The role of vasopressin in light-induced c-Fos expression in the SCN

SCHOOL OF BIOMEDICAL SCIENCES
The role of vasopressin in light-induced
c-Fos expression in the SCN
Jane Chapman
s0942737
Supervisor: Prof Mike Ludwig
This project was carried out jointly with Andrew Allchorne. The report was written
independently, and contains my own work except where otherwise stated.
Signed: _____________________

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Contents
i. Abstract 2
ii. Lay Summary 2
1. Introduction 3
1.1. Co-ordination of circadian rhythms 3
1.2. Entrainment to lighting cycles 5
1.3. Shifting of circadian rhythms with light pulses and
c-Fos expression 6
1.4. Vasopressin and light-induced c-Fos expression 7
2. Materials and Methods 8
2.1. Animals 8
2.2. Procedures 8
2.3. Tissue preparation 8
2.4. Fos protein immunohistochemistry 9
2.5. Immunocytochemical data analysis 9
3. Results 10
3.1. Fos expression in the SCN 10
3.2. Absence of effect of stress on Fos expression in the SCN 13
4. Discussion 14
4.1. Expression of c-Fos in the SCN 15
4.2. Vasopressin has no effect on light-induced c-Fos
expression in the SCN 16
4.3. Stress has no effect on c-Fos expression in the SCN 16
4.4. Future directions 17
5. Conclusion 18
Acknowledgements 18
Reference List 19
Abbreviations
CT circadian time SCN suprachiasmatic
CREB cAMP responsive element binding protein nucleus
GABA ɣ-aminobutyric acid SOM somatostatin
GHT geniculohypothalamic tract VP vasopressin
GRP gastrin-releasing peptide VIP vasoactive intestinal
IGL intergeniculate leaflet polypeptide
ICV intracerebroventricular
LGN lateral geniculate nucleus
NMDA N-methyl-D-aspartate
NPY neuropeptide Y
PACAP pituitary adenylate-cyclase-activating polypeptide
PVN paraventricular nucleus
RGC retinal ganglion cell
RHT retinohypothalamic tract
ROI region of interest

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Abstract
i. Abstract
The suprachiasmatic nucleus (SCN) is responsible for the orchestration of
mammalian circadian rhythms. These rhythms are synchronised (entrained) to
lighting cycles by the activity of retinal ganglion cells which project to the SCN
and alter neuronal activity here. Nocturnal light exposure causes rapid induction
of the immediate early gene c-Fos in the SCN, implicated in shifting these
rhythms. Recently, a population of the retinal projections to the SCN have been
discovered to express the neuropeptide vasopressin (VP). However, the
physiological significance of these VP-releasing neurons remains unknown. In
this study, rats were pre-treated with either a VP antagonist (OPC21268) or
vehicle prior to a 1-hour light exposure during the end of the subjective night
phase of the rat. Light-induced Fos immunoreactivity was seen in rat SCN from
each treatment group, with no significant decrease in Fos labelling from those
given the VP antagonist. This suggests that VP released from these retinal
neurons is not essential for the generation of light-induced c-Fos expression in
the SCN and therefore, in synchronising the SCN to light cues.
ii. Lay Summary
Many features of mammalian physiology and behaviour display circadian
rhythmicity, including sleep, physical activity and hormone levels. These self-
sustained biological rhythms are controlled by a system that includes a master
pacemaker (or clock). This creates an endogenous periodicity of around 24
hours, and a mechanism of entrainment, which adjusts this rhythmic period to
exactly 24 hours using lighting cycles from the outside world to synchronise it’s
phase to the solar day. This mechanism of entrainment functions by shifting the
clock daily, mainly from exposure to light between dusk and dawn. This
circadian system is controlled by the suprachiasmatic nucleus (SCN), found at
the base of the brain in a region called the hypothalamus. Destruction of this
nucleus leads to a disrupted sleep-wake cycle.
The SCN contains a network of nerve cells which fire in a circadian rhythm,
generated by cycles of gene expression in the individual nerve cells. These
firing rates can be altered and molecular rhythmic phases can be shifted by light
stimulation if presented at the appropriate time. The SCN receives information
about light through the retina in the eyes, which contain specialised
photosensitive ganglion cells. These retinal cells can project directly to the SCN
or indirectly via a relay centre found in the thalamus of the brain called the
lateral geniculate nucleus (LGN). These projections entrain this master clock
with retinal ganglion cell release of excitatory neurotransmitters such as
glutamate and pituitary adenylate-cyclase-activating polypeptide (PACAP). The
SCN nerve cells which receive retinal innervations have the capacity for light-
induced gene expression. Light turned on at night, especially in the later part
activates SCN nerve cells to fire action potentials which can be seen by
expression of a certain gene called c-Fos to produce its protein product Fos.

