The orexins and appetitive motivation in the rat
Shaun Khoo
A thesis in fulfillment of the requirements for the degree of
Doctor of Philosophy
School of Psychology
Faculty of Science
July 2017
PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet
Surname or Family name: Khoo
First name: Shaun Other name/s: Yon-Seng
Abbreviation for degree as given in the University calendar: PhD
School: Psychology Faculty: Science
Title: The orexins and appetitive motivation in the rat
Abstract 350 words maximum: (PLEASE TYPE)
Addiction is a chronic relapsing disorder characterised by compulsive drug seeking that persists despite adverse consequences. One popular, and widely heralded therapeutic target, is the orexin system, a hypothalamic neuropeptide system involved in arousal, appetite and reward. The motivational activator theory, the first coherent account of orexin’s role in appetitive motivation, predicts that orexin contributes to appetitive behaviour, including drug seeking, when the reinforcer is highly salient, available under a high unit-cost, or when reward seeking cue- driven. The present study used the dual orexin receptor antagonist, TCS 1102, a commercially available compound related to the clinically approved insomnia drug, suvorexant. TCS 1102 was administered intracerebroventricularly to rats trained to self-administer food, 4% alcohol beer or intravenous nicotine in a variety of self-administration and relapse-like paradigms. While TCS 1102 was able to reduce orexin-A-induced increases in consumption of normal grain pellets, it did not affect FR1, FR5, or FR10 self-administration of palatable food or cue+prime reinstatement of palatable food seeking. Orexin neuron activation measured by c-Fos/orexin-A immunohistochemistry showed that while orexin neurons were robustly recruited under behavioural testing conditions, there was no specific activation of these neurons during cue-induced reinstatement. For alcoholic beer self-administration, TCS 1102 did not affect FR1 self-administration or reacquisition of alcoholic beer. For nicotine self adminstration, FR1 self-administration, cue- induced and nicotine-primed reinstatement was not affected by TCS 1102. However, there was a small, transient effect on cue+prime nicotine reinstatement after a chronic, but not brief, period of self-administration. These results show that TCS 1102 is not effective in reducing appetitive motivation for palatable food, alcoholic beer or nicotine. Moreover, orexin neurons may be recruited by operant or appetitive contexts, but may not be necessary for appetitive motivation. This is the first time that the effects of a dual orexin receptor antagonist have been examined over a range of conditions of reinforcement in accordance with specific predictions of the motivational activator theory. Furthermore, these results suggest that orexin receptor antagonism is unlikely to be an effective addiction pharmacotherapy.
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Date ……………………………………………...... Table of Contents
Acknowledgements ...... ii
Abbreviations ...... iii
List of Figures ...... vii
List of Tables ...... ix
Chapter 1. Introduction ...... 1
1. The Orexin/Hypocretin System ...... 3
2. The Orexins and Animal Models of Addiction ...... 21
3. Selecting Orexin Antagonists for Behavioural Studies ...... 42
4. Aims and Hypotheses ...... 58
Chapter 2. Methods and Results ...... 61
Experiment 1. Orexin-A-induced Feeding ...... 61
Experiment 2. TCS 1102 and Orexin-A-induced Feeding ...... 68
Experiment 3. Palatable Food Self-Administration and Reinstatement...... 73
Experiment 4. Varied Unit Costs for Palatable Food ...... 80
Experiment 5. Neuronal Activation During Reinstatement ...... 89
Experiment 6. Beer Self-Administration and Reacquisition ...... 98
Experiment 7. Nicotine Self-Administration and Reinstatement ...... 107
Experiment 8. Cue/Prime Compound Reinstatement and FR5 ...... 120
Chapter 3. General Discussion ...... 126
References ...... 154
i
Acknowledgements
I would like to acknowledge my supervisor, Prof. Gavan McNally, who believed in my project even when I didn’t. My unofficial supervisor, Dr Kelly Clemens, welcomed me into her lab to run the nicotine and some food experiments and always tried to help me be a better scientist. I am grateful to my co-supervisor and Head of School, Prof. Simon
Killcross, who was very supportive of the PhD student community in the School. I would like to thank my colleagues and friends in the McNally Lab for being there all the time: Dr Asheeta Prasad, Dr Joanna Yau (especially for help with microscopy and photoshop), Dr Auntora Sengupta, Dr Philip Jean-Richard Dit Bressel, Eun A (Lucy)
Choi (especially for help with immunohistochemistry), and Gabrielle Gibson. In the
Clemens/Westbrook-Holmes Lab, I would like to thank: Dr Matthew Castino
(especially for the catheters), Dr Dominic Tran, Dr Justine Fam, Dr Nathan Holmes, and
Prof. Fred Westbrook. Special thanks are due to Anne Rowan and Lydia Williams, who looked after the colony rooms and were always available with advice on how to keep happier rats, and to Jonathan Solomon, Marty Sebastian, John Bolzan, Rahatir Rashid and Shanta Jayawardana of the Technical Support Unit who provided us with technical support. I would like to acknowledge my supervisors and colleagues in the School of
Psychology with whom I worked in other roles including, A/Prof. Branka Spehar,
Jessica Kirkman, and Jenna Zhao (Peer Mentoring Program) and Dr Jenny Richmond,
Dr Marios Panayi, and Helena Pacitti (Tutoring). I am thankful to my past supervisors,
Prof. Andrew Lawrence and Dr Robyn Brown with whom I continued to publish during my PhD. Personally, I’d like to thank Belinda Lay for showing me what a ‘mung fau num jim’ is and my mum for being relatively restrained in asking, “When are you going to graduate/get a job?” Finally, I’d like to thank the rats, who can’t read, but have shown me time and time again which species is more intelligent. ii
Abbreviations
2HPβCD, 2-hydroxypropyl)-β-cyclodextrin
Acb, nucleus accumbens
AcbC, nucleus accumbens core
AcbSh, nucleus accumbens shell
ACEC, Animal Care and Ethics Committee aCSF, artificial cerebrospinal fluid
AKAP, A-kinase anchor protein
Amy, Amygdala
ATP, adenosine triphosphate
BLA, basolateral amygdala
BNST, bed nucleus of the stria terminalis
Ca2+, calcium
CART, cocaine and amphetamine regulated transcript
CeA, central amygdala
CIR, cue-induced reinstatement
CPA, conditioned place aversion
CPP, conditioned place preference
CRF, corticotropin releasing factor
CSF, cerebrospinal fluid iii
Abbreviations
DAG, diacylglycerol
DGL, diacylglycerol lipase
DMH, dorsomedial hypothalamus
DMSO, dimethyl sulfoxide
ERK, extracellular signal-regulated kinase
FRn, Fixed ratio of n responses
HCRT, hypocretin gene (human)
Hcrt, hypocretin gene (rat/mouse)
GABA, γ-amino butyric acid
GABAA, γ-amino butyric acid type A (receptor)
GIRK, G-protein coupled inwardly rectifying potassium channel
GPCR, G-protein coupled receptor
IP3, inositol triphosphate
LC, locus coeruleus
LH, lateral hypothalamus
LV, lateral ventricle
MCH, melanin concentrating hormone mTOR, mammalian target of rapamycin
MW, molecular weight
NCX, sodium-calcium exchanger iv
Abbreviations
NI, nucleus incertus
OX, orexin
OX-A, orexin-A
OX-B, orexin-B
OX1, orexin-1 (receptor)
OX2, orexin-2 (receptor)
PA, phosphatidic acid
PeF, perifornical hypothalamus
PD, Parkinson’s disease
PFC, prefrontal cortex
PIP2, phosphatidylinositol bisphosphate
PIP5K, phosphatidylinositol phosphate 5-kinase
PKA, protein kinase A
PKC, protein kinase C
PLA2, phospholipase A2
PLC, phospholipase C
PLD, phospholipase D
PR, progressive ratio
PV, paraventricular thalamus
SEM, standard error of the mean v
Abbreviations
TPSA, topological polar surface area
TRPC3/6, transient receptor potential canonical cation channel subfamily C members 3 and 6
VIn, variable interval with an average of n seconds
Vitamin E-TPGS, D-α-tocopherol polyethylene glycol 1000 succinate
VP, ventral pallidum
VTA, ventral tegmental area
XLogP, octanol/water partition coefficient
vi
List of Figures
Figure 1. Orexin neurotransmission and signaling*
Figure 2. Microinjection studies and opioids†
Figure 3. Microinjection studies and alcohol seeking†
Figure 4. Microinjection studies and cocaine seeking†
Figure 5. Microinjection studies and natural reinforcers†
Figure 6. The effect of orexin-A on operant free-feeding
Figure 7. Effect of the dual orexin receptor antagonist, TCS 1102, on orexin-A-induced feeding
Figure 8. Effect of TCS 1102 on palatable food self-administration in sated and hungry rats
Figure 9. Effect of ICV TCS 1102 on cue and primed reinstatement for palatable food
Figure 10. TCS 1102 does not affect palatable food self-administration at multiple unit costs
Figure 11. TCS 1102 had no effect on progressive ratio responding
Figure 12. Comparison between single orexin receptor antagonists on FR10 self- administration
Figure 13. Effect of cue-induced reinstatement for palatable food on orexin neuron activation
Figure 14. Representative photomicrographs of orexin-A and c-Fos immunoreactivity
Figure 15. The effect of TCS 1102 on 4% alcohol beer self-administration vii
List of Figures
Figure 16. Effect of ICV TCS 1102 on reacquisition of beer
Figure 17. Effect of ICV TCS 1102 on nicotine self-administration
Figure 18. Effect of ICV TCS 1102 on nicotine self-administration, using a Vitamin E-
TPGS vehicle
Figure 19. Effect of ICV TCS 1102 on reinstatement of nicotine seeking
Figure 20. Effect of ICV TCS 1102 on cue/prime compound reinstatement of nicotine seeking
Figure 21. Effect of ICV TCS 1102 on FR5 self-administration
* Figure 1 is adapted from Khoo (2014) under a Creative Commons Attribution 4.0
International license (CC BY 4.0).
