The Impact of Prolonged Anandamide Availability by Anandamide Transport
Inhibition on Nausea-Induced Behaviour in Rats and Vomiting in Shrews
(Suncus murinus)
By
Lesley D O’Brien
A Thesis
presented to
The University of Guelph
In partial fulfillment of requirements
for the degree of
Master of Science
in
Psychology
Guelph, Ontario, Canada
© Lesley D O’Brien, July, 2013
ABSTRACT
THE IMPACT OF PROLONGED ANANDAMIDE AVAILABILITY BY ANANDAMIDE
TRANSPORT INHIBITION ON NAUSEA-INDUCED BEHAVIOUR IN RATS AND
VOMITING IN SHREWS (SUNCUS MURINUS)
Lesley D O’Brien Advisor: University of Guelph, 2013 Professor L. A. Parker
Considerable evidence supports anandamide (AEA) as an important mediator in the regulation of
nausea and vomiting. The present study investigates the effect of inhibiting a protein reported to
mediate AEA transport, FLAT (FAAH-1-like AEA transporter), on nausea and vomiting and the
neural correlates of AEA regulated nausea in the visceral insular cortex (VIC). The systemic
administration of the AEA transport inhibitor ARN272 was evaluated in LiCl-induced
conditioned gaping in rats, and vomiting in shrews. The effect of intra-cranial administration of
ARN272 into the VIC was also investigated using LiCl-induced conditioned gaping in rats.
Systemic administration of ARN272 dose-dependently suppressed LiCl-induced conditioned
gaping in rats, and was reversed by CB1 receptor antagonism with SR141716. Systemic
administration of ARN272 also attenuated vomiting in shrews. Delivery of ARN272 into the
VIC produced no effect on LiCl-induced conditioned gaping in rats. These results suggest that
preventing the cellular reuptake of AEA through transport inhibition tonically activates CB1
receptors to regulate toxin-induced nausea, but that this is not AEA regulated within the VIC.
iii
ACKNOWLEDGEMENTS
There are a number of people whose contributions and support have made the completion of my
Masters thesis possible, and to whom I am greatly indebted.
To Dr. Cheryl Limebeer, whose skill, experience, and time, during all the laboratory
work was readily and freely shared; and very much appreciated. To Dr. Erin Rock who was also
always willing to share her knowledge and experience. To my advisory committee member Dr.
Boyer Winters, thank you for all your objective and valuable feedback.
To my brother Craig, who has always had an inexplicably unrelenting belief in me,
whose love and support is a beautiful gift. To my father, Dennis, and his wife, Maricel, whose
seemingly endless generosity and support have made this work possible. To my Cosworth Dog,
who has kept my feet and my heart warm for the last 6 years as well as during the writing of this
thesis. Also, to Mr. Simon Hughes, who indirectly made the completion of my Masters possible
at all through his tutoring and support during the first years of my return to higher education.
To my advisor, Dr. Linda Parker, a very special thank you for being the mentor that you
are; supportive, encouraging, always my advocate. Know that working with you these last few
years has forever changed the trajectory of my life, and I shall remain; forever grateful.
iv
Table of Contents
Page
ACKNOWLEDGEMENTS iii
TABLE OF CONTENTS iv
LIST OF FIGURES vii
GENERAL INTRODUCTION 1
Endocannabinoid System: Composition and Function 1
Anandamide Synthesis 1
A localized action 2
Cannabinoid receptors 2
Moving Anandamide 3
The Anandamide Transport Controversy 3
FAAH-like Anandamide Transporter (FLAT) 5
Anandamide Transport Inhibition 5
The Measurement of Nausea in Rats 6
Conditioned Taste Avoidance 6
Conditioned Gaping 7
Regulation of Nausea and Vomiting by the Endocannabinoid System 8
Anandamide’s Role in Nausea and Vomiting 9
The Anatomy of Nausea 10
Peripheral Nervous System (PNS) Versus Central Nervous System (CNS) 10
The Insular Cortex 11
Present Study 12
v
PART I - SUBMITTED ARTICLE IN THE BRITISH JOURNAL OF PHARMACOLOGY 15
Summary 16
Introduction 18
Methods 21
Animals 21
Drugs 22
Apparatus 23
Procedure 24
Experiment 1: Potential of ARN272 to attenuate LiCl-induced 24
conditioned gaping, and reversal of ARN272-suppressed gaping by
SR141716
Experiment 2: Effect of systemic administration of ARN272 on 25
LiCl-induced vomiting in shrews
Behavioral Measures 25
Data Analysis 26
Results 26
Experiment 1: Systemic ARN272 suppressed LiCl-induced conditioned 26
gaping in rats, and was reversed by the CB1 receptor antagonist SR141716
Experiment 2: Systemic ARN272 reduced LiCl-induced vomiting in shrews 27
Discussion 27
References 31
Figure Legends 37
vi
PART II – THE NEURAL CORRELATES OF NAUSEA MEDIATION BY 41
ANANDAMIDE TRANSPORT INHIBITION IN THE VISCERAL INSULAR CORTEX
Summary 41
Introduction 42
Method 45
Animals 45
Drug Treatments 46
Surgical Implantation of the Intracranial Guide Cannula 46
Behavioral Measures 47
Behavioral Apparatus 47
Behavioral Procedures 47
Histology 49
Data Analysis 49
Results 49
Discussion 50
References 54
GENERAL DISCUSSION 62
Anandamide: Neuromodulator of Nausea and Vomiting 62
Anandamide Deactivation and Movement 64
Measuring Nausea 66
Anandamide in the CNS 68
Concluding Remarks 70
REFERENCES 72
vii
List of Figures
Page
GENERAL INTRODUCTION
A Orofacial musculature activated by rat gaping reaction and shrew retch 14
SUBMITTED ARTICLE IN THE BRITISH JOURNAL OF PHARMACOLOGY
Experiment 1: Potential of ARN272 to attenuate LiCl-induced conditioned gaping, and reversal
of ARN272-suppressed gaping by SR141716
1 Number of gapes by rats on drug-free test day 38
2 Volume of saccharin consumed by rats at 30 and 120 minutes 39
Experiment 2: Effect of systemic administration of ARN272 on LiCl-induced vomiting in shrews
3 Vomiting episodes displayed by S. murinus 40
PART II - THE NEURAL CORRELATES OF NAUSEA MEDIATION BY ANANDAMIDE
TRANSPORT INHIBITION IN THE VISCERAL INSULAR CORTEX
1 Number of gapes by rats on drug-free test day 58
2 Volume of saccharin consumed by rats at 30 and 120 minutes 59
3 Representative photomicrograph of a VIC bi-lateral cannula track 60
4 Traces of infusion sites in the Visceral (granular) IC 61
GENERAL DISCUSSION
B Anandamide translocation and action locations 71
1
General Introduction
The psychoactive properties of the Cannabis sativa plant have been documented by
scientists since the early 1800’s (Christison, 1848; Inglis, 1845; Oliver, 1883). Despite this long
history, knowledge of how the body’s own endogenous cannabinoid system mediates these
effects has grown only somewhat recently in the preceding two decades. Following the
identification of the main active constituent of the Cannabis plant, ∆9-tetrahydrocannabinol or
THC (Gaoni & Mechoulam, 1964), came the discovery of its target, the cannabinoid receptors
(Matsuda, Lolait, Brownstein, Young & Bonner, 1990; Munro, Thomas & Abu-Shaar, 1993),
and then the endogenous cannabinoid ligands, the endocannabinoids (Devane et al., 1992;
Mechoulam et al., 1995; Sugiura, et al. 1995). The endocannabinoid system has since been
found to play a neuromodulatory role (Di Marzo, Melck, Bisogno & De Petrocellis, 1998),
regulating physiological outcomes in a wide range of areas for example; pain and inflammation
(Hohmann & Suplita, 2006), obesity (Matias & Di Marzo, 2007), and emesis (Parker, Rock, &
Limebeer, 2011). The experiments conducted for this thesis address the regulation of nausea and
vomiting by the endocannabinoid system, and in particular by the first endogenous cannabinoid
ligand to be characterized, anandamide, named from the Sanskrit word ananda, meaning bliss
(Devane et al., 1992).
Endocannabinoid System: Composition and Function
Anandamide Synthesis. To date five endocannabinoids have been extracted and
identified, anandamide (Devane et al., 1992), 2-arachidonoylglycerol or 2-AG (Mechoulam et al,
1995; Sugiura, 1995), noladin ether (Hanus et al., 2001), virodhamine (Porter et al., 2002), and
N-arachidonoyldopamine (Huang et al., 2002). Of these endocannabinoids, anandamide is the
best understood in the context of nausea and vomiting. The evolutionary history of lipid
2
messengers can be found in the fatty acid ethanolamides of plant cell membranes, released to
trigger stress and immune system responses (Chapman, 2000; Piomelli, 2003). Mammalian cells
have conserved this lipid signalling mechanism with anandamide being the ethanolamide of
arachidonic acid (Piomelli, 2003). Anandamide synthesis can be induced in two ways, Ca2+ entry
into neurons (Giuffrida et al., 1999) and the activation of G-protein-coupled receptors (Ferrer et
al., 2003; Giuffrida et al., 1999). Anandamide originates from a phosolipid precursor, N-
arachidonoyl-phosphatidly-ethanolamine (NArPE), which is converted to anandamide through a
total of four possible routes (Di Marzo, 2008; Liu et al., 2008). While the details of each route
are outside of the scope of the topic at hand, their existence coupled with evidence that
anandamide can also serve as a substrate for cyclooxygenase-2 (COX2) enzymes to produce
prostaglandins (Yu, Ives, & Ramesha, 1997), serves as an example of the intricacy of the
biosynthesis of anandamide and of its multifaceted functionality.
A localized action. Endocannabinoids have been found to act on their receptors locally,
likely as a result of their lipid composition and thus their highly hydrophobic nature (Di Marzo &
Petrosino, 2007). Wilson and Nicoll (2001) reported that endocannabinoids were only found to
travel up to 20 µm from their release sites before being eliminated. In contrast to classical
neurotransmitters, endocannabinoids are not stored in secretory vesicles, but are produced on
demand (Di Marzo, 2008). One way this production may be facilitated is from endocannabinoid
precursor molecules found in neuronal plasma membranes, allowing them to leave the cell as
soon as they are formed (Piomelli, 2003).
Cannabinoid receptors. Cannabinoid 1 receptors (CB1) and CB2 receptors are the most
widely studied of the cannabinoid molecular targets, with CB1 receptors being considered the
most ubiquitous G-protein coupled receptor in the mammalian brain (Piomelli, 2003). Originally,
3
CB1 receptors were considered to be present only in the central nervous system (CNS) and CB2
receptors in the peripheral nervous system (PNS). However, CB2 receptors have since been found
to be present in the CNS (Van Sickle et al., 2005; Onaivi et al., 2008); though at lower levels
than CB1 receptors (Mechoulam & Parker, 2012), and CB1 receptors have also been found to be
present in peripheral organs (Mechoulam & Parker, 2012). Both CB1 and CB2 receptors
participate in retrograde signalling in the CNS, in that endocannabinoids synthesized and
released from post-synaptic neurons bind to receptors on pre-synaptic neurons and thus inhibit
neurotransmitter release (Di Marzo & Petrosino, 2007; Pertwee, 2006).
