The function of anxiety fear related genes in animal models 2

1

Chapter 1: Introduction

CHAPTER 1: INTRODUCTION
1. Anxiety and fear
1.1. Definition
Fear is usually defined as a state of the organism elicited by a threatening and
clearly identifiable stimulus that prepares the organism to cope with the danger, and
subsides shortly after its offset. Anxiety is defined as a state of the organism that is
characterized by many of the same signs and symptoms of fear. However, it usually
is not clearly associated with a single eliciting stimulus, and may last for longer
periods of time once activated (Charney and Deutch, 1996).
Some authors argue that external stimuli are insufficient to distinguish fear
and anxiety. According to them fear is related to action, and particularly to escape
and avoidance. When the action is blocked, fear is turned into anxiety. Therefore
fear is an avoidance motive. If there were no restraints, internal or external, fear
would support the action of fight or flight. Anxiety can be defined as unresolved
fear, or, alternatively, as a state of undirected arousal following the perception of
threat. The alarm or primary anxiety may be channelled or resolved into fear, if
escape is selected as the action option after completely controlled processing of
stimulus situation (Lang et al., 2000).
In this work, anxiety will be differentiated from “fear” on the basis of two
above cited characteristics. First, the two are differentiated by the presence (in fear)
or the absence (in anxiety) of a specific stimulus that elicits the affective response.
Second, fear is conceptualized as the motivator of a set of coping responses to events


Chapter 1: Introduction

2

that provide more or less direct threats to the survival or well-being of the organism,
whereas anxiety occurs when the situation is too uncontrollable to permit active
coping behaviors (Ohman and Mineka, 2001). However, it must be emphasized that
anxiety and fear are normal reactions to danger. When anxiety and fear are more
recurrent and persistent than what is reasonable under the circumstances, and when
they impend normal life, an anxiety/fear disorder exists (LeDoux, 1995).

1.2. Anxiety disorders
According to the estimation of ADAS (Anxiety Disorders Association of
America, 2002), anxiety disorders are the most common mental illness in the U.S.
with 19.1 million (13.3%) of the adult U.S. population (ages 18-54) affected.
Characteristics that distinguish abnormal from adaptive anxiety include:
(1) Anxiety out of proportion to the level of threat
(2) Persistence or deterioration without intervention (> 3 weeks)
(3) Symptoms that are unacceptable regardless of the level of threat,
including recurrent panic attacks, severe physical symptoms and abnormal beliefs
such as thoughts of sudden death
(4) Disruption of usual or desirable functioning
The five anxiety disorders are identified as: Panic Disorder, ObsessiveCompulsive Disorder, Post-Traumatic Stress Disorder, Generalized Anxiety
Disorder and Phobias (including Social Phobia, also called Social Anxiety Disorder).
The significance of anxiety/fear in health and disease is well recognized
today, but its underlying molecular and neurobiological mechanisms are not well


Chapter 1: Introduction

3

understood. Anxiety/fear is a complex emotional state that cannot be reduced to

imbalances of a single neurotransmitter; however, preclinical and clinical evidence
has suggested the involvement of noradrenaline (NA), benzodiazepine, serotonin (5hydroxytryptamine, 5-HT), dopamine neuronal systems in anxiety and fear
development. And a lot of neurotransmitters are responsible for this process as well.
Among them, a prominent participation of cholecystokinin (CCK) (Griebel, 1999;
Woodruff et al., 1991), 5-HT (Blanchard et al., 1988; Graeff et al., 1996) and
corticotropin-releasing factor (CRF) (Eckart et al., 1999; Takahashi, 2001) in anxiety
and fear is generally acknowledged today.

2. Cholecystokinin (CCK) and its receptors
2.1. CCK: a neurotransmitter
2.1.1. Discovery of CCK and its molecular forms
CCK was first identified and characterized in the gastrointestinal tract as a
hormone with a major role in regulating the control of gut motility, pancreatic
secretion, and gall bladder contractions. In the brain, CCK is also one of the most
widely distributed peptides, where it acts as a neurotransmitter. CCK fulfills the
criteria for a neurotransmitter in the CNS: it is synthesized and stored in nerve
terminals and cell bodies, it is released by depolarization, specific receptors and
antagonists exist, and it influences the firing rate of other central neurons.
This peptide, initially characterized as a 33-amino-acid sequence, is present
in a variety of biologically active molecular forms derived from a 115-amino-acid
precursor molecule (prepro-CCK) (Deschenes et al., 1984), such as CCK-58, CCK-


Chapter 1: Introduction

4

39, CCK-33, CCK-22, sulfated CCK-8 [Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-PheNH2] and CCK-7, unsulfated CCK-8 and CCK-7, CCK-5, and CCK-4 (Trp-MetAsp-Phe-NH2) (Fig. 1-1). Several molecular CCK forms, ranging from 4 to 58 amino
acids, are found in the brain and peripheral organs. The most abundant fragment in
the brain is the sulfated octapeptide CCK-8S. The tetrapeptide CCK-4 is an

important and the shortest biologically active form occurring in the brain as well.