† Figures 2 – 5 contain material which is adapted from Khoo, Gibson, Prasad and
McNally (2017) which is reproduced with permission from John Wiley and Sons
(License Number 4038961122335, 30 January 2017).
viii
List of Tables
Table 1. Summary of orexin antagonists used in studies of appetitive motivation
Table 2. Physicochemical properties associated with drug-like compounds
Table 3. Synthetic orexin antagonist affinities and potencies
ix
Chapter 1. Introduction
Addiction is a chronic relapsing disorder characterised by compulsive drug seeking despite adverse consequences and may involve tolerance, withdrawal and cravings (American Psychiatric Association, 2013; World Health Organization, 1992).
Drugs, alcohol, and tobacco are responsible for over 9 million deaths worldwide each year (Lim et al., 2012). This health burden is also increasing. Between 1990 and 2010, the rankings of drugs, alcohol, and tobacco as risk factors for death and disability all increased (Lim et al., 2012). The economic costs associated with drugs, alcohol, and tobacco are usually estimated to be around 2 – 3% of gross domestic product (Rehm et al., 2009; Single, Robson, Xie, & Rehm, 1998). The cost of tobacco use is particularly high, as it is the leading cause of preventable disease in North America (Danaei et al.,
2009; Hughes, 2016; Lim et al., 2012). Economists estimate that for every dollar spent on tobacco control, 15 dollars are saved in costs such as medical expenses and lost productivity (Chattopadhyay & Pieper, 2012).
Relapse rates in addiction remain high even with current best-practice therapies
(Cahill, Lindson-Hawley, Thomas, Fanshawe, & Lancaster, 2016; Miller, Walters, &
Bennett, 2001). While there are constantly new therapeutics under development for addiction (Kim & Lawrence, 2014; Xi et al., 2016), there are currently no approved pharmacotherapies available for cocaine, methamphetamine or cannabis (Pierce,
O’Brien, Kenny, & Vanderschuren, 2012) and where there are pharmacotherapies they have important limitations. For example, alcoholism is treated using acamprosate, which may be ineffective apart from its pairing with calcium, as shown by the inability of the sodium salt of acamprosate to exert effects in animal models and its effects in human patients correlate with their plasma calcium levels (Spanagel et al., 2014).
Disulfiram is very effective in reducing alcohol intake but suffers from poor compliance 1
Chapter 1. Introduction
(Krampe & Ehrenreich, 2010; Neto, Lambaz, & Tavares, 2007) and naloxone and naltrexone offer only moderate efficacy (Ray, Chin, & Miotto, 2010). The dominant first-line therapy for tobacco, nicotine replacement therapy, has failure rates of up to
90% (Benowitz & Peng, 2000). The nicotinic receptor partial agonist, varenicline, is the most effective pharmacotherapy for tobacco smoking and doubles the chances of quitting compared to placebo (Cahill et al., 2016), but this is still relatively modest given the low rate of success of smokers to begin with. In fact, tobacco and alcohol use have the lowest rates of spontaneous remission (Heyman, 2013; Lopez-Quintero et al.,
2011) and so an improved understanding of the neurobiological factors behind these addictions is particularly important to develop improved therapies.
This thesis is concerned with the role of the orexin/hypocretin system in appetitive motivation, including food, alcohol, and nicotine seeking because of its potential as a therapeutic target.
Hence, this introduction reviews key features of this neuropeptide system. First, an overview of the orexin/hypocretin system is provided, including its possible roles in various pathophysiologies. Next, the role of the orexin/hypocretin system in appetitive motivation and drug addiction is considered, including the results from behavioural pharmacological studies in animals and a discussion of the motivational activator theory, the first coherent theoretical account of the orexin/hypocretin system’s function.
Finally, the pharmacological tools that have been used to study the orexin/hypocretin system are reviewed in terms of their selectivity profiles, their physicochemical properties, and their suitability for use in microinjection studies.
2
Chapter 1. Introduction
1. The Orexin/Hypocretin System
One possible therapeutic target for addiction is the orexin/hypocretin system.
The orexins are a neuropeptide system involved in arousal, appetite, and reward
(Sakurai, 2007). Orexin neurons originate exclusively from the lateral hypothalamus
(LH), perifornical hypothalamus (PeF) and dorsomedial hypothalamus (DMH; Baldo,
Daniel, Berridge, & Kelley, 2003; Elias et al., 1998; Nambu et al., 1999; Peyron et al.,
1998). Orexin fibres project widely throughout the neuraxis, including to key mesocorticolimbic reward regions, such as the ventral tegmental area (VTA), nucleus accumbens (Acb) and prefrontal cortex (PFC; Baldo et al., 2003; Peyron et al., 1998).
The orexin/hypocretin system was discovered simultaneously by two research groups and while the orexin nomenclature produces marginally more results in PubMed, the hypocretin and orexin terms are now used to refer to the genes and their protein products respectively (de Lecea et al., 1998; Gotter, Webber, Coleman, Renger, &
Winrow, 2012; Sakurai et al., 1998).
Orexin signalling. The orexins are two neuropeptides, orexin-A (OX-A) and orexin-B (OX-B), which are 33 and 28 amino acid peptides derived from a single precursor, prepro-orexin (de Lecea et al., 1998; Sakurai et al., 1998). As shown in
Figure 1, the orexin precursor peptide is encoded by the hypocretin gene (Human:
HCRT, Rat/Mouse: Hcrt) which encodes for both OX-A and OX-B peptides to be cleaved from separate sites of the prepro-orexin peptide (Sakurai et al., 1999).
The orexin neuropeptides bind to two G-protein coupled receptors (GPCRs), the orexin-1 (OX1) and orexin-2 (OX2) receptors. Both OX1 and OX2 receptors are excitatory (de Lecea et al., 1998; Sakurai et al., 1998) and promiscuous (Khoo &
Brown, 2014; Kukkonen, 2014; Kukkonen & Leonard, 2014). The signal transduction
3
Chapter 1. Introduction
mechanism was shown early on to be heavily dependent on calcium (Ca2+) from both extracellular (Ammoun et al., 2003; Holmqvist et al., 2005; Zhu et al., 2003) and intracellular stores (Magga et al., 2006; Smart et al., 1999), suggesting coupling to Gq second messenger pathways. Extracellular Ca2+ crosses the membrane via diacylglycerol (DAG)-dependent TRPC3/6 channels and the associated sodium-calcium exchanger (NCX; Louhivuori et al., 2010; Näsman et al., 2006; Peltonen et al., 2009) in order to mobilise inositol phosphate (Johansson, Ekholm, & Kukkonen, 2007; Lund et al., 2000) and activate phospholipase C (PLC). The orexin signalling cascade also extends to paracrine endocannabinoid signalling via the PLA2 and PLC-DGL
(diacylglyceral) pathways (Turunen, Ekholm, Somerharju, & Kukkonen, 2010;
2+ Turunen, Jäntti, & Kukkonen, 2012). In addition to Gq and Ca -mediated signalling,
OX1 receptors associate with Gs and Gi signalling pathways (Magga et al., 2006), which makes them sensitive to pertussis toxin (Holmqvist et al., 2005).
