The Role of the 5-HT3 Receptor in the Central Nervous System

(1)

The Role of the 5-HT3 Receptor in the Central

Nervous System

March 2012–August 2012

Joren Zandstra, student ID 5922283

MSc in Brain and Cognitive Sciences,

University of Amsterdam, neuroscience track

Supervisor

– Prof. dr. Berend Olivier; UU

Co-assessor

– Dr. Hans van Hooft; UvA

August 10, 2012

Abstract

The role of the 5-HT3 receptor in the central nervous system is dis-cussed. A global introduction describing the functional properties of the 5-HT3 receptor, its expression, and its variants is included. The role of central 5-HT3 receptors is discussed regarding the topics aggression, anxi-ety, depression, drug addiction, satianxi-ety, schizophrenia, and sleep disorder. For each topic animal and human research is discussed, including the pre-sumed mechanisms in which the 5-HT3 receptor is involved, the clinical relevance of drugs targeting the 5-HT3 receptor, and current issues with the research. A conclusion is provided summarizing the findings, attempt-ing to deduce general conclusions about 5-HT3 receptor function, 5-HT3 receptor clinical relevance, and outlining possible directions of future re-search.

(2)

1 Introduction

Serotonin or 5-hydroxytryptamine (5-HT) is a neurotransmitter with very wide application. Receptors for 5-HT are found in both the central nervous sys-tem (CNS, including cerebral cortex, olfactory syssys-tem, hippocampus, amygdala, basal ganglia, central gray, mesencephalic reticular formation, and dorsal raphe

nucleus (DRN; partially expressed there by glutamatergic neurons); (Monti,

2011)) and peripheral nervous system (PNS), and these receptors have been

im-plicated in numerous conditions for almost 20 years, such as anxiety, depression,

schizophrenia, and drug abuse (Greenshaw, 1993; Lucki, 1998). 5-HT acts on

the 14 members strong 5-HT receptor family. Since the discovery of what

cur-rently is called the 5-HT receptor in 1957 (reprinted in Gaddum and Picarelli

(1997)), various agonists and antagonists for the whole family of receptors have

been found. In the early 1990s, the discovery of specifically the 5-HT3 receptor sparked a wave of reports on possible treatments of all sorts. One decade later a review of specifically the anxiolytic properties of 5-HT3 receptor antagonists byOlivier et al.(2000) concluded that “5-HT3 receptor antagonists do not rep-resent a breakthrough in the treatment of various anxiety disorders, as initially suggested”. Presently it has been two decades since these promises of novel therapeutic applications, and for the first few compounds the 20-year patents are about to run out. In that light, this review will attempt to summarize what is known of the 5-HT3 receptor, what its functions are within the CNS and which agonists and antagonists have, in retrospect, fulfilled their bright promises. The functions discussed are aggression, anxiety, depression, (drug) addiction, satiety, schizophrenia, and sleeping disorders.

This review is mainly focused on the central nervous system presence of 5-HT3 receptors so it omits the consistent application in combating postoper-ative nausea and cancer chemotherapy, as well as its use in treating irritable

bowel syndrome. The sections on satiety (subsection 3.6) and sleep disorders

(subsection 3.8) do feature research on peripheral 5-HT3 receptor action but they try to extract what is known about central 5-HT3 receptor function. The topics of nociception and memory with respect to 5-HT3 receptors are omitted here due to time constraints.

2 Methods

For this literature review, the PubMed database1_{was searched for articles}

con-cerning the 5-HT3 receptor and its various involvements. The search terms used

are available inAppendix A; for each of the following subsections the article

pro-gression from raw search results to review-included is available in Appendix B.

1

(4)

3 The

5-HT3 receptor and its function in the

CNS

3.1 5-HT3 receptor structure, function, and classification

Serotonin is a ubiquitous transmitter, and its receptors are expressed through

the body (Hoyer et al., 1994). A whole family of serotonin receptors exist

(5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT6, and 5-HT7) although not all of them occur in humans and some have their own subtypes. The 5-HT3 receptor alone is a ligand-gated ion channel where the other types all are G-protein coupled

receptors (Hoyer et al.,1994,2002). Proximally, 5-HT3 receptors induce influx

of Ca2+ _{(as well as Na}+ _{and K}+_{), depolarizing neuronal membranes.}

Dis-tally, 5-HT3 receptors control complex regulation of neurotransmitter release, both inhibiting and increasing release of 5-HT as well as dopamine, the two neurotransmitters that will be discussed most in this review. Further effects comprise cholinergic, dopaminergic, noradrenergic, and GABAergic signalling,

as reviewed inFink and G¨othert(2007). Notably, cells expressing 5-HT3 in rat

brain are mostly GABA-containing cells (Bloom and Morales,1998).

The 5-HT3 receptor itself consists of five subunits (5-HT3A–5-HT3E) around a central pore (Davies et al., 1999; Dubin et al., 1999; Holbrook et al.,

2009; Miyake et al., 1995; Niesler et al., 2003), most well-researched of which

are 5-HT3A and 5-HT3B (Davies et al., 1999; Dubin et al., 1999), both

lo-cated on 11q23.1-q23.2 (Davies et al., 1999; Miyake et al., 1995; Weiss et al.,

1995). A long and a short (truncated) splice variant of 5-HT3A have

re-cently been dubbed 5-HT3AL and 5-HT3AT, respectively (Br¨uss et al., 2000;

Niesler et al.,2007), both of which combine with 5-HT3A into functional

recep-tors (Rajkumar and Mahesh, 2010). Subunits 5-HT3C, 5-HT3D, and 5-HT3E

were mapped to 3q27.1 (Niesler et al., 2003), and isoforms for 5-HT3D and

5-HT3E have been found as well (Niesler et al.,2007). It should be noted that

not all species with 5-HT3 express all five of these 5-HT3 subunits (Barnes et al.,

2009;Miyake et al.,1995). For reference,Figure 1shows the pentameric struc-ture of the receptor subunits and the overall strucstruc-ture of the receptor.

The 5-HT3A subunit is the only subunit capable of forming functional

monopentameric receptors (Davies et al.,1999;Dubin et al.,1999).

Heteropen-tameric functional receptors can be composed of any combination of these subunits with the condition that they contain at least two 5-HT3A subunits (Niesler et al., 2007). However, receptor properties may vary markedly

be-tween the various subunit compositions (Niesler et al.,2007). To give one

exam-ple, monopentameric 5-HT3A receptors have lower single channel conductance,

greater Ca2+ _{permeability, and slower kinetics of activation, deactivation, and}

sensitization compared to 5-HT3AB heteropentameric receptors (Davies et al.,

1999;Niesler et al.,2003;Walstab et al.,2008). Because of these varying prop-erties, results of 5-HT3 receptor function research may be unclear because the various receptor compositions are often not explicitly differentiated. Where possible this review indicates the receptor subtypes in the referenced literature.

See alsoYamada et al.(2006) andYaakob et al. (2011) for reviews of HTR3A,

HTR3B, HTR3C, and HTR3E SNPs, which may cause receptor properties dif-ferent from wildtype even with identical subunit composition.

(5)

Figure 1. Schematic structure of the pentameric 5-HT3 receptor, consisting of a combination of five subunits (5-HT3A–5-HT3E, where at least 2 5-HT3A subunits are required to form functional receptors (Yaakob et al.,2011)) around a central pore. One subunit has been removed in the picture to reveal the cation channel lumen. Arrows point at binding sites for agonists, competitive/non-competitive antagonists and positive modulators. The orthosteric ligand binding site is enlarged to show the contributing binding loops of adjacent subunits. The four transmembrane domains (TMs) of a single subunit, TM 2 lining the channel pore, are also shown. Figure and part of the caption adapted from

Walstab et al.(2010).

expressed in human brain (hippocampus, caudate nucleus, putamen, nu-cleus accumbens, amygdala), rat brain (nunu-cleus of the solitary tract (NTS),

dorsal motor nucleus of vagus, area postrema, limbic regions (e.g.

amyg-dala, hippocampus, frontal, entorhinal cortex), raphe nucleus, and olfac-tory bulb), and mouse brain (nucleus of vagus nerve and (to a lesser ex-tent) amygdala, hippocampus, entorhinal cortex), as well as peripheral regions

in those species (Rajkumar and Mahesh, 2010). However, medial prefrontal

5-HT3 receptors tend to differ from peripheral receptors and cell line

recep-tors (Rajkumar and Mahesh,2010), leading to differential pharmacological

re-sponses depending on the area exposed to drugs.

