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The handle http://hdl.handle.net/1887/20930 holds various files of this Leiden University
dissertation.
Author: Houben, Mathias Bernard (Thijs)
Title: Regulation of behavioral activity by the suprachiasmatic nuclei
Issue Date: 2013-06-06
Chapter 1
General introduction
1
Preface
General introduction
Since the earliest days of life on earth, living creatures have been exposed to a
dynamic, constantly changing environment. Only those organisms with the necessary
attributes to adapt were able to reproduce successfully and spread their genes forward
in the tree of life. Many of the challenges facing living organisms from those early
days up to the present are due to chance and unpredictable. A number of
environmental factors is not governed by chance however, but change in a cyclic and
predictable pattern because they are driven by the daily rotation of the earth around
its axis and its yearly trajectory around the sun. The day-night rhythms in the amount
of light, temperature, humidity and many other parameters require constant
adaptation of the organism in order to survive. A plant that is dependent on sunlight
as the energy source for photosynthesis should store enough energy during the day to
drive vital processes during the night. A nocturnal rodent that forages for food has to
make sure that it is safely back in its burrow and away from open fields before the sun
rises in the morning. The adaptive value of predicting and anticipating cyclic
environmental changes led to the evolution of a built-in timekeeping mechanism, a
biological clock, that keeps track of the time of day and is present in nearly every
form of life.
A second environmental rhythm that is equally essential to survival is the 365 day
seasonal rhythm caused by the rotation of the earth around the sun. The yearly cycle
in the duration of day and night and the amount of solar energy reaching the earths’
surface leads to strong seasonal differences in temperature, weather patterns and food
availability. This requires organisms to time reproduction, energy expenditure and
other physiological functions to the right time of year. And just as with 24 hour
rhythms, anticipation of these seasonal changes greatly improves the chances for
survival and reproduction. Plants should blossom in the right season to allow seeds to
fully ripen before the winter. Animals should time conception of their offspring so
they are born when there is enough food available to feed them. Although the yearly
cycle of the seasons occurs on a very different timescale as the daily cycle of light and
darkness, it is also measured by the biological clock that keeps track of time of day by
measuring the seasonally changing duration of daylight. There are many examples
showing that life has adapted to a rhythmic world and how nearly every form of life is
constantly anticipating and changing to prepare for the next phase of the cycle and for
the next season.
Although humans, through the invention of alarm clocks and electric lighting, are no
longer fully dependent on the solar light-dark cycle for timing their activity and rest,
they too possess a built-in biological clock driving their daily physiological and
behavioral rhythms. As we progress during the day, we eat, drink, and perform a wide
8
Circadian rhythms
The first evidence for the presence of an internal timekeeping mechanism was
presented in a letter published in 1729 by the French geophysicist Jean-Jacques
d'Ortous de Mairan (4). In this letter, he described a simple experiment performed on
the Mimosa plant, that has the interesting property that it folds its leaves during the
night and opens them in the daytime. De Mairan aimed to test if the leaf movement
was a response to the presence of light during the day, and placed the plant in a dark
closet to see if it would keep its leaves in the night position. To his surprise, he found
that the plant continued to open and close its leaves in the absence of any light.
Although his experiment was far from perfect, it did provide the first known evidence
that organisms have a built-in clock that continues ticking in constant conditions. An
idea that has been confirmed in many plant and animal species since then, from the
cellular level (gene expression, cell division) to physiological processes (hormone
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1
General introduction
array of physical and mental activities. As we rest and sleep during the night, we give
our body and brain a precious time-window to recover, process the nutrients and
experiences of the previous day and allow our physiological systems to prepare for the
next active period. Throughout this repeating daily cycle, it is our biological clock
system that coordinates that our physiological, mental and metabolic systems are at all
times optimally prepared for the changing demands posed by the daily rhythm of our
lives. In recent years, the importance of this endogenous clock system for health and
the association of rhythm disturbances with disease have received growing attention.
From clinical practice in many types of disease, it is evident that disturbances of the
sleep-wake rhythm occur as illness develops or progresses over time. In the case of
psychiatric disorders with an episodic nature such as schizophrenia, bipolar disorder
or depression, disruption of the sleep-wake cycle often precedes or coincides with
episodes of increasing severity of the symptoms (1-3). Although the causality in the
association between disease and disturbed rhythms is uncertain in many cases, there is
an increasing recognition that through nighttime light exposure, jet lag and shift work,
the urban lifestyle of the modern 24-7 society exposes our endogenous biological
clock system to disruptive inputs that can impair its’ function and may ultimately
compromise our health.
This thesis consists of two parts. The first part consists of five studies in which we
used the mouse as a model organism for investigating the mechanisms that enable
mammals to modify their behavior and physiology in order to deal with daily and
seasonal rhythms. The second part consists of three experimental studies in which we
explore the relation between disruption of the circadian system and diseases, including
psychiatric disorders, metabolic disease and renal failure.
General introduction
Figure 1. Circadian rhythm in behavioral
activity
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Waking and sleeping in humans and animals is
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scheduled according to the time of day. In
laboratory animals, we can measure the timing
4
of behavioral activity by placing a movement
5
sensor or a running wheel in the cage of the
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animal. Here, the activity of a laboratory mouse
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(Mus musculus, C57Bl6 strain) in a running wheel
was recorded during 18 days and plotted as an
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actogram. In an actogram, each line shows 24
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hours of behavioral activity, and subsequent
10
days are plotted below each other. For the first 9
11
days, The animal was exposed to an artificial 24
hour day, consisting of 12 hours of light (white
12
background) alternated by 12 hours of darkness
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(grey background). As mice are nocturnal
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animals, they sleep during most of the light
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phase and run in their wheel in the darkness.
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After day 9, the lights are kept off and the
animal is left in continuous darkness. Although
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the animal can no longer tell the time of day
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from the light in its’ environment, the rhythm in
0
4
8
12
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20
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behavioral activity continues. Without any
Time [hours]
external time-source, activity starts slightly
earlier each day, and the ‘free-running’ rhythm
obtains a period that is slightly shorter 24 hours.