Most of the above studies were completed on the OX1 receptor, but the OX2 receptor has been shown to exhibit similar signalling properties. It signals through Gs and Gi pathways (Karteris, Randeva, Grammatopoulos, Jaffe, & Hillhouse, 2001). This appears paradoxical, because of the opposing actions of Gs and Gi pathways, but the
OX-A-mediated inhibition of Forskolin-induced cAMP production is sensitive to pertussis toxin (Urbańska et al., 2012; Zhu et al., 2003), suggesting that Gi signalling is indeed involved. Gi signalling in both receptors also produces an increase in G-protein coupled inwardly rectifying potassium channel (GIRK) signalling following low concentrations of OX-A, but at higher concentrations OX-A inhibits GIRKs (Hoang,
Bajic, Yanagisawa, Nakajima, & Nakajima, 2003). Both receptor subtypes are thought to signal through Gq, as discussed in the previous paragraph and evidenced by a role of extracellular signal-related kinase (ERK) signalling in OX2 receptor signal transduction 4
Chapter 1. Introduction
ERK (Tang et al., 2008). Receptor recycling in both cases occurs via β-arrestins, but the
OX2 receptor is recycled back to the membrane more slowly than the OX1 receptor
(Dalrymple, Jaeger, Eidne, & Pfleger, 2011; Jaeger, Seeber, Eidne, & Pfleger, 2014).
The OX1 and OX2 receptors are differentially distributed throughout the brain which suggests functional heterogeneity. The OX1 receptor is the predominant orexin receptor in the prefrontal cortex, ventral pallidum (VP) and locus coeruleus (LC), while the OX2 receptor is the most common receptor in the nucleus accumbens shell (AcbSh) and LH (Ch’ng & Lawrence, 2015; Cluderay, Harrison, & Hervieu, 2002; Hervieu,
Cluderay, Harrison, Roberts, & Leslie, 2001; Marcus et al., 2001; Trivedi, Yu, MacNeil,
Van der Ploeg, & Guan, 1998). There is also behavioural evidence that the two receptors have some differing roles, with the OX1, but not OX2, receptor involved in cue-induced cocaine and ethanol reinstatement (Brown, Khoo, & Lawrence, 2013;
Lawrence, Cowen, Yang, Chen, & Oldfield, 2006; Smith, See, & Aston-Jones, 2009).
Recent advances in sleep research suggest that OX2 receptor antagonism produces sleep without disturbing the balance between rapid eye movement and non-rapid eye movement sleep, while co-administration of an OX1 antagonist promotes sleep but disturbs this balance (Dugovic et al., 2014; Roecker et al., 2014). The specific roles of these receptors in arousal and appetite differ, but activation tends to increase arousal and appetite while antagonism tends to decrease arousal and appetite.
The complexes formed by orexin receptors suggest that the orexin receptors may have some complementary functionality, but also indicate interactions with other signalling systems. The OX1 receptor has been shown to exist primarily as a homodimer, with OX-A promoting higher order complexes and OX1 antagonists promoting the monomeric form (Xu, Ward, Pediani, & Milligan, 2011). Early evidence demonstrated that the cannabinoid CB1 receptor interacted with the OX1 receptor 5
Chapter 1. Introduction
(Hilairet, Bouaboula, Carrière, Le Fur, & Casellas, 2003) and studies in Chinese hamster ovary cells have shown that the OX1 and OX2 form heterodimers with each other and with CB1 receptors (Jäntti, Mandrika, & Kukkonen, 2014). Complexation between the OX1 and CB1 receptors results in greater internalisation of both receptors, an effect that can be reversed by treatment with either the selective CB1 antagonist rimonabant (also called SR141716A) or the selective OX1 receptor antagonist SB-
674042 (Ellis, Pediani, Canals, Milasta, & Milligan, 2006). There has also been evidence that there are interactions between OX1 and corticotropin releasing factor
(CRF) 1 receptors (Navarro et al., 2015). These different orexin receptor complexes suggest that the two orexin receptors are likely to have complementary functions, but also that they may interact with other systems that regulate, appetite, arousal, and stress.
Neurons which release orexins are also neurons which release other neurotransmitters. The dominant cotransmitter is dynorphin, which is present in over
90% of orexin neurons (Chou et al., 2001) in the same synaptic vesicles (Muschamp et al., 2014). Neurotensin is also present in over 80% of orexin neurons (Furutani et al.,
2013) and glutamate is present in over 60% of orexin neurons (Henny, Brischoux,
Mainville, Stroh, & Jones, 2010; Rosin, Weston, Sevigny, Stornetta, & Guyenet, 2003).
There is mixed evidence that GABA (γ-amino butyric acid) is present in orexin neurons because anatomical studies have had mixed findings regarding the presence of
GABAergic markers (Harthoorn, Sañé, Nethe, & Heerikhuize, 2005; Rosin et al., 2003).
Studies of orexin-GABA interactions have shown orexins modulate GABAergic neurons (Thorpe, Doane, Sweet, Beverly, & Kotz, 2006), but have only reported hypothalamic GABA neurons are intermingled with orexin neurons (Hassani, Henny,
Lee, & Jones, 2010). It is therefore generally accepted that orexin neurons are also
6
Chapter 1. Introduction
dynorphin, neurotensin, and glutamate neurons. This issue will also be addressed in the
General Discussion.
There are numerous compounds available that function as antagonists at the
OX1, the OX2, or both the OX1 and OX2 (dual orexin receptor antagonists). The most commonly used are the OX1 receptor antagonist SB-334867, the OX2 receptor antagonist TCS-OX2-29, and the dual orexin receptor antagonists almorexant and suvorexant. The advantages and limitations of these, and other, orexin receptor antagonists, are reviewed in detail in section 3 of this introduction.
7
Chapter 1. Introduction
Figure 1. Orexin neurotransmission and signalling. The orexins are encoded by the hypocretin gene and cleaved from a single precursor peptide in neurons that frequently co-express dynorphin and glutamate. OX-A is non-selective for the two orexin receptors, while OX-B is selective for the OX2 receptor. There are a variety of small molecule antagonists, targeting one or both receptors. Intracellular signalling cascades are primarily excitatory, dominated by the Gs and Gq second-messenger pathways and their downstream effects. Abbreviations: AKAP, A-kinase anchor protein, ATP, adenosine triphosphate, DGL, diacylglycerol lipase, HCRT, hypocretin, IP3, inositol triphosphate, mTOR, mammalian target of rapamycin, OX, orexin, PA, phosphatidic acid, PIP2, phosphatidylinositol bisphosphate, PIP5K, phosphatidylinositol phosphate
5-kinase, PKA, protein kinase A, PKC, protein kinase C, PLA2, phospholipase A2,
PLC, phospholipase C, PLD, phospholipase D, TRPC3/6, transient receptor potential canonical cation channel subfamily C members 3 and 6. Adapted from Khoo (2014). 8
Chapter 1. Introduction
Functions and pathophysiology of the orexin/hypocretin system. Despite being a relatively small population of neurons, numbering approximately 6,000 in the rat (Allard, Tizabi, Shaffery, Ovid Trouth, & Manaye, 2004) and 70,000 in humans
(Thannickal et al., 2000; Thannickal, Nienhuis, & Siegel, 2009), the orexin/hypocretin system has several important functions in arousal, appetite and reward. There have been studies examining the role of the orexin/hypocretin system in pathophysiologies as diverse as narcolepsy, Parkinson’s disease (PD), cancer, cachexia, and anxiety. The orexin/hypocretin system is highly conserved between species. Human and rat prepro- orexin sequences share 83% homology and their OX1 and OX2 receptors share 94% and
95% homology respectively (Sakurai et al., 1998). This high degree of conservation extends across many different species, including fish, which share half of the OX-A sequence with mammals despite having an extra 15 - 18 amino acid spacer sequence
(Wong, Ng, Lee, Ng, & Chow, 2011). The conservation of the orexin/hypocretin system throughout evolutionary history suggests that its important neurobiological functions have also been conserved between species allowing for generalisability between animal models and clinical conditions.