2.1.2. Distribution of CCK-related peptides in the central nervous system
CCK is very abundant in the brain, more than in the gut. CCK levels are very
high (> 4 ng CCK/mg protein) in cerebral cortex, caudate-putamen, hippocampus,
and amygdala, while thalamus, hypothalamus and olfactory bulb are lower (1–2
ng/mg protein). The pons, medulla and spinal cord have even lower levels (<1 ng/mg
protein), while CCK is barely detectable in the cerebellum (Beinfeld et al., 1981).
The anatomy of central CCKergic projections is very complex and has been
the subject of intense investigation since its discovery. The ability to visualize CCK
mRNA has provided much additional information about the location of CCK cell
bodies. Many brain regions like the cortex and hippocampus contain both CCKpositive interneurons and a mixture of afferent and/or efferent cells and terminals.
Certain regions like the striatum, nucleus accumbens and olfactory tubercle have
abundant CCK terminals, but few CCK-positive cells.
CCK cells are abundant in three sexually dimorphic nuclei in the rat
forebrain (the central part of the medial preoptic nucleus, the encapsulated part of the


Chapter 1: Introduction

5

bed nucleus of the stria terminals, and the posterodorsal part of the media nucleus of
the amygdala). CCK mRNA levels are altered by estrogen in these areas.

2.2. CCK receptors
2.2.1. Characterization of two CCK receptor subtypes: CCK1 and CCK2
Receptors for CCK have been pharmacologically classified on the basis of
their affinity for the endogenous peptide agonists CCK and gastrin, which share the
same COOH-terminal pentapeptide amide sequence but differ in sulfation at the sixth

(gastrin) or seventh (CCK) tyrosyl residue. Two types of CCK receptors: CCKA and
CCKB, have thus been distinguished.
A nomenclature committee of the International Union of Pharmacology
(IUPHAR) has changed the name of the CCK A and B receptors to CCK1 and CCK2
receptors, respectively. Although many researchers expressed their opposition to this
change and felt that the old system was working well (A for alimentary and B for
brain), both nomenclatures currently co-exist in the literature. This new
nomenclature has been adopted in our present study.
The CCK1 receptor was first characterized using pancreatic acinar cells
(Sankaran et al., 1980), whereas the CCK2 receptor, with a different pharmacological
profile, was discovered in the brain (Innis and Snyder, 1980). The gastrin receptor
mediating acid secretion in the stomach was initially thought to constitute a third type
of high-affinity receptor on the basis of its location and small differences in affinity
for CCK and gastrin-like peptides (Song et al., 1993). However, subsequent cloning
of gastrin and CCK-B receptors revealed their molecular identity.


6

Chapter 1: Introduction

CCK1 and CCK2 receptor types have been shown to differ by their relative
affinity for the natural ligands, their differential distribution, and their molecular
structure. Both of them have equally high affinity for CCK 8 sulfate, while they
differ substantially for unsulfated CCK peptides, gastrin and amidated peptides
shorter than CCK 7, like CCK 4 and CCK 5 (also called pentagastrin). The CCK1
subtype, typically found in the pancreas, is relatively specific for sulfated CCK 8;
unsulfated CCK 8, CCK 4 and gastrin are 2–3 orders of magnitude less potent. For
the CCK2 subtype, the difference in potency between sulfated CCK 8, unsulfated
CCK 8, gastrin and CCK 4 is about one order of magnitude. The distribution of

CCK1 and CCK2/gastrin receptors is tissue dependent.

2.2.2. Distribution of CCK receptors
A. Distribution in Central Nervous System
Specific CCK-binding sites were demonstrated in membranes from brain
homogenates almost two decades ago (Innis and Snyder, 1980; Saito et al., 1980).
Since then, numerous studies using autoradiography and, more recently, in situ
hybridization and immunocytochemistry have investigated the regional distribution
and specific cellular localization of CCK receptors throughout the neuraxis. Early
studies used radioligands such as

125

I-CCK-33,

125

I-CCK-8, [3H]pentagastrin,

[3H]CCK-8, [3H]CCK-4 or [3H]Boc[Nle28,31]CCK27-33 that do not distinguish
between the two CCK receptors. In general, these studies performed in several
species (e.g., rat, guinea pig, monkey, humans) showed high densities of CCKbinding sites in several areas, including the cerebral cortex, striatum, olfactory bulb