Often-used 5-HT3 receptor antagonists include ondansetron, tropisetron (or ICS205930), and bemesetron (or MDL72222); mirtazapine is a broad 5-HT3 antagonist that also includes adrenergic α2-autoreceptor and α2-heteroreceptor antagonist and 5-HT2 antagonism. Functional 5-HT3 activity has been proven in structurally dissimilar antidepressants (tricyclides, selective serotonin reup-take inhibitors (SSRIs), noradrenalin reupreup-take inhibitors (NRIs),

noradrener-gic/specific serotonergic antidepressants; (Rajkumar and Mahesh, 2010)), the

(6)

as ethanol (5-HT3A homopentameric receptors only; Davies (2011); Johnson

(2004);Lovinger(1999)) and cocaine (Davidson et al.,2002), hinting at several mechanisms of receptor modulation. A word of caution about the drugs above is warranted. After years of research, the specificity of even selective 5-HT3

receptor antagonists has been called into question (Faerber et al., 2007). For

example, ondansetron, bemesetron, and tropisetron block acetylcholine-induced α9α10-nicotinic receptor currents to the same extent as 5-HT3 receptor

cur-rents (Rothlin et al., 2003). Even when drugs are known for having multiple

interactions, for example mirtazapine (affinities as above) and cyamemazine (antagonist to dopamine D2 receptors and 5-HT2A, 5-HT2C, and 5-HT3 re-ceptors), care is not always taken. Publications seem to sometimes cherry-pick a possible 5-HT3 receptor interaction for its novelty value, only to omit con-trol experiments that show 5-HT3 as the actual mechanism involved in the particular effect researched. Accordingly, this review attempts to annotate re-search with cautionary notes when it omits control experiments for mirtazapine, cyamemazine, or drugs that are otherwise conceivably involved in other ways than 5-HT3 receptor interactions.

Another problem in drug research (and in practical medicine, of course) is the blood-brain barrier (BBB). In both animal and human research, drugs are often administered systemically, making the assumption that systemic drugs pass the BBB in order to exert their effects centrally if CNS effects are the focus of research. However, for both 5-HT agonists and 5-HT antagonists, it is not generally true that they pass the BBB at effective levels. While some

publications mention easy passage of both 5-HT agonists (Campiani et al.,1999)

and 5-HT3 antagonists (Walstab et al., 2010) through the blood-brain barrier,

a cursory search on literature on this topic did not reveal a wealth of results. Note that for the topic of satiety this problem is partially circumvented: some of the involved brain areas are outside the BBB, namely the area postrema, medial nucleus of the solitary tract, and dorsal motor nucleus of the vagus.

(7)

3.2 Aggression

The involvement of 5-HT with aggression has been firmly established for a long

time (e.g. Kostowski et al.(1975)), and with it the actions of the 5-HT3

recep-tor. However, the specific role of 5-HT3 is not clear cut. Some studies have

found no link between 5-HT3 receptor and aggressive behavior (S´anchez et al.,

1993; White et al., 1991), others have found a variably exacerbating or

atten-uating influence of 5-HT3 receptor (e.g. McKenzie-Quirk et al. (2005)). The

research, and some of these inconsistencies, are discussed below.

3.2.1 Animal research into aggression

Animal aggressive behavior with respect to 5-HT3 receptors has been studied in mice, rats, and hamsters. Animal models of aggression revolve mostly around the resident-intruder paradigm, where the tested animal is confronted with an age- and size-matched ‘intruder’ in either the animal’s home cage (familiar terri-tory for him, unfamiliar territerri-tory for the intruder) or a neutral cage (unfamiliar territory for both). In this situation, the two animals may interact either ag-gressively (e.g. attacks, bites, pursuits, threatening behavior) or socially (e.g. smelling, sniffing), or display non-social activity (e.g. solitary behaviors such as digging, scratching, jumping, lying down), or any mix of the previous behavior over time. Aggressive behavior is scored over some time, and this is a measure of the aggression displayed.

White et al. (1991) and S´anchez et al. (1993) showed that 5-HT3 receptor

antagonist zacopride ((0.0001–10) mg kg−1_{IP, 60 minutes before testing) did not}

inhibit isolation-induced aggression in mice when looking at attack latency and total fighting time. Later research repeated these findings with

apomorphine-induced and cocaine-apomorphine-induced aggression in hamsters (Ricci et al., 2004a) but

added to them: latency to attack and bite initiation and contact time were in-deed unaffected by 5-HT3 antagonism, but attack and bite types and frequencies were greatly decreased.

In contrast, McKenzie-Quirk et al. (2005) found 5-HT3 antagonists to

un-equivocally reduce aggression (attack bites, tail rattles, threats) in two popu-lations of mice. In male CFW mice in a resident-intruder paradigm where the

5-HT3 antagonists ondansetron ((0.01,0.1,1.0) mg kg−1 _{IP) and zacopride ((0.1–}

17) mg kg−1 _{IP; the higher two doses of 10 mg kg}−1 _{and 17 mg kg}−1 _affected

locomotion) administered 30 minutes before intruder introduction reduced ag-gressive behavior. In male B6SJL/F2 mice overexpressing 5-HT3, alcohol intake

did not affect intruder-directed aggression, and zacopride ((1,10,56) mg kg−1_IP)

reduced aggression in wt mice but not in 5-HT3 overexpressing mice.

Gao and Cutler(1992a,b) used the 5-HT3 receptor antagonist BRL46470A

at 2.5 mg kg−1_{and (25,2.5) µg kg}−1_{IP. In the first experiment (}_{Gao and Cutler}_,

1992a), it increased aggressive behavior (aggressive grooms, attacks, bites, chases, offensive upright postures), but decreased non-social exploratory

ac-tivity. In the second experiment (Gao and Cutler,1992b), mouse social

interac-tions were scored both in a neutral cage and in a home cage. In the neutral cage, BRL46470A increased social acts and digging at the expense of exploration. In the home cage, social investigation was increased at the expense of non-social activity. Notably, partners to BRL46470A mice spent significantly less time ag-gressively/in flight. In both experiments, 5-HT3 receptor-mediated anxiolytic

(8)

effects (cf. subsection 3.3) most likely affected the aggressive measures. In the first experiment, less anxiety about the possible repercussions of aggressive be-havior might lead to more aggression. At the same time, less anxiety might lead to more social behavior of the mouse in both his home cage and the neutral cage, which in turn would explain the finding in the second experiment that partner mice had to spend less time fighting/fleeing. Indeed, the ratio of flight to received aggression remained the same so the BRL46470A mice were not hindered in their aggressive ability, just in their aggressive tendency.

Rudissaar et al.(1999) scored aggression in male adult Wistar rats treated

with apomorphine (0.5 mg kg−1_{IP for 10 days) and pre-treated with DSP-4}

(N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine hydrochloride; 50 mg kg−1 _{21 days}

before apomorphine) to accelerate apomorphine-induced aggression onset

with-out altering its effect size (Rudissaar et al., 1999). DSP-4 ptreatment

re-sulted in aggression unmodulated by either 5-HT3 receptor agonism (1-PBG

(3.0,30) mg kg−1_{, m-CPBG (1.0,10) mg kg}−1_{, IP) or antagonism (tropisetron}

0.3 mg kg−1_{, bemesetron (0.4,4.0) mg kg}−1_{, IP). In control rats, aggression was}

attenuated by both the agonist 1-PBG and the antagonists bemesetron and tropisetron, the latter only in combination with citalopram, an antidepressive SSRI. The unexpected result of 5-HT3 receptor agonism-induced aggression re-duction is explained by peripheral action of 1-PBG overshadowing its central

5-HT3 agonism (Higgins et al., 1993;Rudissaar et al., 1999). Another

remark-able result is that tropisetron needs an SSRI challenge to show aggression atten-uation. This is due to the fact that apomorphine competes with tropisetron for

binding sites, and the administered dose of 0.3 mg kg−1 _{is reportedly too small}

to displace apomorphine on 5-HT3 receptors.