Without a daily ‘resetting’ of the clock by the external light-dark cycle, it generates a period close to, but
not exactly, 24 hours in constant conditions. Therefore, the rhythm is called a ‘circadian’ rhythm (from
the latin ‘circa’ = about, and ‘dies’ = day). Unpublished data.
1
Time [days]
1
levels, body temperature, metabolism), to behavior (sleeping/waking, cognitive
performance). A common aspect of these rhythms is that although they continue
under constant conditions, their period deviates slightly from 24 hours if there are no
environmental changes that indicate the time of day. When a mouse has a running
wheel in its cage, it will very precisely start running at the same time each night, and
will continue running for a large percentage of time until the end of the night (Figure
1). If the mouse with the wheel is then placed in constant darkness, the running wheel
rhythm will remain very strong, but the animal will start and stop running a fixed
number of minutes earlier each day. Without an external rhythm, the endogenous
clock continues ticking, but with a period that is (in case of the mouse) slightly shorter
than 24 hours. After the mouse in the wheel, such rhythms that start several minutes
earlier or later each day (depending on the species) are said to be ‘free-running’. This
observation of the endogenous biological clock running several minutes faster or
slower than 24 hours if conditions are constant prompted researcher Frans Halberg to
call these rhythms “circadian”, from latin “circa” (about) and “dies” (day).
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Entrainment of circadian rhythms
B Delay
No Shift
C
D
Advance
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2
3
1
Phase shift (hours)
Time (Days)
A
5
7
9
*
*
**
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Advance
0
-1
-2
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0
Active period
Delay
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8
12
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Time (hours)
20
24
0
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8
12
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Time (hours)
20
24
0
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Time (hours)
20
24
-3
6
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22
0
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Circadian Time (circadian hours)
Figure 2. Entrainment of circadian rhythms: the Phase Response Curve
Much has been learned about the synchronization of the biological clock to the environmental light-dark
cycle by placing animals in constant darkness and measuring the effect of brief light pulses on the timing
of their behavior. Here, three actograms are shown of a mouse in constant darkness. On day 7, each
animal was exposed to 30 minutes of light (red asterisk) at different times relative to their behavioral
activity. A. When the light pulse is timed during the resting period, the timing of behavioral activity on
the days following the pulse is not shifted. B. When the light pulse occurs in the first hours after the
animal woke up and became active, the activity in the days following the pulse starts later; a phase delay has
occurred. C. When the light pulse occurs towards the end of the active phase, the activity on subsequent
days starts earlier. To visualize this phase advance, a red line was fitted through the start of activity on the
days preceding the light pulse, and a blue line was fitted through the start of activity on the days
following the light pulse. D. By performing many such phase shifting experiments with light pulses at
different times, a phase response curve (PRC) can be constructed that plots the average shift in behavior as a
function of the time of the pulse (10). Here, the PRC of the mouse is drawn schematically. The time of
the pulse is determined in Circadian Time (CT), in which CT12 is defined as the start of behavioral activity
on the day of the pulse, and one circadian hour equals the total duration of the free-running period
(about 23.5 hours in mice), divided by 24. As is evident from the actograms in B and C as well as the
PRC in D, mice respond with large phase delays to light pulses at the start of the active period and with
much smaller phase advances to light pulse at the end of the active period. Unpublished results.
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1
General introduction
The endogenous circadian clock needs to adjust to the external cycle to keep accurate
track of time. How the endogenous circadian clock stays synchronized, or entrained,
to the 24 hour environment became a main question in the mid twentieth century,
culminating in the first international conference on biological rhythms in Cold Spring
Harbour in 1960. Progress in the field was driven by the systemic exploration of the
properties of animal circadian rhythms, led by a number of key researchers, including
Jürgen Aschoff, Rutger Wever, Patricia deCoursey and Colin Pittendrigh. In a range of
species, including birds, insects and rodents, they recorded and manipulated behavioral
rhythms in the field and in the lab. In doing this, they established and described a
number of key general properties of circadian rhythms (5, 6). One is that light has a
strong effect on the timing, or phase, of circadian rhythms and is the most important
signal responsible for keeping the internal biological clock synchronized to the 24
hour environment (7-9). By keeping the animals in constant conditions, and testing
the effect of single pulses of light on the behavioral rhythms it was shown that the
effect of light on circadian rhythms is dependent on the phase of the biological clock
(10, 11). In the evening and beginning of the night, light delays (slows down) the
biological clock, causing the animal to wake up and start activity later on the days after
1
General introduction
the light pulse (Figure 2). In the end of the night and the beginning of the morning, a
light pulse has the reverse effect and advances (speeds up) the clock, leading to earlier
times of waking up and falling asleep in the days after a pulse. During the day, light
pulses have little to no phase-shifting effect on the clock. The time-dependent effect
of light on the clock can be summarized in a so-called phase-response curve and
explains how exposure to environmental light in the morning and evening keeps the
biological clock adjusted to the environment.