7

Chapter 1: Introduction

and tubercle, and certain amygdaloid nuclei. Moderate levels were observed in the
hippocampus, claustrum, substantia nigra, superior colliculus, periaqueductal gray

matter, and pontine nuclei. Low densities were reported in several thalamic and
hypothalamic nuclei and in the spinal cord.
With the advent of specific radioligands that could differentiate between the
two types of CCK receptors, it has become apparent that CCK1 and CCK2 receptors
exhibit a sometimes overlapping, yet distinct, distribution throughout the CNS. The
vast majority of CCK receptors in the CNS are of the CCK2 type, with CCK1
receptors restricted to rather discrete regions. The precise anatomical localization of
the two CCK receptor types serves to provide morphological substrates for many of
the diverse functions attributed to neural CCK, including involvement in feeding,
satiety, cardiovascular regulation, anxiety, pain, analgesia, memory, neuroendocrine
control, osmotic stress, dopamine-related behaviors, and neurodegenerative and
neuropsychiatric disorders (Crawley and Corwin, 1994).
B. Distribution in Gastrointestinal and Other Systems
In the gastrointestinal tract and other peripheral systems, CCK1 receptors are
present in pancreatic acinar cells, chief cells and D cells of the gastric mucosa,
smooth muscle cells of the gallbladder, pyloric sphincter, sphincter of Oddi, some
gastrointestinal smooth muscle and enteric neuronal cells, and anterior pituitary
corticotrophs (Wank et al., 1994). CCK1 receptors can also be expressed in several
tumors,

including

pancreatic

adenocarcinomas,

meningiomas,

and

some

neuroblastomas, as well as in certain pancreatic carcinoma, neuroblastoma, and lung
cancer cell lines. Furthermore, CCK1 receptor mRNA has been found in esophageal,


Chapter 1: Introduction

8

gastric, and colon cancers (Clerc et al., 1997). On the other hand, peripheral CCK2
receptors are located in smooth muscle cells throughout the gastrointestinal tract
(including the gallbladder), parietal, enterochromaffin-like, D cells and chief cells of
the gastric mucosa, myenteric plexus neurons, pancreatic acinar cells, monocytes,
and T lymphocytes. Tumors and tumor cell lines expressing CCK2 receptors include
medullary thyroid, gastric, colon, ovarian and small cell lung carcinomas,
astrocytomas, and certain pancreatic and lung cancer cell lines (Reubi et al., 1997).

2.2.3. The molecular biology of the CCK1 and CCK2 receptors
In April 1992, the cloning of the rat pancreatic CCK1 receptor (Wank et al.,
1992) and the canine parietal gastrin receptor (Kopin et al., 1992) were reported.
Both appear to be classical seven-domain, membrane-spanning receptors. The CCK1
and CCK2 receptors are 48% identical to each other and code for a protein of about
450 amino acids (Fig. 1-2; 1-3). They contain potential sites for N-linked
glycosylation and serine phosphorylation. Unlike other neurotransmitter receptor
systems (e.g., acetylcholine, serotonin and somatostatin), the CCK system, with only
two receptor subtypes, appears to be a model of simplicity.
The human CCK1 receptor gene has been localized to chromosome 4 using a
panel of human/hamster hybrid DNAs. The mouse CCK1 receptor gene has been
mapped to a syntenic region on chromosome 5 using a wild × inbred backcross panel

of mice [(BALB/cAN × Mus spretus) F1 × BALB/cAN]. This region of mouse
chromosome 5 is syntenic with human chromosome 4p16.2-p15.1 (Huppi et al.,
1995). The human CCK1 receptor was further mapped to 4p15.1-p15.2 using


Chapter 1: Introduction

9

fluorescence in situ hybridization. The rat CCK1 receptor gene has been localized to
a syntenic region on chromosome 14 by fluorescence in situ hybridization
(Takiguchi et al., 1997).
The human CCK2 receptor has been localized to chromosome 11 in humans
and a syntenic region on chromosome 7 in the mouse using a panel of
human/hamster hybrid DNAs (Huppi et al., 1995). Fluorescence in situ hybridization
of human metaphase chromosomal spreads has further localized the human CCK2
receptor gene to the distal short arm of chromosome 11 (11p15.4). The colocalization
of the CCK1 receptor gene with the dopamine D5 receptor gene at 4p15.1-p15.3 and
of the CCK2 receptor gene with the gene encoding the dopamine D4 receptor at
11p15.4-p15.5 is especially interesting in view of the coexistence of CCK and
dopamine in midbrain neurons and the regulation of mesolimbic dopaminergic
pathways by both CCK1 and CCK2 receptors.

2.3. Function of CCK-related peptides and CCK receptors
Over the last 20 years, numerous and extensive studies on the functional
significance of CCK peptides in the CNS have been published. It was demonstrated
that biologically active CCK peptides in the brain are involved in the regulation of
feeding, the control of learning and memory, behavioral expression of anxiety and
panic attacks, mediation of painful stimuli, and modulation of dependence and
withdrawal processes as well as functions controlled by the dopaminergic,

serotonergic, and opioid systems. The referred interactions of CCK with classical


Chapter 1: Introduction

10

transmission systems have been found in regions both with and without neuronal colocalization with other transmitters (Crawley and Corwin, 1994).