Ricci et al.(2004a,2005) applied the 5-HT3 receptor antagonist tropisetron

((0.01–1.20) mg kg−1_{IP) and agonist m-CPBG ((5.0–15.0) mg kg}−1_{IP) to}

ham-sters that had been cocaine-treated throughout adolescence to stimulate of-fensive aggression. Using a resident-intruder paradigm, tropisetron alone

dose-dependently reduced cocaine-induced aggression (Ricci et al.,2004a,2005).

Re-ceptor agonist m-CPBG did not increase the overall aggression, but this might be a ceiling effect after the adolescence cocaine treatment. Instead, m-CPBG

seemed to reduce attack latency (Ricci et al., 2004a, 2005). In contrast, in

saline control hamsters, m-CPBG induced highly escalated levels of

aggres-sion where tropisetron had no effect (Ricci et al., 2005). Administering

m-CPBG prior to tropisetron required a 4 times higher dose of tropisetron to

block aggression in cocaine-treated hamsters (Ricci et al., 2004a). 5-HT3

re-ceptor densities (Ricci et al., 2004a) in cocaine-treated hamsters were

signif-icantly elevated in several brain regions. From the data it was concluded

that 5-HT3 receptors are involved in high-level specific aggression (e.g. flank bites), but not in more generic aggression (e.g. upright postures). Additionally, 5-HT3 receptor receptor localization hints towards a link to GABA subsystems where perhaps 5-HT3 receptor receptors augment GABA inhibition of other

aggression-suppressing 5-HT1 and 5-HT2 receptor subsystems (Cervantes et al.,

2010;Fink and G¨othert,2007;Ricci et al.,2005).

Cervantes and Delville (2009) found similar results comparing high-aggressive and high-aggressive hamsters, a behavioral difference where low-aggression hamsters are less impulsive and wait for higher-payout longer-delay lever presses. 5-HT3 receptor receptor densities were mostly similar in high-aggression hamsters compared to low-high-aggression hamsters except for the

(9)

me-dial amygdala (part of a reciprocal neural network associated with aggression (Delville et al.,2000)) and the nucleus accumbens and limbic prefrontal cortex,

two areas associated with some forms of impulsive behaviors (Cardinal, 2006;

Robinson et al.,2008). Further study (Cervantes et al.,2010) found that offen-sive aggression in hamsters was facilitated by 5-HT3 receptor activation. Using

the resident-intruder paradigm and tropisetron (0.3 mg kg−1 _{IP) as 5-HT3}

re-ceptor antagonist, impulsive agonistic aggression and non-aggressive impulsive behavior were both reduced, the latter to low-aggression hamster levels. The proposed mechanism for these findings is that aggressive animals have both low 5-HT levels and low 5-HT3 receptor levels. In addition, GABA inhibitory

ter-minals also contain 5-HT3 receptor (Fink and G¨othert, 2007), so tropisetron

could remove GABA-ergic inhibition of 5-HT release, thereby further depleting 5-HT levels (Cervantes et al.,2010).

3.2.2 Human research into aggression

In humans, research into aggression has met with results contrary to animal

findings. Wetzler et al.(1991) reports negative results for 5-HT-related

aggres-siveness in humans. The 5-HT receptor agonist meta-chlorophenylpiperazine

(m-CPP; 0.25 mg kg−1_{) failed to affect anger in either direction in all subjects,}

both depressed patients, panic disorder patients, previous suicide attempted patients, and controls, as measured on the Profile of Mood States (POMS) anger/hostility scale. In addition, both outwardly and inwardly directed aggres-sive patients did not differ from controls either in cortisol or prolactin levels, and m-CPP-induced hormone release was unrelated to aggression. However, it should be noted that m-CPP is not by any means a specific 5-HT3 receptor ago-nist, and its effects at 5-HT2 and other 5-HT receptors may well overshadow its

5-HT3 specificity. Genetically, Melke et al.(2003) showed that a C178T

poly-morphism in the HTR3A gene promotor has significant association with anxiety in women. The 178T allele correlated with lower scores in fear of uncertainty, anticipatory worry, as well as lower scores for indirect aggression, verbal aggres-sion, and irritability. In vitro this T allele caused 5-HT3 receptor overexpresaggres-sion, implying that 5-HT3 receptor activation reduces aggression in humans.

3.2.3 Conclusion

In summary, 5-HT3 receptors play a role in aggression in rodents (rats, mice, hamsters) but their role in humans is tentative. In animals, agonism of the 5-HT3 receptor increases aggressive tendencies whereas antagonism inhibits them. Even though ceiling effects exist for total aggression when aggression is induced prior to agonist/antagonist administration, agonists like m-CPBG facilitate displaying this aggression. Antagonists to the 5-HT3 receptor can counteract alcohol- and cocaine-induced aggression, but baseline aggression is not reduced. A confounding factor in this research could be that 5-HT3

re-ceptor antagonists may also have an anxiolytic effect (see subsection 3.3), and

this may either facilitate aggression (less apprehension about its results) or in-hibit it (more social behavior due to lessened anxiety towards intruders). In humans, 5-HT modulation failed to affect aggression in one 1991 study, and only a tentative link between 5-HT3 receptor expression and anxiety was shown in a 2003 study. Obviously, animal studies do not fully translate to humans,

(10)

due perhaps to different expression areas, densities, or pharmacokinetic action (cf. subsection 3.1).

In general, a mitigating role is proposed for 5-HT3 receptors where their ac-tion is through other aggression-modulating subsystems rather than directly causing or inhibiting aggression. As said, it should be further investigated whether this model is correct, and whether it translates to humans at all.

(11)

3.3 Anxiety

In the early nineties the then-new 5-HT3 receptor antagonists were hailed for

their promising anxiolytic properties (Barrett and Vanover, 1993). Over the

next two decades, research has found new evidence of 5-HT3 receptor involve-ment in anxiety but its role is still inconclusive. Animal and human research

both report contrary findings even within paradigms and antagonists (Ye et al.,

2001), and the truth appears far more complicated than one single anxiety

mechanism singly moderated by a single 5-HT receptor. Additional problems are the co-morbidity of anxiety with depression to the point where it is a

clin-ical challenge to distinguish between the two diagnoses (Fawcett and Barkin,

1998a, p. 123), adding some overlap between the research in this section and in subsection 3.4, and the implication of interaction of 5-HT3 receptors with

5-HT1A receptors on GABAergic synapses (Fink and G¨othert, 2007).

Func-tionally, 5-HT3 activation on those synapses enhances GABAergic inhibition (Bloom and Morales, 1998; Fink and G¨othert, 2007), which in turn inhibits long-term potentiation in cornu ammunis 1 (CA1) and CA3, implying a role

in anxiety disorders and schizophrenia (cf. subsection 3.7).

3.3.1 Animal research into anxiety

Animal research has used several behavioral research paradigms. Most seen are the elevated plus maze, the light/dark box, social interaction tests, and several punishment-related behaviors and extinctions. In the elevated plus maze (EPM) an animal is placed in the center of a +-shaped maze elevated above the floor, with two opposite arms closed (walls on three sides, open topped) and the two remaining arms open (no walls). Rodents have a natural aversion to open spaces, and presumably anxious rodents will spend more time investigating the closed arms than the open ones. In the light/dark box, the natural aversion towards brightly lit places is tested. An animal is put into a cage with two compartments, one brightly lit and one dark(er), with more time spent in the light compartment as a measure of lower anxiety. A variant on this is the open field test, where a brightly lit open cage will show anxious animals exploring the center less, the walls more, and moving less total distance. Social interaction tests measure the time spent interacting with an unfamiliar cage-mate in familiar/unfamiliar cages under intense/dim lighting. Again, more anxious animals will interact less with cage mates, and even less still in unfamiliar brightly lit cages. Lastly, punishment is used in models where an animal is first conditioned to associate a certain environment or stimulus (audio pulse, lights) with for example an electric shock. After conditioning, it can be measured how much anxiety an animal shows by freezing after exposure to the conditiuned stimulus of shock context, or how long it takes to unlearn the pairing of context/stimulus. More anxious animals display longer retention of the association, and more and longer fearful behavior when faced with conditioned stimuli or contexts.