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Recently, a population of the retinal projections to the SCN have been found to
be vasopressin (VP)-expressing neurons. However, the physiological
significance of these VP neurons is unknown. VP is a neuropeptide that can
modulate a number of physiological effects and can alter the firing rate of
neurons. It is also shown to have a diurnal pattern of expression in a population
of VP-expressing SCN neurons. We hypothesised VP released by retinal
afferents into the SCN mediates the c-Fos expression seen when light is
exposed at the appropriate time. This could account for the synchronisation to
light cues, enabling mammals to anticipate environmental changes.
To test this hypothesis, we pre-treated rats to a VP receptor antagonist before
giving a light pulse at night. The antagonist was to prevent any VP from
activating its receptor on nerve cells in the SCN. Immunohistochemistry was
used to visualise Fos protein and the number of Fos-positive cells were counted
in the SCN. Our results show wide variability of Fos protein levels in the SCN,
with no significant decrease in rats given the VP antagonist. This suggests that
VP released from retinal nerve cells into the SCN is not essential for the
generation of light-induced c-Fos expression found here.
1. Introduction
1.1. Co-ordination of circadian rhythms
Mammalian physiology and behaviour are organised by biological clocks,
allowing mammals to synchronise to and anticipate environmental changes1
.
This is achieved by the circadian system which creates and maintains 24-hour
rhythms of biological processes and synchronises (entrains) to external time
cues such as the solar day. Multiple circadian oscillators are scattered
throughout the body, with circadian clocks ticking away within single cells and
organs. For the circadian timing system to function properly, all these clocks
must be synchronised with each other and to the 24-hour day2
. Recognition for
the importance of circadian regulation in health has been growing in recent
years, with implications not only in sleep disorders, but also in cancer, diabetes
and bipolar disorder3
.
The orchestration of this circadian programme comes from a master clock
within the anterior hypothalamus, called the suprachiasmatic nucleus (SCN)4
.
Lesions of the SCN abolish circadian rhythms of behaviour in hamsters5
and
transplantation of SCN neural grafts restores circadian fluctuations to
arrhythmic animals whose own nuclei have been ablated6
. The SCN is
composed of two small wing-like structures, each containing a network of
approximately 10,000 neurons7
. Individual neurons within these nuclei contain
molecular clocks8
with self-sustained circadian rhythms in protein levels of key
clock components (such as Period gene expression of the PER protein),
powered by autoregulatory transcription-translation feedback loops9
. Single
dissociated neurons from rat SCN in culture display circadian firing patterns that
are independently phased10
. However, the coupling of these individual SCN
neurons achieves synchronous, high amplitude and precise neuronal firing
frequencies, proving connectivity between these single neurons is essential for
appropriate time-keeping.

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Figure 1. Organisation of the
different neuronal populations in
the rat SCN [adapted from the
Paxinos & Watson atlas and
7
].The left SCN displays the
different phenotypic regions. The
SCN shell is composed primarily
of vasopressin (VP) and
somatostatin (SOM) neurons
whereas the core contains
vasoactive intestinal polypeptide
(VIP) and gastrin-releasing
peptide (GRP). The right SCN
depicts subregions of different
Period gene expression. Some
cells (orange) have rhythmic gene
expression while others (grey) can
demonstrate light-induced gene
expression. Some cells (red) have
rhythmic gene expression that is
in antiphase to the pattern seen in
the shell; 3V=third ventricle,
OX=optic chiasm.
The structure of the SCN is heterogeneous and traditionally divided into two
anatomical subdivisions: a ventrolateral ‘core’ which lies alongside the optic
chiasm and receives photic input, and a dorsomedial ‘shell’, which partially
encases and receives input from the core. Neurons throughout the nuclei can
be defined by neurochemical content (Fig. 1). Anatomy and neuropeptides vary
across species11
and there is variability along every axis which makes functional
examination of individual neurons and subregions of the SCN in controlling
mammalian circadian rhythms, challenging12
.
Expression profiles of PER protein demonstrate that circadian rhythms are
generated in the SCN from two populations of neurons: those in the
dorsomedial region which have a robust autonomous ability of expression with
no light response and those in the ventrolateral region with a weak autonomous
expression and a strong response to light13
. This expression pattern is similar to
endogenous circadian rhythms of peptides (such as VP and SOM), which peak
in the daytime and trough in the night-time. VP has been extensively studied in
the SCN but its role is still unclear. The VP V1a receptor found on both VPergic
and VIPergic SCN neurons also show a diurnal rhythm of expression under
light/dark conditions14
. This expression is 12 hours out of phase to VP
expression, with peak levels being reached at midnight. VP application to SCN
neurons in vitro led to increased firing rates in approximately 50% of the
neurons and application of VP antagonist resulted in phase-dependent
decreased activity15
, suggesting this peptide acts as a tonic excitatory input,
circadianly regulated. Most neuropeptides in the SCN are co-localised with
GABA and the majority of synapses here are GABAergic16
. The effects of GABA
vary depending on where it acts in the SCN. In the shell, this neurotransmitter is
excitatory but in the core it is inhibitory17
.