Sleep, narcolepsy, and insomnia. The most prominent function of the role of orexin/hypocretin system is in sleep, wakefulness, and circadian rhythms
(Mileykovskiy, Kiyashchenko, & Siegel, 2005; Nattie & Li, 2012; Siegel, Moore,
Thannickal, & Nienhuis, 2001). Orexins are thought to promote arousal because they are at their highest levels during the day and their levels fluctuate with seasons, increasing as days lengthen (Boddum, Hansen, Jennum, & Kornum, 2016). People with narcolepsy, characterised by excessive sleepiness during the day and frequently accompanied by cataplexy (sudden muscle weakness), have a reduced number of orexin neurons (Thannickal et al., 2000) and the condition can be induced in animal models by 9
Chapter 1. Introduction
selective ablation of orexin neurons (Tabuchi et al., 2014). Further, it has been shown that orexin neurons regulate serotonergic neurons in the dorsal raphe and noradrenergic neurons in the locus coeruleus to prevent narcolepsy (Hasegawa, Yanagisawa, Sakurai,
& Mieda, 2014).
The role of the orexin/hypocretin system in narcolepsy was also simultaneously discovered by two groups (Chemelli et al., 1999; Lin et al., 1999). The Hcrtr2 gene had actually been discovered much earlier by Baker, Foutz, McNerney, Mitler, and Dement
(1982) who were selectively breeding for an autosomal recessive mutation in canine narcolepsy. Their candidate gene, canarc-1, was later discovered by Lin et al. (1999) to
-/- encode the OX2 receptor. Meanwhile, Chemelli et al. (1999) developed a Hcrt mouse line that showed narcoleptic symptoms, demonstrating that the absence of orexin produces narcolepsy. However, a key difference between the canine models and human narcolepsy is that the canine model involves a mutation that affects the OX2 receptor while human narcolepsy involves a reduction in the number of orexin neurons
(Thannickal et al., 2000).
It has long been speculated that narcolepsy may be caused by autoimmune depletion of orexin neurons (Thannickal et al., 2000) and there have been more recent findings that this may indeed be the case. In humans, the brains of narcoleptic patients do not show evidence of genetic abnormalities in the orexin/hypocretin system (Peyron et al., 2000). This finding has been replicated by Khatami et al. (2004) who found no evidence of hypocretin mutations in twins with narcolepsy and cataplexy. A lack of hypocretin genetic abnormality, combined with the gliosis observed in the hypothalamus by Thannickal et al. (2000), suggests that the loss of orexin neurons occurs during the patient’s lifetime. One gene in humans that is commonly associated with narcolepsy, HLA-DQB1*0602 (Khatami et al., 2004), provides some insight into 10
Chapter 1. Introduction
possible autoimmune mechanisms because it encodes a human leukocyte antigen and is highly prevalent (98%) in people with narcolepsy, but quite rare in people without narcolepsy (Tafti et al., 2014). The HLA-DQB1*0602 gene has recently been shown to be able to promote CD4-mediated inflammation without loss of orexin neurons, but which may contribute to orexin cell death caused by CD8 T cells (Bernard-Valnet et al.,
2016). Moreover, the HLA-DQB1*0602 allele has been shown to be associated with
H1N1 vaccine and flu-associated increases in narcolepsy in Europe (Dauvilliers et al.,
2013) and China (Han, Lin, Li, Dong, & Mignot, 2013), suggesting that an immune response is the cause of orexin cell depletion (Arango, Kivity, & Shoenfeld, 2015;
Liblau, Vassalli, Seifinejad, & Tafti, 2015). While orexin-based therapies have not yet been developed for narcolepsy, orexin antagonists have been developed for the treatment of insomnia (Brisbare-Roch et al., 2007; Hoever et al., 2012; Kuduk et al.,
2015), culminating in the approval of suvorexant for the treatment of insomnia (Yang,
2014).
Surprisingly little is known about the pathophysiological contribution of the orexin/hypocretin system to insomnia. Overexpression of Hcrt in zebrafish produces an insomnia phenotype (Prober, Rihel, Onah, Sung, & Schier, 2006) and the loss of the orexin receptor in zebrafish results in short, fragmented sleep (Yokogawa et al., 2007).
Selective lesions of neurons expressing Hcrtr2 (OX2 receptors), produces insomnia in rats (Gerashchenko, Blanco-Centurion, Miller, & Shiromani, 2006). However, justification for the use of orexin receptor antagonists in clinical trials for insomnia have largely focussed on the role of the orexins in arousal (Hagan et al., 1999; Herring et al.,
2012) and their normal patterns of release across circadian rhythms (Hoever et al., 2012;
Kiyashchenko et al., 2002). Few studies have examined whether orexin peptides are elevated in clinical cases of insomnia and are isolated to case reports which have 11
Chapter 1. Introduction
actually found normal cerebrospinal fluid (CSF) levels of OX-A (Schenck, Bundlie,
Mignot, & Mahowald, 2003). Normal CSF levels of OX-A have also been reported for fibromyalgia, which is characterised by chronic fatigue and insomnia (Taiwo et al.,
2007). A small (approximately 15%) increase in OX-A levels in lumbar CSF has been reported in a small sample of patients with restless leg syndrome (n = 16), which produces sleep disturbances and insomnia, compared to controls (Allen, Mignot, Ripley,
Nishino, & Earley, 2002). Another study has reported a similar 15% elevation in plasma
OX-A levels, unassociated with hypocretin polymorphisms, in insomnia patients compared to controls recruited from the same hospital with normal sleep (Tang et al.,
2017), but it appears that this study may not have used healthy controls. So, while it is now known from clinical trials that orexin receptor antagonists increase sleep, primarily by increasing time spent in rapid eye movement sleep (Herring et al., 2012), the precise nature of the orexinergic dysfunction or abnormality in insomnia is not fully understood.
Orexins and Parkinson’s disease. A role for the orexin/hypocretin system in PD has been found by some studies, but it is controversial. Patients with PD often experience problems with sleep, including sleep onset, insomnia, and fragmentation, with prevalence of 30 – 59% based on interviews with physicians and increasing as the disease progresses (Chahine, Amara, & Videnovic, 2017). It was therefore hypothesised that there may be a deficit in orexinergic signalling during PD because of the emerging understanding of the role of orexins in sleep. In support of this hypothesis, Drouot et al.
(2003) found that there were lower levels of circulating OX-A in the CSF of PD patients compared to patients with other neurological conditions. Strikingly, nearly half of their sample of PD patients had no OX-A in ventricular CSF while the lowest level in a control patient was nearly 100 pg/mL. However, their results have been controversial 12
Chapter 1. Introduction
because of their use of ventricular CSF and non-healthy controls (Maeda, Nagata,
Kondo, & Kanbayashi, 2006). Other studies have also reported a mix of normal and low
OX-A levels (Asai et al., 2009; Compta et al., 2009; Maeda et al., 2006; Overeem et al.,
2002; Poceta, Parsons, Engelland, & Kripke, 2009). Other studies have found that PD and low levels of OX-A occurred only rarely in PD, but are more common in other neurological conditions, such as progressive supranuclear palsy (Yasui et al., 2006).
While the reason for the differing results is not clear, Compta et al. (2009) have pointed out that all of the studies which have taken lumbar CSF have shown normal OX-A levels, while all of the studies which have taken ventricular CSF have shown decreased
OX-A levels. Drouot et al. (2011) have also now found that ventricular CSF OX-A concentrations have no correlation with daytime sleepiness. CSF OX-A levels in PD patients are therefore inconclusive and different lines of evidence are required to establish a role for the orexin/hypocretin system in PD.
Anatomical, genetic and pharmacological lines of evidence more strongly suggest that there is some correlation between degeneration of the orexin/hypocretin system and PD, but do not establish causality. Fronczek et al. (2007) reported finding orexin cell loss in the hypothalamus of PD patients, including the presence of Lewy bodies. Thannickal, Lai, and Siegel (2007) also found reduced numbers of orexin cells in post-mortem brain tissue and furthermore found depletion of melanin concentrating hormone (MCH) neurons. Genetic correlation studies have shown that patients who reported experiencing sudden onset sleep were more likely to have a polymorphism in their HCRT gene (Rissling et al., 2005). However, none of these studies demonstrates a causal link between orexin cell loss or dysfunction and PD. Additionally, reduced OX-
A levels might be a result of PD medications rather than a cause or factor in PD.