2.3.1. Role in anxiety
The initial suggestion that the CCK system might be involved in anxiety
came from experiments of Fekete et al. (1981), who reported that injection of CCK8
into the central nucleus of the amygdala enhances behavioral arousal and fear
motivation of rats. Since this report, evidence from different standard animal models
of anxiety has accumulated to suggest that CCK-related peptides are anxiogenic after
peripheral or intracerebral administration. Subsequent clinical studies demonstrated
that bolus injections of the CCK2 receptor agonist CCK4 or pentagastrin provoke
panic attacks in patients with panic disorders (Bradwejn et al., 1990) or in healthy
human subjects (Bradwejn et al., 1991; McCann et al., 1994), suggesting that
endogenous CCK system may be altered in panic disorder and contributes to
pathological anxiety.
In experimental psychopharmacology, anxiety-related effects can only be
determined by comparing drug effects in different animal models. The i.p. injection
of BOC-CCK4 produced anxiogenic effect in Wister and Lister rats in conflict test,
elevated plus maze, light/dark test and ultrasonic vocalization test (Rex et al., 1994).
CCK4 was proved to be anxiogenic in Wistar rats in acoustic startle reflex
(Vaccarino et al., 1997) and explotation box (Matto et al., 1997). In addition, CCK4
administered i.v. to African green monkeys has strong and dose-related effects on
behaviors thought to reflect anxiety and panic (Palmour et al., 1992). Derrien et al.

Chapter 1: Introduction

11

(1994) showed the anxiety-like behavior in Wistar rats in the elevated plus maze and
light/dark test after the i.p. injection of CCK agonists BC 197 and BDNL. Caerulein
is anxiogenic in the elevated plus maze in rats and mice (Mannisto et al., 1994;
Singh et al., 1991). CCK8s or CCK8us induced anxiety-like behavior in acoustic
startle reflex, elevated plus maze, four-hole box, marble burying test, light/dark test
and open field (Biro et al., 1993; Belcheva et al., 1994).
However, the anxiogenic effects of CCK peptides in animals have not been
observed by all investigators, and some results have been highly variable and
sometimes contradictory. The heterogeneity of response produced by CCK
administration can be explained by the fact that in some studies, different CCK
fragments have been infused in different brain areas in order to delineate the
anatomical substrate of CCK-inducing anxiogenic-like effects. The local application
of CCK4 in the basolateral amygdala produced an increase in the startle response
after acoustic stimulation, while perfusion in the nucleus accumbens did not modify
basal startle amplitude (Vaccarino et al., 1997). Studies with CCK8s also yielded a
different profile in different regions. The local application of CCK8s produced
anxiogenic-like effects in the elevated plus-maze when perfusion was performed in
the amygdala (Belcheva et al., 1994), posterior nucleus accumbens (Dauge et al.,
1990) and dorsal periaqueductual gray matter, but not in the anterior nucleus
accumbens (Dauge et al., 1990). Thus, the effects of CCK compounds could vary
considerably because of existing differences in the CCK fragments, brain areas of
injection, distribution and binding characteristics of CCK receptor types and/or
affinity states among species. Although negative findings have been obtained in

Chapter 1: Introduction

12

some anxiety models, it is noteworthy that anxiogenic effects of CCK-like peptide
have been reported, in the great part, in different animal species.
The hypothesized role for the CCK neuronal system in anxiety is further
upheld by studies with CCK antagonists. Between the two CCK receptors: CCK1 and
CCK2, CCK2 receptors have primarily been implicated in the control of exploratory
behavior and the development of anxiety. This is proved by the above introduction
that these CCK-like peptides, mainly CCK2 or CCK1/CCK2 agonists, when
administered systemically or intracerebrally, produced anxiogenic-like effects in
different animal species, including mouse, rat, guinea pig, cat, and monkey
(Blommaert et al., 1993; Harro et al., 1993). In addition, these effects could be
blocked by CCK2 antagonists, e.g. LY288513, which attenuated anxiety behavior in
animals when applied alone. CI-988 showed anxiolytic effect in conflict test,
elevated plus maze, light/dark test and social interaction test (Costall et al., 1991;
Bickerdike et al., 1994; Hughes et al., 1990). The selective CCK2 receptor antagonist
L-365,260 not only decreased the anxiety-like behavior in animal models, but also
blocked the effect of caerulein in elevated plus maze (Mannisto et al., 1994). Acute
treatment with L-365,260 was also reported to block CCK4-induced panic attacks in
panic disorder patients (Bradwejn et al., 1994) and pentagastrin-induced panic
symptoms in healthy volunteers (Lines et al., 1995). Costall et al. (1991)
demonstrated PD135158 induced the anxiolytic behavior in rats in elevated plus
maze. In response to the work implicating CCK in anxiety and fear behaviors, recent
anxiolytic drug development effects have focused on the therapeutic potential of
CCK2 antagonists. Potent and selective CCK2 antagonist compounds, such as CI-988


Chapter 1: Introduction

13

(Park-Davis) and L-365,260 (Merck) have shown considerable promise as novel
anxiolytic agents in some standard preclinical (Singh et al., 1991) and clinical tests
(Bradwejn et al., 1994).