Higgins et al. (1991) tested ondansetron ((10–100) ng), tropisetron ((10– 100) ng), GR65630 ((1–10) ng) and bemesetron ((100–1000) ng) on male hooded Lister rat CNS using an implanted cannula above the dorsal raphe nucleus (DRN) and both amygdalae. In the DRN, none of the drugs influenced social interaction (SI), although 5-HT increased SI under high lighting/unfamiliar cage (HLU) but not low lighting/familiar cage (LLF) conditions. In the amygdala,

(12)

all drugs increased HLU SI but not LLF SI, showing that the amygdala may be a disinhibitory, possibly anxiolytic locus for 5-HT3 receptor antagonist ac-tion. Increased 5-HT as well as decreased 5-HT3 receptor action (leading to

lower 5-HT, cf. subsection 3.1) may contribute to lowered anxiety. Silva et al.

(1993) found that BRL46470A (0.286 pmol and 0.858 pmol) in male Wistar rat

amygdalae significantly increased the latency to step back down from a cage elevation onto a floor they had been conditioned to associate with a foot shock, significantly indicating an anxiogenic effect. However, since no additional anxi-ety measures were included this is a tentative interpretation; memory-effects

might be the cause instead. Additionally, in shock-associated behavior

re-search the intensity of the training stimulus matters (Castej´on and Cubeddu,

1998; Nevins and Anthony, 1994). Nevins and Anthony (1994) found that male CD rats reacted differently to 5-HT3 receptor antagonists ondansetron

((0.001–1.0) mg kg−1_{), granisetron ((0.001–1.0) mg kg}−1_{), R-zacopride ((0.0001–}

1.0) mg kg−1_{), as well as to diazepam (an anxiolytic; (0.32–3.2) mg kg}−1_),

de-pending on the shock intensity. Training with 0.5 mA foot shocks showed

decreased startle intensity with diazepam but not with any 5-HT3

antago-nist except the highest dose of (R)-zacopride. Training with 0.25 mA foot

shocks allowed all compounds to reduce startle intensity for all doses except

the lowest. Similarly (Castej´on and Cubeddu, 1998), hungry pigeons trained

on a lever that either dispensed food freely (white light) or food punished with shocks (red light) were found to be unresponsive to either diazepam

((1,1.5) mg kg−1_{), buspirone ((0.3,1) mg kg}−1_{), or ondansetron (100 µg kg}−1_IV)

when trained with high-intensity shocks. Low-intensity shocks allowed these drugs to increase the amount of shocks they would tolerate in order to get food (Castej´on and Cubeddu,1998).

Schreiber et al.(1998) found male Wistar rat ultrasonic vocalizations (USV) dose-dependently reduced by selective serotonin reuptake inhibitors (SSRIs),

an effect not blocked by 5-HT3 receptor antagonist ondansetron (0.1 mg kg−1

IP) nor increased by 5-HT3 agonist m-CPBG (30 mg kg−1 _IP). _{Eguchi et al.}

(2001) found that 5-HT3 in these rats did modulate HLU SI and EPM

behav-ior. MCI-225 (a noradrenalin reuptake inhibitor with 5-HT3 receptor

antag-onism; 10 mg kg−1 _{PO) and ondansetron (1 mg kg}−1 _{PO) both increase HLU}

SI and EPM open arm entry. m-CPBG (1 mg kg−1 _{IP) does block MCI-225}

and ondansetron-induced increase of SI but does not decrease SI by itself.

An-other EPM study (Zhang et al.,2001) found that male ICR mice given DAIZAC

(desamino-3-iodozacopride, a selective high-affinity 5-HT3 receptor antagonist;

0.05 mg kg−1_{) chose to enter the open arm more, given equal arm entries. Brain}

analysis for DAIZAC-like activity showed that higher activity correlated with more open arm entries.

Hensler et al.(2004) found that alcohol-preferring FH/Wjd rats2_{spend more}

of their time in open arms and have more open arm entries than ACI/N

con-trol rats. Administration of bemesetron (3 mg kg−1 _{IP) had no effect on FH}

rats but had an anxiolytic effect on ACI rats. However, this lower FH anxi-ety is housing- and substrain-dependent, and FH rats showed different overall locomotor activity on the EPM.

Genetically, 5-HT3 action has met with similarly conflicting results on both

2

These rats, also fawn-hooded rats, have a central 5-HT dysfunction. This leads among other things to higher voluntarily alcohol consumption than ACI/N strain rats (Hensler et al.,

(13)

conditioned and unconditioned paradigms.

In conditioned paradigms, Harrell and Allan (2003) found 5-HT3

overex-pressing (5-HT3-OE) B6xSJL mice in their forebrain (including hippocampus, required for contextual fear conditioning) but not in their amygdala (required for both contextual and cued fear conditioning) to be more susceptible to

fear-conditioned context freezing. In contrast, Bhatnagar et al.(2004b) found that

5-HT3A knockout (5-HT3A-KO) C57B1/6J mice tended towards higher context freezing also and did show significantly higher tone freezing. Additionally,

on-dansetron ((0.5,1.0) mg kg−1_{IP) decreased both 5-HT3-OE and wt mice context}

freezing down to levels different from vehicle (Harrell and Allan,2003). It was

further noted that actual 5-HT3 expression was up in hippocampal GABAergic

interneurons as well as principal excitatory neurons (cf. (Mikics et al., 2009)

below).

In unconditioned paradigms, the results argue as well. In the EPM,

Harrell and Allan(2003) found that 5-HT3-OE mice had more open arm entries, open arm time, and center time compared to wt mice, implying an anxiolytic

effect of 5-HT3 upregulation. Likewise, 5-HT3A-KO mice (Bhatnagar et al.,

2004b;Kelley et al.,2003) also showed more open arm time and higher distance travelled compared to wt mice, implying an anxiolytic effect of 5-HT3A removal (i.e. extreme downregulation). In contrast, 5-HT3-KO C57BL/6J mice show

the same novelty-suppressed feeding behavior as wt mice (Smit-Rigter et al.,

2010, but note that anxiety is merely a control measure). Surprisingly, the

same knockout mice seem to differ between themselves as well, despite their

supposedly identical 5-HT3 null phenotype. In the light/dark box,Kelley et al.

(2003) mice showed less anxiety and Bhatnagar et al. (2004b) mice the same

anxiety as wt mice. In the open field test/novelty interaction, Kelley et al.

(2003) mice approached a foreign object in an open field more than wt mice

and Bhatnagar et al. (2004b) mice showed the same anxiety as wt mice in a

purely open field test. To further confuse the data, Bhatnagar et al. (2004a)

has found that full knockout of the 5-HT3 receptor results in mouse behavior that differs by sex. C57B1/6J mice subjected to a forced swim test (where they are put in a water-filled tub with no landing places) show no knockout ef-fect in male mice but increased immobility and decreased swimming in females. Similarly, in a defensive withdrawal test male 5-HT3-KO mice respond more anxiously but female 5-HT3-KO mice respond less anxiously. Curiously, gene knockout appears to give female 5-HT3-KO mice the same behavior as wt male mice.

With respect to these conflicting findings, Kelley et al. (2003) conclude

that their consistent genetic anxiolytic effect between three paradigms (EPM, light/dark, novelty seeking) implicates pharmacological differences between drugs as the cause for the inconsistent results reported so far, rather than paradigm differences. However, the point remains that at least in humans,

5-HT3A is required to form functional 5-HT3 receptors (subsection 3.1). How

mouse 5-HT3 receptors are formed needs to be research further, since with the current results, compensatory effects cannot be ruled out.

It has been proposed that unconditioned paradigms such as SI and EPM

tend to be more sensitive to 5-HT3 antagonists (Eguchi et al.,2001).

Further-more, 5-HT3 antagonist DAIZAC appeared to reduce passive avoidance of the open arm when the animal was in the central compartment without affecting active avoidance of that arm when the animal was in the exposed condition

(14)

(Zhang et al.,2001, p. 576). This perhaps indicates that there are several anxi-ety mechanisms, with 5-HT3 receptor antagonists affecting more the initial anx-ious choice to venture into the unknown and less the subsequent anxanx-ious choice to stay there. It is likely that 5-HT3 antagonists act differentially on more

than one subsystem at once (Anttila and Leinonen, 2001; Bourin et al., 2004;

Delgado et al.,2005;Faerber et al.,2007;Greenshaw,1993). In support of this,

Bourin et al.(2004) reviewed cyamemazine, an antipsychotic and dopamine D2 and 5-HT2A, 5-HT2C, and 5-HT3 receptor antagonist. Acute but not chronic cyamemazine was anxiolytic in the light/dark box and chronic but not acute

cyamemazine was anxiolytic in the EPM.Delgado et al.(2005) showed an

anxi-olytic effect of CSP-2503 (an 5-HT1 agonist with 5-HT2A and 5-HT3 antagonist properties; (2.5–20) mgkg SC) on light/dark box paradigm and and social

inter-action. Note however thatBourin et al.(2004) does not review articles explicitly

controlling for which receptor effect is at play. Since the affinity of cyamemazine for 5-HT3 (Ki = 75 nM) is lower than its affinity to both 5-HT2A and 5-HT2B (Ki = 1.5 nM and Ki = 12 nM, respectively), non-5-HT3 effects can reasonably be expected.