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1.2. Entrainment to lighting cycles
An essential feature of circadian rhythmicity is the capability of the clockwork to
synchronise with environmental stimuli18
, the most potent entraining signal
being light from the outside world4
. Light information reaches the SCN via two
visual projections, one directly from the retina and the other indirectly via the
intergeniculate leaflet (IGL) of the lateral geniculate nucleus (LGN) (Fig. 2). The
‘shell’ of the SCN receives far less innervation from these major input
pathways19
. Photic entrainment is seen as small phase-shifts of physiological
and behavioural rhythms which reflect the difference between the endogenous
free-running period of the SCN and that of the environmental light/dark cycle20
.
Figure 2. Schematic representation of the retinal input pathways to the SCN [adapted from21
].
Light signals received by retinal ganglion cells (RGCs) in the eye are transmitted to SCN
neurons via the retinohypothalamic tract (RHT)22
and to the IGL which further project to the SCN
via the geniculo-hypothalamic tract (GHT). RGCs release glutamate (Glu) and pituitary
adenylate-cyclase-activating polypeptide (PACAP)19
, while both the SCN and IGL release
GABA. The SCN controls output rhythms through diffusible molecules from SCN neurons, in
addition to directly targeting other brain areas, such as the paraventricular nucleus (PVN) and
the IGL.
Intrinsic RGCs are photosensitive due to the photopigment melanopsin23
and
innervates the light-responsive ‘core’ of the SCN24
. The release of PACAP at
SCN neurons has a modulatory role by enhancing the effects of glutamate.
Glutamate acts at its receptors to depolarize the membrane and cause an influx
of calcium which ultimately phosphorylates and activates CREB to induce
transcription2
. Glutamate is essential for light-induced phase-shifts as giving a
glutamate receptor (NMDA) antagonist after the photic stimulus prevents this
shift25
. RGCs also target other areas of the brain such as the IGL, intercalated
between the dorsal and ventral LGN26
. This structure is implicated in both photic
and non-photic control of circadian rhythms27
, including neuropeptide Y (NPY)
signalling from the IGL to the SCN via the GHT28
. Retinal information entering
the ventral side of the SCN daily synchronises to the environmental photoperiod
and creates rhythmic high neuronal firing during the day and low firing during

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the night. Intact neuronal networks in the IGL demonstrate similar patterns of
rhythmic action potentials to those seen in the SCN29
.
Coupling in the SCN is altered by light input, as seen by in vitro SCN neurons
from mice kept on a light/dark cycle demonstrating different phase distributions
to mice kept in constant darkness30
. Light-induced phase-shifts of clock gene
expression and neuronal firing rhythms in the SCN are high threshold
responses which can integrate photic input over substantial periods22
. Constant
light desynchronises SCN neurons and reveals multiple circadian oscillators
within these nuclei31
. Hamsters housed in constant light display independent
oscillatory cycling of gene expression in the left and right SCN32
, providing
evidence for these bilateral nuclei to function independently from each other. In
addition, shifts in the light/dark cycle also perturbs coupling of the SCN. An
advanced or delayed light/dark cycle (jet lag) shows properties of resetting in
subregions in the SCN. A large shift in this environmental cycle causes
molecular circadian rhythms to shift rapidly in the SCN core and later in the
dorsal shell (after receiving coupling signals from the core)33
. This phase
shifting and the rate of resetting can further differ along the rostral-caudal axis
of the SCN34
. After a 6-hour advance of the light/dark cycle, the initial widely
distributed phases require at least 8 days to fully resynchronise35
.
1.3. Shifting of circadian rhythms with light pulses and c-Fos expression
In rodents, light pulses also shift the phase of the circadian locomotor rhythm
with a magnitude that varies depending on the time point the pulse is applied36
.
Brief exposure to light during the first part of an animal’s night (or the subjective
night if the animal has been housed in constant darkness), causes a phase-
delay of their SCN-driven behaviours, such as the sleep/wake cycle and
locomotor activity. By contrast, light exposure during the second part of the
(subjective) night causes a phase-advance and exposure during the middle of
the night or subjective day has no effect37
. This demonstrates that the biological
clock reacts differently to light, depending on its internal state.
Although the molecular mechanism for entrainment by light is unknown, the
activation of immediate-early genes such as c-Fos in the SCN probably plays a
vital role38
. This functional indicator of neuronal activation has altered
expression in mammalian neurons in response to multiple stimuli39
, including
light and even stress40
. For example, neurons in the paraventricular nucleus
(PVN), a crucial structure in the stress-response, display increased c-Fos
protein expression after acute and chronic stress41
. Interestingly, the
hypothalamic PVN receives afferent inputs from many brain regions, including
the SCN and projects VP fibers back, extending ventrally into the SCN and
concentrating in the dorsomedial region42
.
In addition to phase-shifts, exposure of rats and hamsters to pulses of light
causes a dramatic elevation of SCN c-Fos mRNA43
, mostly found in the ventral
retino-recipient zone of the nucleus20
(Fig. 3). However, this elevation is only
seen when light pulses are administered during the subjective night, as pulses
given during most of the subjective day neither induces c-Fos expression or
phase-shifts. Furthermore, a study using a 15-minute light stimulus during the
second part of the night induced 4.2 times more SCN profiles immunoreactive
for c-Fos than light given during the first part of the night44
. In addition, a period