Anandamide has been found to interact with a variety of receptors, of which CB1 and CB2
receptors have been most widely studied. Interestingly, anandamide has demonstrated greater
affinity (how tightly a ligand binds to a protein; e.g., lower Ki) for both CB1 and CB2 receptors
than 2-AG, whereas 2-AG shows a greater efficacy (the maximal response produced by a ligand;
e.g., higher Vmax), (Di Marzo, 2008). Anandamide has also been shown to interact with non-
cannabinoid receptors such as the transient receptor potential, vanilloid subtype I (TRPV1),
which binds the endocannabinoid at an intracellular site (Starowicz, Nigam & Di Marzo, 2007).
Other orphan G-protein-coupled receptors such as GPR55 may also be involved in anandamide
signalling though it appears to be cell type and tissue dependent (Sharir & Abood, 2010).
Moving Anandamide
The Anandamide Transport Controversy. It makes logical sense that in order to
terminate endocannabinoid action, which utilizes both a highly localized form of neural
communication and a short duration of action, an efficient method of removing signaling
molecules from the synaptic cleft would be necessary. The specific mechanism of how
anandamide is internalized into the post-synaptic neuron has long been the subject of
4
considerable and heated scholarly debate. What is not under question is the two-fold process by
which endocannabinoids are terminated: cellular reuptake and subsequently, cellular
degradation. In the case of the latter, anandamide intracellular hydrolysis is known to be
mediated by fatty acid amide hydrolase, FAAH-1 (Cravatt et al., 1996; McKinney & Cravatt,
2005). The issue of contention concerns the former, how anandamide re-enters the post synaptic
cell.
The discovery that the reuptake of anandamide does not require cellular energy (Beltramo
et al., 1997; Hillard et al., 1997) possibly prompted and lent weight to hypotheses which
explained reuptake as being driven by FAAH metabolism through passive membrane diffusion
(Glaser et al., 2003). The ability of anandamide to passively diffuse across lipid membranes does
not however rule out the possibility that the process could be accelerated by a selective carrier
system (Piomelli, 2003). In the absence of specific molecular characterization, proponents of an
active anandamide transporter have provided other supporting evidence (Ligresti et al., 2004).
Indirect observations of support include; cells which do not express FAAH are still able to take
up anandamide (Day et al., 2001), anandamide reuptake inhibitors enhance effects of presynaptic
CB1 receptors but inhibit effects of TRPV1 receptors found on the cytosolic side of membranes
(De Petrocellis et al., 2001), and lipopolysaccharides have been found to inhibit FAAH
expression without affecting anandamide reuptake (Maccarrone et al., 2001). There is also
evidence to suggest that an anandamide transporter may exert its effects bi-directionally,
facilitating both release and reuptake, such that when anandamide reuptake inhibitors are
injected inside the cell they are found to block retrograde activation of CB1 receptors due to the
inhibition of anandamide release (Gerdeman, Ronesi, & Lovinger, 2002).
5
FAAH-like Anandamide Transporter (FLAT). The molecular identity of such an
anandamide transporter was recently reported by Fu et al. (2012). The protein in question was
found to be an isoform of the FAAH-1 molecule, and as such was named FLAT, FAAH-1-like
anandamide transporter (Marsicano & Chaouloff, 2012). As a variant of the FAAH-1 molecule,
FLAT has been found to bind anandamide selectively, but not other structurally similar
molecules such as 2-AG, and without enacting any catalytic activity (Fu et al., 2012). Fu et al.
(2012) found that anandamide transport inhibitors, AM404 and OMDM-1 displaced the binding
of anandamide to FLAT, providing further support for its role in anandamide translocation. The
authors also found that the FAAH inhibitor, URB597, exhibited no effects with regard to
anandamide binding, providing support for the lack of amidase activity by FLAT (Fu et al.,
2012). Also, an overexpression of FLAT was found to be in concert with a substantial elevation
of extracellular anandamide, suggesting that FLAT may be a mechanism for anandamide release
as well as reuptake (Fu et al., 2012).
Anandamide Transport Inhibition. As previously mentioned, there are already several
compounds available which act as anandamide transport inhibitors. The challenges associated
with many such compounds in selectively assessing anandamide transport, are their diverse off
target effects. By way of an example, the widely used anandamide reuptake inhibitor AM-404
has been found to elicit off target effects at CB1 receptors (Beltramo et al., 1997), to activate
TRPV1 receptors (De Petrocellis et al., 2000), and to effect FAAH inhibition (Fowler et al.,
2004; Hillard et al., 2007). Whereas, no such off target effects have yet to be reported from one
anandamide transport inhibitor, OMDM-1, the ability of this compound to inhibit anandamide
transport does however seem to be cell type dependent (Fowler et al., 2004). Thus, making
6
inferences from behavioral data to endocannabinoid action becomes fraught with potential
pitfalls as to whether effects are due to cellular uptake alone or also catabolism.
Fu et al (2012) searched for and identified, from a library of 4.3 million compounds, a
molecule which selectively interferes with FLAT activity, ARN272. As a competitive antagonist,
ARN272 demonstrated no inhibitory activity on endocannabinoid metabolizing enzymes, and
when administered systemically was found to increase plasma anandamide without changing
levels of other structurally analogous lipids, 2-AG, oleoylethanolamide (OEA), and
palmitoylethanolamide (PEA) (Fu et al., 2012). The authors also gathered behavioral data
demonstrating attenuated responses to acute and inflammatory pain in mice when ARN272 was
delivered systemically and centrally (Fu et al., 2012). Thus ARN272 may be a useful tool in
differentiating the functions of anandamide from other lipid messengers, as well as assessing the
inhibition of anandamide deactivation without impacting hydrolysis.
The Measurement of Nausea in Rats
The rat is an animal physiologically incapable of vomiting. Although rodents lack an
emetic reflex, their gastric afferents respond in the same manner to physical and chemical
(intragastric copper sulphate and cisplatin) stimulation that precedes vomiting in ferrets,
presumably resulting in nausea that precedes vomiting (Billig et al., 2001; Hillsley & Grundy,
1998). Indeed, 5-HT3 receptor antagonists that block vomiting in ferrets also disrupt this
preceding neural afferent reaction in rats, suggesting that the rat detects nausea, but that the
vomiting reaction may be absent in this species.
Conditioned Taste Avoidance. The conditioned taste avoidance measure has often been
used to evaluate the nauseating potential of drugs in rats. Conditioned taste avoidance is simply a
measure of to what extent a taste previously paired with an emetic agent is avoided through the
7
amount a rat drinks in a consumption test (Parker et al., 2008). Problematic evidence for
conditioned taste avoidance as a model of nausea in rats has been found, in so much as, rats not
only avoid tastes paired with nauseating drugs, but they also avoid tastes paired with drugs they
choose to self-administer (Berger, 1972; Reicher & Holman, 1977). As well, anti-emetic drugs
do not generally interfere with the establishment of conditioned taste avoidance, at least not
when the taste avoidance is based on a high dose of an emetic drug (for review see Parker et al.,
2008). Therefore, conditioned taste avoidance is not a selective measure of nausea in rat.
Conditioned Gaping. Rats display a distinctive pattern of disgust reactions, most
prominently conditioned gaping reactions (Parker et al., 2011), as measured by the taste
reactivity test (Grill & Norgren, 1978). Despite the rat inability to vomit, similar orofacial
musculature is activated by their gaping reaction as the orofacial vomiting reaction in species
capable of vomiting (Travers & Norgren, 1986), see Figure A. These disgust reactions occur
when intraorally infused with a bitter tasting solution of quinine, as well as when infused with a
sweet solution that has been previously paired with a drug which produces vomiting in species
capable of such (Parker et al., 2008). Indeed, unlike conditioned taste avoidance, only acutely
administered drugs with emetic properties produce such conditioned disgust responses when
paired with a flavored solution or a contextual stimulus (for review see Parker et al., 2008).
Moreover, and unlike conditioned taste avoidance, anti-emetic drugs, such as ondansetron
(Limebeer & Parker, 2000), 8-OH-DPAT (Limebeer & Parker, 2003) and cannabinoids (Parker
et al., 2011) consistently prevent the establishment of nausea-induced conditioned gaping in rats.
However these same anti-nausea drugs have not been found to interfere with quinine induced
unconditioned gaping (Limebeer & Parker, 2000), suggesting the attenuated gaping was specific
to the induction of nausea and not the production of the behavioral response. Therefore, nausea-
8
induced gaping reactions in rats are a useful tool in investigating the role of the anandamide in
nausea regulation.
Regulation of Nausea and Vomiting by the Endocannabinoid System
Cannabinoid based medicines have been available in North America for the treatment of
chemotherapy-induced nausea and vomiting since the 1970’s and early 1980’s (Parker et al.,
2011). Several studies comparing phytocannabinoid (plant-derived) ∆9-THC oral administration
(Marinol, Dronabinol) with the then available anti-emetic dopamine antagonists concluded THC
was at least as effective as standard anti-emetic treatment (Carey et al., 1983; Tramer et al.,
2001). The anti-emetic properties of cannabinoid drugs have been found to extend beyond
humans to other emetic species such as ferrets (Simoneau et al, 2001; Van Sickle et al, 2001),
cats (McCarthy & Borison, 1981), and the house musk shrew Suncus murinus (Kwiatkowska et
al, 2004; Parker et al, 2004). Specifically in the house musk shrew, ∆9-THC has been found to
dose-dependently suppress cisplatin-induced vomiting (Kwiatkowska et al, 2004).
In an extension from the effects of phytocannabinoids, recent research has also linked
lower endocannabinoid blood concentration levels to individuals who experienced greater
motion sickness during flight parabolic manoeuvers (Chouker et al., 2010). As such, the
contributions of the endocannabinoid system in the regulation of nausea and vomiting seem to be
pervasive given the physiological differences in the CNS vomiting centers versus inputs from the
inner ear involved in motion sickness.
In the 1990’s more effective anti-emetic treatments than the dopamine antagonists were
discovered, resulting in the “gold standard” anti-emetic treatment for chemotherapy-induced
nausea and vomiting, the combination of 5-HT3 antagonists, NK1 antagonists (neurokinin-1 is a
G protein-coupled receptor acted on by tachykinins, a family of neuropeptides) and
9
dexamethasone (e.g., Campos et al, 2001). Although this treatment combination is highly
effective in the reduction of vomiting, it is less effective in reducing nausea (acute, delayed or
anticipatory) in chemotherapy patients (Campos et al, 2001). Indeed nausea is often considered
to be the most distressing symptom of chemotherapy patients in the advent of better control of
vomiting (e.g. Hickok et al, 2003). Considerable preclinical evidence indicates that cannabinoids
may be useful in this regard (see Parker et al, 2011).
Anandamide’s Role in Nausea and Vomiting. There is a considerable body of evidence
among animal models to suggest that anandamide plays a role in the regulation of nausea and
vomiting. The administration of exogenous anandamide has been found to have anti-emetic
properties in the least shrew (Darmani, 2002) and in ferrets (Van Sickle et al., 2005). Although
the administration of exogenous anandamide was not found to have any impact on vomiting in
the house musk shrew (Parker et al, 2009a), prolonging the activity of endogenous anandamide
through inhibiting its degradation did reduce vomiting in the house musk shrew (Parker et al,
2009a) and in the ferret (Sharkey et al., 2007). Also in the house musk shrew, inhibiting the
activity of the FAAH enzyme with URB597 non-selectively reduced toxin-induced vomiting
from both cisplatin and nicotine (Parker et al, 2009a). Prolonging the action of anandamide by
the inhibition of its degradation has also been shown to attenuate nausea-induced responding.
The FAAH enzyme inhibitor URB597 has been shown to interfere with LiCl-induced
conditioned gaping reactions in rats (Cross-Mellor et al., 2007).