The molecular mechanisms at the basis of circadian rhythmicity
The 1953 discovery that DNA is the carrier of our genetic blueprint started the era of
molecular genetics (12). Using constantly improving analysis methods, scientists
throughout the life sciences set forth to make sense of the vast amount of
information stored in the genome, and to understand the connections and
mechanisms connecting this information to the properties of cells, tissues and the
organism as a whole. In 1971 the DNA revolution caught up with chronobiology for
the first time in a publication describing the discovery of a gene in the fruit fly
(Drosophila melanogaster), that is involved in generating and regulating circadian rhythms
(13). Fruit flies are a favorite organism for molecular biologists because they are fast
breeders and have DNA that can easily be manipulated and investigated. Like many
other insects, fruit flies start their life as larva that metamorphose into an adult fly in
the pupal stage. They emerge from the pupa in an event called eclosion. The time at
which the adults emerge from the pupa has a strong circadian rhythm that could be
easily measured using devices and techniques developed in the lab of Colin
Pittendrigh. In the experiments, random DNA damage was induced in many fruit flies
before mating, followed by a screen for abnormalities in the circadian rhythm in
eclosion time of the offspring. With this strategy, the researchers discovered that
damage in different parts of a specific gene on the flies X chromosome led to strong
changes in the circadian rhythm of the offspring. One group had a very fast clock of
19 hours, one group a very slow clock of 28 hours, and the third group had no
circadian rhythm at all in constant conditions. These rhythm changes were present not
only in the time of eclosion but also in the behavior of larva and adults. The newly
discovered gene was called “Period” and was the first of many similar genes
discovered in many species of animals, plants, fungi and bacteria. Together, these
genes form the essential basis for the intracellular mechanisms responsible for
generating the basic day-night rhythm that allow organisms to be prepared at all times
for the challenges posed by living in a rhythmic world.
In the 4 decades since the discovery of the first clock gene, an enormous amount of
detail has been unraveled about the genes, molecules and their interactions that make
up the molecular circadian clock. Despite numerous differences between species, a
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The suprachiasmatic nuclei
In stark contrast with the accumulating knowledge on the general properties of
circadian rhythms and their entrainment, very little was known in the mid twentieth
century about the underlying mechanisms of the biological clock and where it was
located. It took until 1972 before the first evidence emerged that the mammalian
biological clock is in fact located in a specific anatomical substrate. This evidence
consisted of two papers in which brain lesions were made in a small region at the
bottom of the rat hypothalamus, directly above the optic chiasm, the area where the
optic nerves reach the brain. In the first study, a lesion of this suprachiasmatic area
caused the disappearance of the behavioral rhythms in drinking and running in a
running wheel (14). The second study investigated the daily rhythm in corticosterone
production by the adrenal gland, and found that this endocrine rhythm was absent in
animals with a suprachiasmatic lesion (15). While these lesion studies showed that an
intact suprachiasmatic nucleus is necessary for the expression of circadian rhythms, it
was not yet conclusive evidence that the SCN is also the location of the biological
clock itself. It took several follow up studies to make this point as well. First of these
was the 1979 paper by Inouye and Kawamura (16). In this study, they implanted
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1
General introduction
uniform principle has emerged that explains how different sets of clock genes and
their products interact in a very similar way that makes them function as a pacemaker,
generating a rhythm of approximately 24 hours. The unifying principle is the concept
of a transcription-translation feedback loop that has been found at the heart of the
intracellular clock in all investigated organisms. In mammals, a set of 5 genes, Period
1,2,3 and Cry1 and 2, all contain so called e-box elements in their promotor region.
One CLOCK protein can bind together with one BMAL protein to form a dimer that
binds to this e-box and activates transcription of the Per and Cry genes during the
day. Over the course of the day, PERIOD and CRY proteins accumulate and also
bind together to form dimers. These dimers then find their way to the nucleus, where
they interact with the CLOCK/BMAL dimers and inhibit the transcription of the
Period and Cry genes during the night. At the end of the night, degradation of
PERIOD and CRY proteins diminish the number of inhibitory dimers, giving the
CLOCK/BMAL dimers the opportunity to initiate a new cycle. The abundance of
BMAL and CLOCK is also rhythmic and regulated in an additional regulatory loop
that is thought to stabilize and enhance the rhythm of the Period/Cry feedback loop.
In this secondary loop, Bmal1 and 2 transcription is inhibited during the day by REVERBα binding to ROR elements in the Bmal promotor. REV-ERBα levels are
controlled by the CLOCK/BMAL complex are lower and the inhibitory effect on
Bmal transcription is counteracted by RORα.
1
General introduction
electrodes in the rat brain to record the electrical activity of SCN neurons in awake
animals. They found that the SCN electrical activity level shows a clear circadian
rhythm, with high activity during the day and low activity in the night. The rhythm
continued when the animals were placed in constant darkness and persisted even
when they surgically severed the connections between the SCN and the rest of the
brain, showing that the rhythm was not dependent on synaptic input from other brain
regions. In 1982, three different labs independently managed to extract a thin slice
containing the suprachiasmatic nucleus from the rat brain, keep this brain slice alive,
and use it in an in vitro preparation to do electrophysiological recordings (17-19). All
studies showed that the SCN is capable of generating a circadian rhythm in electrical
activity even when it is completely isolated from rest of the body. The case for the
SCN as the location for the biological clock was made complete by an elegant
transplantation study, published in 1990 (20). The study made use of the discovery of
a spontaneous genetic mutation in hamsters, that caused hamsters with this ‘tau’
mutation to have a very fast clock, with a period of 22 hours in constant darkness
(21). In the study, SCN lesions were made in normal hamsters with a 24 hour rhythm
and afterwards, the SCN from ‘tau’ mutant hamsters was transplanted into the SCN
lesioned normal hamsters. The result of this treatment was that circadian rhythms
were restored in the lesioned hamsters, but they now expressed a 22 hour rhythm
showing that the period of the rhythm is determined by the SCN. Together, these
studies indicate that the SCN is the location of brain clock responsible for generating
circadian rhythms.
Anatomical location of the SCN
The suprachiasmatic nuclei are located at the bottom of the brain (Figure 3), directly
on top of the location where the optic nerves coming from the eye reach the brain
and within the hypothalamus (23). Anatomically, the hypothalamus is part of the
diencephalon, an ancient vertebrate brain structure connecting the more rostral
midbrain and hindbrain to the cerebrum, the most frontal part of the brain (24). The
nuclei of the hypothalamus form a central command structure responsible for
regulating a number of key physiological and behavioral systems including
reproduction, body temperature, metabolism and hunger, water balance & thirst, and
activity and rest. The hypothalamus contains mechanisms for sensing the bodies’
temperature, water content and metabolic energy levels. In a very broad sense, the
functions of the hypothalamus all have to do with maximizing the animals chances of
survival and reproduction by balancing the animals behavior and physiology to each
other and adjusting both to the environmental demands. Most functions regulated by
hypothalamic nuclei display a strong circadian rhythm, an observation that closely
14
matches the location of the SCN at the base of the hypothalamus, as well as its strong
connectivity to the nearby nuclei (25).