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of 5 minutes of light is sufficient to induce an increase of c-Fos mRNA in the
SCN of golden hamsters, with highest levels occurring 30 minutes after the
onset of photic stimulation45
. Blocking c-Fos accumulation in rat SCN with
antisense oligonucleotides can prevent light-induced phase shifts46
, consistent
with the evidence that c-Fos has a principal role in resetting the circadian clock
with photic-stimulation. Interestingly, the phase-dependence of light-induced c-
Fos expression in the SCN is not seen in the IGL, which expressed the early
gene marker regardless of circadian time47
.
Figure 3. Expression of
c-Fos in the SCN after a
light pulse during the late
night48
. Hamsters were
housed under light/dark
conditions, given a 5min
light pulse and
immunostained for c-
Fos. A) Dark control, B)
perfused 45min after
light pulse, C) perfused
80min after light pulse
and D) 90min after light
pulse. Approx. SCN
boundaries are encircled
by dotted lines, defined
by counterstain. c-Fos
expression was initially
localised to the ventral
SCN but later found
throughout the nuclei;
Scale bars=100µm.
1.4. Vasopressin and light-induced c-Fos expression
VP is a neuropeptide involved in a wide range of physiological effects49
, and in
the SCN known to be synthesised and secreted in a circadian pattern. It has
also been demonstrated that VP is present in mammalian retina, with levels
varying in a diurnal rhythm and which can be modified by light50
. Recently, a
population of retinal afferents that project to the SCN have been discovered to
express VP [Ludwig et al., unpublished]. However, the physiological
significance of this is still unknown. This preliminary data suggests a potential
mechanism whereby VP could mediate the light-induced c-Fos expression in
the SCN. There is clear importance in this retinal pathway for co-ordinating
synchronisation of circadian rhythms to the solar day-night cycle. In the present
study, we hypothesised that VP is involved in mediating light-induced c-Fos
expression in the SCN. To test this, we pre-treated rats to a VP antagonist prior
to light stimulation and later, used immunohistochemistry to visualise Fos
protein in the SCN. By comparing the number of Fos-positive cells in rats pre-
treated with antagonist to treated with vehicle, we could examine the effects of
VP on light-induced c-Fos expression in rat SCN and a reduction of c-Fos
expression would implicate the involvement of VP-expressing neurons the
retina projects to the SCN.

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2. Materials and Methods
2.1. Animals
Twenty adult male Sprague-Dawley rats weighing ~200g were used. Animals
were housed to Home Office standards and had free access to food and water
throughout, except during the photic-induction period and were individually
housed in clear plastic cages. Animals were gradually entrained to an altered
light/dark cycle (lights on 1300; lights off 0100) over one week, the lighting
schedule being altered by 1h per day until the new schedule was reached. For
the next two days, rats were handled/habituated in light (after 1300). After this,
rats were handled daily in the late morning in darkness under red light, up until
the end of the second week. This ensured rats were on their new light/dark
cycle more than 7 days before surgery. Animals were divided into 3 treatment
groups; those that received either a) VP antagonist, b) vehicle or c) light control.
2.2. Procedures
In the third week, 14 rats were anaesthetised with isoflurane and an
intracerebroventricular (ICV) cannula implanted during the light phase (1300 to
1800). The co-ordinates of the cannula relative to bregma were -0.6mm
rostrocaudal, +1.6mm lateral, 4.5mm deep. Immediately after surgery, rats were
given an analgesic (Rimadyl) and another second dose of Rimadyl given 24h
later. Rats were continued to be handled and after 8 days, ICV injections and
perfusions were carried out. This time interval from surgery was to reduce any
effect the anaesthesia may have had on the circadian cycle. Rats received an
ICV injection of either the VP antagonist (OPC21268; 50ng in 2µl saline plus
0.1% DMSO) or vehicle (1030 to 1045) under red light, 15min before onset of
light exposure for 1h (during the late part of their subjective night, 2h before
they were scheduled to wake). For the light pulse, each cage was transferred
from a dark room to a brightly-lit room (>1000 lux). After 60min of light
exposure, animals were deeply anaesthetised with sodium pentobarbital
(130mg/kg I. P. approx.)
2.3. Tissue preparation
Deeply anaesthetised animals were transcardially perfused slowly for tissue
fixation with physiological saline (0.9% NaCl) followed by 800ml of cold, 4%
paraformaldehyde in a 0.1M phosphate buffer (pH 7.3). Following perfusion and
before dissection, a small amount (approx. 2µl) of dye was injected through the
cannula into the brain. This confirmed the precise location of the cannula.
Brains were removed, post-fixed in 4% paraformaldehyde and stored at 4°C
overnight in 15% sucrose in 2% paraformaldehyde. They were then
cryoprotected (to prevent freezing damage to the tissue) in 30% sucrose until
sections were cut (at least 24h). Free-floating coronal brain sections (40µm in
thickness) were cut through the SCN using a freezing microtome. SCN slices
were taken, using the atlas of Paxinos and Watson for reference and sections
were stained for c-Fos expression.

