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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
__________________________________________________________________ i
Keywords
Keywords: intrinsically photosensitive Retinal Ganglion Cells (ipRGC),
melanopsin, pupil light reflex, post-illumination pupil response, cone photoreceptor,
rod photoreceptor, circadian rhythm, melatonin
Abstract
__________________________________________________________________ ii
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
__________________________________________________________________ iii
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
__________________________________________________________________ iv
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
__________________________________________________________________ v
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
__________________________________________________________________ vi
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
__________________________________________________________________ vii
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
__________________________________________________________________ viii
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
__________________________________________________________________ ix
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
__________________________________________________________________ x
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
__________________________________________________________________ xi
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
__________________________________________________________________ xii
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
__________________________________________________________________ xiii
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
__________________________________________________________________ 1
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
__________________________________________________________________ 2
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
__________________________________________________________________ 3
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
__________________________________________________________________ 4
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
__________________________________________________________________ 5
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).
Literature Review Chapter 2
__________________________________________________________________ 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 &
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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).
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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
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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).
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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.
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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,
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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
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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
Literature Review Chapter 2
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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).
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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).
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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
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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
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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
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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.
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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
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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
Experimental Methods Chapter 3
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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).
Chapter 3 Experimental Methods
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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.
Experimental Methods Chapter 3
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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
Chapter 3 Experimental Methods
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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.
Experimental Methods Chapter 3
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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.
Chapter 3 Experimental Methods
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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.
Experimental Methods Chapter 3
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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.
Chapter 3 Experimental Methods
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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)
Experimental Methods Chapter 3
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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.
Chapter 3 Experimental Methods
__________________________________________________________________ 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.
Experimental Methods Chapter 3
__________________________________________________________________ 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.
Chapter 3 Experimental Methods
__________________________________________________________________ 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.
Experimental Methods Chapter 3
__________________________________________________________________ 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
Chapter 3 Experimental Methods
__________________________________________________________________ 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.
Experimental Methods Chapter 3
__________________________________________________________________ 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
Chapter 3 Experimental Methods
__________________________________________________________________ 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
Experimental Methods Chapter 3
__________________________________________________________________ 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,
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
__________________________________________________________________ 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
measured via salivary melatonin.
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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
__________________________________________________________________ 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).
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
__________________________________________________________________ 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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
__________________________________________________________________ 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
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
__________________________________________________________________ 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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
__________________________________________________________________ 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,
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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.
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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.
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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.
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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,
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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).
Group Model Individual Models
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.
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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.
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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Table 4.4. Participant (n = 11) post-illumination pupil response amplitudes for the 488 nm and
610 nm stimuli.
Group Model Individual Models
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.
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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).
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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.
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
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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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
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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
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
__________________________________________________________________ 71
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.
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
__________________________________________________________________ 72
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
Chapter 4 Investigation of circadian variation of the ipRGC Pupil Response
__________________________________________________________________ 73
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
Investigation of circadian variation of the ipRGC Pupil Response Chapter 4
__________________________________________________________________ 74
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
__________________________________________________________________ 75
Chapter 5.
Conclusions and Future Studies/Directions
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
Conclusions and Future Studies/Directions
__________________________________________________________________ 76
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
measured via salivary melatonin.
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
Conclusions and Future Studies/Directions
__________________________________________________________________ 77
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
Conclusions and Future Studies/Directions
__________________________________________________________________ 78
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)
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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
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
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
c) Have to get up to use the bathroom
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
d) Cannot breathe comfortably
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
e) Cough or snore loudly
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
f) Feel too cold
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
Appendices
__________________________________________________________________ 110
During the past month, how often have you had trouble sleeping because you…
g) Feel too hot
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
h) Had bad dreams
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
i) Have pain
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
j) Other reason(s), please describe
_____________________________________________________________________
_____________________________________________________________________
How often during the past month have you had trouble sleeping because of this?
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
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”)?
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
8. During the past month, how often have you had trouble staying awake while driving, eating
meals, or engaging in social activity?
Not during the Less than Once or Three or more
past month ____ once a week ____ twice a week ____ times a week ____
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
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
nts to remain awake.
Appendices
__________________________________ 113
Light exposure (yellow
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
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)
Group Model Individual Models
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)
Group Model Individual Models
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)
Group Model Individual Models
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.