Michinaga, Hisatsune, Isohama, and Katsuki (2010) showed that the PD medication 13
Chapter 1. Introduction
ropinirole depletes OX-A in slices, but does not cause cell death. Models of pharmacologically-induced PD show that MPTP depletes dopamine neurons without affecting orexin neurons (Bensaid et al., 2015) but that also OX-A is neuroprotective against the oxidative stress induced in dopamine neurons by MPP+ (Feng et al., 2014).
Thus, the available evidence for a role in PD is generally correlative, but there may be some role for the orexin/hypocretin system in PD symptoms or as target of PD-related degeneration or medication side-effects.
Orexins and anxiety. Several studies have reported a role of the orexin/hypocretin system in anxiety-like behaviour. Suzuki, Beuckmann, Shikata,
Ogura, and Sawai (2005) showed that central administration of OX-A increased the amount of time rodents spent in the dark compartment of the light-dark test or the closed arms of an elevated plus maze. In these behavioural assays, time spent in the dark or in the protected closed arm of the plus maze is indicative of anxiety because the light compartment or open arm leaves the animal exposed. Consistent with this, microinjections of OX-A or OX-B into the paraventricular thalamus (PV) increases the amount of time rats spend in the closed arms of the elevated plus maze, while intra-PV
OX2 receptor antagonism using TCS-OX2-29 reduced the amount of time spent in the closed arm (Li et al., 2010). Similar results have been obtained in hamsters following microinjections of OX-A into the central amygdala (CeA) or suprachiasmatic nucleus
(Alò et al., 2016; Avolio, Alò, Carelli, & Canonaco, 2011), in rats following microinjection of OX-A into the bed nucleus of the stria terminalis (BNST; Lungwitz et al., 2012), and in goldfish following central administration of OX-A (Nakamachi et al.,
2014). Moreover, it is thought that these effects are mediated by GABA signalling via
GABAA (γ-amino butyric acid type A receptor) as well as α and β adrenergic signalling
14
Chapter 1. Introduction
because antagonism of these receptors blocks the OX-A-induced increase in anxiety- like behaviour on the elevated plus maze (Palotai, Telegdy, & Jászberényi, 2014).
Examination of the role of the orexin/hypocretin system in fear and anxiety using other models has yielded results that are mostly consistent with an anxiogenic role for the orexins. Orexins are required for panic responses in rats, and humans with panic disorder show upregulated levels of orexin in their CSF (Johnson et al., 2010). In mice, orexin signalling has been shown to facilitate the acquisition of fear to a conditioned cue and context, while OX1 receptor antagonism facilitates extinction (Flores et al.,
2014). In rats, optogenetic activation of orexin neurons decreases social interaction, suggesting increased anxiety (Heydendael, Sengupta, Beck, & Bhatnagar, 2014). OX1 receptor antagonism using SB-334867 also reduces defecation in rats following carbon dioxide exposure (Johnson et al., 2012b) and dual orexin receptor antagonism reduces immobility in rats that have received an episode of footshocks in both an open field and footshock-associated context (Chen et al., 2014). Anatomically, it has been shown that orexin neurons in the DMH and PeF are associated with responding to anxiety. Injection of FG-7142, an inverse agonist of the GABAA receptor, induces anxiety/panic-like behaviour in the rat and increased c-Fos expression in the DMH and PeF (Johnson et al.,
2012a). Its effects are also attenuated by SB-334867 (Johnson et al., 2012a).
There are some studies which have provided more mixed results for a role of the orexins in anxiety. Singareddy, Uhde, and Commissaris (2006) found that central administration of OX-A or OX-B reduced the startle response of rats in response to a loud noise rather than amplifying it. Dual orexin receptor antagonism in the PV has no effect on cue or context-conditioned fear in the rat (Dong, Li, & Kirouac, 2015) and
Staples and Cornish (2014) found that SB-334867 reduced avoidance of cat odour, but had no effect on the anxiety-like behaviour of rats in an elevated plus maze. In humans, 15
Chapter 1. Introduction
Ozsoy, Olguner Eker, Abdulrezzak, and Esel (2017) reported a positive association between childhood trauma and serum levels of OX-A. However, Strawn et al. (2010) found a reduced level of OX-A in the CSF of combat veterans with PTSD. Consistent with Strawn et al. (2010), Hcrt-/- mice show increased levels of anxiety as measured by time spent in the centre of the open field test and normal fear learning to cue or context- associated footshocks (Khalil & Fendt, 2017). One study has also suggested that there may be different roles for the OX1 and OX2 receptors in anxiety because Hcrtr1 knockdown in the basolateral amygdala (BLA) had no effect on anxiety in chronically stressed mice, but Hcrtr2 knockdown increased anxiety-like behaviour in an open field test (Arendt et al., 2014). This result is more surprising because it is also not consistent with multiple previous studies that have implicated OX1 receptors in anxiety-like behaviour (Johnson et al., 2012a; Johnson et al., 2012b; Staples & Cornish, 2014). The role of the orexin/hypocretin system in anxiety is therefore equivocal because there are multiple studies in humans and animals that demonstrate an anxiogenic effect, an anxiolytic effect, or no effect of orexin signalling.
Orexins, cachexia, and cancer. Deficiencies in orexin functioning have also been suggested as a potential target for treatments relating to cancer and associated progressive wasting (cachexia). Very soon after the discovery of the orexin/hypocretin system, Inui (1999) suggested that the orexin/hypocretin system could be a possible target for the treatment of cachexia because of its role in appetite and feeding. Cachexia affects approximately half of cancer patients and involves fatigue, loss of appetite, adipose tissue and muscle mass (Tisdale, 1997; Vanhoutte et al., 2016). Cachexia is often associated with increased catecholamine turnover (Tisdale, 1997), while the orexin/hypocretin system has been shown to suppress catecholamine synthesis in cell culture (Nanmoku et al., 2000). Hypothalamic orexin neurons have also been shown to 16
Chapter 1. Introduction
increase glucose uptake by skeletal muscle and increase insulin sensitivity (Shiuchi et al., 2009). Exogenous OX-A has been reported to reduce pain from rheumatoid arthritis in rats, while promoting weight gain (Mohamed & El-Hadidy, 2014) and to provide analgesia in mice following chemotherapy (Toyama, Shimoyama, & Shimoyama,
2017). In mice, a cocktail of chemotherapy drugs including cyclophosphamide, adriamyclin, and 5-fluorouracil, reduced voluntary locomotor activity and orexin neuron activation after a single dose (Weymann, Wood, Zhu, & Marks, 2014). Similar effects have been reported in rats following lipopolysaccharide or tumour-induced inflammation (Grossberg et al., 2011). In both mice and rats, normal levels of activity were restored following central administration of OX-A, suggesting that an orexin agonist or mimetic could have therapeutic potential (Grossberg et al., 2011; Weymann et al., 2014). However, Tanaka et al. (2015) have challenged these findings with their study showing that postnatal genetic ablation in orexin/ataxin-3 mice resulted in reduced wakefulness the day after acute administration of lipopolysaccharide relative to wild-type mice. Although the effects reported by Tanaka et al. (2015) are small, their work suggests that there may be side-effects to orexinergic pharmacotherapies for cachexia because these may reduce the quality of rest. Unfortunately, small molecule agonists or orexin potentiators have only attracted minor interest and serendipitous discoveries (Lee, Reddy, & Kodadek, 2010; Turku et al., 2016), suggesting that orexin- based pharmacotherapies for cachexia will not be developed for several years.
Some studies have even suggested a role for the orexin/hypocretin system directly in various cancers, but with mixed results. In pheochromocytomas, a type of adrenal tumour, exogenous OX-A and OX-B can promote catecholamine secretion
(Mazzocchi et al., 2001). In adrenocortical adenomas, OX-A and OX-B raised proliferation rates and OX-A but not OX-B increased cortisol secretion (Spinazzi, 17
Chapter 1. Introduction
Rucinski, Neri, Malendowicz, & Nussdorfer, 2005). However, other studies have suggested a cancer suppressing role for the orexins. For example, Rouet-Benzineb et al.