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2.4. Fos protein immunohistochemistry
Free-floating sections were washed with PB-T (phosphate buffer (PB 0.1M, pH
7.4) with 0.3% TRITON X-100) for 4x15min at room temperature and incubated
in 0.3% H2O2 in 0.1M PB for 20min. Sections were washed with PB-T as before
and then with 0.1M PB for 5min at room temperature. They were then incubated
with 3% normal horse serum in 0.1M PB-T for 60min at room temperature and
then with an anti-Fos polyclonal antibody (Calbiochem) raised in rabbit, used at
1: 20 000 for 60min at room temperature, then for 40hr at 4°C. A negative
control used sections that omitted the primary antibody; a positive control for
Fos used a brain from a hypertonic rat (intra peritoneal 3.5M NaCl injection)
with sections containing the SON and PVN. This is because hypertonic salt
solution stimulation is known to induce c-Fos immunoreactivity in magnocellular
neurons in these regions51
. Following incubation with the primary antibody,
sections were rinsed in PB-T for 8X5min and incubated with a biotinylated anti-
rabbit secondary antibody (Vector Labs Elite), used at 1: 500 in 3% normal
horse serum in PB-T for 60min at room temperature. After incubation with
secondary antibody, sections were rinsed in PB-T 3x5min and incubated at
room temperature for 1h with an avidin-biotin-peroxidase complex (Vectastain
Elite ABC Kit, Vector Labs). Immunoreactivity was later revealed after
incubation with ABC reagents, washing with PB for 2x10min, and rinsing in
0.5M Tris buffer (pH 7.6) for 5min at room temperature. Sections were
incubated in 0.015% H2O2-Ni-DAB solution for approx. 5-8min on an orbital
shaker, for the DAB-Ni reaction to produce black deposits. The DAB reaction
was stopped by washing in 0.5M Tris buffer (pH 7.6) for 5 min at room
temperature. Sections were lastly washed in 0.1M PB wet-mounted onto slides,
allowed to dry, counterstained with nuclear fast red, dehydrated through a
series of alcohols and xylene, mounted with DPX and cover-slipped.
2.5. Immunocytochemical data analysis
The study was ‘blinded’ to prevent any bias in the way the data were obtained.
Images of Fos immunoreactivity in the SCN (bregma 0.92mm to –1.40mm)
were taken using a Leica microscope and analysed using ImageJ software. The
region of interest (ROI) was drawn in ImageJ as area/units2
and the numbers of
c-Fos positive cells in this area were quantified using ImageJ software with a
threshold set at 45-80. This threshold was chosen by comparing automated and
manual cell counts for various thresholds and choosing the one that gave
comparable results for both. For objective comparisons, cell counting was
carried out using the same magnification and equal grey scale settings for
correction of background staining. Manual counting was performed where
appropriate and the number of Fos-positive cells for each rat was expressed as
the mean number of positive cells/unit area. Fos immunoreactivity in the PVN
(bregma -0.80mm to -2.12mm) was qualitatively measured. Results are
expressed as the mean ± S.E.M. Data on Fos-positive cell counts were
compared across the three groups using a one-way ANOVA followed by a post
hoc Tukey's multiple comparisons test. The level for statistical signiﬁcance was
set at p< 0.05.

