and melatonin

Appendices

__________________________________________________________________ 125

Figure 7.6. Individual post-illumination pupil response (488 nm) and salivary melatonin data

and models for the 11 participants recorded over 20 - 24 hours. Each panel shows the response

of one of the 11 participants. Pupil light reflex recorded for the 488 nm, 10 sec, 14.2

log photons.cm-2.s-1 stimulus. Pupil diameter (blue filled circles) was determined using custom-

designed software (mean of 2 measurements ± SD). Pupil data and normalised salivary melatonin

(open triangles) modelled with skewed baseline cosine function (Equation 4.1) (pupil, blue line;

melatonin, black dashed line). Pupil diameter is shown in mm on the left axis and normalised

salivary melatonin on the right axes.

intrinsically photosensitive melanopsin retinal ganglion...

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