Download Introduction 9 INTRODUCTION Circadian rhythms in physiology and

Introduction
9
INTRODUCTION
Circadian rhythms in physiology and behavior can be found throughout the plant
and animal kingdom and are generated in individual organisms by an
endogenous circadian pacemaker. In mammals, the main circadian pacemaker
is located in the suprachiasmatic nuclei (SCN) at the base of the hypothalamus.
First indications were provided in 1972, when it was shown that lesions of the
SCN abolish the adrenal corticosterone rhythm, as well as rhythms in drinking
and locomotor activity. In 1990, it was demonstrated that transplantation of the
SCN into an arrhythmic SCN-lesioned animal restores rhythmicity with a period
that is characteristic of the donor, providing unambiguous evidence for the
location of the mammalian circadian pacemaker. Since then, it has become
clear that circadian rhythm generation occurs within individual SCN cells. The
cell-autonomous mechanism of rhythm generation results in the presence of
multiple, single cell oscillators within the SCN.
Circadian rhythms need to be correctly phased to the external environment.
This is achieved by synchronization to the environmental light-dark cycle, which
occurs as a consequence of the phase shifting effects of light. Photic
information is transmitted from the retina in the eye to the SCN via a specialized
pathway, which terminates predominantly in the ventral SCN region. This
suggests that the SCN is a heterogeneous structure and that the multiple, single
cell oscillators within the ventral SCN may be functionally different from those in
the dorsal SCN.
In the past decade, increasing evidence has appeared for the presence of
autonomous oscillators outside the SCN in the central nervous system and also
in peripheral tissues. These oscillators may or may not act as local pacemakers.
For a proper functioning of the circadian system, the multiple oscillators within
and outside the SCN need to be mutually synchronized. Interactions are
thought to exist within the SCN, between the two SCN nuclei and between the
SCN and the periphery.
It is now well-established that the generation of circadian rhythmicity in single
cells occurs at the molecular level and is based on a transcriptional-translational
feedback loop, consisting of several clock genes and their protein products. On
the other hand, the presence of a multitude of circadian oscillators, as well as
the occurrence of coupling between them, suggests that other characteristics of
the circadian timing system may be generated at the neuronal network or tissue
level. The aim of this thesis was to obtain insight in the hierarchical organization
of the circadian timing system and to determine whether certain attributes of the
system arise at the tissue level. The first chapter of this thesis is a review
discussing the presence and characteristics of multiple oscillators in several
vertebrate and invertebrate animal species. Moreover, the existence and
mechanisms of coupling within the circadian timing systems of these species
are compared. In the experiments described in chapters 2 through 6, the
responses of different components of the system to several types of phase
10
Introduction
resetting stimuli are investigated and the interactions between a number of
them are characterized. The individual chapters are outlined below.
Phase resetting of the circadian timing system is noticeable as a phase
advance or delay in the onset of behavioral activity. However, it has been
observed that light-induced phase resetting of the activity offset shows different
kinetics than that of the onset. This phenomenon is most obvious following light
pulses that are administered during the late subjective night. The activity offset
responds with a substantial phase advance, the full magnitude of which is
reached almost immediately. In contrast, the activity onset shows transient
phase resetting with the size of the phase shift increasing in the course of
several cycles. As a result, the length of daily activity changes temporarily, but
generally returns to baseline values in the course of several days. A model has
been proposed suggesting that the mammalian circadian pacemaker consists of
two coupled circadian oscillators that control either the activity onset or offset
and that respond differently to light. It is not clear whether the systems
responsible for the different phase shifting kinetics of the activity onset and
offset are located within the SCN or whether the difference is a result of
complex downstream responses. The experiments described in chapter 2 were
designed to further characterize the systems controlling the behavioral activity
onset and offset. In addition to light, several other stimuli, collectively referred to
as non-photic stimuli, are able to affect the phase of overt circadian rhythms. In
order to investigate the responses of the activity onset and offset to non-photic
stimuli, their phase shifts were examined following administration of the opioid
agonist fentanyl and the benzodiazepine midazolam.
The SCN receives several neuronal projections from the brain, such as a
serotonergic projection from the raphe nuclei and a projection from the
intergeniculate leaflet that contains, among others, neuropeptide Y and
enkephalins, which are endogenous opioids. Many neurotransmitters present in
the afferents of the SCN have phase shifting effects on behavioral rhythms and
their actions on molecular and electrophysiological processes in the SCN have
been relatively well described. A previous study from our lab demonstrated that
the opioid agonist fentanyl induces phase shifts in hamster wheel running
activity rhythms. These data suggest that the hamster circadian timing system
can be modulated by opioidergic input, but do not reveal the site of action, e.g.
whether opioids act directly on the SCN in inducing phase shifts. In the
experiments described in chapter 3, we investigated whether fentanyl affects
the neuronal discharge rate and clock gene expression in the SCN. Moreover,
the existence of a putative interaction in the SCN between light input and
opioids was investigated at the behavioral and the molecular level.
It has become clear that certain types of behavior affect the mammalian
circadian system. For example, several hours of wheel running activity during
the subjective day may induce phase advances in locomotor activity rhythms.
Interestingly, behavioral activity can be accompanied by a suppression of SCN
neuronal activity, suggesting that behavioral activity also has acute effects on
Introduction
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the SCN. It has been unresolved whether sleep is one of the behavioral
processes that affect the biological clock. Some indications for such an effect
have come from the phase shifting effect of sleep deprivation on locomotor
activity rhythms. In the experiments described in chapter 4, the presence of a
correlation between vigilance state and short-term changes in the discharge
rate of SCN neurons was explored through simultaneous recording of the
electroencephalogram (EEG) and SCN neuronal activity. When this correlation
was established, the presence of a causal relationship between these
phenomena was tested using selective sleep deprivation experiments.
After having analyzed the effects of several non-photic stimuli on the circadian
pacemaker, chapters 5 and 6 focus on the photic input. The effects of short light
pulses on the SCN are relatively well described. For example, light pulses
induce sustained changes in SCN neuronal activity and, after administration
during the subjective night, they result in a change in the expression level of
several clock genes in the SCN. In a study by Yamazaki and coworkers (2000),
animals were exposed to another photic stimulus, a phase shift of the light-dark
cycle, and the effects on the rhythms in clock gene expression in the SCN were
investigated. It was demonstrated that the Per1 bioluminescence rhythm phase
shifted immediately upon a 6-hour phase advance or a 6-hour phase delay of
the light-dark cycle. This was surprising, since the behavior of most laboratory
rodents adjusts relatively slowly to a shifted cycle. The findings suggest that the
molecular clock in the SCN and overt locomotor rhythmicity can dissociate
temporally. The experiments described in chapters 5 and 6 were designed to
investigate at which level this dissociation occurs. In chapter 5, the phase
resetting characteristics of locomotor rhythms were compared to those of clock
gene expression and electrical activity rhythms in the SCN. In chapter 6, the
phase resetting kinetics of the electrical activity rhythms in the dorsal and
ventral parts of the SCN were studied. These experiments were performed
using intact SCN slices and slices that were bisected to physically separate the
ventral and dorsal SCN regions. The effect of longterm application of the
GABAA antagonist bicuculline on the electrical activity patterns in these regions
was investigated, as well as the immediate effects of short-term bicuculline
application.