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3. Results
3.1. Fos expression in the SCN
Following the 1-hour light exposure during the subjective night phase of the rat,
immunohistochemistry revealed robust c-Fos expression in the SCN (Fig. 4D).
In order to investigate the role of VP in this expression, Fos-positive cells were
counted in rat SCN with and without pre-treatment to the VP antagonist. The
atlas of Paxinos and Watson was used to ensure only sections containing the
SCN had cells counted from (Fig. 4B had; 4A, 4C had not). There was
considerable variability of c-Fos expression observed from individual rats (Figs.
5 & 7) and expression was not significantly different between the treatment
groups (p=0.88; antagonist 0.703±0.244 versus vehicle 0.867±0.602, p=0.95
and light control 0.598±0.140, p=0.9793; data are means±S.E.M; n = 6; Fig. 6).
As the SCN is a small structure, it is unlikely that all sections counted contained
the nucleus. Therefore, the section with most c-Fos expression was taken from
each rat and the mean number of Fos-positive cells in each SCN was
calculated for each treatment group (antagonist = 49.5±18.412; vehicle =
52.5±28.739 and light control = 45.167±11.285; Fig. 8). Again, there was no
significant difference between groups (p > 0.05), supporting our results.
These results indicate that the effect of light on c-Fos expression is unlikely to
be caused by the release of VP from retinal projections to the SCN.
Figure 4. Expression of c-Fos in the SCN.
(A-C) Photomicrographs of serial coronal sections through
the SCN; Scale bars = 500µm.
(A) Rostral of the SCN, bregma= -0.80mm. (B) Location of
the SCN, bregma= -1.30mm. (C) Caudal of the SCN,
bregma= -1.60mm. (D) Inset from B of SCN. Arrows indicate
a selection of Fos-positive cells. Scale bar = 50µm; 3V = third
ventricle, OX = optic chiasm.

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Figure 5. Number of Fos-positive cells per unit area in individual rats (animal number/number of
sections analysed) from the 3 treatment groups. Light control group had no surgery before
photic induction. Error bars = S.E.M.
-0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
NumberofFos-positivecells/unitarea
Antagonist Vehicle Light Control
Rat number and n number in each treatment group
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
MeannumberofFos-positivecells/unit
area
Treatment
p = 0.95
p = 0.9793
p = 0.8745
Figure 6. Mean
number of Fos-
positive cells per unit
area in each
treatment group.
Error bars = S.E.M.
There is no significant
difference between
groups (p>0.05). n=6
for each condition.

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Figure 7. Variability of light-induced Fos immunoreactivity within the SCN of various rats. (A, B)
Rats given an ICV of VP antagonist. (C, D) Rats given an ICV of vehicle. (E, F) Control rats
where only light stimulation was given and had no surgery. Rat 12 (A) had a low level of c-Fos
expression, whereas Rat 4 (B) had a high level. Rat 14 (C) had a high level of c-Fos
expression, whereas Rat 5 (D) had a low level. Rat 16 (E) had a high level of c-Fos expression,
whereas Rat 20 (F) had a low level; 3V = third ventricle, OX = optic chiasm. Scale bars =
100µm.

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Treatment Rat number and n number SCN c-fos PVN c-fos
2 (n=8) + -
4 (n=9) ++ ++
6 (n=8) +++ +++
9 (n=10) +++ +
11 (n=10) + ++
12 (n=8) + +++
3 (n=9) + -
5 (n=5) + -
8 (n=5) +++ ++
10 (n=11) ++ +
13 (n=8) + +
14 (n=10) + +++
15 (n=9) ++ +
16 (n=11) + +
17 (n=7) +++ ++
18 (n=8) ++ +++
19 (n=10) + -
20 (n=11) + -
Antagonist
Vehicle
Light Control
3.2. Absence of effect of stress on Fos expression in the SCN
In order to ensure that the
results of our SCN studies were
not affected by stress of the
animals, it was deemed
necessary to investigate stress-
induced PVN c-Fos expression.
We hypothesised that there
would be a correlation between
c-Fos expression in the PVN
and SCN, indicating that some
SCN c-Fos expression was
affected by stress. Variable c-
Fos expression in the PVN was
quantified across rats from the
three treatment groups which
did not seem to correlate with c-
Fos expression found in the
SCN (Figs. 9 & 10). These
results indicate that the lack of
effect of VP on light-induced c-
Fos expression appears to be
valid and SCN expression was
unlikely to be affected by stress.
0
10
20
30
40
50
60
70
80
90
MeannumberofhighestFos-positive
cells
Treatment
Figure 9. Table of results comparing c-Fos
expression in the SCN and PVN of individual rats.
(- = no staining, + = low staining, ++ = moderate
staining, +++ = high staining).
Figure 8. Mean
number of Fos-
positive cells in
each treatment
group with only
sections containing
the highest number
of Fos-positive cells
taken for each rat.
Error bars = S.E.M.
No significant
difference between
the treatment
groups (p>0.05).
n=6 for each
condition.

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Figure 10. Examples of light-induced and stress-induced Fos immunoreactivity within the SCN
and PVN respectively of the same rat. Each rat was given an ICV injection of VP antagonist
prior to photic-induction. (A, B) Rat 4 has high expression of c-Fos in the SCN but not PVN. (C,
D) Rat 6 has high expression of c-Fos in both the SCN and PVN. (E, F) Rat 12 has low
expression of c-Fos in the SCN but high expression in the PVN; 3V = third ventricle, OX = optic
chiasm. Scale bars in A, C & E = 100µm, Scale bars in B, D & F = 200 µm
4. Discussion
This study investigated the role of vasopressin (VP) in light-induced c-Fos
expression in the SCN. We hypothesised that VP mediates the c-Fos
expression in the SCN seen when the rat is exposed to light at the appropriate
time. To test this, rats were pre-treated with either a VP antagonist or vehicle
prior to a 1-hour light pulse given 2 hours before the rats were scheduled to
wake. Immunohistochemistry was carried out to visualise Fos protein and the