(2004) showed that OX-A, via the OX1 receptor, induced apoptosis in colon cancer and neuroblastoma cells. Similar growth suppressing and pro-apoptotic effects have been reported by the same group for rat pancreatic cells (Voisin, Firar, Avondo, & Laburthe,
2006). This group subsequently attributed the pro-apoptotic activity of OX1 receptors to their immunoreceptor tyrosine-based inhibitory motif, which is present in the OX1 receptor and if it is removed through mutation, prevents the pro-apoptotic effects of
OX1 receptor activation (El Firar et al., 2009; Voisin, El Firar, Rouyer-Fessard, Gratio,
& Laburthe, 2008). However, the cancer and tissue type are likely to be important because studies that have found cancer-promoting effects of orexins have been done in adrenal tumours (Mazzocchi et al., 2001; Spinazzi et al., 2005), while studies in colon cancer and neuroblastoma have shown cancer-suppressing effects (Rouet-Benzineb et al., 2004; Voisin et al., 2006). For prostate cancer, a recent paper even argues that there is no orexin effect and that previous results are due to contamination (Szyszka et al.,
2015). Nonetheless, it is more plausible for orexin-based pharmacotherapies to target the appetitive functions of orexin, rather than potential cancer and disease-related pathophysiologies.
Orexins, appetitive motivation, and energy balance. Appetitive motivation is a key function of the orexin/hypocretin system and disorders of appetitive motivation are among the best established examples where an excess of orexin signalling is thought to be important (Ripley et al., 2001). The orexin peptides were shown early on to stimulate feeding, because central administration of both OX-A and OX-B increase food consumption in rats (Sakurai et al., 1998). Expression of the peptides increases in anticipation of feeding (Akiyama et al., 2004; Mieda et al., 2004) or during food 18
Chapter 1. Introduction
deprivation (Karteris et al., 2005), and chronic central administration of OX-A increases feeding during the light phase (Yamanaka, Sakurai, Katsumoto, Masashi, & Goto,
1999). OX-A also counteracts the effects of satiety signals, like cholecystokinin
(Asakawa et al., 2002) and OX1 antagonism using SB-334867 reduces total food intake in ob/ob (leptin-deficient obese) mice (Haynes et al., 2002). However, studies of OX-A- induced feeding have found that total food intake remains unchanged because of a compensatory reduction in feeding after the initial increase (Ida, Nakahara, Katayama,
Murakami, & Nakazato, 1999; Yamanaka et al., 1999) and recent results show that orexin-deficient narcolepsy patients are resistant to the sensory-specific satiety produced by consumption of a salty and sweet snack (van Holst et al., 2016). This finding of reduced satiety in orexin deficient people appears to contrast with the well- established finding that OX-A promotes feeding and opposes satiety signals. Some insight into this discrepancy comes from a recent fibre photometry study assessing calcium transients from OX neurons in the awake, freely moving mouse. This study showed that orexin cells decrease in activity with feeding onset, regardless of whether the food is caloric or the animal is hungry or sated (González et al., 2016). This implies that under physiological conditions, it is not just the level of orexin but also the pattern of OX neuronal activity that is important. The orexin/hypocretin system may not simply be an appetite promoting neuropeptide system, but one that regulates appetitive behaviour in a time, activity, and concentration-dependent manner.
The orexin/hypocretin system is now thought to promote feeding as a regulator of energy homeostasis. Genetic ablation of orexin neurons in an orexin/ataxin-3 transgenic mouse line reduces the arousal, exploratory, and foraging response to fasting that is seen in wild-type mice (Yamanaka et al., 2003). Paradoxically, this mouse line shows both a reduction in feeding and an increase in bodyweight (Hara et al., 2001). 19
Chapter 1. Introduction
This may be because OX-A also promotes energy expenditure (Lubkin & Stricker-
Krongrad, 1998) by activating the sympathetic nervous system and mobilising endogenous glucose production (Yi et al., 2009). Glucose can, along with leptin, inhibit the activity of isolated orexin neurons (Yamanaka et al., 2003), a feedback loop that signals via leptin receptor-containing neurotensin neurons (Goforth, Leinninger,
Patterson, Satin, & Myers, 2014). Thus there is evidence that the orexin/hypocretin system both promotes food consumption and energy expenditure and is part of the neural circuitry that regulates feeding and appetite. Other aspects of the role of orexins in appetitive motivation will be discussed in Section 2. The Orexins and Animal Models of Addiction.
Orexin function and neuroanatomy. The different functions of the orexin system in various physiological processes and disorders implies that there may be different neuroanatomical networks involved. Microinjections of OX-A to the anterior lateral hypothalamus or to the nucleus accumbens stimulates feeding (Thorpe et al.,
2006; Thorpe & Kotz, 2005), but accumbal OX-A also stimulates locomotor activity
(Thorpe & Kotz, 2005). It has been shown in zebrafish that only a small subpopulation of orexin neurons express neurotensin receptors (Levitas-Djerbi, Yelin-Bekerman,
Lerer-Goldshtein, & Appelbaum, 2015), indicating that orexin neurons form parts of multiple disparate neural circuits. Indeed, early studies of orexin receptor distribution reported the presence of high density OX1 receptors in regions such as the tenia tecta and indusium grisium (Trivedi et al., 1998), which are parts of the supracallosal gyrus that have no reported role in arousal, appetite, or reward. These disparate networks present a potential confound for studies involving systemic or central administration of orexin peptides or antagonists because it is possible that there may be orexin in neural networks with unknown or different behavioural functions. However, in order to 20
Chapter 1. Introduction
examine whether the orexins are a potential therapeutic target, it is necessary to examine the effect of orexin antagonists on the whole brain because it best models how an orexin antagonist may be used in the clinic.
Other functions of the orexin system. There are several other roles for the orexin system and theories of its operation that are beyond the scope of this thesis. For example, it has been theorised that the orexins are important for normal motor function
(Siegel, 2004) and that it has a close interaction with the histamine system in the aetiology of sleep disorders and sleep disruptions in other disorders such as PD (Shan,
Dauvilliers, & Siegel, 2015). Orexins have also been implicated in hypertension (Huber,
Chen, & Shan, 2017), sensing acidity and carbon dioxide (Johnson et al., 2012b;
Williams, Jensen, Verkhratsky, Fugger, & Burdakov, 2007), thermoregulation (Kuwaki,
2015; Takahashi et al., 2013), sexual behaviour (Muschamp, Dominguez, Sato, Shen, &
Hull, 2007), and apoptosis (Voisin et al., 2006). Orexin receptors are also present in several organs in the periphery including the spleen, liver, and bone marrow (Regard,
Sato, & Coughlin, 2008) where it may have metabolic circadian and rheostatic roles
(Greene et al., 2016; Wei et al., 2014).
2. The Orexins and Animal Models of Addiction
The role of the orexin/hypocretin system has been examined for multiple reinforcers in several different kinds of animal models of addiction. A role for the orexin/hypocretin system has been shown in both Pavlovian and operant paradigms for drugs of abuse such as cocaine, opioids, alcohol, and nicotine. The results of these studies have informed the development of the motivational activator theory, the first coherent theory of the general function of the orexin/hypocretin system in appetitive motivation. Microinjection studies have also identified important roles for orexinergic
21
Chapter 1. Introduction
projections to several regions of the mesocorticolimbic pathway, implicating the orexin/hypocretin system in the processing of the rewards. The findings from these preclinical animal studies suggest that the orexin/hypocretin system may be a potential therapeutic target for multiple drugs of abuse, but further preclinical studies are required.
Animal models of addiction. There are several animal models of addiction that rely on different aspects of physiology or psychology. Physiological dependence can be induced in rodents by chronic administration of a drug, such as by implantation with a morphine pellet (Georgescu et al., 2003) or chronic intermittent exposure to ethanol vapour (Lopez, Moorman, Aston-Jones, & Becker, 2016). This allows experimenters to observe effects on subsequent behaviours, such as withdrawal or drug self- administration behaviour. However, more commonly, experimenters use models which are based on Pavlovian and operant conditioning processes. These include the Pavlovian conditioned place preference (CPP) model and various operant self-administration and reinstatement paradigms.