2.3.2. Role in memory process
There is increasing preclinical evidence that the CCK system may play a role
in memory processes. The presence of CCK is conspicuous in brain regions
suspected to underlie memory functions, including the hippocampal formation,
amygdaloid nuclei, and cerebral cortex. It has been suggested that CCK1 and CCK2
receptors have different roles in learning and memory functions. In particular, a
balance between CCK1 receptor-mediated facilitatory effects and CCK2 receptormediated inhibitory effects on memory retention has been postulated (Lemaire et al.,
1992).
However, there are conflicting reports on the effects of CCK2 receptor
agonists in animal models of memory. For instance, in the two-trial memory task
based on exploration of novelty, it has been shown that BC 264 enhanced spatial
working memory, supporting the cognitive-enhancing properties of this agonist,
whereas BC 197 was found to induce an amnesic effect, a result in good agreement
with the memory deficit obtained with CCK-4 (Daugé and Léna, 1998). Treatment
with BC 264 has also been described to elicit prominent hypervigilance in monkeys
and to increase behavioral arousal in rats. Although some groups have found that
these peptides enhance memory (Gerhardt et al., 1994), others have reported that
selective CCK2 receptor agonists (e.g., CCK-4, BC 264) impair memory (Katsuura


Chapter 1: Introduction

14

and Itoh, 1986; Daugé et al., 1992; Lemaire et al., 1992). Factors that potentially
contribute to discrepant findings include differences in experimental paradigms,
dosage, and mode of drug administration. Another possible explanation of the
discrepant findings on the role of CCK receptors in memory function might be due to
the heterogeneity of CCK receptors.
To date, only a few studies have been devoted to the effects of CCK receptor
agonists on human memory. In one study, the administration of the nonselective
CCK receptor agonist ceruletide had no demonstrable effect on recent or remote
memory, although at higher doses it produced mild sedation. On the other hand,
electrophysiological investigations of event-related brain potentials showed that
ceruletide improved selective attention in healthy volunteers (Schreiber et al., 1995).
Ceruletide has also been reported to improve cognitive processing in young, but not
in elderly, healthy subjects (Dodt et al., 1996). Shlik et al. (1998) found that the
continuous administration of the selective CCK2 receptor agonist, CCK-4, produced
impairments in cognitive tests of free recall and recognition, although it had no
effect on psychomotor performance. The results of this study suggest that CCK-4
may exert a negative influence on memory consolidation and retrieval.

3. 5-hydroxytryptamine (5-HT) and its receptors
3.1. Introduction
Since its discovery over 50 years ago, 5-HT has been the topic of intense
research activity, which has led to the discovery of a range of potential drug targets:
the receptors, the metabolising and synthetic enzymes, the uptake sites. Some have


Chapter 1: Introduction

15

been exploited successfully and drugs are available and some have delivered
compounds currently under preclinical or clinical evaluation. The disorders treated
by drugs modulating 5-HT function cover a wide range, from chemotherapy-induced
emesis to depression.
The early research on 5-HT was focused on its functions in peripheral tissues,
and it was not until much later that its function as a neurotransmitter in the brain was
demonstrated. This was a major turning point in the discovery of therapeutic agents
that modify 5-HT function. There are some very important peripheral therapeutic
applications of 5-HT research, but it is a fact that a large proportion of the research
to exploit 5-HT pharmacology for therapeutic benefit has focused on its CNS
functions.
5-HT has been shown to have a multitude of different physiological actions,
and this is not surprising given the nature of the 5-HT neuronal system and the
variety of different 5-HT receptors (Barnes and Sharp, 1999). The 5-HT neurons
originate in the hindbrain in a relatively circumscribed area, but they send
projections to most parts of the brain. Couple that with the multiple ways that 5-HT
can exert its action through many different receptors and it becomes clear why the
pharmacology of 5-HT in the CNS is so complex. Furthermore, 5-HT is known to
interact with other neurotransmitter systems, and the literature is particularly rich in
publications in the interactions of 5-HT systems with dopaminergic systems.

3.2. 5-HT and anxiety


Chapter 1: Introduction

16

It is widely accepted that 5-HT is involved in the regulation of anxiety;
however, there is no agreement on whether 5-HT enhances or, conversely, decreases

anxiety. The former hypothesis is largely based on earlier experiments in which the
effect of drugs acting non-selectively on brain 5-HT mechanisms were measured in
animals under punishment or other approach/avoidance conflict situations (Graeff
and Schoenfeld, 1970; Tye et al., 1977; Wise et al., 1972). For example, in animal
conflict tests (after 24-h water deprivation period, the drinking attempts in rats were
punished with an electric shock), drugs or brain lesions that reduce 5-HT output have
a benzodiazepine-like anxiolytic effect. Moreover, microinjection of 5-HT receptor
antagonists into the amygdala releases punished behavior (increased the number of
shocks), whereas 5-HT receptor activation increases response suppression. These
results support the classical view that 5-HT is anxiogenic. In contrast to conflict
tests, however, models in which animals actively escape or avoid aversive brain
stimulation point to an anxiolytic role for 5-HT. Clinical evidence is also
controversial. For instance, benzodiazepine (BZD) anxiolytics are supposed to
alleviate anxiety, at least in part, by decreasing 5-HT release (Wise et al., 1972).
However, antidepressant drugs are beneficial in several anxiety disorders when
chronically administered. Yet, this drug regimen is likely to enhance, rather than
impair, 5-HT neurotransmission (Artigas, 1993; Blier et al., 1987).