General introduction
A
1
B
C
V3
OC
Suprachiasmatic Nuclei
Figure 3. The suprachiasmatic nuclei. The SCN are located at the base of the brain, in an area called
the hypothalamus. A. Schematic view of the mouse brain from the side. When the brain is cut at the red
line, the SCN (red structures) become visible when seen from the front, as is depicted in figure B. C.
Microscopic image of a mouse brain section, showing the hypothalamic area, demarcated by the rectangle
in figure B. In the middle of the picture is the ventricle (V3). At the bottom, the white structure (OC) is
the optic chiasm, which is the place where the optic nerves enter the brain. Immediately on top of the
optic chiasm are the bilateral suprachiasmatic nuclei. In this image, the SCN were stained using an
antibody against AVP, a neuropeptide expressed by SCN neurons. A,B redrawn after Paxinos & Franklin
(22), C. unpublished result.
Light input to the SCN
The eyes are connected to the brain via the optic nerves that enter brain at the optic
chiasm, located at the base of the hypothalamus. The eyes contain specialized neurons
within the retina that are sensitive to light. These photoreceptive rods and cones
release neurotransmitters onto nearby retinal ganglion cells and react to light by
changing their membrane potential and decreasing the amount of neurotransmitter
they release. The ganglion cells have long axons projecting to the brain within the
optic nerves, and react to the decrease in transmitters by increasing the rate of action
potentials they generate. In the last decade, it has become clear that apart for the rod
and cone cells that are essential for vision, the eyes contain a second group of lightsensitive cells. This second group consists of a small percentage of retinal ganglion
cells that contain melanopsin, a photopigment that is most sensitive to blue light
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1
General introduction
(26-29). Mice with a genetic defect causing the loss of rods and cones are completely
blind, but retain non-visual responses to light such as the restriction of pupil size,
suppression of nocturnal melatonin release and entrainment to the 24 hour light-dark
cycle (30-32). Different from the rhodopsins and photopsins found in rods and cones,
melanopsin allows these ganglion cells to respond in a different way to light that
makes them ideally suited to act as a sensor that measures the intensity of the light
coming into the eye. Where rods and cones respond almost instantly to changes in the
amount and color of the light, their opsins are also quickly bleached by prolonged
light exposure. This bleaching makes their responsiveness decrease over time, as
evidenced by the amount of time needed to adjust your eyes to sudden darkness. In
contrast, when melanopsin containing ganglion cells are exposed to light, they increase
their activity much more gradually, reaching a sustained level of activity that reflects
the amount of light to which the cells are exposed. The role of melanopsin containing
ganglion cells as illumination sensors is reflected by the function of the brain regions
they innervate. Where regular retinal ganglion cells transmit signals from rods and
cones to the visual cortex via the lateral geniculate nucleus, axons from melanopsin
containing ganglion cells innervate several nuclei in the hypothalamus and thalamus.
This specific pathway is called the retino-hypothalamic tract (RHT) and connects the
melanopsin containing ganglion cells in the retina with the SCN, the intergeniculate
leaflet (IGL) and the olivary pretectal nucleus (OPN). These are the nuclei that are
essential for regulating circadian rhythms (SCN and IGL) and the pupillary light reflex
(OPN) (33, 34). Besides the direct innervation of the SCN by the RHT, light
information has second, indirect way of reaching the SCN. This secondary light input
originates from the IGL, which has efferent fibers projecting to the SCN that release
neuropeptide Y and contribute to the response of the SCN to photic stimuli (33).
Axons of the melanopsin containing ganglion cell of the RHT heavily innervate the
ventral part of the SCN. When light hits the retina, these axons release a mixture of
glutamate and Pituitary adenylate cyclase- activating peptide (PACAP) onto the ventral
SCN cells. Glutamate is an excitatory transmitter that increases the firing rate of the
cells in the ventral SCN (35, 36). Since SCN cells have a maximal firing rate, the
excitatory effect of light on SCN activity is most pronounced during the night, when
most cells are not generating spontaneous activity (37). Light exposure in the eye
during the night leads to a firing rate increase in the SCN and a shift of the circadian
rhythm if the exposure is strong enough and occurs in the early or late night. Both
SCN activation and phase shifts can be suppressed by using blockers that interfere
with the glutamate receptor found on SCN neurons (38, 39). The neuropeptide
PACAP that is co-released with glutamate appears to augment and reinforce the effect
of glutamate in the SCN. Although excitation of SCN cells and phase shifting can still
16
take place when PACAP is absent or blocked, both are decreased in the absence of
PACAP (40-42).
Apart from photic input from the eye and IGL, the SCN also receives inputs from
other regions of the brain. These inputs allow the SCN to respond to other cues than
light, including activity (43), feeding (44) and sleep deprivation (45). The signaling of
such information about the the behavioral state of the animal to the SCN is called
‘non-photic’ input and involves several pathways. One of these originates in the
brainstem raphe nucleus, which connects to many areas in the brain and releases
serotonin, a central neurotransmitter involved in the regulation of behavior and
mood. One of its’ targets is the SCN, where it releases serotonin whenever an animal
is active (46). In the SCN, serotonin alters the sensitivity of SCN neurons to light by
modulating glutamate release from the RHT and altering the postsynaptic sensitivity
to glutamate (47, 48). Furthermore, serotonin release can induce phase shifts that
mimic the phase shifts that are induced by non-photic stimuli applied during the
resting phase of nocturnal rodents, when light has no effect on the clock (figure 2).