In CPP, an animal is trained to make a context-drug association. During acquisition, the animal is given an injection of drug and confined to one of two connected compartments, defined by a variety of visual, olfactory or tactile cues
(Hoffman, 1989). The animal is also given injections of vehicle and confined to the other compartment. On test, the animal is allowed to move between the two compartments and the amount of time spent in each compartment is recorded as a measure of the animal’s preference for the drug. The associations between the drug and context can also be extinguished by non-reinforced pairings of the context and then reinstated using a priming injection of drug, as done by Shoblock et al. (2011). This allows CPP paradigms to provide some insight into the contextual associations with the 22
Chapter 1. Introduction
hedonic properties of a drug but the main disadvantage is that the drugs are experimenter-administered and so they do not model the voluntary behavioural processes in addiction.
Operant models of drug seeking and drug taking provide a richer behavioural paradigm for the study of the role of orexins in addiction. In the operant chamber, animals are able to voluntarily seek drug (or non-drug) rewards by performing an instrumental response, such as lever pressing or nosepoking. In the case of alcohol or beer, the reward can then be taken by drinking from a magazine (McGregor & Gallate,
2004; Spanagel, 2003). For other reinforcers, such as cocaine, heroin, or nicotine, the drug is delivered intravenously by a surgically implanted catheter (Macnamara, Holmes,
Westbrook, & Clemens, 2016; Schmeichel et al., 2015; Stefanik, Kupchik, Brown, &
Kalivas, 2013). Experimenters can vary the number of responses required for each reward, also known as the schedule of reinforcement, most commonly using fixed ratio
(FR) or progressive ratio (PR) schedules (Arnold & Roberts, 1997; Richardson &
Roberts, 1996). As their names imply, an FR schedule involves a fixed number of responses for each reward delivery, while a PR requires more responses for each subsequent reward. It is typically thought that FR schedules model drug-taking behaviour, while PR schedules model motivation because a more motivated animal will be willing to respond more times to obtain a single bolus of reward. However, high FR schedules also model motivation because rats must be willing to make multiple responses to obtain each reward and PR schedules also model drug-taking because responses are reinforced.
Operant models of relapse. Once animals have acquired operant self- administration behaviour, they can be taken through processes of extinction and reinstatement that model relapse behaviour in humans. During extinction, operant 23
Chapter 1. Introduction
responding typically has no consequences and so the animal rapidly learns to suppress their operant drug seeking behaviour. Drug-associated cues, which are presented alongside drug delivery during acquisition and self-administration, are typically never presented during extinction. However, these cues retain the ability to increase responding when they are presented during a cue-induced reinstatement session where they function as a conditioned reinforcer (Davis & Smith, 1976; Perry, Zbukvic, Kim, &
Lawrence, 2014). Reinstatement can also be precipitated by other stimuli, such as a priming injection or delivery of the drug (de Wit & Stewart, 1981; de Wit & Wise,
1977) or stress (Mantsch, Baker, Funk, Le, & Shaham, 2016; Shaham & Stewart, 1996).
Different methods of precipitating reinstatement can also be combined, for example, combining a footshock with cue presentation (Buffalari & See, 2009). Renewal, or context-induced reinstatement, can also be used to assess relapse-like behaviour in animals by conducting extinction in a distinct context (defined by different olfactory, tactile, and/or visual cues) and then returning the animal to the training context
(Crombag, Bossert, Koya, & Shaham, 2008; Crombag & Shaham, 2002).
Reinstatement and renewal are thought to occur because extinction involves new learning that does not erase the acquisition memory, allowing it to subsequently be unmasked during reinstatement or renewal (Bouton, 2002; Bouton, Todd, Vurbic, &
Winterbauer, 2011). When learning an operant task, animals form associations between the required response (e.g. lever press or nosepoke), the reinforcer (e.g. food or drug), and any cues that accompany the procedure. Extinction of the operant response has been shown to occur because the extinction context forms an inhibitory association with the operant response (Rescorla, 1993, 1997; Todd, 2013; Todd, Vurbic, & Bouton, 2014), unlike for Pavlovian conditioning where the context gates retrieval of an extinction association. However, for operant behaviours, extinction training decreases responding 24
Chapter 1. Introduction
because the context acts directly to inhibit the operant response. If the animal is removed from the extinction context, or if the animal is presented with the reinforcer or a reinforcer-associated cue, the inhibition is removed and reinstatement occurs in the testing context.
Regardless of the psychological processes underlying relapse-like behaviour, there is good correspondence between relapse-promoting conditions in animals and humans (Spanagel, 2003), resulting in widespread use of reinstatement and renewal as preclinical models of addiction.
Neural reward circuitry. The neural circuitry underlying reward and addiction has been studied using both Pavlovian and operant models of relapse and reward seeking. Lesions to the lateral hypothalamus abolish intracranial self-stimulation (Olds
& Milner, 1954). The reward system, which evolved to respond to natural rewards such as food and sex (Kelley & Berridge, 2002), is now thought to encompass several brain regions including the VTA, Acb, PFC and ventral pallidum (Khoo, Gibson, Prasad, &
McNally, 2017). In the classical mesocorticolimbic dopamine pathway thought to mediate reward, the VTA is the source of dopamine release in response to drugs of abuse (Di Chiara & Imperato, 1988). The Acb shell and core receive dopaminergic inputs and have differing roles in behaviour (Meredith, Baldo, Andrezjewski, & Kelley,
2008), with the shell having a stronger role in context-induced reinstatement (Cruz et al., 2014a; Hamlin, Blatchford, & McNally, 2006; Hamlin, Clemens, & McNally, 2008;
Hamlin, Newby, & McNally, 2007; Khoo et al., 2017) while the core has a greater role in cue, stress and drug-primed reinstatement (Bossert, Marchant, Calu, & Shaham,
2013; Marchant, Li, & Shaham, 2013). The PFC is similarly thought to have subregions with distinct functions, with the prelimbic cortex implicated in precipitating reinstatement while the infralimbic cortex is involved in extinction (Kalivas & Volkow, 25
Chapter 1. Introduction
2005; Peters, Kalivas, & Quirk, 2009; Peters, LaLumiere, & Kalivas, 2008). Other regions, such as the VP, have also been shown to be involved in various forms of reinstatement (McFarland & Kalivas, 2001; Perry & McNally, 2013; Torregrossa &
Kalivas, 2008), but the mechanisms through which these effects are mediated are less well understood (Khoo et al., 2017).
Orexin antagonist studies and the motivational activator theory. Once orexin antagonists were developed, they were rapidly applied to animal models relating to drugs of abuse. The selective OX1 receptor antagonist, SB-334867, was the first small molecule antagonist developed to target the orexin/hypocretin system (Porter et al., 2001; Smart et al., 2001). SB-334867 has subsequently been used to reduce operant cocaine, heroin, alcohol, and nicotine self-administration and reinstatement (Harris,
Wimmer, & Aston-Jones, 2005; Hollander, Lu, Cameron, Kamenecka, & Kenny, 2008;
Lawrence et al., 2006; Smith & Aston-Jones, 2012; Smith et al., 2009). A variety of
OX2 receptor antagonists have also been used against cocaine (Smith et al., 2009), heroin (Schmeichel et al., 2015), alcohol (Brown et al., 2013; Shoblock et al., 2011), and nicotine (Uslaner et al., 2014). A more limited number of studies have involved the use of the dual orexin receptor antagonist, almorexant, against cocaine (Steiner,
Lecourt, & Jenck, 2013b), alcohol (Srinivasan et al., 2012), and nicotine (LeSage,
Perry, Kotz, Shelley, & Corrigall, 2010).
The motivational activator theory has been proposed as an explanation for the role of the orexin/hypocretin system in reward. The theory, based largely on results from cocaine studies, states that the orexin/hypocretin system responds when highly motivated behaviour is required, which occurs when reward seeking is under the control of conditioned cues or stress, when rewards are available under a high unit-cost, or when the reward is highly salient or physiologically relevant (James, Mahler, Moorman, 26
Chapter 1. Introduction
& Aston-Jones, 2017; Mahler, Moorman, Smith, James, & Aston-Jones, 2014; Mahler,
Smith, Moorman, Sartor, & Aston-Jones, 2012). The orexin/hypocretin system would therefore be expected to be involved in self-administration and reinstatement for rewards that are either drugs of abuse or palatable foods high in fat and/or sugar. This is generally consistent with reported effects in the literature, with a few exceptions which are discussed below.