3.3. 5-HT receptor subtypes
To date, 7 different 5-HT receptors and more than 14 subtypes have been
identified, some with splice variants and others with isoforms created by mRNA


Chapter 1: Introduction

17

editing. Furthermore, some of the 5-HT receptor isoforms and variants differ in their
neuroanatomical distribution. There are very few highly selective receptor agonists
or antagonists for these individual receptor subtypes. Animal and clinical data have

shown that 5-HT1A, 5-HT1B, 5-HT2A and 5-HT3 receptors are all involved in the
control of anxiety. Identification of many different serotonin receptor subtypes and
the development of selective agonists and antagonists for each of these various 5-HT
receptor subtypes, have provided the basis for an improved understanding of the dual
role of 5-HT in anxiety.

3.3.1. 5-HT1 receptors
5-HT1 sites have been further subdivided into 1A, 1B, 1C, 1D and 1E
subtypes. The 5-HT1A and 5-HT1B receptors have been proved to be present in the
mammalian brain. Numerous clinical and preclinical studies have confirmed the
anxiolytic properties of 5-HT1A receptor agonists, such as buspirone (De Vry et al.,
1992), which are effective in the treatment of GAD (generalized anxiety disorder).
These substances act at 5-HT1A receptors, located both presynaptically (as
somatodendritic autoreceptors in midbrain raphe) and postsynaptically (with the
highest expression occurring in forebrain limbic structures) (Griebel, 1995; Palacios
et al., 1990). Agonist activation of presynaptic 5-HT1A receptors reduces 5-HT cell
firing, synthesis and release (Sinton and Fallon, 1988; Sprouse and Aghajanian,
1987), while activation of postsynaptic receptors typically results in neuronal
inhibition (Van den Hooff and Galvan, 1992).


Chapter 1: Introduction

18

Several agonists, such as 8-OH-DPAT and the slightly less potent
pyrimidinylpiperazine derivatives, buspirone, ipsapirone and gepirone, show
reasonable selectivity for the 5-HT1A receptor as do a limited number of recently
developed antagonists, such as WAY100635 (Zifa and Fillion, 1992). The 5-HT1A
antagonists display intrinsic activity by themselves, which can potentially complicate

interpretation of combined agonist/antagonist effects. Nevertheless, `silent doses'
(i.e., doses lacking behavioral effects) of 5-HT1A antagonists have been shown to be
useful for characterizing the receptor-specificity of 5-HT1A agonist effects (Hogg et
al., 1994; Remy et al., 1996).
Three groups reported the generation of 5-HT1A knockout mice in 1998
(Parks et al., 1998; Heisler et al., 1998; Ramboz et al., 1998). Each of the three
research groups found a similar spontaneous behavioral phenotype, characterized by
elevated anxiety. In addition, although all research groups used open-field
exploratory behavior as a model for assessing anxiety, two groups, using other
behavioral models, the elevated zero or plus maze and a novel object test, confirmed
that these mice had elevated anxiety. The mechanism of both the increased anxiety
and reduced immobility in the antidepressant or stress (forced swim or tail
suspension) test models is likely to be a consequence of increased 5-HT availability
resulting from the lack or reduction in presynaptic cell body 5-HT1A autoreceptor
negative feedback function.
A high density of 5-HT1B sites is found in the basal ganglia (particularly the
substantia nigra, globus pallidus, caudate putamen, ventral pallidum and
entopeduncular nucleus), but also many other regions such as hippocampus and


Chapter 1: Introduction

19

cortex (Bruinvels et al., 1993). Data from many studies suggest that 5-HT1B
receptors are located on terminals presynaptically and postsynaptically relative to the
5-HT neurons where they play the role of both 5-HT autoreceptors and 5-HT
heteroreceptors. The former control the release of 5-HT while the latter control the
release of non-serotonin neurotransmitters. Some reports have suggested the
presence of 5-HT1B receptors in the dorsal raphe nucleus, which could play a

modulatory role in 5-HT release in this brain region (Moret and Briley, 1997). So
far, little information is available concerning the role of 5-HT1B receptor in anxietyrelated behaviors. Some data has suggested that 5-HT1B has opposite function to 5HT1A in animal models.
Various agonists that exhibit a certain selectivity for 5-HT1B receptors, such
as RU 24969, TFMPP and eltoprazine, show anxiogenic-like activity in animal
models, such as the shock probe conflict procedure, the social interaction test and the
elevated plus-maze test both in rats (Pellow et al., 1987) and in mice (Benjamin et
al., 1990). By using more selective antagonists, Chopin et al. (1998) have shown the
possible implication of 5-HT1B receptors in anxiety. The 5-HT1B/D and 5-HT1B
receptor antagonists, respectively, GR 127935 and SB 224289, increase the amount
of time spent in the light chamber in the two-compartment paradigm in mice,
suggesting the drugs to exert anxiolytic-like activity in this anxiety model.
5-HT1B "knockout" mice have been produced by specific ablation of the 5HT1B receptor gene by homologous recombination. Ramboz et al. (1996) have found
that there is no difference in the level of anxiety between mutant and wild-type mice
as judged by their activity in the light/dark model. In contrast, 5-HT1B knockout mice