Mikics et al. (2009) found that mice without the cannabinoid CB1 gene (CB1-KO) show anxiety-related effects in a complicated way. In an open field test, CB1-KO mice are no different from wt NMRI mice, even with m-CPBG

((1,3,5) mg kg−1_{IP) challenge. On the EPM, CB1-KO mice show reduced open}

arm time and entries. m-CPBG ((3,10) mg kg−1_{) had no further effect on NRMI}

mice but was anxiolytic in CB1-KO mice, though not fully restoring wt behav-ior. The authors propose CB1-mediated retrograde cannabinoid signaling as an important mechanism that is not often controlled for, causing perhaps some

of the inconsistent results so far (cf. (Olivier et al., 2000); m-CPBG action

(Eguchi et al.,2001;Schreiber et al.,1998); 5-HT3 receptor deletion or overex-pression (Bhatnagar et al., 2004b;Kelley et al.,2003)).

3.3.2 Human research into anxiety

Anxiety in humans comprises generalized anxiety disorder (GAD), phobia, post-traumatic stress disorder (PTSD), panic disorder, and obsessive-compulsive dis-order (OCD), the latter of which is not treated by classic anxiety drugs such

as diazepam (Hewlett et al.,2003). Apart from ethical considerations

preclud-ing most control conditions, research is difficult due to patients with anxiety

often having co-morbid depression (Walstab et al., 2010) and cognitive factors

playing a role in the anxiety (Harmer et al., 2006).

Ondansetron (0.15 mg kg−1 _{IV) was tested on its interaction with oral}

d-amphetamine (Grady et al., 1996). d-amphetamine has a self-reported

activa-tion/euhoria and anxiogenic effect, and ondansetron did reduce this self-reported anxiety but only in people that self-reported increased activation/euphoria.

Hewlett et al. (2003) found ondansetron (1 mg t.i.d.) to lower Yale-Brown Ob-sessive Compulsive Scale (YBOCS) scores by upwards of 28%. Further, they suggest that SRI-induced anxiogenesis may be due to increased 5-HT levels and

activated 5-HT3 receptors (cf. Faerber et al.(2007,2004)). Concurrent 5-HT3

antagonist treatment might increase SRI effectiveness in treating anxiety symp-toms.

In one interesting study, (Harmer et al.,2006) subjected healthy volunteers

(15)

categoriza-tion and memory test, and facial expression recognicategoriza-tion test. No subjective mood or anxiety changes were reported, and no effects were found for facial ex-pression recognition or emotional categorization or memory. However, emotion-potentiated startles (blink reactions to positive/negative/neutral pictures) were abolished by ondansetron. Since facial expressions require more interpretative and strategic processing than emotion-potentiated startle responses, this may reveal a stratification of cognitive anxiety effects. Indeed, ondansetron may

af-fect more cognitive anxiety efaf-fects to a lesser extent (Harmer et al., 2006, p.

23).

Mirtazapine an antidepressant (mean dose 23 mg day−1_{), has been found to}

lower Hamilton rating scale for depression (HAMD) scores in 249 anxiety

pa-tients (controls n = 246; Nutt (1998)), and HAMD and YBOCS scores in 2

out of 6 subjects with OCD at 45 mg day−1 ₍_{Koran et al.}_, ₂₀₀₁_{). Pre-surgical}

mirtazapine lowered anxiety scores in female patients (Anttila and Leinonen,

2001; Chen et al., 2008). It was also active in first-week treatment of DSM-IV major depression patients with co-morbid generalized anxiety disorder (Anttila and Leinonen,2001). However, mirtazapine is known as an antagonist for adrenergic α2-autoreceptors and α2-heteroreceptors and 5-HT2 receptors as

well as for 5-HT3 receptors (Anttila and Leinonen, 2001). Hence, the results

above may not be due to 5-HT3-specific action at all.

Next to ondansetron and mirtazapine there have been few studies on other

drugs. Reviewing cyamemazine for human use,Bourin et al.(2004) found that

anxious-depressive syndrome patients improved their HAMD anxiety scores. Tropisetron was reported as a reductor of GABAergic synaptic transmission,

where classical anxiolytics act as GABA agonists (Faerber et al.,2007,2004).

3.3.3 Conclusion

5-HT3 receptors have been solidly implicated in anxiety, but the precise mech-anism by which anxiety is influenced is not yet known.

Animal research tends to find either an anxiolytic or no effect from 5-HT3 re-ceptor antagonists. Contrasting findings include an anxiogenic effect of 5-HT3 receptor agonists in mice without a cannabinoid-1 receptor, and susceptibil-ity to anxiety in 5-HT3-OE mice. Additionally, it is strange that same-strain 5-HT3-KO mice would differ between to experimental groups, as they did in three paradigms. Explanations for these disparaging results include both the animals and the drugs. On the animal side, there is the possible involvement of wholly unreported mechanisms (e.g. that anxiolytic effects of 5-HT3 receptor

ligands may depend on CB1 receptors; cf. Mikics et al. (2009)), and hitherto

unexplored species-, strain-, and situation-related factors influencing receptor

expression and function (Mikics et al., 2009). On the drug side, many

com-pounds have activity other than just the 5-HT3 receptor, including ondansetron,

tropisetron, mirtazapine, and cyamemazine (Faerber et al.,2007). (See also the

short discussion insubsection 3.1.) In addition, 5-HT3 antagonist enantiomers

may behave differently from each other (zacopride, for one;Greenshaw(1993)),

and a bell-shaped dose-response curve (Faerber et al., 2007, 2004;Greenshaw,

1993) makes it hard to compare contrasting findings, necessitating the tracking

of dosages and administration methods across experiments.

Human research so far seems promising, finding 5-HT3 antagonists effective in reducing anxiety overall. However, complications in humans include the facts

(16)

that anxiety may play at different cognitive levels and that it may be co-morbid with other severe afflictions. In addition, the same sex-differentiated results

for 5-HT3 receptor function as found in mice (Bhatnagar et al., 2004a) need

to be ruled out, and personal differences as with D-amphetamine-interaction (Grady et al.,1996) need to be fully cleared up before the 5-HT3 is fully under-stood in human anxiogenesis.

(17)

3.4 Depression

Depression is projected to be the world’s second largest single cause of lost

productive life years by 2020 (World Health Organization, 2012b). Its lifetime

prevalence is 18% (Fawcett and Barkin, 1998b), and it is often comorbid with

anxiety, drug abuse, and eating disorders (covered in this review;Hammer et al.

(2009);Rajkumar and Mahesh(2010);Walstab et al. (2010)) as well as cancer

and fibromyalgia (not covered in this review;Rajkumar and Mahesh(2010)). Its

main mechanism is presumed biochemical disequilibrium in central

neurotrans-mission pathways (Fawcett and Barkin, 1998b), often treatable with a

combi-nation of antidepressants, psychotherapy, and electroconvulsive therapy. How-ever, cognitive factors and apparent sex-specific higher susceptibility in women (Mahesh et al.,2010) make depression a complex topic of research.

3.4.1 Animal research into depression

Animal models are explicitly about consistent reactions to known antidepres-sants more than behaviors directly shared with depressed humans. The two paradigms most often found in literature on depression are the forced swim test and tail suspension test. In the forced swim test (FST), an animal is placed in a water-filled container without any platforms to land on. The time spent immobile in the water is a measure of depressive-like behavior, with the time spent active sometimes used as a measure of locomotion to control for dimin-ished overall mobility. Antidepressants rather consistently reduce the amount of time that mice are expending the minimum effort to stay afloat, as opposed to actual swimming or to for example pawing at the container-water edge. In the tail suspension test (TST), a mouse is suspended only from its tail and scored for total time spent immobile versus moving. Again, total time spent immobile is a measure of depressive-like behavior, a measure consistently de-creased by antidepressants. While immobile animal behavior is reminiscent of depressive listlessness in humans, neither of these tests are likely to be direct cognitive analogs of depression in people. Hence, their validity as models de-rives mostly from their consistent reaction to antidepressants, often using known antidepressants as positive controls. Subsequently, compounds that influence these paradigms have antidepressant-like effects (i.e. effects akin to the effects of antidepressants), but not necessarily antidepressant effects (i.e. effects that counteract depression).