The mechanism of action by which many phytocannabinoids are thought to act in the
regulation of emesis is CB1 receptor mediated. Molecular studies have found that pre-treatment
with the CB1 receptor agonist ∆9-THC reduces c-fos expression in the dorsal motor nucleus of the
vagus induced by cisplatin pre-treatment (Van Sickle et al., 2001; 2003). The expression of the
10
immediate early gene c-fos is rapidly and transiently induced by a variety of stimuli (Morgan,
1991), thus the interference by ∆9-THC with the cisplatin-induced marker of neuronal activation
demonstrates the involvement of the CB1 receptor in attenuating changes in intracellular activity.
The mechanism by which anandamide exerts its effects is also thought to be CB1 receptor
mediated. Van Sickle et al. (2005) found that the anti-emetic effects of exogenous anandamide
were reversed by a CB1 antagonist (AM251) but not by a CB2 antagonist (AM630). The
suppression of nausea by the FAAH inhibitor URB597 was reversed by the CB1 antagonists AM-
251 and SR141716 in rats (Cross-Mellor et al., 2007) and house musk shrews respectively
(Parker et al., 2009a). Despite anandamide being an endogenous agonist at CB1 receptors
(Devane et al., 1992), this may not be the sole route by which it contributes to nausea regulation.
Endogenous anandamide has been found to exert a more potent pharmacological effect in vivo at
CB1 receptors than pure agonists, but more so in cells that co-express both TRPV1 and CB1
receptors (Hermann et al., 2003). Indeed, Sharkey et al. (2007) found that the anti-emetic
properties of anandamide could be attenuated by TRPV1 antagonists, iodoresiniferatoxin and
AMG9810, in ferrets.
The Anatomy of Endocannabinoid Regulation of Nausea
With the discovery of the endocannabinoid system, not only how, but also where
cannabinoids act to regulate nausea, continues to be explored.
Peripheral Nervous System (PNS) Versus Central Nervous System (CNS). CB1
receptors in both the PNS and the CNS have been shown to be involved in the control of emetic
responses (Darmani & Johnson, 2004; Van Sickle et al., 2001). Recently, Limebeer et al. (2012)
found that the nausea-relieving effects of CB1 receptor agonists were mediated in the CNS as the
peripherally restricted CB1 agonist, CB13, suppressed lithium-induced conditioned gaping in rats
11
when administered centrally but not peripherally. Seemingly paradoxically, the production of
nausea by CB1 inverse agonists appears to be peripherally mediated (Limebeer et al., 2010;
McLaughlin et al., 2005; Parker et al., 2003), in support of this, Limebeer et al. (2010; 2012)
found that the CB1 inverse agonist/antagonist AM-251 did not produce nausea when
administered centrally.
To further explore the nausea-reducing effects of endocannabinoids in the CNS we can
look to areas currently known to play a role in emesis. In vomiting species, the dorsal vagal
complex is thought to be the starting point for a common pathway which includes the area
postrema, nucleus of the solitary tract, and the dorsal motor nucleus of the vagus, all found in the
brainstem. Concurrently, CB1 receptors and the catalytic enzyme of anandamide, FAAH, have
been found in these areas involved in emesis (Van Sickle et al., 2001). Nausea specifically
appears to be mediated in forebrain regions (Limebeer et al., 2004), with much research currently
being focused on the insular cortex (Limebeer et al., 2012; Tuerke et al., 2012).
The Insular Cortex. Visceral sensation has been found to be represented in several areas
of the forebrain, paralimbic and limbic structures; including the insular cortex (Aziz et al., 2000).
It is the interconnection of the insular cortex with limbic structures that is thought to mediate the
affective and cognitive components of visceral sensation in humans (Aziz et al., 2000). The
insular cortex is known to process homeostatic information (Craig, 2002), and is an important
structure for the perception of bodily needs (Contreras et al., 2007). The insular cortex seems to
be activated by states of autonomic arousal, such as increased cardiovascular activity (Critchley
et al., 2000), which often accompany sensations of nausea (Borrison & Wang, 1953).
Furthermore, stimulation of the insular cortex has been shown to produce vomiting in humans
(Fiol et al., 1988; Catenoix et al., 2008) and other animals (Kaada, 1951). Kiefer & Orr (1992)
12
demonstrated that ablation of the insular cortex prevented the nausea-induced behavior of
conditioned gaping in rats.
The insular cortex has also been shown to be involved in the generation of human disgust
reactions (Calder et al., 2007), and the sensation of nausea in humans as well as rats (Contreras et
al., 2007; Penfield & Faulk, 1955). Research using fMRI has shown that the insula is activated
when humans are shown pictures of disgusting foods or are exposed to disgusting odours
(Heining et al., 2003; Wicker et al., 2003). Critchley et al. (2004) also used voxel-based
morphometry from fMRI to show that subjective ratings of visceral awareness in humans
correlate with local gray matter volume in the right anterior insular. Neuronal activation has been
found to increase in the insular cortex following the nausea induced by LiCl administration, as
measured by a significant increase in Fos-immunoreactivity (Fos-IR) expression in rats
(Contreras et al., 2007). Fos-IR is quantified as increases in the numbers of neurons per mm2
expressing c-Fos, the protein product of the immediate early gene c-fos, suggesting increased
cellular activity in the insular cortex in response to the LiCl emetic challenge.
The specific region of the insular cortex responsible for nausea appears to be the visceral
region of the insular cortex (VIC); temporary lesions of this region interfered with LiCl-induced
malaise (Contreras et al., 2007). Furthermore, intracranial administration of the anti-emetic drug
ondansetron delivered to the visceral region, but not the gustatory region of the insular cortex
interfered with the establishment of the nausea-induced behavior of gaping (Tuerke et al., 2012).
Present Study
It is hypothesized that the inhibition of anandamide transport will produce a prolonged presence
of anandamide in the synapse. As such, the prolonged synaptic availability of anandamide was
expected to regulate toxin-induced nausea in rats, and vomiting in the house musk shrew by
13
indirect agonism of, and extended activation at, cannabinoid receptors. It is also hypothesized
that the VIC is a forebrain region necessary for the production of nausea sensations, specifically
regulated by anandamide activation of cannabinoid receptors in this area.
In Part I, Experiment 1 investigated the potential of ARN272 to attenuate LiCl-induced
conditioned gaping, and reversal of ARN272-suppressed gaping by antagonism of the CB1
receptor by SR141716. Also in Part I, Experiment 2 evaluated the effect of systemic
administration of ARN272 on LiCl-induced vomiting in shrews. Part II investigated the neural
correlates of nausea mediation by central administration of ARN272 directly to the visceral
insular cortex (VIC) and its impact on LiCl-induced conditioned gaping in rats.
14
Figure A
Orofacial characteristics of the rat gape and shrew retch
This figure demonstrates the similar orofacial musculature activated by the rat gape (left) and the
retch of the emetic shrew (right).
15
Manuscript submitted to: British Journal of Pharmacology
Prolonged anandamide availability by anandamide transport inhibition attenuates nausea-induced
behaviour in rats, and vomiting in shrews (Suncus murinus)
L D O’Brien1, C L Limebeer1, E M Rock1, G Bottegoni2, D Piomelli2,3, and L A Parker1
1Department of Psychology and Collaborative Neuroscience Program University of Guelph,
Guelph, ON, Canada, 2Drug Discovery and Development, Instituto Italiano di Technologia,
Genova, Italy, and 3Department of Anatomy & Neurobiology, University of California at Irvine,
USA.
Correspondence: Linda A. Parker, Department of Psychology, University of Guelph, Guelph,
ON, N1G 2W1, Canada. Email: [email protected].
16
Summary
Background and Purpose
To understand how prolonged synaptic availability of anandamide impacts the regulation of
nausea and vomiting and the receptor level mechanism of action involved. In light of recent
characterization of an anandamide transporter, FAAH-1-like anandamide transporter (FLAT), to
provide behavioral support for anandamide cellular reuptake as a facilitated transport process.
Experimental Approach
The systemic administration of the anandamide transport inhibitor ARN272 was used to evaluate
the prevention of LiCl-induced nausea-induced behaviour (conditioned gaping) in rats, and LiCl-
induced emesis in shrews (Suncus murinus). The mechanism of how prolonging anandamide
availability acts to regulate nausea in rats was explored by the antagonism of CB1 receptors with
the systemic co-administration of SR141716.
Key Results
The systemic administration of ARN272 produced a dose-dependent suppression of nausea-
induced conditioned gaping in rats, and produced a dose-dependent reduction of vomiting in
shrews. The systemic co-administration of SR141716 with ARN272 (at 3.0 mg.kg-1) in rats
produced a complete reversal of ARN272-suppressed gaping at 1.0 mg.kg-1. SR141716 alone did
not differ from VEH.
Conclusions and Implications
These results suggest that prolonging the availability of anandamide through transport inhibition
tonically activates CB1 receptors and as such produces a type of indirect agonism to regulate
toxin-induced nausea and vomiting. The results also provide behavioral evidence in support of a
facilitated transport mechanism used in the cellular reuptake of anandamide.
17
Keywords
Endocannabinoid, anandamide, nausea, gaping, vomiting, FAAH-1-like anandamide transporter,
FLAT, CB1, ARN272, taste reactivity.
Abbreviations
ARN272, [(4-(5-(4-hydroxy-phenyl)-3,4-diaza-bicyclo[4.4.0]deca-1(6),2,4,7,9-pentaen-2-
ylamino)-phenyl)-phenylamino-methanone]; CB1, cannabinoid 1 receptor; CCAC, Canadian
Council on Animal Care; CTA test, Conditioned Taste Avoidance test; DRN, dorsal raphe
nucleus; FAAH, fatty acid amide hydrolase; FLAT, FAAH-1-like anandamide transporter; OEA,
oleoylethanolamide; PEA, palmitoylethanolamide; ∆9-THC, ∆9-tetrahydrocannabinol; TR, Taste
Reactivity
18
Introduction
The Cannabis sativa plant has been known for centuries to exert therapeutic effects in the
treatment of nausea and vomiting. More recently cannabinoid agonists such as ∆9-
tetrahydrocannabinol (∆9-THC) have been found to be as effective as anti-emetic dopamine
antagonists in human clinical trials (Carey et al., 1983; Tramer et al., 2001). The anti-emetic
properties of cannabinoid agonists have been found to extend beyond humans to other emetic
species, attenuating vomiting in ferrets (Simoneau et al., 2001; Van Sickle et al., 2001), cats
(McCarthy & Borison, 1981), and the house musk shrew Suncus murinus (Kwiatkowska et al.,
2004; Parker et al., 2004). Despite this long history, knowledge of how the endogenous
cannabinoid system mediates nausea and vomiting is still incomplete.
Comparable to the effects of plant-derived cannabinoids, there is a body of evidence
among animal models implicating the endocannabinoid anandamide, as important in the
regulation of nausea and vomiting. The administration of exogenous anandamide has been found
to have anti-emetic properties in the least shrew (Darmani, 2002) and in ferrets (Van Sickle et
al., 2005). Deactivation of anandamide occurs through intracellular hydrolysis and is known to
be mediated by the enzyme fatty acid amide hydrolase, FAAH (Cravatt et al., 1996; Desarnaud
et al., 1995; McKinney & Cravatt, 2005). As such, prolonging the activity of endogenous
anandamide through the inhibition of its degradation has also been found to reduce vomiting,
specifically in the house musk shrew (Parker et al., 2009) and in the ferret (Sharkey et al., 2007).
FAAH inhibition has also been shown to attenuate nausea-induced responding, interfering with
conditioned gaping reactions in rats (Cross-Mellor et al., 2007). These findings suggest that
anandamide acts within the endocannabinoid system to regulate both nausea and vomiting.