Chapter 1: Introduction

20

are less "anxious" compared to wild-type mice in the open-field, the ultrasound
vocalisations and the elevated plus-maze (Zhuang et al., 1999). These data in general
lend support to the notion that a 5-HT1B receptor antagonist is likely to exert an
anxiolytic activity.

3.3.2. 5-HT2 receptors
To date, three 5-HT2 receptor subtypes have been characterized, namely the
5-HT2A, 5-HT2B, and 5-HT2C receptors. The 5-HT2A receptor is predominantly
located in the cortex, basal ganglia, and in some components of the limbic system
(including hippocampus, septum and amygdala) (Zifa and Fillion, 1992). The highest

expression of 5-HT2C receptors is found in choroid plexus, with lower but substantial
densities found in parts of the limbic system (as above) and basal ganglia (Wright et
al., 1995). Finally, a report suggests that, unlike the 5-HT2A and 5-HT2C subtype, the
5-HT2B receptor has a highly restricted distribution in rat brain, with the highest
expression occurring in lateral septum, dorsomedial hypothalamus and medial
amygdaloid nucleus (Duxon et al., 1997). The number of 5-HT2 receptor binding
sites is decreased in rat frontal cortex following chronic administration of most
antidepressant drugs and increased after repeated eletroconvulsive shocks (Sugrue,
1983).
Pharmacologically, few ligands selectively discriminate between the three 5HT2 receptor subtypes, making it extremely difficult to evaluate their individual
functional roles. Nevertheless, the combined use of agonists and/or antagonists with
varying affinities for each of the 5-HT2 receptor subtypes might permit cautious


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21

insight into the functional roles played by each. Initial clinical trials using 5-HT2
receptor antagonists (e.g., ritanserin) indicate that they may be useful for the
treatment of generalized anxiety disorder, and perhaps some phobias (Taylor et al.,
1985). Of the various compounds with antagonist properties at the 5-HT2A receptor
tested in the clinic, serazepine (CGS-15040A) showed efficacy in a multicentre trial
in GAD. Effects appeared primarily related to the psychic components of anxiety
(Katz et al., 1993).
The data regarding the 5-HT2B receptor in anxiety is very rare. One report
suggested that the injection of 5-HT2B receptor agonist BW 723C86 into the medial
amygdaloid nuclei increased the total interaction time of a pair of male rats in the
social interaction test. The increase in social interaction was prevented by
pretreatment with the 5-HT2C/2B receptor antagonist SB 200646A, which did not alter

behavior when given alone. The results are consistent with the proposal that
activation of 5-HT2B receptors in the medial amygdala induces anxiolysis in the
social interaction model (Duxon et al., 1997)
More recently, a series of compounds that are selective antagonists at the 5HT2C receptor were published. Of these compounds, SB-243213 has been shown to
be active in animal models of anxiety following acute and chronic administration
(Wood et al., 2001). Additionally, Deramciclane (EGIS 3886), a novel 5-HT2C
receptor antagonist, is currently in late phase II trials for the treatment of anxiety. It
appeared to have an anxiolytic effect in a variety of tests on animal models,
including punished drinking tests, social interaction tests and maze tests in rats and
marble burying tests in mice. It does not have a muscle relaxant effect but appears to


Chapter 1: Introduction

22

improve the escape performance of rats in a learned helplessness model, indicating
that it may also have potential for the treatment of depression (Pälvimäki et al.,
1998).
To investigate 5-HT2C function, 5-HT2C-knockout mice were generated by
introducing a nonsense mutation into exon 5 of the X-chromosome gene encoding
the 5-HT2C receptor. Mice lacking 5-HT2C receptors also displayed impaired
performance in the Morris water maze (Tecott et al., 1998). This deficit was not
associated with a generalized learning impairment, but rather with a highly selective
deficit in long-term potentiation confined to the dentate gyrus and not other
hippocampal regions. As information processing within the dentate gyrus has also
been associated with exploratory behavior, these mice were tested in several models
of exploratory, fear-related behaviors. 5-HT2C-/-mice were found not to differ from
their normal littermates in contextual fear conditioning, but did exhibit a threefold
reduction in emergence neophobia (entry into a brightly lit open field). The data

confirms and extends some of the previous findings of the role of 5-HT2 receptors in
anxiety.