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number of Fos-positive cells in rat SCN. Our results refute the hypothesis,
which demonstrate no significant difference in SCN c-Fos expression between
rats given the VP antagonist or vehicle. These results implicate VP released
from retinal afferents into the SCN does not mediate light-induced c-Fos
expression.
4.1. Expression of c-Fos in the SCN
Light stimulation given during the subjective night phase of the rat, induced c-
Fos expression in the SCN (Fig. 4D). These results are in agreement with other
studies demonstrating a phase-dependent photic induction of this immediate-
early gene in rat52
and hamster43
. As Fos induction provides a useful marker of
photic activation of cells53
, this provides evidence that the >1000-lux light
stimulation was sufficient to induce the c-Fos gene in the SCN. This appears
likely as light intensities of 300-lux exposed for 15 minutes has been shown to
cause expression of c-Fos54
. However, in these studies, expression appeared to
be higher than reported here, usually reaching over 150 Fos immunoreactive
cells within the SCN of hamster53
and mice55
. This number is greater even
compared to our sections containing the greatest Fos immunoreactivity from
rats (Fig. 8). One possible explanation could be due to the time point when
animals received the light-pulse or the type of species used. In the previously
mentioned study, they gave a 1-hour light stimulus at circadian time (CT) 20,
two hours before we did. Furthermore, another experiment reported peak c-Fos
expression in mice to occur when the light pulse was delivered at CT 1856
,
whereas we exposed the light pulse at CT 22. However, the mean number of
light-induced Fos immunoreactive cells in this experiment (see Fig. 6) were
similar as in other reports using Sprague-Dawley rats57
such as this one.
Note that Fos immunoreactivity was heavily concentrated in the ventrolateral
region of the SCN (Figs. 7B, C & E), consistent with numerous reports58, 47
. In
addition, labelled cells were sometimes found to be scattered dorsally, as far as
the PVN and to adjacent thalamic areas as have been previously described43
.
This pattern of labelling corresponds to the terminal endings of retinal afferents
forming the RHT and which has been traced by cholera toxin-conjugated
horseradish peroxidase59
. This is consistent with evidence that the ventral zone
is the retino-recipient area of the SCN14
and confirms that our rats were
neurophysiologically responsive to the light stimulation administered. The
occasionally spotted c-Fos expression in the dorsal SCN is similar to a study
using Wistar rats and which found levels of c-Fos protein to peak around
dawn52
. This zone is only weakly innervated by the retina in rats, but is an area
known to display spontaneous Fos immunoreactivity with a rhythm that differs to
light-induced Fos immunoreactivity52
. However, these results do contrast with
another study which found no evidence of dorsal SCN c-Fos expression48
.
In this study, the number of Fos-positive cells varies considerably between
individual rats (Figs. 5 & 7); with sometimes much lower light-induced c-Fos
expression than seen in other studies, as previously discussed. Variability and
lack of relation between light stimulation and c-Fos expression could be due to
a weak coupling of the SCN to the entraining light/dark cycle prior to the pulse
of light60
. This would mean that retinal illumination could potentially be at a time
in certain rat’s circadian cycles when light is incapable of altering gene

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expression and influencing entrainment. This is highly plausible as there is
known to be individual differences in entrainment patterns20
. Interestingly, some
rats also indicated differential c-Fos expression between their two nuclei. This is
possibly a result from the two paired SCN functioning independently from each
other which has previously been shown32
.
4.2. Vasopressin has no effect on light-induced c-Fos expression in the SCN
Although there were great individual differences between rats, the present study
clearly shows no significant difference of c-Fos expression between the
treatment groups (Fig. 6). Earlier experiments that have also manipulated the
light-induced c-Fos response include ventricular administration of the glutamate
antagonist (MK-801)61
, noradrenaline reuptake inhibitor (atomoxetine)62
and the
serotonin mixed agonist/antagonist (NAN-190)55
, all of which suppressed
induction of c-Fos in the SCN. The response from these drugs collectively point
to the role of their respective ligands in mediating the effects of light on SCN
cells and modulating the photic induction pathway. Interestingly, the
pharmacological manipulation with atomoxetine and NAN-190 found the down-
regulation of c-Fos induction to be accompanied with enhancement of photic
phase-shifting. This is evidence that an enhanced response in behaviour is not
necessarily accompanied with an increase in photic-induced c-Fos55, 63, 64
,
demonstrating a miscorrelation between the magnitude of phase-shift and
amount of c-Fos expressed. Moreover, another study found pre-treatment of
NAN-180 in hamsters before a light pulse to have no effect on SCN c-Fos
expression compared to the vehicle control65
. One possible reason for the
contrasting effects from this serotonin agonist/antagonist may be due to species
differences between mouse and hamster62
. Our findings that c-Fos was
expressed regardless of whether animals had been pre-treated with the VP
antagonist or not, argues against the involvement of VP on light-induced
expression, at least during the latter part of the night phase. However, one
possibility that shouldn’t be overlooked is that the VP antagonist may have
altered c-Fos expression kinetics but at a time that differed to the single time-
point investigated. In future studies it would be of interest to examine the effect
of the VP antagonist on behavioural circadian rhythmicity, to study the effects
on phase-shifting and to make use of other animals to investigate any species
differences in VP effects.
4.3. Stress has no effect on c-Fos expression in the SCN
We investigated whether stress could be attributable for aberrant results of SCN
c-Fos expression, as previous reports have linked stress with disrupting
circadian rhythms66
. As it is known that the PVN is a key component in
regulating the stress response and stress causes increased c-Fos expression
here67
as well as in the SCN68
, we examined the PVN for Fos immunoreactivity.
Interestingly, light microscopy has also revealed SCN efferent neurons to
contact interneurons in and around the PVN to influence a circadian release of
stress hormones69
. The same sections used to investigate the SCN were
qualitatively analysed in the PVN (Fig. 9) which found variability in c-Fos
expression but which was not correlated to that seen in the SCN (Fig. 10).