Besides serotonin, behavior-associated neuropeptide Y (NPY) release in the SCN by
fibers from the intergeniculate nucleus also induces non-photic-like phase shifts, that
can be blocked by pre-treating the SCN with an NPI blocker (49). A third signaling
pathway that can be considered ‘non-photic’, involves several hormones for which
SCN cells express receptors. These hormones that can phase-shift SCN cells or
modulate their light-sensitivity, include melatonin (50, 51), estrogen (52) and
testosterone (53).
Finally, the SCN also receives information about sleep and vigilance state. Where
previous studies had shown that locomotor activity can alter SCN electrical activity
levels (54, 55), combined EEG and SCN electrical activity recordings showed that
SCN electrical activity is also modulated by sleep states (56). Specifically, REM sleep
increases SCN electrical activity, while non-REM sleep suppresses it. The anatomical
pathways that mediate these specific sleep effects on the SCN likely involve sleepregulatory areas such as the nucleus basalis the pedunculopontine tegmental nucleus
and the laterodorsal tegmental nucleus but also the serotonergic projections from the
raphe nuclei.
Anatomical and functional organization of the SCN
Anatomical studies of the SCN in different mammalian species have revealed that
within the SCN, subregions exist in which neurons differ in their properties and
function. Communication in the circadian system can be conceptually divided into
17
General introduction
Non-photic input to the SCN
1
1
General introduction
four different aspects: input to the SCN from the eye, input from other brain regions,
communication between different cells and regions within the SCN, and the output
communication of the SCN to other parts of the brain. Each of these
communication pathways utilizes a distinct set of neurotransmitters and peptides.
Nearly all neurons of the SCN release Gamma-Amino Butyric Acid (GABA) as their
principle neurotransmitter, but regional differences exist within the SCN in the type of
neuropeptides that are expressed and release by the SCN neurons (23). Although
much remains unknown about the precise roles of the different peptides, it appears
that the presence of certain peptides demarcates two main parts of the SCN that each
have a specific functional role. The first of these is the so-called core or ventromedial
part of the SCN that lies immediately on top of the optic chiasm. This core region
contains neurons that express vasoactive intestinal peptide (VIP) and gastrin-releasing
peptide (GRP). The neurons of the core region are densely innervated by axons
terminals of retinal ganglion cells that release glutamate and PACAP when light
activates the retina. Thus, a main function of the core region appears to process
photic information from the eye and use this to entrain the circadian rhythm of the
SCN to the environmental light-dark cycle. Apart from photic input from the retina,
the ventral SCN is also innervated by serotonergic fibers from the raphe nuclei and
NPY-releasing fibers from the IGL, enabling the SCN to integrate both photic and
non-photic environmental timing cues (57). The VIP positive neurons of the core
region project to dorsal SCN neurons and to other hypothalamic areas. Mutant mice
that lack either VIP or its receptor show strong deficits in entrainment and have
difficulty in maintaining a circadian rhythm in constant darkness (58, 59). Thus, VIP
seems to play an essential signaling role in keeping SCN neurons synchronized to each
other and the outside world. The dorsal part of the SCN contains neurons expressing
arginine vasopressin (AVP), a substance mainly involved in communication timing
signals to downstream targets of the SCN that regulate metabolism (60). For the
communication of timing information between the ventral and dorsal SCN regions,
GABA has been shown to be especially important in a study investigating the
adaptation of the rat SCN to a 6 hour delay of the light-dark cycle, an adaptation
comparable with a transcontinental flight from western Europe to New York (61).
Immediately following such a shift, brain slices containing the SCN were prepared
from the brains of the rat, in which the electrical activity rhythm of the SCN was
recorded. Instead of the single daily electrical activity peak in the SCN from unshifted rats, the SCN rhythm now displayed a double peak. The first peak was timed in
the middle of the day relative to the original light-dark cycle. The second peak
corresponded to the middle of the day in the new, 6 hours delayed day. Separating the
slices into a dorsal and ventral SCN containing part with a knife-cut showed that the
shifted peak originated in the ventral SCN, while the dorsal SCN showed an unshifted
18
Electrophysiological properties of SCN neurons
The rhythms generated by the intracellular molecular clock are translated into a
rhythmic output signals at the membrane of SCN neurons. These neurons have been
found to spontaneously generate action potentials at frequencies of 10 to 15 spikes
per second (63, 64). Neurons of the SCN spontaneously generate action potentials for
about 3 to 5 hours each day (65-67). Generating this rhythm is cell-autonomous
property that does not rely on interactions between neurons. This has been shown in
experiments in which SCN neurons were taken out of the brain and investigated in
vitro. When these cells were cultured with little or no connectivity to other cells, they
were still capable of generating a circadian rhythm in electrical activity (68). As all
excitable cells in the body, the generation of an action potential in an SCN neuron
requires that the membrane potential is increased up to the threshold potential at
which enough voltage gated sodium channels open to initiate an action potential. Like
other neurons, SCN neurons fire when their membrane potential is sufficiently
depolarized by axonal input from other neurons as illustrated by experiments showing
that SCN cells start firing when the retina is exposed to light (69, 70). Apart from
these induced potential changes, neurons of the SCN have the uncommon property
that during several hours each day, their membrane potential spontaneously
depolarizes toward the threshold potential, causing the cell to generate action
potentials. This time-window of spontaneous electrical activity falls during the day for
most SCN cells, causing a strong circadian rhythm in the electrical activity level of the
SCN that can be detected using implanted electrodes in the SCN (Figure 4). Although
the intracellular molecular clock is ticking inside nearly every cell in the body, SCN
neurons are unique in their ability to communicate this intracellular rhythm to other
cells by coupling the molecular clock to the membrane potential. Although many
mechanistic details remain unknown, it appears that in the SCN, the expression of
several ion channels that modulate membrane potential is under circadian control (71).