19
Anandamide is known to be an endogenous agonist at Cannabinoid 1 (CB1) receptors
(Devane et al., 1992). The mechanism by which anandamide exerts its anti-emetic and anti-
nausea effects is thought to be CB1 receptor mediated. The anti-emetic effects of exogenous
anandamide administration have been found to be reversed by the CB1 antagonist, AM251 (Van
Sickle et al., 2005), and the suppression of nausea by the FAAH inhibitor URB597 was reversed
by the CB1 antagonists AM251 and SR141716 in rat (Cross-Mellor et al., 2007) and the house
musk shrew respectively (Parker et al., 2009).
The specific mechanisms behind how anandamide signalling is terminated are still
unfolding. How anandamide re-enters the post synaptic cell appears to conform to a two-fold
process, cellular re-uptake and subsequently cellular degradation. Previously, cellular reuptake
has been hypothesized as occurring through either passive membrane diffusion driven by FAAH
metabolism (Glaser et al., 2003), or by some previously unknown selective carrier system
(Hillard et al., 2007; Ligresti et al., 2004). In support of the latter, Fu et al. (2012) recently
reported the molecular identity of a facilitated anandamide transport mechanism, FAAH-1-like
anandamide transporter (FLAT). As an isoform of the FAAH molecule, FLAT was found to bind
anandamide selectively, but not other structurally similar molecules such as 2-AG, and without
enacting any catalytic activity (Fu et al., 2012). Concurrently, Fu et al. (2012) identified a
competitive antagonist of FLAT, ARN272. The ability to inhibit FLAT activity through the
anandamide transport inhibitor ARN272 offers a new tool to investigate the neurobiological
effect of prolonging the synaptic availability of anandamide in vivo, in models of nausea and
vomiting.
While emetic species are used to explore the regulation of vomiting, the subjective
experience of nausea requires more consideration in animal models. Conditioned taste avoidance
20
(CTA) is a measure which has often been used to evaluate the nauseating potential of drugs in
rats (the extent to which a taste previously paired with an emetic agent is avoided through the
amount a rat drinks in a consumption test). However problematic evidence for conditioned taste
avoidance as a model of nausea in rats has been found. Anti-emetic drugs do not generally
interfere with the establishment of conditioned taste avoidance, (for review see Parker et al.,
2008). Also, rats not only avoid tastes paired with nauseating drugs, but they also avoid tastes
paired with drugs they choose to self-administer (Berger, 1972; Reicher & Holman, 1977).
Therefore the CTA test cannot be considered a selective measure of nausea in rat. Physiological
regulation of nausea can be studied, however, in the rat using their distinctive pattern of disgust
reactions, most prominently conditioned gaping reactions (Parker et al., 2011). Despite the rat
inability to vomit, the detection mechanism of nausea is still present, with similar orofacial
musculature being activated by the gaping reaction as the orofacial vomiting reaction in emetic
species (Travers & Norgren, 1986). Conditioned gaping reactions occur both when intraorally
infused with a bitter tasting solution of quinine, as well as when exposed to cues (taste or
context) previously paired with a drug which produces vomiting in emetic species (Parker et al.,
2008). Moreover, and unlike conditioned taste avoidance, only drugs with emetic properties
produce conditioned gaping reactions when paired with a flavor or contextual stimulus, and anti-
emetic drugs consistently prevent the establishment of nausea-induced conditioned gaping in rats
(Limebeer & Parker, 2000; Limebeer & Parker, 2003). Therefore conditioned gaping can be used
as a selective measure of nausea in rat.
The experiments reported here investigate the impact of prolonging the synaptic
availability of anandamide through anandamide transport inhibition on the endocannabinoid
systems regulation of nausea and vomiting. Experiment 1 evaluated the potential of systemic
21
administration of ARN272 to attenuate LiCl-induced conditioned gaping in rats and the potential
of the CB1 receptor antagonist/inverse agonist SR141617 to reverse the ARN272-suppressed
conditioned gaping response. The extent to which rats avoided a taste paired with the nausea
inducing agent LiCl was also assessed using a Conditioned Taste Avoidance measure, to re-
assert the selectivity of conditioned gaping in the measurement of nausea. Experiment 2
evaluated the potential of systemic administration of ARN272 to regulate LiCl-induced vomiting
in the house musk shew (Suncus murinus). Here we provide behavioural support for anandamide
reuptake occurring through a facilitated transport mechanism from the ARN272-suppressed
gaping in rats and attenuated vomiting in shrews. As such it is reasoned that prolonging the
synaptic availability of anandamide augmented its action in areas of the central nervous system
where anandamide is endogenously released, to tonically activate CB1 receptors and extend the
anti-emetic action of anandamide.
Methods
Animals
All animal care and experimental procedures complied with the recommendations of the
Canadian Council on Animal Care (CCAC) and were approved by the Animal Care Committee
of the University of Guelph. ARRIVE guidelines were consulted (Kilkenny et al., 2010). A total
of 58 naïve Male Sprague-Dawley rats (Charles River Lab, St. Constrant, QC, Canada) were
used for assessment of anti-nausea-induced behaviour. Rats were single-housed in 48 x 26 x 20
cm shoebox cages in a colony room at an ambient temperature of 21°C. All animals were
maintained on a reverse light/dark cycle (7:00am lights off; 7:00pm lights on) with free access to
food (Iams rodent chow, 18% protein) and tap water, except during testing which occurred
during the dark cycle. All animals were provided with environmental enrichment from two clean
22
paper towels (replenished weekly during cage changes) and a soft plastic container 14 cm long
and 12 cm in diameter.
A total of 21 Suncus murinus, house musk shrews, were bred and raised in a colony at the
University of Guelph. Shrews were single-housed in cages at an ambient temperature of 21˚C on
a 14/10 light dark schedule (lights off at 2100 h). Shrews were tested during their light cycle,
between 0900h and 1400h. Both males (42.9g-53.0g) and females (26.1g-32.9g) were used and
equally distributed among the groups, with subjects ranging from 98 days to 814 days of age.
The sexes did not significantly differ in vomiting frequency in any analysis; therefore, males and
females were pooled in all reported analyses. The shrews had previous emetic experience with
the limitation of a minimum of 3 weeks recovery between treatments.
Drugs
The anandamide transport inhibitor, ARN272 (Danieli Piomelli, University of California
Irvine/Istituto Italiano di Tecnologia), was prepared in a vehicle solution (VEH) of 1:1:8
PEG400, Tween, and physiological saline, respectively for all experiments. All systemic
injections were administered i.p.. In Experiment 1, ARN272 was delivered to rats at
concentrations of 0.1 mg.ml-1 (0.1 mg.kg-1 dose), 1 mg.ml-1 (1 mg.kg-1 dose), and 3 mg.ml-1 (3
mg.kg-1 dose), and at a volume of 1 ml.kg-1, chosen on the basis of previous experiments
performed by Fu et al. (2012) where 1 mg.kg-1 increased plasma anandamide levels 2 hours post
administration. In Experiment 2, ARN272 was delivered to shrews at concentrations of 3.0
mg.ml-1 (9.0 mg.kg-1 dose) at a volume of of 3 ml.kg-1, and 3.0 mg.ml-1 (18 mg.kg-1 dose) at a
volume of 6 ml.kg-1, chosen on the basis of previous experiments where Suncus murinus required
a dose increase by at least a factor of 3 times an effective dose in rats (Kwaitkowska et al., 2004;
Parker et al., 2004).
23
The concentration of SR141716 (SR; Sequoia Research Products Ltd, UK) in Experiment
1 was delivered at 1.0 mg.ml-1 (1.0 mg.kg-1 dose) at a volume of 1 ml.kg-1. The dose of 1.0
mg.kg-1 SR141716 was chosen based on prior effectiveness in reversing the breakpoint and
reinstatement of nicotine self administration (Forget et al., 2009), while also being found not to
potentiate the effects of emetic agents unlike doses of SR141716 at 2.5 mg.kg-1 or higher (Parker
et al., 2003).
The LiCl drug treatment 0.15M (Sigma) used in all experiments was prepared in sterile
water and administered at volumes of 20 ml.kg-1 (127 mg.kg-1) in rats in Experiment 1 (Limebeer
& Parker, 2000), and 60 ml/kg (390 mg.kg-1) in shrews in Experiment 2 (Rock et al., 2011).
Apparatus
The Taste Reactivity (TR) chamber consisted of a clear Plexiglas box (29 x 29 x 10 cm) resting
on a glass surface. Two 60W lights suspended from the apparatus illuminated the chamber. A
mirror mounted at a 45 degree angle below the glass surface facilitated viewing of the ventral
surface of the rat, specifically any orofacial responses. Each rat, prior to being placed in the
chamber, was connected to an infusion pump (KDS100; KD Scientific Inc., Holliston, MA,
USA) via a section of PE 90 tubing attached to its intra-oral cannula which ran through a hole in
the lid of the TR chamber. All orofacial and somatic responses were recorded during the session
via a video camera (Sony DCR-HC28 Handy Cam) connected directly to a desk top PC using
Roxio Videowave Premiere Suite 8 video capture program.
Vomiting in shrews was measured in a clear Plexiglas chamber (22.5x26x20 cm)
illuminated by a 60 W light suspended from the chamber’s floor. A mirror was mounted at a 45°
angle beneath the chamber floor, which allowed for clear viewing of the ventral surface of the
shrew, and an observer counted the number of vomiting episodes.
24
Procedure
Experiment 1: Potential of ARN272 to attenuate LiCl-induced conditioned gaping, and reversal
of ARN272-suppressed gaping by SR141716. All rats were surgically implanted with intra-oral
cannula under isoflurane anaesthesia as described by Limebeer et al. (2010). Following recovery
from surgery (3 days), rats received a single adaptation trial to habituate them to the chamber and
the infusion procedure. During the adaptation trial rats were placed individually in the TR
chamber and received a 2 min intra-oral infusion of water (reverse osmosis water infused at 1
ml/min). On the following day, rats received the first of two conditioning trials (separated by 72
hr). On each conditioning trial, rats received a pretreatment injection of ARN272 or VEH 120
minutes prior to the conditioning trials. During conditioning trails, rats were intra-orally infused
with a saccharin solution (0.1%) for 2 min (1 ml/min) and orofacial and somatic reactions were
recorded on video. Immediately following the saccharin infusion the rats were injected with LiCl
(0.15M) or Saline, and then returned to their home cage. Two additional groups were added
(after ARN272 at 3.0 mg.kg-1 attenuated gaping) where a pretreatment of ARN272 at 3.0 mg.kg-1
or VEH was given 120 min prior, and with SR141716 30 min prior, to each conditioning trial.
The groups were: VEH-Saline, n=9; VEH-LiCl, n=8; 0.1 mg.kg-1 ARN272-LiCl, n=9;
1.0 mg.kg-1 ARN272-LiCl, n=8; 3.0 mg.kg-1 ARN272-LiCl, n=8; 1.0 mg.kg-1 SR-3.0 mg.kg-1
ARN272, n=8; 1.0 mg.kg-1 SR-VEH, n=8.
Seventy-two hours following the second conditioning trial, the rats received a drug-free
TR test. During the TR test, rats were re-exposed to a 2 min intra-oral infusion of saccharin
solution and their orofacial and somatic responses again recorded. All video recordings were
later scored by a rater blind to the experimental conditions using ‘The Observer’ (Noldus
Information Technology Inc., Leesburg, VA, USA).