of 23
Sections containing different amounts of c-Fos expression (i.e. high with VP
antagonist and low with vehicle or light control) displayed both high and low
levels of Fos immunoreactivity in the PVN. These results suggest that the
effects from stress cannot account for a specific effect on SCN Fos
immunoreactivity. It is not surprising that expression was seen in some sections
of the PVN, as rats were ‘restrained’ by holding during ICV injections, a known
mediator of stress70
. Our results are consistent to other studies which found
restraint stress to induce a c-Fos response, primarily located in the medial
parvocellular area and the lateral subdivision of the caudal PVN71
. In addition,
reduced c-Fos expression in stress-exposed rats might be explained by the
involvement of oxytocin (OT). OT-expressing neurons are found in the
magnocellular area of the caudal PVN72
and OT locally infused into the PVN
has been demonstrated to inhibit c-Fos activation in response to a stressor73
.
As there was no correlation between Fos immunoreactivity in the SCN and
PVN, quantitative analysis was not pursued.
One limitation in the experiment was the difficulty in defining the borders of the
SCN and PVN and also in distinguishing which cells were Fos-positive. Even
although every attempt was implemented to ensure the ROI drawn was
bordering the SCN, there is still a chance of error which may have skewed the
results that presented the number of cells per unit area. This can be improved
by using a different counterstain to the nuclear fast red used here. Previous
experiments have delineated the SCN by staining with 4′, 6-diamino-2-
phenylindole (DAPI)74
or staining for Nissle substance with cresyl violet (Fig. 3.),
which can be used for future experiments.
4.4. Future directions
One possibility for the decreased Fos immunoreactivity seen in this report could
be due to some rats having not entrained properly to the new light/dark cycle.
Therefore, the light pulse may have been given at a phase in the circadian
rhythm during which light is incapable of entrainment and causing increased c-
Fos expression. SCN c-Fos induction is known to be related to the magnitude of
the daily phase shift needed for stable entrainment20
. Because of individual
differences in entrainment patterns, future experiments could find the
endogenous free-running periods of the rat circadian rhythms and from this, the
daily phase shifts required for stable entrainment could be calculated. To
ensure animals had appropriately adjusted to the new light/dark cycle,
behavioural activity from individual animals could be analysed.
Another future experiment could explore if the light stimulation given did in fact
mediate VP release from retinal afferents into the SCN. As it is unknown
whether VP neurons originating in the SCN are affected by light stimuli75
, we
would selectively ablate these using a transgenic rat line in which the human
diphtheria toxin receptor was inserted into the VP promoter region and locally
infuse diphtheria toxin into the SCN. This would ensure measurement of VP
levels were a result of retinal VP release and not SCN VP release. We would
use light-stimulation to activate RGCs and simultaneously measure release of
VP in the SCN by microdialysis. Furthermore, if VP is shown to be released in
response to stimulation, in vivo electrophysiological recordings from SCN cells
could be used to determine if retinal VP alters cell activity here.

of 23
5. Conclusion
In summary, exposing a 1-hour light pulse during the subjective night phase of
rats pre-treated with a VP antagonist or vehicle resulted in no significant
difference in c-Fos protein expression in the SCN. These findings suggest that
VP is not involved in modulating light-induced c-Fos expression and therefore,
must be the actions of other mediators to induce circadian clock resetting. This
mechanism is essential in enabling mammals to synchronise to and anticipate
environmental changes.
Acknowledgements
I wish to thank Andrew Allchorne for expert technical assistance and advice and
Professor Mike Ludwig for his support and perseverance.

of 23
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