Importantly, the coupling between molecular clock and membrane potential appears
19
1
General introduction
peak. When the same procedure was done 6 days after the shift of the light-dark cycle,
the entire SCN had completely shifted and again displayed a single electrical activity
peak in the middle of the shifted day. Thus, the cells of the ventral SCN that receive
retinal input shift immediately, while dorsal SCN cells that receive less direct retinal
input take a couple of days to fully adapt to such a shift. Blocking the GABA-a
receptor had the same effect as making the knife-cut between the ventral and dorsal
SCN. Furthermore, the data showed that at certain times of the day, GABA can have
an excitatory effect on cells in the dorsal SCN. This finding was later confirmed in a
study by Choi et al., (62) and is surprising because GABA is generally regarded as a
purely inhibitory neurotransmitter in the adult brain.
1
to be bidirectional: studies have shown that blocking the electrical rhythm of SCN
neurons appears to suppress the intracellular molecular clock (72).
General introduction
Network properties of the SCN
The SCN contain approximately 10000 neurons in each nucleus (23). These neurons
form a network by means of direct synaptic connectivity between them. The network
of SCN neurons in turn is part of a bigger network, receiving input from the eye and
from other brain regions, and sending output to other parts of the brain. Although
SCN neurons are capable of generating circadian rhythms in firing frequency when
communication between them is absent or impaired, the rhythms generated by
individual cells are imprecise and variable in period (68, 73, 74). Communication
within the SCN network appears to add a level of robustness to the SCN clock,
allowing it to compensate for the effects of mutations in the Per1 or Cry1 clock genes.
Although dissociated SCN neurons and peripheral cells from animals carrying these
mutations became arrhythmic, SCN-containing brain slices that preserved the network
connectivity remained strongly rhythmic (75). In normal animals, the rhythm
produced by the intact SCN is much more precise and stable when these neurons are
connected in a network where the rhythmic activity of each neuron is transmitted to
connected SCN cells by neurotransmitters that are released from its axon terminals
during each action potential. Apart from this neurochemical communication, some of
Figure 4. Electrical activity rhythm in the suprachiasmatic nuclei in freely moving mice
The neurons of the SCN spontaneously generate action potentials during part of the day, causing a
circadian rhythm in spontaneous electrical activity that can be measured by surgically implanting a
recording electrode into the SCN. Here, two examples are shown of a 102 hour (4.25 days) recording in
the SCN of a freely moving mouse. Action potentials from neurons close to the electrode are detected in
the signal, counted, and stored on a computer. The black dots indicate the number of action potentials
per second. The behavioral activity of the animals was recorded simultaneously and is depicted below the
electrical activity (vertical black lines). The background color indicates the light that is present in the
recording cage, white indicates lights were on, grey indicates that lights were off. The animal in example
A. was exposed to an artificial 24 hour day, while the animal in example B. was recorded in constant
darkness. Note that electrical activity is high during the day and low during the night, while behavioral
activity in the nocturnal mice shows a reverse pattern. The recording in B shows that the rhythm in SCN
electrical activity continues under constant conditions, in line with the behavior (see also Figure 1.).
Unpublished data.
20
SCN Output mechanisms and pathways
In order to regulate the diverse physiological and behavioral rhythms, the circadian
timing signal generated by the SCN needs to be communicated to other brain areas
and organs. Axons from SCN neurons terminate in several brain regions. Besides
direct axonal projections that communicate directly with postsynaptic neurons via
neurotransmitters, the SCN also releases several neuropeptides that can communicate
circadian time to target neurons even without direct synaptic connections. In an
elegant experiment, Silver et al. (81) used tau mutant Hamsters with a 22 hour clock
and made them arrhythmic by lesioning the SCN. Then they took SCN tissue from
hamsters with a normal, 24 hour period, placed the tissue inside a capsule made of
semipermeable polymer. After implanting the capsule in the SCN-lesioned animals,
they observed that these animals became rhythmic again, with a 24 hour period. As
the polymer capsule allows free diffusion of soluble compounds but prevents the
21
1
General introduction
the SCN neurons are also electrically coupled by gap junctions that allow an action
potential generated by one cell to directly spread onto the membrane of the
connected cell. The function of the communication between the cells of the SCN is
to synchronize the rhythm of the individual cells to each other and to the outside
world. To achieve this synchronization, SCN cells are capable of adjusting the phase
of their circadian rhythm in response to the signals they receive from the network.
Output pathways from the SCN contain axons from multiple neurons. The circadian
pattern in neurotransmitter release from these pathways is therefore determined by
the sum of the circadian release pattern of al individual neurons involved in
generating the output. SCN slice recordings show that individual neurons of the SCN
generate action potentials for only 3 to 5 hours each day, and are silent for the
remaining hours. In contrast, SCN population recordings show a much longer
duration of high electrical activity that reflected the full duration of the previous lightdark cycle (65-67). Thus, the SCN network can track seasonal changes in day-length by
distributing the timing of the short activity pattern of individual neurons over the day
to ensure that the duration of high population activity accurately reflects day-length
(76). In a study testing the role of VIP in this ‘photoperiodic encoding’, VIP-deficient
mice were unable to adapt to different day-lengths, indicating that VIP plays an
essential role in the synchronization process that allows the SCN to adapt to seasonal
changes in day-length[Lucassen et al., in press]. In these mice, circadian rhythmicity is
weakened by a genetic defect, but similar deficits in network synchronization may also
occur as a consequence of aging, when VIP levels in the SCN have been shown to
decrease (77, 78). The importance of synchronization of the SCN network is further
supported by studies showing that exposing animals to constant light severely disrupts
synchronization of SCN neurons and leads to disrupted behavioral rhythms (79, 80).
1
General introduction
outgrowth of nerve fibers, this study convincingly indicated that the SCN releases a
diffusible signal that is sufficient to drive a circadian rhythm in running wheel activity.