Variations on quinoxalin-2-carboxamides, all 5-HT3 receptor-antagonists,

showed promise as antidepressants in Swiss albino mice in a FST (Mahesh et al.,

2010), exceeding the effects of ondansetron. QCM-13

(N-cyclohexyl-3-methoxyquinoxalin-2-carboxamide) was tested on Swiss albino mice and Wistar

rats (Gupta et al.,2011). In both FST and TST, QCM-13 ((2,3) mg kg−1 _IP)

reduces immobility time comparably to positive controls, and administered

to-gether QCM-13 increases the effect of both positive controls. Devadoss et al.

(2010) tested QCF-3 ((4-benzylpiperazin-1-yl)(quinoxalin-2-yl) methanone) on

Swiss albino mice and olfactory bulbectomized (OBX) Wistar rats. OBX rats display hypo-serotonergicity, a depression-like state consistently reversed by

an-tidepressants. QCF-3 ((1,2,4) mg kg−1 _{IP) decreased FST and TST immobility}

in mice, although slightly less so than positive controls. In OBX rats, chronic

(18)

reduced the OBX-typical hypermobility in the open field test, although again in both tests the positive control proved more effective. Bis-selenide

((Z)-2,3-bis(4-chlorophenylselanyl)prop-2-en-1-ol) tested on Swiss mice ((0.1–5) mg kg−1 _PO)

showed antidepressive effects in both FST and TST (Jesse et al.,2010).

Inter-action effects with antagonists for various neurotransmitter systems revealed no interaction with noradrenergic or dopaminergic agents. Curiously, ondansetron (5-HT3 receptor antagonist), ketanserin (5-HT2A/2C antagonist), and PCPA (5-HT synthesis inhibitor) all reverse the antidepressive effect of bis-selenide in TST (but not the open field test or FST) at a TST-ineffective dose.

Further animal research includes the 5-HT knockout mice by

Bhatnagar et al.(2004a, cf. subsection 3.3) that revealed sex-differentiated re-sponses for depression. In FST, second-day immobility was increased in female KO mice whereas there was no effect of genotype in male mice. (Interestingly, female KO mice ended up at levels of immobility similar to male wt mice, but whether or not this result has meaning was not discussed.) Sex-differentiated 5-HT3 receptor action is relevant with regards to higher depression incidence

in women (Mahesh et al., 2010). Electroconvulsive shocks (ECS) altered

5-HT3 receptor function in two studies of rat CA1 hippocampal neurons (Ishihara and Sasa, 1999, 2001). Spontaneous post-synaptic potentials were increased by 5-HT3 receptor activation (through 5-HT-induced GABA release). This 5-HT3 receptor-mediated effect was potentiated by ECS but antagonized by the 5-HT3 receptor antagonist LY278,584. Additionally, 5-HT3 receptor

antagonism reverses learned helplessness in rats (Rajkumar and Mahesh,2008)

and potentiates antidepressant-like effects of serotonin and norepinephrine

reuptake inhibitors and SSRIs in FST (Rajkumar and Mahesh,2010).

3.4.2 Human research into depression

As mentioned, animal depression models are chosen for their reaction to known antidepressants more than their likeness to human depression. Indeed, the rel-evance to depression of TST and FST specifically has been called into

ques-tion (Jesse et al., 2010). Furthermore, not all antidepressants work on both

paradigms: bupropion for example has no effect on FST behavior but does on

TST behavior in Swiss mice (Devadoss et al., 2010; Gupta et al.,2011)). Since

animal results do not transfer to humans with confidence, human depression re-search sticks closer to substances known to be safe (ondansetron, mirtazapine) and emphasizes genetics research to better anticipate depression.

Case studies provide circumstantial evidence of 5-HT3 receptor involve-ment in depression. Mirtazapine reduced depressive symptoms in 19

individ-uals at an effective dose of 30 mg (n = 11) or 45 mg (n = 8) (Thompson,

2000). Another 19 participants retained their depression remission on

mirtaza-pine ((7.5–45) mg day−1_{) after switching from SSRIs in an open label study}

(Gelenberg et al., 2000). Reviews provide more solid evidence, finding mir-tazapine beating placebo and tying with amitriptyline in a treatment review

of moderate-to-severe depression (n = 4500; (Fawcett and Barkin,1998b)) and

depression cormorbid with anxiety (n = 293; (Fawcett and Barkin, 1998a)).

It reduced anxiety and sleep disturbances (n = 495; (Nutt, 1998)), and

Anttila and Leinonen(2001) found it as effective as other antidepressants (ami-tiptyline, clomipramine, doxepin, fluoxetine, paroxetine, citalopram, and ven-lafaxine). However, mirtazapine is often seen as mainly adrenergic

(19)

antidepres-sant. Since 5-HT3 involvement was not explicitly controlled for, these findings may not prove to be 5-HT3 receptor-related upon closer study.

Genetically, HTR3A and HTR3B have both been mapped to the location

of depression susceptibility genes (Walstab et al., 2010). Gatt et al. (2010a)

investigated the depression-associated SNPs HTR3A C42T and brain-derived neurotrophic factor (BDNF) p.V66M (c.G196A) in 363 volunteers. The C allele of C42T reduces HTR3A expression and the V allele of V66M reducing BDNF secretion. Both polymorphisms compounded early life stress (ELS)-dependent effects that increase depression risk. The CC genotype of the HTR3AC42T mutation leads to an increase of depressed mood, higher scores on the depres-sion and anxiety stress scale (DASS-21), and loss of gray matter in

hippocam-pal structures (Gatt et al., 2010b). Under ELS, this loss of gray matter is

ex-tended to frontal cortices. Yamada et al.(2006) studied a Japanese population

comprising 180 unrelated female patients (99 with bipolar disorder, 81 unipo-lar major depressive disorder) on HTR3A and HTR3B. For HTR3A, 3A13 (a tetranucleotide repeat marker) was associated with bipolar but not unipo-lar disorder. For HTR3B, SNP 3B08 (G462, p.A154A) was associated with combined mood disorders and with unipolar illness alone. Haplotype block analysis further revealed a cluster associated with female unipolar illness,

in-cluding p.Y129S (c.C178T; cf. subsection 3.1,subsection 3.6). In a follow-up,

Krzywkowski et al. (2008) investigated this p.Y129S mutation. 129Y is more frequent in female unipolar patients, and 129S is the norm in mouse, rat,

fer-ret, guinea pig, dog, and chimpanzee (Krzywkowski et al., 2008). In humans,

the frequency of the Y/Y genotype is lower than the combined Y/S, S/S

fre-quency in three of five populations studied (Krzywkowski et al., 2008, suppl.

figures). 5-HT3AB129s has a 20-fold lower deactivation and 10-fold lower

de-sensitization than (wt) 5-HT3AB129y receptors. Additionally, 5-HT3AB129s

increases the maximum 5-HT response in FLIPR membrane potential assay (Krzywkowski et al.,2008) and calcium influx assay (Walstab et al.,2008), but not the potency for both 5-HT and tropisetron in a fluorescence-based assay,

nor does surface expression differ by genotype (Krzywkowski et al., 2008). In

general, artificial mutations (Y129A,C,D,E,F,H,L,N,S,W) all tend to increase

maximum 5-HT response without affecting EC50. Through research as

dis-cussed above, a better understanding is gained of how exactly genes change function given certain mutations. In turn, this understanding may lead to a better understanding of the role of unaltered receptors in the CNS, as well as a better understanding of disease mechanisms related to those mutations.