25
Following the TR test, the rats were returned to their home cages and at 16:00 h their
water bottles were removed to begin a water deprivation regime in preparation for the
Conditioned Taste Avoidance tests (CTA test). At 08:00 h the following morning the rats
received a one-bottle test in which a graduated tube of 0.1% saccharin solution was placed on the
home cage, and the amount consumed was recorded at 30 and 120 minute intervals. A one-bottle
test was used as there is evidence to suggest it is more sensitive in detecting between group
differences in strength of taste avoidance than a two-bottle test where both water and saccharin
are made available, (Batsell & Best, 1993).
Experiment 2: Effect of systemic administration of ARN272 on LiCl-induced vomiting in
shrews. Each shrew was offered 4 meal worms (Tenebrio sp.) in its home cage 15 minutes prior
to pretreatment injections. The shrews received pretreatment injection of ARN272 120 minutes
prior to behavioral testing (VEH, n=10; 9.0 mg.kg-1, n=6; 18.0 mg.kg-1, n=5). Immediately prior
to behavioral testing the shrews were injected with LiCl (0.15M) and then placed in the TR
chamber for 45 min. An observer counted the number of vomiting episodes. A vomiting episode
is defined as abdominal contractions and expulsion of gastric fluid.
Behavioral Measures
In Experiment 1, video recordings were scored for the number of gaping reactions (rapid, large
amplitude opening of the mandible with retraction of the corners of the mouth) during the 2 min
infusions. During the CTA test, the mean cumulative amount of saccharin consumed was
measured at 30 min and 120 min. In Experiment 2, the frequency of vomiting episodes was
scored live during the 45 min period post LiCl administration.
26
Data Analysis
In Experiment 1, the number of gapes exhibited by rats on the drug-free test trial was entered
into a one-way ANOVA and analyzed with the group as the between subjects factor. For the
CTA measure, the mean cumulative volume of saccharin consumed across drug pretreatment
groups was analyzed using 2 separate one way ANOVA’s at each of the two time points, 30 and
120 minutes. Bonferroni post hoc comparison tests were conducted for all statistically significant
effects. In Experiment 2, the number of vomiting episodes was entered into a one-way ANOVA
and analyzed with the drug pretreatment as the between subjects factor. Planned comparisons
were conducted. Statistical significance was defined as p<.05.
Results
Experiment 1: Systemic ARN272 suppressed LiCl-induced conditioned gaping in rats, and was
reversed by the CB1 receptor antagonist SR141716.
Gaping Measure. The systemic administration of ARN272 produced a dose-dependent
suppression in nausea-induced conditioned gaping in rats, effects which were reversed by
pretreatment with the CB1 receptor antagonist SR141716. Figure 1 presents the mean number of
gapes on the drug free test day by drug pretreatment group. The one way ANOVA revealed a
significant effect of drug pretreatment, F(6, 51)=10.83, p<.001; subsequent post hoc Bonferroni
tests revealed that ARN272 3.0 significantly attenuated gaping compared to all groups other than
VEH-SAL (p’s<.01), which also differed from all other groups (p’s<.01).
CTA Measure. All pretreatment groups demonstrated greater taste avoidance than the
VEH-Saline group at both time intervals (30, 120 min) in that less saccharin was consumed.
There were no saccharin consumption differences specifically between the pretreatment
conditions that received LiCl, at any of the time intervals. Figure 2 presents the mean cumulative
27
amount of saccharin consumed by the various drug pretreatment groups. The one way ANOVA’s
revealed significant differences in the mean cumulative amount of saccharin consumed at 30
minutes, F(6, 51)=34.66, p<.001, and at 120 minutes, F(6, 51)=27.66, p<.001. Subsequent post
hoc Bonferroni tests revealed the VEH-SAL group as drinking significantly more saccharin than
all other groups (p’s<.001) at each time period.
Experiment 2: Systemic ARN272 reduced LiCl-induced vomiting in shrews.
Vomiting Measure. The systemic administration of ARN272 produced a dose-dependent
reduction of vomiting in shrews. Figure 3 presents the mean number of LiCl-induced vomiting
episodes for shrews pretreated with VEH, ARN272 9.0 and ARN272 18.0. The one way
ANOVA revealed a significant effect of drug pretreatment, F(2, 18)=3.75, p<.05, with planned
comparisons revealing that ARN272 18.0 significantly attenuated vomiting as compared to VEH
(p<.05).
Discussion
Consistent with the reported anti-emetic effects of increasing anandamide availability through
FAAH inhibition in shews (Parker et al., 2009) and ferrets (Sharkey et al., 2007), so prolonging
anandamide availability through transport inhibition reduced vomiting in the house musk shrew.
The present study’s findings were also consistent with existing evidence that increased
anandamide availability through FAAH inhibition attenuates nausea-induced responding in rats
(Cross-Mellor et al., 2007) in that inhibiting FLAT also produced a suppressive effect of
conditioned gaping.
Fu et al. (2012) have reported greater anandamide accumulation in cells expressing
FLAT as compared to controls, and an elevation in extracellular anandamide in cells which
overexpress FLAT, suggesting a bi-directional mechanism of anandamide translocation. As such
28
it is hypothesized that FLAT inhibition acted to regulate nausea in rats and vomiting in shrews
by prolonging the synaptic availability of locally and endogenously produced anandamide. The
extended agonist action of anandamide at CB1 receptors was evidenced by the reversal of
ARN272-suppressed gaping by a CB1 receptor antagonist. Endocannabinoids have a short
duration of action (Di Marzo, 2008) and a highly localized form of neural communication
(Wilson and Nicoll, 2001), being produced on demand as required. As such, it is logical that the
termination of endocannabinoid signalling would need to be as efficient a process as the one
required for its activation. Here the behavioral evidence suggests that the efficiency of
anandamide synaptic removal occurs via a facilitated transport system, through FLAT.
The challenges associated with the many available compounds which pharmacologically
target anandamide transport are their diverse off target effects, most notably at higher
concentrations, FAAH inhibition (Hillard et al., 2007). The inhibition of FLAT by ARN272 does
appear to be selective in that the compound produced only a weak and incomplete inhibition of
FAAH in vitro, and had little to no inhibitory effect on other endocannabinoid metabolizing
enzymes such as monoacylglycerol lipase, (Fu et al., 2012). There are further benefits to using
indirect agonism by a selective transport inhibitor as compared to FAAH inhibition to understand
the role of anandamide within the endocannabinoid system. The catalytic activity of the FAAH
enzyme has been found to impact other N-acylethanolamides as well as anandamide; such as
oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), (Bracey et al., 2002; Kathuria et
al., 2003), bioactive molecules that act on non-cannabinoid receptors. As such the use of FAAH
inhibition to isolate anandamide mediated effects remains problematic.
Existing evidence to suggest that anti-nausea treatments do not interfere with conditioned
taste avoidance learning in rats (Rabin & Hunt, 1983; Rudd et al., 1998; Limebeer & Parker,
29
2000; Limebeer et al., 2012) was supported in that pretreatment with ARN272 did not attenuate
CTA responding. Only conditioned disgust, the gaping reaction specifically produced by the
nausea induced by LiCl, was blocked by pretreatment with ARN272. As such the present
findings suggest that ARN272 is not interfering with learning per se; instead it interfered with
LiCl-induced nausea selectively necessary for the production of gaping reactions, but not CTA
(see Parker et al., 2009).
How the prolonged availability of anandamide is acting to regulate nausea and vomiting
may be through the interaction of the endocannabinoid system with the 5-hydroxytryptaminergic
system (Kimura et al., 1998). Low doses of ∆9-THC and the 5-HT3 receptor antagonist
ondansetron which have been found to be ineffective alone, when combined, suppress cisplatin-
induced vomiting in the house musk shrew (Kwiatkowska et al., 2004). Additionally,
administration of ∆9-THC has been found to reduce the ability of 5-HT3 receptor agonists to
produce emesis in the least shrew (Darmani & Johnson, 2004). The dorsal vagal complex, a
critical termination site for vagal nerve afferents in the brain stem, is densely populated with both
CB1 receptors and 5-HT3 receptors (Himmi et al., 1998). It has been hypothesized that
cannabinoids act at CB1 presynaptic receptors to inhibit the release of 5-HT (Schlicker &
Kathmann, 2001). As such, the dorsal raphe nucleus which provides the majority of 5-HT
innervation to the forebrain (Rock et al., 2011), and forebrain areas implicated in nausea
regulation such as the visceral insular cortex (Limebeer et al., 2012; Tuerke et al., 2013), may be
targeted as future sites to investigate the neural correlates of anandamide transport inhibition on
nausea and vomiting.
The use of endocannabinoid transport inhibitors have potential not only as a means to
further elucidate the role and function of the endocannabinoid system, but also as therapeutic
30
agents. The indirect agonism produced by FLAT inhibition occurs preferentially in brain areas
where receptors are targeted by locally acting endogenous anandamide, augmenting the body’s
own regulatory processes. Indirectly enhancing anandamide action in tissue where synthesis,
release, and degradation is already occurring, could provide a safer and more selective action
than direct agonists (Di Marzo, 2008).
The present experiments suggest that prolonging the availability of anandamide through
transport inhibition tonically activates CB1 receptors to regulate nausea and vomiting, and
provides in vivo support for a facilitated transport mechanism used in the cellular reuptake of
anandamide.
Acknowledgements
This research was supported by grants from the Natural Sciences and Engineering Research
Council of Canada (NSERC-92057) to LAP, and the National Institute on Drug Abuse
(DA012413) to DP.
Conflicts of interest
None.
31
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37
Figure Legends
Figure 1. Mean (+ SEM) number of gapes by rats on drug-free test day, in Experiment 1, by each
of the groups. VEH-SAL (n=9), VEH-LiCl (n=8), ARN272 0.1 mg.kg-1 (n=9), ARN272 1.0
mg.kg-1 (n=8), ARN272 3.0 mg.kg-1 (n=8), ARN272 3.0 mg.kg-1 + SR 1.0 mg.kg-1 (n=8), VEH +
SR 1.0 mg.kg-1 (n=8). ***P<0.001 indicates that group ARN272 3.0 gaped less than VEH,
ARN272 0.1, SR 1.0, and ARN272 3.0 + SR 1.0, and that group VEH-SAL gaped less than all
other groups. The number of rats that gaped in each group is indicated above each bar.
Figure 2. Mean (± SEM) cumulative volume of saccharin consumed by rats 24 hr after TR test
day and immediately following water restriction, at 30 and 120 minutes, in Experiment 1.
***P<0.001 indicates that all pretreatment groups consumed less saccharin than the VEH-Saline
group at both time intervals, and there were no saccharin consumption differences between the
LiCl pretreatment conditions at any of the time intervals.
Figure 3. Mean (± SEM) number of vomiting episodes displayed by S. murinus during the 45
minute post LiCl administration observation period. S. murinus were given the following
pretreatments prior to LiCl treatment administration: VEH (n=10), ARN272 9.0 mg.kg-1 (n=6),
ARN272 18.0 mg.kg-1 (n=5). *P<0.05 indicates that group ARN272 180.0 vomited significantly
less than VEH. The number of shrews that vomited in each group is indicated above each bar.
38
Figure 1
39
Figure 2
40
Figure 3
41
PART II
The neural correlates of nausea mediation by anandamide transport inhibition in the visceral
insular cortex
42
Summary
Background and Purpose
To investigate the neural correlates of nausea meditation by anandamide through prolonged
synaptic availability in the visceral insular cortex (VIC).
Experimental Approach
A micro-infusion of the anandamide transport inhibitor ARN272 was delivered bilaterally to the
visceral insular cortex via an intracranial guide cannula to investigate LiCl-induced nausea-
induced behaviour (conditioned gaping) in rats.
Key Results
The central administration of ARN272 into the visceral insular cortex produced no change in
conditioned gaping behaviors across drug pretreatment groups.