Importantly, these diffusible signals only restore the circadian rhythm in behavioral
activity, but not the daily neuro-endocrine rhythms in hormones such as
corticosterone, and melatonin, for which synaptic output from the SCN is essential
(82). Since the discovery that the SCN releases humoral factors than drive behavioral
rhythms, several of such substances have been identified. The first of these was
transforming growth factor alpha (TGF-α). TGF-α is rhythmically produced by the
SCN with a peak during the sleep phase, and the epidermal growth factor (EGF)
receptors that respond to TGF-α are present in the sub-paraventricular zone (sPVZ)
(83). The sPVZ is an area that is densely innervated by SCN neurons and an essential
relay station in the circadian regulation of behavioral activity (84). Infusion of TGF-α
in the third ventricle inhibited behavioral activity and mice with a defective EGF
receptor displayed excessive daytime locomotor activity and failed to suppress activity
when exposed to light. A second SCN-released factor that controlled behavioral
activity was prokineticin 2 (PK2) (85, 86). Like TGF-α, PK2 release peaks during the
resting phase and suppresses behavioral activity when infused into the third ventricle.
The receptors for PK2 are expressed in several SCN-innervated regions, but unlike
the receptor for TGF-α not in the sPVZ. Another difference with TGF-α signaling, is
that the PK2 receptor is also present in neurons in the dorsal SCN where PK2 has an
excitatory effect (87, 88). Mutants missing PK2 or a functional PK2 receptor have
severely disrupted circadian rhythms (89, 90). The third SCN-released humoral factor
was cardiotrophin-like cytokine (CLC), which is released by a subpopulation of SCN
vasopressin neurons and peaks during the sleep phase (91). CLC receptors are located
along the the third ventricle and, like TGF-α and PK2, locomotor activity was
inhibited when CLC was injected into the ventricle. Conversely, locomotor activity is
increased when CLC receptors are blocked during the sleep phase.
The last SCN-released humoral factor mention is AVP, which was in fact the first
rhythmic, SCN-released substance to be discovered. In a 1981 paper (92), Reppert et
al., described a strong rhythm in AVP in the cerebrospinal fluid in cats. In spite of this
discovery, AVP was soon discarded as an important SCN output factor because of the
subsequent discovery that the “Brattleboro” strain of rats was perfectly capable of
generating circadian rhythms in behavior, even though they carried a genetic mutation
that prevented the expression of AVP (93). It took until 1992 to reinstate AVP as a
relevant SCN output, when it was shown that the rhythmic AVP release by the SCN is
coupled to the daily rhythm in blood corticosterone level by inhibiting the
hypothalamic nuclei that regulate corticosterone release by the adrenal gland (94).
22
Outline of this thesis
The experimental chapters of this thesis can be divided in two parts. In the first part,
consisting of chapters 2 to 5, we used electrophysiological recordings in the mouse
SCN to investigate the regulation of behavior by the SCN. The second part,
consisting of chapters 6 to 8, investigates how disturbances of the circadian system
are related to changes in behavioral activity and disease.
Part I - Regulation of behavioral activity by the SCN
In chapter 2, we investigated how changes in the neuronal network of the SCN allow
animals to adapt their daily behavioral activity pattern to seasonal differences in day
23
1
General introduction
Anatomical studies have shown that efferent connections from the SCN originate in
both ‘core’ VIP and ‘shell’ AVP positive SCN neurons. The distribution of VIP and
AVP positive axons overlaps, and they jointly terminate in several brain areas in the
hypothalamus, thalamus and basal forebrain (23). Areas innervated by the SCN
include the sub-paraventricular zone (sPVZ), essential for circadian behavioral
rhythms(84), the sleep-regulating ventro-lateral preoptic area (VLPO) (95), the
preoptic areas in the anterior hypothalamus that regulate body temperature (96) and
the dorsomedial hypothalamic nucleus (DMH) that regulates metabolism and
corticosterone levels.
A major function of the SCN is to coordinate that metabolic processes in the body
are adjusted to the demands posed by the daily rhythms in behavior and food intake.
During the active phase, the body has increased demands for glucose to power muscle
and brain activity. In animals and humans, the body is prepared for waking up by the
daily peak in the release of corticosteroid hormones by the adrenal glands. In the
hours before waking up, corticosteroid levels in the blood start to increase, causing an
increase in the blood glucose level that prepares the body for activity. The rhythmic
release of corticosteroids is driven by the SCN via a dual pathway: (1) AVP, released
by the SCN during the day into the SPVZ and DMH regulates the release of
Corticotropin-releasing hormone (CRH) by the paraventricular nucleus, the releasing
factor that stimulates the pituitary to release adrenocorticotrophic hormone (ACTH)
into the bloodstream; (2) The SCN modifies the sensitivity of the adrenal glands for
ACTH via a multisynaptic neuronal connection through the autonomic nervous
system (97, 98). Apart from the regulation of blood glucose levels via the
corticosterone pathway, the SCN influences glucose production by the liver via the
sympathetic nervous system and the orexin system (99-101) and modulates the release
of insulin by the adrenal glands via the parasympathetic system (99).
1
General introduction
length. We exposed animals to short day photoperiods to simulate winter and long day
photoperiods to simulate summer. After animals had adapted their behavior to the
artificial summer and winter days, we recorded the SCN neuronal population activity
pattern in the SCN in vivo and in vitro and found differences between short and long
days. Using a detailed analysis of the activity pattern of both individual SCN neurons
and small subpopulations, we tested whether the observed changes in the SCN output
pattern is a consequence of changes of the activity pattern of individual neurons, or a
result of alterations in synchronization in the SCN network.