3.3.3. 5-HT3 receptors
The highest density of 5-HT3 binding sites is found in the area postrema,
with lower but significant densities also found in limbic structures such as the
septum, hippocampus and amygdala (Zifa and Fillion, 1992). Agonist activation of
the 5-HT3 receptor results in rapid neuronal depolarization, which can be blocked by
a number of selective 5-HT3 antagonists (e.g., tropisetron, ondansetron, GR38032F,


Chapter 1: Introduction

23

MDL72222). Preliminary clinical trials using 5-HT3 antagonists (e.g., ondansetron
and tropisetron) indicate that they may be useful in the treatment of generalized
anxiety disorder (Kunovac and Stahl, 1995).
Infusions of ondansetron, granisetron and zacopride into basolateral
amygdala produced selective anxiolytic effects in the elevated plus-maze (Tomkins
et al., 1990) as did infusions of GR38032F and tropisetron into central amygdala in
the mouse light–dark exploration test (Costall et al., 1989). These latter effects were
similar in magnitude to those induced by diazepam. The injection of ondansetron,
tropisetron, MDL72222, granisetron, and GR65630 also significantly increased rats'
social interactions in the high light/unfamiliar test condition, with a magnitude equal
to or greater than that of flurazepam (Higgins et al., 1991). Anxiolytic doses of these
5-HT3 antagonists did not affect locomotor activity in the high light/unfamiliar
condition, or social interaction in the less aversive, low light/familiar condition.
Conversely, selective anxiogenic effects were demonstrated in the light–dark test and
social interaction following intra-amygdala infusions of 2-methyl-5-HT (Costall et

al., 1989; Higgins et al., 1991). Importantly, the rank order potencies of the 5-HT3
antagonists in the amygdala (i.e., granisetron>tropisetron=GR 65630>ondansetron>
MDL72222) agreed well with their relative affinities for the 5-HT3 binding site, and
with their relative potencies for inhibiting 5-HT-induced depolarizations of the rat
vagus nerve. These correlations provide strong evidence that the anxiolytic effects of
5-HT3 antagonists in the amygdala were mediated at the 5-HT3 receptor (Higgins et
al., 1991).


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24

On balance, the anxiolytic effects of 5-HT3 antagonists were reasonably
consistent across a variety of behavioral tests, although there is some suggestion that
5-HT3 receptors in different regions of the brain might mediate different fear
behaviors. For example, 5-HT3 antagonists had anti-conflict effects in the
hippocampus but not in the amygdala, and increased social interaction in the
amygdala but not in the raphe. Confirmation of these dissociations may provide
important information on understanding the exact role 5-HT3 plays in anxiety and
fear development.

3.3.4. Other 5-HT receptors
The role of the other 5-HT receptors in anxiety and fear disorders is so far
poorly understood. At present, there are limitations to pharmacological
characterization of 5-HT receptor subtypes in the brain of animals, since agonists,
antagonists and antibodies to distinguish all 5-HT receptor subtypes are not yet
available.
Receptors for 5-HT6, like those for 5-HT4 and 5-HT7, stimulate adenylate
cyclase activity. This effect can be blocked by antidepressant and antipsychotic

drugs such as amitriptyline, clomipramine, clozapine and loxapine in HEK293 cells
stably transfected with 5-HT6 receptors (Sleight et al., 1996). The prominent
localization of 5-HT6 mRNA in the striatum, nucleus accumbens, olfactory tubercle
and substantia nigra, together with its high affinity for both typical and atypical
neuroleptics, has led to speculation that this receptor might be one of the target sites
of action for antipsychotic agents. In a microdialysis experiment, 5-HT release in the


Chapter 1: Introduction

25

prefrontal cortex elicited by conditional fear stress (but not the comparable 5-HT
release elicited by the control condition, unconditional footshock) was eliminated by
the 5-HT6 antisense oligonucleotide osmotic minipump pretreatment regimen
(Yoshioka et al., 1998). This data suggests the possibility that 5-HT6 receptors might
be involved in behavioral disorders and in the mechanism of 5-HT-modulating
drugs, including antidepressants, which are effective in their treatment.
5-HT7 receptors have been identified in rodent and human brain and, like 5HT6 receptors, they share high binding affinity for antidepressant and antipsychotic
drugs. Rat frontal cortical astrocyte 5-HT7 receptors responded to the administration
of the antidepressant amitriptyline with an enhanced camp response to 5-HT
(Yoshioka et al., 1998). This enhancement was reduced by either a 5-HT7 receptor
antisense oligonucleotide or a non-5-HT subtype-selective antagonist, methiothepin.
In general, the large amount of 5-HT receptors and subtypes as well as their
interaction with other neurotransmitter systems could be responsible for the
incredibly diverse actions of serotonin.

4. Corticotropin-releasing factor (CRF) and its receptors
4.1. Structure and distribution of CRF-like peptides
CRF exists as a 41-amino-acid polypeptide in a large variety of mammalian

species that is synthesized in the hypothalamus and mediates the release of
adrenocorticotropic hormone (ACTH) from the anterior pituitary (Spiess et al., 1981;
Vale et al., 1981). It is generated by cleavage of the C-terminus of pre-proCRF, the
196-amino-acid precursor (Dautzenberg and Hauger, 2002). 29 different peptides