Conclusions and Implications
Despite existing evidence to implicate the endocannabinoid system in the regulation of nausea-
induced conditioned gaping within the VIC, these results suggest that nausea may not be
anandamide regulated within this area.
43
Introduction
The neuroanatomy of nausea and vomiting does not appear to conform to a straightforward
concept of one solitary vomiting center, but rather there appear to be groups of organized neural
pathways activated in sequence (Hornby, 2001). The coordination of various autonomic
processes involved in nausea and vomiting occur at the level of the hindbrain, specifically at the
medulla oblongata. Although coordination occurs in the brainstem, lesions to the dorsomedial
medulla have been shown not to prevent the induction of emesis (Miller et al, 1994), providing
evidence to support such an integrated system.
Areas of importance for the production of emesis within the hindbrain start within the
dorsal vagal complex, an area in the brainstem which contains the area postrema and the nucleus
tractus solitarius (NTS). The area postrema is still outside of the blood brain barrier and as such
uses chemosensitive receptors to detect blood-born emetic agents, which it then relays
information about to the adjacent NTS (Hornby, 2001). Afferent input into the NTS also arrives
directly via the vagal nerve (Saper, 1982), which relays signals related to intestinal luminal
contents and gastric tone (Hornby, 2001). The dorsal vagal complex also contains areas involved
in controlling physiological processes such as swallowing, respiration, and tone/motility of the
stomach (Hornby, 2001), as well as efferent output neurons which control the muscles required
for the production of emesis.
The projections originating in visceral organs ascend from the dorsal vagal complex via
the parvocellular nuclei of the lateral and medial ventroposterior thalami, and terminate in the
forebrain; specifically in the insular cortex (Cechetto & Saper, 1987). Stimulation of the insular
cortex has been found to elicit the sensation of nausea in humans (Penfield & Faulk, 1955), and
produce vomiting in cats (Kaada, 1951). The insular cortex is subdivided based on its degree of
44
granularity (Mesulam & Mufson, 1982). Afferent projections from the visceral organs
specifically terminate in the posterior granular cortex, or also known as the visceral insular
cortex (VIC), (Cechetto & Saper, 1987; Allen et al., 1991). It is hypothesized that the insular
cortex is the central forebrain projection of the NTS (Stephani et al, 2011).
The VIC is potentially an area of importance in conditioned disgust reactions (Limebeer
et al., 2012). Conditioned gaping in rats being a measure of such conditioned disgust, Kiefer and
Orr (1992) found that ablation of the insular cortex eradicated conditioned disgust reactions to
LiCl in rats. Despite a lack of conditioned disgust reactions, rats with insular cortex ablation
were still able to gape in response to a bitter quinine solution (Kiefer & Orr, 1992), suggesting
that the insular cortex is important for the production of nauseous sensation not the behavioral
gaping response itself. Yet more recently, and more specifically to the VIC, Tuerke et al. (2012)
found that the classical anti-emetic agent, ondansetron, when microinfused directly into the VIC
attenuated LiCl-induced conditioned gaping reactions in rats, without modifying the non-
selective behavior of LiCl-induced taste avoidance. The VIC therefore seems implicated in
conditioned disgust reactions through conditioned gaping in rats, and as such can then be
hypothesized to be an area linked to the generation and regulation of nausea.
There is also evidence to suggest that the endocannabinoid system is involved in the
nausea regulation occurring within the VIC. Limebeer et al. (2012) found that a CB1 receptor
agonist, HU-210, when delivered directly to the VIC suppressed LiCl-induced conditioned
gaping in rats, without modifying LiCl-induced taste avoidance. Knowledge of exactly how the
endocannabinoid system is working to agonise CB1 receptors in the VIC is however, incomplete.
Anandamide, as an endogenous agonist of CB1 receptors (Devane et al., 1992), is a potential
candidate for the endogenous ligand responsible for attenuating nausea-induced responding
45
within the VIC. Indeed, when the action of anandamide is prolonged systemically by
administration of the FAAH inhibitor, URB597 (Cross-Mellor et al., 2007) or by administration
of the anandamide transport inhibitor, ARN272 (Chapter 1), the nausea-induced conditioned
gaping reaction was attenuated. It is hypothesized that intra-cranial administration of ARN272
directly to the VIC will attenuate LiCl-induced conditioned gaping in rats.
The experiments described below investigated the neural correlates of nausea mediation
by anandamide via central administration of the anandamide transport inhibitor, ARN272,
directly to the visceral insular cortex (VIC) and its impact on LiCl-induced conditioned gaping in
rats. The extent to which rats subsequently avoided a taste paired with the nausea inducing agent
LiCl was also assessed using a Conditioned Taste Avoidance (CTA) measure.
Method
Animals
All animal care and experimental procedures complied with the recommendations of the
Canadian Council on Animal Care (CCAC) and were approved by the Animal Care Committee
of the University of Guelph.
A total of 50 naïve Male Sprague-Dawley rats (Charles River Lab, St. Constrant, QC,
Canada) were single-housed in 48 x 26 x 20 cm shoebox cages in a colony room at an ambient
temperature of 21°C. All animals were maintained on a reverse light/dark cycle (7:00am lights
off; 7:00pm lights on) with free access to food (Iams rodent chow, 18% protein) and tap water,
except during testing which occurred during the dark cycle. All animals were provided with
environmental enrichment from two clean paper towels (replenished weekly during cage
changes) and a soft plastic container 14 cm long and 12 cm in diameter. Animals were
46
euthanized after testing by Euthansol (Intervet Canada Corp., Kirkland, QC, Canada) followed
by transcardial perfusion (see Histology section).
Drug Treatments
The anandamide transport inhibitor, ARN272 (provided by Danieli Piomelli, University of
California Irvine/Istituto Italiano di Tecnologia), was prepared in a vehicle solution (VEH) of
1:1:8 PEG400, Tween, and physiological saline respectively. Centrally administered ARN272
was prepared in the same vehicle solution as when systemically administered, with a
concentration of 0.03 µg/µL, 0.3 µg/µL, and 3.0 µg/µL, and was delivered bilaterally at rate of
0.5 µL/min.
The LiCl drug treatment 0.15M (Sigma) was prepared in sterile water and administered at
the volumes of 20 ml/kg (127 mg/kg), on the basis of its effectiveness in producing conditioned
gaping in rats (Limebeer & Parker, 2000).
Surgical Implantation of the Intracranial Guide Cannula
Rats were implanted with an intracranial guide cannula as described by Limebeer et al. (2012).
Rats were anaesthetized with isoflurane gas, Carprofen was administered (5 mg/kg, i.p.), and a
strip of skin was shaved between the ears 2.5 cm long. The shaved area was cleaned, and a
topical anaesthetic (0.1 mL, s.c.; Marcaine, Hospira, Montreal, QC, Canada) was injected on
either side of the skull. Verification of surgical plane of anaesthesia was demonstrated by a lack
of withdrawal reflex response in the hindlimb, decreased muscle tone, and a slow, regular
respiratory response. Stabilization was achieved using the flat skull position (Paxinos and
Watson, 1998) in the stereotaxic frame and the skull was exposed. The stainless steel guide
cannula (22G, 6 mm below pedestal) was lowered to surgically implant bilateral indwelling
guide cannula in the VIC (arms set at divergent 10° angle, relative to Bregma: AP -0.5; LM ±5.0;
47
DV -4.5 from the skull). The guide cannulae were stabilized by six screws secured in the skull
and with the application of an acrylic dental cement cap. A stainless steel obturator was then
inserted into each guide cannula to maintain patency. Following removal from the stereotaxic
frame, rats were surgically implanted with intraoral cannula (see intraoral cannula surgery
description in Part I). Rats were then placed in a heated recovery area and monitored until they
were ambulatory, at which time they were returned to the colony room. Rats were given a total
of 14 days recovery time; with a second dose of carprofen (0.1 mg/kg) twenty-four hours after
surgery, and their health monitored for the first 3 consecutive days post-surgery during which
time intra-oral cannulae were flushed once a day with chlorohexidine.
Behavioral Measures
All video recordings and live behaviors were scored using the Observer (Noldus Information
Technology, Sterling, VA, USA) event recording programme. The number of gaping reactions
(rapid, large amplitude opening of the mandible with retraction of the corners of the mouth) was
counted during the 2 minute infusions.
Behavioral Apparatus
Intra-cranial micro-infusions were conducted using a 28 G injector that extended 2 mm beyond
the guide cannula tip. The injector was connected to micro-infusion pump (KDS101; KD
Scientific Inc.) using Tygon tubing with a 0.1905 mm inner diameter (Cole-Parmer, Vernon
Hills, IL, USA). The Taste Reactivity apparatus was identical to that used in Part I.
Behavioral Procedures
Of the total 50 rats implanted with intracranial guide cannulae, 18 were removed due to improper
placements leaving 32 in the data analysis (see figure 3 for a representative bi-lateral cannula
placement in the VIC, and see figure 4 for specific bilateral placement of cannula tips). The 32
48
rats had been randomly assigned to one of 4 pretreatment groups; group VEH (n=9), group 0.03
µg/µL ARN272 (n=7), group 0.3 µg/µL ARN272 (n= 8), and group 3.0 µg/µL ARN272 (n=8),
and the pretreatment was delivered bilaterally at rate of 0.5 µL/min with group designation being
denoted by the pretreatment infusion.
The experiment began 2 weeks after surgeries were completed and consisted of the Taste
Reactivity (TR) adaptation, conditioning and a test, the timing and series of which was identical
to Part I. Rats received a pretreatment micro-infusion of ARN272 (VEH, 0.03, 0.3, or 3.0 µg/µL)
1 hour prior to both conditioning trials. All micro-infusions were of 2 minute duration, with the
injector left in place for 1 min post-infusion before removal from the guide cannula. The
obturator was replaced after each micro-infusion. During conditioning trials, as in Part I, rats
were intra-orally infused with a saccharin solution (0.1%), and immediately following such were
injected with 20 ml/kg of LiCl (0.15M), and then returned to their home cage. The second
conditioning trial was performed 72 hours later with an identical procedure to the first, and the
drug-free TR test was performed with an identical procedure as in Part I.
Immediately following the TR test the rats were returned to their home cages, and at
16:00 h their water bottles were removed to begin a water deprivation regime in preparation for
the Conditioned Taste Avoidance tests (CTA test). At 08:00 h the following morning the rats
received a one-bottle test, which is more sensitive in detecting between group differences in taste
avoidance strength than a two-bottle test where both water and saccharin are available (Batsell &
Best, 1993). During the one-bottle CTA test, a graduated tube of 0.1% saccharin solution was
placed on the home cage, and the amount consumed was recorded at 30 and 120 minute
intervals.
49
Histology
Verification of cannula placement was determined by histological evaluation of tissue. The rats
were deeply anaesthetized using an 85 mg/kg injection of Euthansol (Intervet Canada Corp.,
Kirkland, QC, Canada), and then a transcardial perfusion was performed with PBS (0.1M) and
4% formalin. The brains were removed and stored at 4°C in 4% formalin solution for 48 hr, after
which they were placed in a 20% sucrose solution overnight at room temperature. The brains
were then sliced in 50 µm sections using a CM1850 Leica cryostat and relevant sections were
mounted on gelatin-subbed glass microscope slides. The tissue was then stained with thionin 24
hr later and examined for accurate cannula placement using a Leica MZ6 Stereomicroscope with
a Leica DFC420 Digital Camera and Leica Application Suite software. Any rat with inaccurate
cannula placement was excluded from the data analysis.