The experiments described in chapter 3 investigate how SCN activity is related to the
time of the transition between the active and rest period. SCN neuronal activity
displays a 24 hour rhythm with high activity in the day. Towards the end of the day the
neuronal activity level starts to decrease, reaching a trough activity level in the early
night, from where it will start increasing again in the late night and early morning. This
circadian rhythm in electrical activity is communicated to other brain regions and leads
to a division of behavioral activity into an active (waking) and an inactive (sleeping)
phase. In mice as well as in humans, a transition between the two phases occurs in the
morning and in the evening. To investigate at what precise level of SCN activity the
transitions between the waking and resting phase occurred, we implanted mice with
microelectrodes in the SCN and performed long-term, simultaneous recordings of
SCN neuronal activity and behavioral activity. Using the obtained dataset, we analyzed
what SCN firing rate levels correspond to the onset of active period and the onset of
the resting period. These in vivo recordings were performed under three different day
lengths, to investigate whether there are seasonal differences in the SCN firing rate
level at the time of the transition between activity and rest.
Chapter 4 investigates how behavioral activity influences the waveform of SCN
neuronal activity. Although previous studies have presented evidence that behavioral
activity can modulate SCN firing rate levels, it was not known whether this effect
depends on specific types and/or intensities of behavior. It was also not clear what
the function of these feedback effects may be and whether they have a meaningful
impact on the waveform of the SCN output rhythm. To address these questions, we
used longterm in vivo SCN recordings in combination with video observation of the
animals behavior. Using this data, we analyzed if the presence and strength of SCN
MUA changes depends on specific types or intensity of behavior. Furthermore, we
performed a set of mild manipulations, to see whether evoked behavioral activity
would have a similar effect on SCN firing rate compared to spontaneous activity.
After observing that the SCN neuronal activity rhythm governs the transition between
the resting and active period and thereby determines the daily duration of the activity
period, we investigated in chapter 5 whether the SCN may also be involved in
regulating the level of activity within the active period. To test this hypothesis, we
24
Part II - Deterioration of circadian rhythms and disease
In chapter 6, we collaborated with the research group of professor Albrecht in
Fribourg (Switzerland) to investigate the relationship between the circadian system
and the mesolimbic reward system. The reward system of the brain consist of several
subcortical structures that are connected in a network that gets activated whenever an
animal has a rewarding stimulus, e.g. finding food or having intercourse. The main
neurotransmitter in this system is dopamine. The reward system in general, and
specifically dopamine, have been linked to psychiatric disorders such as depression
and also to substance abuse and addiction. Several lines of evidence suggest a link
between the circadian system and mesolimbic dopamine neurotransmission.
Depression in patients often coincides with rhythm and sleep disturbances (2, 102,
103), and mutations in clock genes lead to alterations in the response to cocaine (104).
Little was known however, about the mechanisms linking the circadian system and the
dopaminergic reward system. We investigated a possible link between per2 gene
expression and the regulation of striatal dopamine levels through mono-amine oxidase
A (MAOA), the enzyme responsible for the degradation of dopamine. Using per2
expression analysis we were able to demonstrate that per2 regulates MAOA
expression, leading to increased dopamine release in the mesolimbic reward system of
per2 mutant mice. We used behavioral tests and in vivo electrophysiological recordings
in the striatum to explore the consequences of the altered dopamine levels in these
mice and observed behavioral changes and altered electrophysiological responses to
antidepressant drugs.
There is evidence that circadian rhythm disturbances are related to metabolic
disorders such as type 2 diabetes. Several studies investigating this link have shown
metabolic abnormalities in mice carrying mutations in clock genes (105-107).
Furthermore, a recent study found that a specific destruction of SCN tissue causes
significant metabolic abnormalities and hepatic insulin resistance (108). Importantly,
several studies show that SCN function deteriorates during aging (109, 110), leading to
a decrease in amplitude of the rhythmic timing signal generated by the SCN. In
25
1
General introduction
exposed animals to short (22 hours) or long (26 hour) light-dark cycles. Using
implanted microelectrodes, we observed that such changes in the light-dark cycle
resulted in considerable changes in the waveform of the SCN firing rate rhythm. If
SCN neuronal activity levels are in fact involved in regulating the level of behavioral
activity, the observed changes in the circadian waveform of SCN neuronal activity
should result in changes in the behavioral activity distribution within the active period,
which we tested in our dataset.
1
General introduction
chapter 7, we investigated whether a decrease in SCN amplitude affects metabolism
and may contribute to the increased prevalence of type 2 diabetes mellitus in elderly
people. To test the effects of SCN amplitude on metabolism, we disrupted the
circadian rhythm of adult mice by exposing them to constant light. Using implanted
micro-electrodes in the SCN, we observed that this treatment led to a reduction of
amplitude of the SCN neuronal activity rhythm comparable to the amplitude
reduction observed during aging. The metabolic effects of constant light treatment
were investigated using metabolic cage assays and hyperinsulinemic-euglycemic clamp
analysis. An additional metabolic challenge was provided to part of the animals by
feeding them with a high-fat diet. The results showed that disrupting SCN amplitude
leads to body weight gain and a loss of circadian rhythms in metabolism and insulin
sensitivity.
Whereas chapters 6 and 7 investigated the adverse effects of genetic and
environmental disruption of circadian rhythms on disease susceptibility, it may also be
that disease-related physiological changes adversely affect the function of the
circadian system. For instance in patients with renal failure, sleep disorders are
frequently observed. In chapter 8 we collaborated with the department of nephrology
in the LUMC to investigate whether the circadian rhythm of behavioral activity is
affected in a mouse model for chronic renal failure (CRF). In these mice, a surgical
procedure was used to disrupt kidney function, causing significantly increased serum
urea levels and anemia compared to sham-operated mice. Both mice with disrupted
kidney function as well as sham-operated mice were housed under constant darkness
while their behavioral activity was monitored in order to test the capacity of the
circadian system to maintain a circadian cycle of activity and rest under physiological
conditions mimicking chronic renal failure in patients. The results showed that the
circadian rest-activity cycle was not impaired in the CRF animals compared to shamoperated mice, indicating that uremic toxins do not impair the function of the
circadian pacemaker in the SCN.
26
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