3.4.3 Conclusion

In animals, the forced swimming test (FST) and tail suspension test (TST) show that 5-HT3 receptor antagonists (QCM-13, QCF-3, bis-selenide, tropisetron, and ondansetron) induce a reduction in immobility and for FST an increase in swimming behavior (FST). They share this effect with humeffective an-tidepressants. Confusingly, indirect action of 5-HT3 receptor antagonists has been shown to potentiate antidepressant effects of serotonin reuptake inhibitors (Rajkumar and Mahesh, 2010), but also to attenuate the antidepressant

ef-fects of electroconvulsive shocks (Ishihara and Sasa, 1999) and bis-selenide

(Jesse et al., 2010). The definitive mechanism for these counteracting effect has not yet been found. However, it is known that 5-HT3 receptor antagonists

(20)

have depressant-like effects: presynaptic 5-HT3 receptor antagonism suppresses dopaminergic transmission and so inhibits GABA release, inducing GABA-mediated depressant-like effects. Additionally, while depression is associated with low 5-HT, stimulation of 5-HT inhibits ACh release and increased ACh

is possibly beneficial in controlling depression as well (Rajkumar and Mahesh,

2010). Furthermore, these rodent models of depression may not be entirely

valid for human 5-HT3 receptor depressive action. Firstly, immobile behavior in swimming and tail suspension is not obviously analogous to human depressive behavior. Secondly, not all 5-HT3 receptor antagonists work in these paradigms both (e.g. bupropion as mentioned above), implying at least some difference between the two. Thirdly, pharmacokinetics such as the bell-shaped dose re-sponse curves of 5-HT3 receptor antagonists, their antidepressant-like action at lower dose ranges, and interactions with antidepressants at those levels make it hard to separate dose-effects from drug-effects proper.

In humans, 5-HT3 receptor antagonists have been found solidly beneficial to depression, although new 5-HT3 receptor antagonists have been hesitantly tested for the reasons above. Mirtazapine has been an effective singular antide-pressant, although its 5-HT3-specific effect has not been sufficiently separated from its adrenergic and other effects. Additionally, complementary 5-HT3 re-ceptor antagonism may increase the effectiveness of antidepressive medication as has been found in animals. Genetically, the C allele of the HTR3A C42T SNP may increase the impact of early life stress, a known depression risk factor, and reduce gray matter brain mass in frontolimbic regions. HTR3B p.Y129S results in greatly lower deactivation and desensitization speeds in heteromeric 5-HT3AB receptors, and is associated with depression. Future research may focus on the kinetics of 5-HT3 receptor mutations to find effective depression treatments and ultimately understand the biological part of this disease and its relations to drug abuse, anxiety, and eating disorders.

(21)

3.5 Drug Addiction

Addiction is not fully understood yet, but 5-HT involvement in addiction has

long been known (Leonard,1994). The primary biological explanation for

addic-tion is the mesolimbic dopamine pathway, which responds to cocaine, ethanol, amphetamine, opiates, and nicotine, and is an important pathway in reward

circuitry (Engleman et al., 2008). 5-HT has its effects on brain dopamine (cf.

Walstab et al. (2010) for an overview), and specifically 5-HT3 receptor action

on drug addictions is suggested to involve dopamine (Davies,2011) via

projec-tions from the ventral-tegmental area (Engleman et al., 2008; Gorelick et al.,

2004; Hui et al., 1996) to limbic regions such as the nucleus accumbens area (Engleman et al., 2008; Gorelick et al., 2004) and cortical regions such as the

prefrontal cortex (Engleman et al., 2008). 5-HT3 receptor activation also

en-hances GABA release, the principal neurotransmitter for intoxication (Davies,

2011;Lovinger,1999).

Additionally, drugs may act on 5-HT3 directly. Cocaine binds to 5-HT trans-porters and acts as a weak antagonist for 5-HT3 receptors. Conversely, 5-HT3 receptor antagonists can inhibit cocaine-, morphine-, nicotine-, and

ethanol-induced dopamine-efflux stimulation (Davidson et al., 2002; Faerber et al.,

2007,2004;Gorelick et al.,2004;Walstab et al.,2010;Ye et al.,2001). Ethanol potentiates the 5-HT3 receptor, inducing a preference for the open state of the

receptor, presumably via the N-terminal extracellular domain (Davies, 2011;

Johnson,2004;Lovinger, 1999).

3.5.1 Animal research into drug addiction

Five major experimental paradigms in addiction research are drug self-administration (human equivalent: drug seeking), reinstatement model (chronic drug administration followed by a withdrawal period and subsequent reinstate-ment of the drug; human equivalent: relapse), conditioned place preference (animals are given drugs and placebo in different, easily recognized compart-ments and the preference for the drug-associated or placebo-associated com-partment is measured; human equivalent: drug craving), drug discrimination model (whether the animal can distinguish between administration of drug and placebo without external cues; no human equivalent), and behavioral sensiti-zation (drug administration increases or decreases the response to subsequent drug challenges due to modulation of drug responses; human equivalent: pro-gressive intensification of drug-induced rewards and/or incentive motivation) (Gorelick et al., 2004). Self-administration research is sometimes enhanced by allowing drug administration directly to the brain, eliminating orosensory

fac-tors that may influence drug choices (Engleman et al., 2008).

Splitting the results of animal research by substance alphabetically, results have been found mainly for cocaine and ethanol, but also for metamphetamine, morphine, and nicotine.

Cocaine King et al. (1997) investigated cocaine sensitization and tolerance

in Sprague-Dawley rats. Cocaine (14 days of 40 mg kg−1 _{SC b.i.d.} _or

same dose internally via mini-pumps) and a 15.0 mg kg−1 _{IP cocaine}

chal-lenge showed sensitization on the Ellinwood and Balster behavioral rating

(22)

blocked sensitization in the intermittent cocaine group and slightly but sig-nificantly increased sensitization in the continuous cocaine group. Plausibly intermittent administration allowed the concurrent ondansetron to be present for the entire effective duration of cocaine, thereby blocking 5-HT3 receptors in-volved in cocaine sensitization. The same group later showed that ondansetron

also blocks the expression of sensitization, not only its development (King et al.,

1998). Cocaine as above was followed by a seven day withdrawal period with

ondansetron administered during the first five days thereof. A single 15 mg kg−1

IP cocaine challenge on day 7 of withdrawal revealed that intermittent cocaine sensitized (increased behavioral ratings) and continuous cocaine desensitized (hence, tolerance buildup; decreased behavioral ratings) to a challenge.

On-dansetron attenuated both these effects at 0.01 mg kg−1 _{and 1.0 mg kg}−1_{, but}

strangely not at the intermediate 0.1 mg kg−1 _dose.

Ondansetron also reverses expression of repeated cocaine sensitization (Davidson et al., 2002). Sprague-Dawley rats on a seven on, seven off, seven

on, seven off schedule of cocaine (40 mg kg−1 _{SC) were given ondansetron}

(0.2 mg kg−1 _{SC) either in the second dosing period (3.5 hours after cocaine}

administration) or in the second withdrawal period. In both cases ondansetron

reversed the expression of behavioral sensitization upon a 7.5 mg kg−1 _IP

co-caine challenge. Likewise, rats trained on a progressive ratio schedule

(stabiliz-ing at (26–30) mgday−1_{intake) showed no reduced break points (meaning either}

60 minutes without a response or 5 hours elapsing) on withdrawal ondansetron, but concurrent ondansetron reduced breakpoints for up to three days after the last injection. Additionally, post-reinforcement pauses were increased for this group, indicating perhaps a diminished reinforcement value of cocaine. Invoking the opponent process theory of addiction, the authors further hypothesize that

ondansetron counteracts withdrawal effects (cf. Suzuki et al.(1997) below).

In contrast, Szumlinski et al. (2003) found that IP administration of

on-dansetron, Y-25130 and bemesetron 30 minutes before a daily 30 mg kg−1

co-caine injection had no effect on a 14 mg kg−1 _{cocaine challenge 21 days after}

treatment. In fact, bemesetron in that paradigm increased long-term behavioral sensitization, as did ondansetron in continuous cocaine above. Another

exper-iment that administered antagonists tropisetron ((0.001,0.01,0.1) mg kg−1_{) and}

ondansetron ((0.01,0.1,1.0) mg kg−1_{) 30 minutes before a 15 mg kg}−1_{IP cocaine}

challenge in DBA/2N mice also found negative results (Lˆe et al.,1997). Neither

antagonist had an influence on the locomotion scores obtained after this cocaine challenge.