Data Analysis
The number of gapes exhibited by the rats on the drug-free test trial was entered into a one-way
ANOVA and analyzed with the drug pretreatment as the between subjects factor. For the CTA
measure, the mean cumulative volume of saccharin consumed across drug pretreatment groups
was analyzed using 2 separate one way ANOVA’s at each of the two time points, 30 and 120
minutes. Statistical significance was defined as p<.05.
Results
Gaping Measure. The bilateral intra-cranial administration of ARN272 directly into the visceral
insular cortex produced no differences in conditioned gaping across drug pretreatment groups.
Figure 1 presents the mean number of gapes displayed by the various groups. The one way
ANOVA revealed no significant effect of drug pretreatment, F(3, 31)=.45, ns.
50
CTA Measure. There were no significant differences in the amount of saccharin that was
consumed between all pretreatment groups at both time intervals (30, 120 min), demonstrating
no differences in taste avoidance. Figure 2 presents the mean cumulative amount of saccharin
consumed by the various drug pretreatment groups. The one way ANOVA’s revealed no
significant differences in the mean cumulative amount of saccharin consumed at 30 minutes,
F(3, 31)=1.64, ns, or at 120 minutes, F(3, 31)=2.40, ns.
Discussion
Despite evidence in the literature to implicate the VIC in the regulation of nausea-induced gaping
in rats (Limebeer et al., 2012; Tuerke et al., 2012), the inhibition of anandamide transport in the
VIC did not yield any significant interference with gaping at any of the three drug doses. The
insular cortex has previously been linked to nausea in several ways. One such example is through
increases in Fos-ir activity within this region of the insular cortex induced by the blood-born
emetic agent LiCl (Contreras et al., 2007). There is also evidence to suggest that the subdivisions
of the insular cortex have dissociated roles. Disgust seems to be mediated within the VIC; as
measured by conditioned gaping in rats, and conditioned taste avoidance within the agranular or
gustatory insular cortex (GIC), (Limebeer et al., 2012; Tuerke et al., 2012). Further evidence to
support the role of the VIC in nausea production has been found by reversible lidocaine-lesions
of the VIC which interfered with LiCl-induced unconditioned malaise (Contreras et al., 2007), as
measured by rats lying flat on their stomachs (Parker et al., 1984). As such, the VIC continues to
be a specific region of interest in nausea regulation within the insular cortex.
The current evidence which implicates the VIC in nausea regulation points to the
involvement of the serotonergic system, and its possible regulation by the endocannabinoid
system. Forebrain 5-HT appears to be critical for nausea-induced behaviors. Grill and Norgren
51
(1978) demonstrated that decerebrate rats fail to display conditioned disgust reactions to a LiCl-
paired flavor (although the rats showed disgust to bitter quinine), indicating that the forebrain
was necessary for conditioned disgust. Indeed, forebrain 5-HT appears to be critical for the
establishment of these disgust reactions, because Limebeer, Parker and Fletcher (2004)
demonstrated that 5, 7-dihydroxytryptamine (5,7-DHT) lesions of the dorsal and median raphe
nuclei (which provide forebrain 5-HT) prevented the establishment of LiCl-induced conditioned
disgust in rats, without affecting LiCl-induced taste avoidance. More recently, the forebrain
region of serotonin activation crucial for conditioned disgust has been isolated to the insular
cortex, as 5, 7-DHT lesions of this region interfered with the establishment of conditioned digust.
More specifically, serotonergic activation of the VIC appears to be necessary for conditioned
disgust, as the classic anti-emetic agent and 5-HT3 antagonist, Ondansetron, was found to
attenuate conditioned gaping when delivered to the VIC (Tuerke et al., 2012), but not to the GIC.
The endocannabinoid system has demonstrated efficacy at regulating nausea-induced gaping in
the VIC through CB1 receptors, as evidenced by the attenuated gaping to a CB1 receptor agonist
HU-210 delivered to the VIC (but not the GIC), and it’s reversal by the CB1 receptor antagonist
AM251 (Limebeer et al., 2012). CB1 receptors have been found to be located on the axon
terminals of presynaptic neurons (Meyer & Quenzer, 2005), and as such it is hypothesized that
they inhibit the release of 5-HT (Schlicker & Kathmann, 2001), reducing levels of forebrain 5-
HT to prevent the sensation of nausea.
One explanation of why anandamide transport inhibition did not impact conditioned
gaping in the VIC could be that nausea is not anandamide regulated within this area, but may be
2-AG regulated. While the specificity of endocannabinoid signaling is still emerging, there is
already evidence to suggest that endocannabinoids differentially modulate or even co-regulate
52
various physiological processes (Luchicchi & Pistis, 2012). So far, all the evidence supporting
the endocannabinoid systems involvement in nausea regulation in the VIC implicates no specific
endogenous ligand. Both of the two major endocannabinoids, anandamide and 2-AG, have been
found to bind to CB1 receptors (Devane et al., 1992; Mechoulam et al., 1995), though with
different affinities (Pertwee & Ross, 2002; Reggio, 2002). As such there are candidates other
than anandamide capable of driving CB1 receptor nausea attenuating effects. In order for the
indirect agonism produced by prolonging anandamide availability within the synapse to elicit a
behavioral outcome, the endogenous synthesis and release of anandamide within the targeted
brain area must first be established. A future direction to determine if anandamide is present or
elevated in the VIC after ARN272 administration, would be to extract the VIC and analyze the
area for endocannabinoid levels post behavioral testing. Also, co-administration of exogenous
anandamide with ARN272 delivered into the VIC may confirm that ARN272 is capable of
prolonging anandamide availability when centrally administered.
Further recent work has found that systemic administration of the dual FAAH/MAGL
(monoacylglycerol lipase) inhibitor JZL195 attenuates the expression of anticipatory nausea, in
that it suppressed conditioned gaping to a context while also elevating anandamide levels as
determined from whole brain analysis (Limebeer et al., submitted). Although JZL195 is a dual
inhibitor, the authors found it to be acting primarily as a FAAH inhibitor, prolonging
anandamide action by preventing its degradation. Despite the current lack of evidence to suggest
that anandamide is acting to regulate nausea specifically within the VIC, elevated whole brain
anandamide does suggest the utilization of a central mechanism by anandamide.
The VIC and GIC have been found to be functionally distinct with respect to conditioned
taste avoidance, in that lesions to the GIC disrupt CTA (Braun et al., 1972; Roman and Reilly,
53
2007), whereas damage to the VIC have been ineffective in attenuating CTA (Mackey et al.,
1986; Nerad et al., 1996). The finding that anandamide transport inhibition in the VIC had no
impact on conditioned taste avoidance (CTA) supports existing literature in which CB1 receptor
agonists (Limebeer et al., 2012) and 5-HT receptor antagonists (Tuerke et al., 2012) delivered to
the VIC were both ineffective in disrupting CTA.
The role of the insular cortex seems to be rooted in the conscious perception of
physiological homeostasis (Critchley et al., 2004). Indeed, the human disgust literature suggests
that it is this cortical region (albeit anterior IC in humans) that is activated by disgusting scenes,
smells and tastes (Calder et al., 2001; Calder et al., 2007). While evidence of nausea regulation
by the endocannabinoid system points to the sub-region of the VIC, further investigation is
required to reveal the functional roles played by anandamide and other endocannabinoids in this
area.
54
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58
Figure 1. Mean gapes at test day across drug groups, VEH and ARN272 at 0.03, 0.3, and 3.0
µg/kg. Intra-cranial administration of ARN272 delivered directly to the visceral insular cortex
(VIC) produced no significant differences in nausea-induced conditioned gaping in rats. The
number of rats that gaped in each group is indicated above each bar.
59
Figure 2. Mean (± SEM) cumulative volume of saccharin consumed by rats 24 hr after TR test
day and immediately following water restriction, at 30 and 120 minutes. There were no
significant differences in saccharin consumption between any of the pretreatment groups at either
time interval.
60
Figure 3. Representative photomicrograph of a coronal brain section showing a VIC bi-lateral
cannula track and microinjection site.
61
Figure 4. Schematic illustrations of approximate infusion sites (circles) in the VIC in rats (N=32)
on drawings of coronal sections. The numbers indicate A-P coordinates relative to bregma. The
right hemisphere is represented in the left side of each image and the left hemisphere in the right
side. Atlas plates are adapted from Paxinos and Watson, (2007).
62
General Discussion
Anandamide: Neuromodulator of Nausea and Vomiting
The experiments reported in Part I add to the body of knowledge already implicating the
endocannabinoid anandamide in the regulation of nausea and vomiting.
Cannabinoid agonists have long been known to interfere with nausea-induced
conditioned gaping in rats, including ∆9-THC and HU-210 (Parker et al., 2003). The present
finding in Part I, that ARN272 suppressed nausea-induced conditioned gaping in rats, supports
the existing literature which points to anandamide as an important endocannabinoid in the
regulation of nausea and vomiting (Cross-Mellor et al., 2007; Sharkey et al., 2007; Van Sickle et
al., 2005). Cross-Mellor et al. (2007) have however found exogenously administered anandamide
to be ineffective at interfering with conditioned gaping. This contrary piece of evidence is
possibly more representative of how rapidly anandamide is eliminated by hydrolysis without
pharmacological interference, given it’s half-life of less than 5 minutes (Willoughby et al.,
1997). The currently presented reversal of the ARN272-suppressed gaping by a CB1 receptor
antagonist in Part I is also consistent with existing evidence which implicates the CB1 receptor as
a ligand binding site required to regulate nausea and vomiting (Cross-Mellor et al., 2007;
Sharkey et al., 2007; Van Sickle et al., 2005).
Cannabinoid agonists have also been found to be effective in interfering with LiCl-
induced vomiting in shrews, including ∆9-THC in the house musk shrew (Parker et al., 2004),
and ∆9-THC and the synthetic analogs CP 55, 940 and WIN-55 in the least shrew (Darmani,
2001). The present finding from Part I that ARN272 attenuated LiCl-induced vomiting in the
house musk shrew is supported by evidence where inhibition of anandamide degradation through
63
the FAAH inhibitor URB-597 suppressed vomiting in the ferret (Sharkey et al., 2007) and
cisplatin- and nicotine-induced vomiting in the house musk shrew (Parker et al., 2009).
The exogenous administration of anandamide alone has, however, been found to exhibit
only a weak anti-emetic effect in the ferret (Van Sickle et al., 2001), and no evident effect in the
house musk shrew (Parker et al., 2009). The lower or absent antiemetic efficacy of anandamide
in ferrets and shrews could again be linked to the temporal restrictions from anandamide
hydrolysis previously mentioned (Willoughby et al., 1997). Also, Darmani et al. (2005)
previously reported the FAAH inhibitor URB-597 as being ineffective in suppressing cisplatin-
induced vomiting in the least shrew, and also reported it as being emetogenic when administered
alone. However, the URB597 was given at an unusually high dose (10 mg/kg) and only 10 min
prior to the emetogen, a pretreatment interval of 1-2 hr is optimal at a dose of 0.3 mg/kg (Fegley
et al., 2005). Unfortunately no control VEH group was used to compare the URB-597 induced
vomiting to in the 2005 study, while in a separate study, Darmani et al. (2001) reported several
VEH animals as displaying a vomiting response. The use specifically of least shrew may present
challenges to data reliability and interpretation due solely to their small size. While being an
emetic species, the least shrew are typically 4-6g, and thus the stress of systemically
administering any drug to an animal of such size may have emetic-inducing potential. Contrary
to the findings in the least shrew, Parker et al. (2009) have since demonstrated non-selective
attenuation of vomiting in the house musk shrew by URB-597 to both the emetic agents:
cisplatin and nicotine. As such the inability of URB-597 to attenuate vomiting in the least shrew
should be considered with caution.