By itself, chronic 5-HT3 receptor antagonism also induces cocaine

toler-ance (King et al., 2002). Sprague-Dawley were given 14 days of tropisetron

((0,4,8) mg kg−1_day−1_{) and LY278,584 ((0.001,0.01,0.1) mg kg}−1_day−1_{) via}

in-ternal minipumps, followed by seven days of withdrawal, followed by a cocaine

challenge ((7.5,15) mg kg−1_{). The 5-HT3 receptor antagonists reduced}

move-ment score increases for both cocaine challenges (tropisetron (4,8) mg kg−1_),

although LY278, 584 0.001 mg kg−1 _{and 0.01 mg kg}−1 _{outperformed the higher}

dose on the greater cocaine challenge. Hence chronic 5-HT3 receptor antagonists by themselves induce tolerance to cocaine where they antagonize sensitization when given concurrently to cocaine. This may by the same mechanism as chronic cocaine-induced tolerance, due to the weak 5-HT3 antagonist activity of cocaine

itself (Davidson et al.,2002). Additionally, systemic tropisetron and LY278, 584

(23)

account for.

Ricci et al. (2004b) investigated subregion-specific 5-HT3 immunoreactiv-ity (5-HT3IR) during cocaine sensitization. Sprague-Dawley rats on cocaine

(15 mg kg−1 _{IP b.i.d.) were given a 15 mg kg}−1 _{IP cocaine challenge after 2}

or 14 days of withdrawal. 5-HT3-IR was reduced in the intermediate zone of the nucleus accumbens after 2 days but not after 14 days of withdrawal, even though behavioral sensitization did show at the 14 day mark. This nucleus ac-cumbens subregion has been implicated in establishing lever pressing behavior upon microinjection of cocaine, and GABAergic neurons there are suppressed during the induction of sensitization. 5-HT3 antagonist treatment attenuates

locomotor response to cocaine during sensitization (King et al.,1997,1998) but

does not block long-term locomotor response (Ricci et al., 2004b), indicating

that 5-HT3 receptor quantity may not be directly related to long-term cocaine effects.

Genetically,Engel et al.(1998) surprisingly found that forebrain 5-HT3

re-ceptor overexpressing (5-HT3 rere-ceptor-OE) B6SJL mice drank half as much al-cohol in a free access paradigm between water and 10% v/v ethanol, compared to wt mice. This surprising result (overexpression showing the same phenotype as receptor antagonism) could be due to the fact that 5-HT3 receptor overex-pression already increases dopamine release. If the dopamine system is already close to maximum capacity, alcohol would not improve it much, diminishing its rewarding effect. Alternatively, perhaps 5-HT3 receptor overexpression results in downregulation of other dopamine-related elements in other areas,

impair-ing the normal ethanol-dependent dopamine stimulation. Hodge et al. (2008)

found that deletion of mouse Htr3a blunts the induction of cocaine sensitiza-tion. C57BL/4J mice −/− for Htr3a were identically affected by cocaine as were littermate control mice, but showed a reduced cocaine-induced

locomo-tor sensitization after 12 days of 10 mg kg−1 _{IP cocaine injections every other}

day (6 injections total). The Htr3a-KO might might have increased accumbal dopamine release through lack of 5-HT3 receptor-mediated inhibition or 5-HT3 receptor absence-induced lowered glutamate levels. Two confounding factors in the study however were saline significance and 5-HT3 receptor effects other than drug-related ones. Firstly, saline-induced locomotor sensitization differed pre-and post-cocaine, indicating a conditioning or anticipation effect of repeated co-caine injections. Secondly, 5-HT3 receptor overexpressing mice from the same group were shown to exhibit enhanced contextual learning and anxiety-related differences (cf. Kelley et al.(2003) insubsection 3.3), indicating possible differ-ent effects of Htr3a deletion.

Ethanol Research byBeardsley et al.(1994) found that Long-Evans hooded

rats trained to self-administrate ethanol in a fixed ratio 1 (FR1) sched-ule, did not reduce their intake with SC injections of either ondansetron

((0.03–3.0) mg kg−1_{), granisetron ((0.01–1.0) mg kg}−1_{), or SC-51296 ((0.1–}

10.0) mg kg−1_{) 30 minutes. Perhaps free-access and gated access motivation}

have different pathways that differentially involve 5-HT3 receptors.

Addition-ally, reported doses of 10 mg kg−1 _{for ICS 205930 are million-fold higher than}

required for anxiolytic effects, implying possible saturation and effects at other 5-HT receptors. Ethanol-induced locomotion was similarly unaffected by either

(24)

same antagonist doses)). Mhatre et al. (2004) found that free-choice alcohol-preferring Sprague-Dawley rats reduced their alcohol intake with tropisetron

(5.6 mg kg−1 _{IP) and naltrexone ((0.5–10) mg kg}−1_{). A combination of}

nal-trexone 5.6 mg kg−1_{and tropisetron (0.001,0.32,5.6) mg kg}−1_{) decreased alcohol}

intake as effectively as high dose naltrexone but without the high

naltrexone-induced food and water intake depression. As Beardsley et al. (1994) above,

high doses are noted for possible effects on other receptors than 5-HT3 recep-tors.

5-HT3 receptors mediate foot-shock induced reinstatement of alcohol seeking

in male Wistar rats (Lˆe et al., 2006). These rats self-administered 12% v/v

ethanol, and after extinction (PR ratio increases) ondansetron and tropisetron ((0.001,0.01,0.1) mgkg IP both) were given 60 minutes before foot shocks. Both drugs attenuated foot shock-induced reinstatement of alcohol seeking, while having no effect on lever pressing without foot shocks. Possible mechanisms are contrary: there may be increased dopamine release through 5-HT3-antagonist disinhibition of raphe nucleus projections, but also decreased stress-induced and drug-induced dopamine release through 5-HT3 receptor-antagonist reduced

5-HT3 receptor mediation.3

Lynch et al. (2011) compared topiramate (an anticonvulsant;

(5,10) mg kg−1_{), ondansetron ((0.001,0.01) mg kg}−1_{), and their}

combina-tions in rat ethanol intake. Singularly, alcohol-preferring rats (P-rats) showed

an alcohol intake decrease for 10 mg kg−1 _topiramate. _{In combination,}

10 mg kg−1 _{topiramate showed this effect for 1 or 3 days (0.001 mg kg}−1 _and

0.01 mg kg−1_{, respectively). In the same rats after 15 days withdrawal,}

antag-onist administration 30 minutes before reinstating alcohol revealed that both drugs decrease the intake spike (same shape but larger effect in P-rats than Wistar control rats), and their combination increases this effect. The additive effect of the drugs suggests multiple pathways of alcohol intake mediation.

Furthermore, heavier drinking rats showed greater intake decreases. Since

higher drinkers have lower baseline dopamine, presumably this leaves more

room for (5-HT3 receptor-mediated) dopaminergic effects (Lynch et al.,2011).

Genetically, C57BL/6J Htr3a null mice did not differ from wt mice in their

alcohol intake (Hodge et al.,2004), and only wt mice decreased their alcohol

in-take with 5-HT3 receptor antagonist LY278, 584. However, genetic redundancy may cause other genes to take over Htr3a function, and the known high alco-hol drinking in C57BL/6J mice may be mediated through other pathways than

5-HT3 receptor-mediated ones. In a review of cyamemazine, chronic 1 mg kg−1

IP decreased alcohol consumption in rats (Bourin et al., 2004). In mice, both

1 mg kg−1 _{IP on the day of alcohol removal as well as 0.5 mg kg}−1 _IP

chroni-cally during alcohol consumption reduced convulsions in alcohol wirthdrawal.

These effects involve both dopamine and 5-HT actions (Naassila et al., 1998).

Overexpression of 5-HT3 receptors increases sensitivity to low-dose activating

effects of ethanol but not to high-dose sedating effects (Engleman et al.,2008).

Lastly, both chronic ethanol and chronic 5-HT3 receptor antagonist administra-tion produce neuroadaptive changes in the ability of 5-HT3 receptors to regulate

3

It could be hypothesized that diminished drug effectiveness would lead to higher drug intake (to achieve the same effect). However, cognitive factors related to 5-HT3 receptor-antagonism-induced decreased dopamine release could also lead to the behavior of lower drug intake (because “it doesn’t help anyway”). Animal research like the one discussed cannot distinguish these two opposed effects.

The Role of the 5-HT3 Receptor in the Central Nervous System