Overall, the attenuation of nausea and vomiting demonstrated by ARN272 in both rats
and shrews serves to provide a cross species qualitative comparison of prolonged anandamide
64
availability efficacy. Given the findings in Part I, the lack of any interference with gaping when
ARN272 was delivered directly to the VIC in Part II is likely more representative of the
differential regulation of nausea by endocannabinoids in given brain areas.
Anandamide Deactivation and Movement
The experiments reported in Part I also add further illumination to the debate around the
mechanisms by which anandamide action is deactivated. Specifically, the studies provide
behavioral support for anandamide cellular reuptake occurring via a facilitated transport
mechanism.
It has been previously hypothesized that the intracellular hydrolysis of anandamide by
FAAH provides a concentration gradient between interstitual and intracellular environments,
driving transport from the former to the latter (Day et al., 2001). Hillard et al. (2007) reported
evidence to suggest this may not the sole anandamide transport mechanism when they conducted
a comparison of the potency of various compounds to inhibit intracellular accumulation of
anandamide, versus anandamide hydrolysis. They found no correlation in IC50 values (the
concentration of a drug required to inhibit a biological process by half) between the inhibition of
intracellular accumulation of anandamide and anandamide hydrolysis. This finding argues
against FAAH being the primary mechanism by which anandamide is internalized into the post
synaptic cell.
Evidence identifying the location of FAAH itself has also proved revealing regarding its
role in anandamide transport (see Figure B). FAAH is found predominantly within the post
synaptic somatodendritic compartment, the majority of which is found on the cytosolic surface of
smooth endoplasmic reticulum cisternae and embedded in mitochondrial outer membranes
(Gulyas et al., 2004). If FAAH is the primary mechanism of anandamide reuptake into the post
65
synaptic cell, so by the same logic the use of a FAAH inhibitor would collapse the concentration
gradient and prevent the reuptake of anandamide. Yet when AM404, primarily a FAAH
inhibitor, was injected inside striatal neurons, retrograde neuromodulator actions mediated by
presynaptic CB1 receptors were blocked (Gerdeman et al., 2002; Ronesi et al., 2004), ruling out
the exclusive role of FAAH in anandamide transport.
The characterization of an anandamide transporter, FLAT, by Fu et al. (2012) was not
only an important finding itself, but it also enabled the drug discovery of a selective inhibitor
which will no doubt prove a useful tool in the dissection of the mechanism of anandamide
movement in isolation from its degradation. The present finding from Part I, that ARN272
suppressed LiCl-induced conditioned gaping in rats and reduced LiCl-induced vomiting in
shrews, adds behavioral support for anandamide transport as a facilitated process. It is thought
that the inhibition of FLAT produced prolonged availability of anandamide within the synaptic
cleft, to tonically activate CB1 receptors and augment the endogenous regulation of nausea and
vomiting to a LiCl challenge. As such it is hypothesized that FLAT promotes the movement of
anandamide away from its ideal lipophilic membrane bound environment toward commencing
travel in an aqueous environment (see Figure B).
The idea that FLAT instigates lipid travel within aqueous environments (Marsicano &
Chaouloff, 2012) highlights another still unknown area: how anandamide travels through the
cytosol to the endoplasmic reticulum to be degraded, or how it is delivered through aqueous
interstitial fluid to its pre-synaptic CB1 receptor target. Systemic transport of anandamide in the
bloodstream is made possible by its reversible binding to serum albumin (Bojensen & Hansen,
2003), a plasma protein in mammals. A similar chaperone protein transport mechanism may
occur both intra- and extracellularly in the CNS. One suggested candidate for extracellular
66
transport of anandamide are lipocalins (Piomelli, 2003), a family of proteins which transport
hydrophobic molecules, and which are expressed in high levels in the brain (Beuckmann et al.,
2000).
Measuring Nausea
All animals need to be able to discriminate between safe and toxic foods as a matter of survival.
If a novel taste is followed by malaise, so it follows that a learned association will produce an
aversion to, and or an avoidance of, the taste upon re-exposure. As such, biologically relevant
stimuli are more likely to become associated (Revusky & Garcia, 1970). For a considerable time,
researchers have been trying to discern reliable and accurate animal measures of the subjective
experience of nausea. It is within these animal models that the distinction between aversion and
avoidance becomes an important one.
Avoidance is typically measured using Conditioned Taste Avoidance (CTA), where the
extent to which a flavor previously paired with a nausea inducing agent is subsequently avoided
in a consumption test. Aversion is measured using conditioned gaping, where rats are intra-orally
infused with a flavor previously paired with a nausea inducing agent and disgust reactions in the
form of a gaping response are counted. After over half a century of empirical research in
avoidance behaviors, it is now thought that these models differ in two ways: their sensitivity and
their selectivity.
More importantly in the realm of making accurate inferences from behavioral data, there
is evidence to suggest that conditioned gaping is a more selective measure of nausea than CTA.
Conditioned gaping has been found only to be produced by emetic drugs (see Parker et al.,
2009b), and appears only to be attenuated by anti-emetic drugs (Limebeer & Parker, 2000, 2003;
Parker et al., 2008, 2009a). CTA does not follow such a selective pattern. CTA has been found to
67
be induced not only by emetic drugs, but also by rewarding drugs that animals choose to self
administer (Parker, 1995; Reicher & Holman, 1977; White et al., 1977; Wise et al., 1976). CTA
also seems to manifest differentially in species capable versus incapable of vomiting. Shrews
have been found to develop a conditioned taste preference to a taste paired with rewarding drugs
(Parker et al., 2002), opposite to the previously mentioned pattern found in rats. It could be
therefore that CTA is sensitive to any change in drug induced internal state.
The decreased selectivity demonstrated by CTA can also be seen when evaluating the
effect of anti-emetic drugs in the paradigm. There is considerable evidence which points to anti-
emetic drugs not interfering with CTA (Limebeer & Parker, 2000, 2003; Rabin & Hunt, 1983),
or only interfering with very weak LiCl-induced taste avoidance (Balleine et al., 1995; Gorzalka
et al., 2003). However, the same anti-emetic treatments have been found to interfere with
conditioned gaping (for review, see Parker et al., 2009b). As such it is reasoned that anti-emetic
treatments that reduce the impact of nausea prevent gaping, however they are not interfering with
learning as the flavor still signals some kind of change in internal state so that CTA remains
evident.
The present findings in Part I and Part II that ARN272 had no impact on LiCl-induced
CTA support the findings that anti-emetics interfere with conditioned gaping (for review, see
Parker et al., 2009b) but not CTA (Limebeer & Parker, 2000, 2003; Rabin & Hunt, 1983). The
intact CTA responding also serves as evidence that ARN272 is interfering with the subjective
experience of nausea to attenuate LiCl-induced conditioned gaping in Part I, and not learning per
se. A full review of CTA would be no small undertaking, but the points drawn from the literature
serve to indicate that the avoidance of a taste could occur from reasons other than nausea alone,
whereas aversive reactions in the form of gaping to a taste seem to arise solely from the
68
experience of nausea. As such conditioned gaping could be considered a reliable and selective
measure of nausea in rats (Parker, 1998), and CTA a measure of associative learning between
taste and internal state (Parker, 2013 in publication).
Further support for CTA and conditioned gaping as measuring different qualities comes
from their seemingly different neurobiological underpinnings. Rana & Parker (2008) found that
lesions of the BLA attenuated CTA, but not conditioned gaping, whereas ablation of the insular
cortex eliminated conditioned gaping, but not CTA (Kieffer & Orr, 1992). Taste cells in the oral
cavity have been found to follow a taste-pathway to the rostral part of the nucleus of the solitary
tract (Harrer & Travers, 1996), on to the posteromedial parabrachial nucleus, where they then
project to the basolateral amygdala (BLA), as well as the ventral posteromedial nucleus of the
thalamus (VPM), (Norgren, 1995). The VPM has then been found to project directly to the
gustatory insular cortex (GIC), (Pritchard et al., 2000). Both the GIC and the BLA have been
found to be interrelated in the acquisition and retention of CTA (Burešová, 1978; Yamamoto et
al., 1981). Conversely, pathways (as previously described in Part II) involved in the experience
of nausea originate in visceral organs, which then project and terminate in the visceral insular
cortex (VIC). Indeed the double dissociation found by Tuerke et al. (2012) where intra-cranial
administration of the 5-HT3 receptor antagonist Ondansetron to the VIC impaired LiCl-induced
conditioned gaping but not CTA, and administration to the GIC impaired LiCl-induced CTA but
not conditioned gaping; supports the different functionalities of these areas of the insular cortex
with respect to aversion and avoidance.
Anandamide in the CNS
Where in the nervous system anandamide is acting to regulate nausea remains to be ascertained.
In a broader view, there is evidence to suggest that both central and peripheral CB1 receptors
69
maybe involved in the control of nausea and vomiting (Van Sickle et al., 2001; Darmani &
Johnson, 2004). The finding that the peripherally restricted CB1 receptor agonist CB13 interfered
with LiCl-induced conditioned gaping when centrally but not systemically administered
(Limebeer et al., 2012), points to the exploration of mechanisms of endogenous CB1 receptor
agonists in the central nervous system.
The target area of the present study, the insular cortex, is an area already well known to
be connected to nauseous sensation (Penfield & Faulk, 1955), and yet there is little known about
the functional differences of its sub-regions. Despite CB1 receptors being implicated in the
regulation of nausea in the VIC (Limebeer et al., 2012), Part II of the present study found no
evidence to suggest a role for anandamide in the endogenous suppression of conditioned gaping
in this area. There are however, a variety of methodological factors to consider which could
impact the effect, or lack thereof, on conditioned gaping when administering inter-cranial micro-
infusions of ARN272. Manipulations of the drug pretreatment time interval may worth exploring
in future studies, along with adding additional drug dose groups. A further consideration is to
confirm the successful delivery of the drug into the VIC. Quantifying anandamide levels within
the VIC immediately following the conditioning procedure may speak to the drugs appropriate
delivery in the area, or to the presence of endogenous anandamide production within the VIC.
Further studies exploring the nature of other endocannabinoids in the VIC would be of
benefit to ascertain the role of this area with regards to nausea and vomiting. There are certainly
many other areas to be explored in the continued search for the seat, or combined pathways,
through which anandamide acts to regulate nausea and vomiting. One further area which could
be considered of interest in exploring anandamide nausea regulation is the area postrema (AP).
CB1 receptors are extensively found within the AP (Van Sickle et al., 2003), and AP lesions have
70
been found to interfere with LiCl-induced malaise (Bernstein et al., 1992) as measured by rats
lying flat on their stomachs (Parker et al., 1984), as well as LiCl-induced conditioned gaping
(Ossenkopp & Eckel, 1995).
Concluding Remarks
The findings presented add to the body of evidence which points to anandamide as a
neuromodulator of nausea and vomiting, and provides behavioral evidence for the cellular
reuptake of anandamide as occurring through a facilitated transport mechanism. While the
evidence presented potentially relieves FAAH from the sole burden of anandamide signal
termination, it does not rule out the enzyme as a potential contributor. It is still clear that more
aspects of the anandamide transport system remain to be revealed, including how anandamide
travels in aqueous environments. The importance of pharmacological tools such as ARN272 in
gleaning further understanding of mammalian physiology and behavior cannot be understated.
71
Figure B
Anandamide translocation and action locations
This figure represents the proposed participants in the translocation of anandamide and its sites
of action according to the present experiments (adapted from Figure 2 in Ahn et al., 2008;
structural model of FLAT from Fu et al., 2012).
72
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