Orexin antagonist studies have shown that for cocaine, orexin is involved in reinstatement and in self-administration when the unit-cost is high. SB-334867 has been shown to reduce cue-induced reinstatement (Smith et al., 2009), context-induced reinstatement (Smith, Tahsili-Fahadan, & Aston-Jones, 2010b), footshock-stress- induced reinstatement (Boutrel et al., 2005), and cocaine-primed reinstatement in male but not female rats (Zhou et al., 2012a). Operant self-administration of cocaine is also affected by SB-334867 under FR5 (Hollander, Pham, Fowler, & Kenny, 2012;
Muschamp et al., 2014), discrete trial, and PR conditions (España et al., 2010), but not under a less demanding FR1 schedule (Smith et al., 2009; Zhou et al., 2012a; Zhou,
Smith, Do, Aston-Jones, & See, 2012b). OX1 antagonism is also effective in behavioural economics paradigms, where the unit-cost is progressively increased after each reward delivery by decreasing the amount of reward delivered (Bentzley & Aston-
Jones, 2015; España et al., 2010). These findings are consistent with the claim from motivational activator theory that the orexin/hypocretin system is involved when the availability of cocaine is more constrained or when the unit-cost of cocaine is higher.
However, this theory provides no explanation as to why there might be a sex difference in orexin involvement in cocaine reinstatement.
The patterns of effects for other drugs of abuse are consistent to varying degrees with the motivational activator theory. For opioids, although the orexins are involved in 27
Chapter 1. Introduction
morphine withdrawal (Georgescu et al., 2003) and cue-induced reinstatement for heroin,
OX1 receptor antagonism does not reduce heroin-primed reinstatement (Smith & Aston-
Jones, 2012). In contrast with what the motivational activator theory would predict from the cocaine literature, 30 mg/kg i.p. SB-334867 does reduce FR1 heroin self- administration (Smith & Aston-Jones, 2012). The OX2 receptor antagonist, NBI-80713
(up to 30 mg/kg, i.p.), also reduces FR1 self-administration in long (12 h) access sessions but not in short (1 h) access sessions (Schmeichel et al., 2015). These findings are more equivocal because the reinforcer is available under conditions of relatively low effort, but the longer sessions may have given the animal more of an opportunity to develop strong cue-drug associations or greater physical dependence.
The orexin and alcohol literature is also largely consistent with the motivational activator theory, but there are some findings that are not immediately predicted by the theory. The orexins are involved in cue-induced and yohimbine stress-induced reinstatement of alcohol seeking (Jupp, Krivdic, Krstew, & Lawrence, 2011a;
Lawrence et al., 2006; Richards et al., 2008), as predicted, but this does not appear to require the OX2 receptor (Brown et al., 2013). Antagonist effects on self-administration have all been under conditions of FR3 using 10 – 30 mg/kg i.p. SB-334867 (Jupp et al.,
2011a; Lawrence et al., 2006; Richards et al., 2008), 10 mg/kg s.c. of the OX2 receptor antagonist JNJ-10397049 or 100 – 300 µg intracerebroventricular TCS-OX2-29 (Brown et al., 2013; Shoblock et al., 2011), or 15 mg/kg i.p. almorexant (Srinivasan et al.,
2012). Moorman, James, Kilroy, and Aston-Jones (2017) also showed that 10 – 20 mg/kg i.p. SB-334867 selectively reduces FR3 self-administration and reinstatement in high responders for 20% alcohol, as determined by a median split. However, Shoblock et al. (2011) reported no effect of the selective OX1 receptor antagonist, SB-408124 (3 –
30 mg/kg, s.c.), on operant FR3 alcohol self-administration. Anderson, Becker, Adams, 28
Chapter 1. Introduction
Jesudason, and Rorick-Kehn (2014) also present evidence for the involvement of OX1 receptors in 2-bottle free choice consumption, but not in PR, while OX2 antagonism using 30 mg/kg i.p. LSN2424100 does not affect 2-bottle free choice but does reduce
PR responding. Almorexant (10 – 100 mg/kg, i.p.) was shown to affect both 2-bottle free choice and PR. The findings of Anderson et al. (2014) are particularly problematic for the motivational activator theory of orexins in reward because in the 2-bottle free choice assay, alcohol is available with minimal effort. The PR results are also surprising because previous studies with cocaine have shown effects of 30 mg/kg i.p. SB-334867, but not 10 – 30 mg/kg i.p. TCS-OX2-29 (Borgland et al., 2009; España et al., 2010;
Mahler et al., 2012; Smith et al., 2009).
The role of orexin in nicotine seeking is far less well characterised, with only three previously published antagonist studies. SB-334867 has been shown to decrease self-administration under both FR5 and PR conditions (Hollander et al., 2008; LeSage et al., 2010). However, Uslaner et al. (2014) showed that OX2 receptor antagonism reduces cue-induced, but not nicotine-primed reinstatement or FR5 self-administration.
Although the effect of OX1 receptor antagonism on reinstatement has not been tested, the involvement of the OX2 receptor in cue-induced nicotine reinstatement also differs to results from cocaine studies. These results suggest that the role of orexins in nicotine seeking warrants further investigation, but like other drugs, is broadly consistent with the motivational activator theory.
Natural reinforcers. Studies using natural reinforcers, such as sweet or high fat/sugar rewards, show mixed effects of OX manipulations and provide mixed support for the motivational activator theory.
29
Chapter 1. Introduction
On the one hand, administration of 3 ng OX-A into the third ventricle specifically facilitates increases in intake of a high fat (41% kcal), high sugar (45% kcal) food (Clegg, Air, Woods, & Seeley, 2002) and promotes sucrose intake (Benoit,
Clegg, Woods, & Seeley, 2005). Antagonism using 10 mg/kg i.p. SB-334867 reduces
PR responding for high fat chocolate pellets (Borgland et al., 2009). Almorexant (10 –
15 mg/kg, i.p.) has also been shown to reduce 5% sucrose self-administration under an
FR3 schedule (Srinivasan et al., 2012) and, at 300 mg/kg i.p., food self-administration under an FR5 schedule (LeSage et al., 2010). These effects on responding are consistent not only with a role for the OX system in appetitive motivation, but also with the claims of the motivational activator theory. However, several studies have found that OX1 antagonism does not affect normal food self-administration (Hollander et al., 2008;
Hollander et al., 2012; LeSage et al., 2010), PR responding for normal food (Borgland et al., 2009), saccharin self-administration (Shoblock et al., 2011), or cue-induced reinstatement for sweetened condensed milk (Martin-Fardon & Weiss, 2014a). Intra-
PFC OX1 antagonism does not affect cue-induced reinstatement of sucrose and central
OX2 antagonism does not affect FR3 sucrose self-administrations at concentrations that match responding for sucrose to responding for ethanol (Brown et al., 2013; Brown et al., 2016). Moreover, when a dual orexin antagonist (3 – 30 mg/kg oral DORA-22) is used to promote sleep, monkeys retain the ability to wake to chocolate M&M-associated cues (Tannenbaum et al., 2016).
These mixed findings suggest multifactorial determinants of orexin’s role in natural reward seeking. This conclusion is supported by studies on sucrose, saccharin and motivational state. Cason and Aston-Jones (2013b) found that SB-334867 reduces sucrose self-administration and cue-induced reinstatement in rats that were food restricted, but had no effect on rats fed ad libitum. However, for saccharin pellets, this 30
Chapter 1. Introduction
effect was not dependent on motivational state and both food restricted and ad libitum rats showed reduced self-administration as well as cue-induced reinstatement (Cason &
Aston-Jones, 2013a). This is surprising because saccharin has no caloric value and so there should be a greater physiological need for the sucrose pellets. Rats prefer saccharin solutions to have a concentration of 0.2-0.4% (Smith & Sclafani, 2002), which suggests that a 1% saccharin pellet may not have a higher hedonic value than a sucrose pellet. Although the studies reviewed by Smith and Sclafani (2002) were done using aqueous solutions, fast scan cyclic voltammetry in conscious rats shows that they have a greater preference for sucrose pellets than 1.1% saccharin pellets (McCutcheon,
Beeler, & Roitman, 2012). The results of Cason and Aston-Jones (2013a, 2013b) therefore suggest that the involvement of the orexin/hypocretin system is determined by motivational state and other properties of the reinforcer which likely include, but are not limited to, its hedonic value.
Orexin neuroanatomy and addiction. Neuroanatomical and microinjection studies have demonstrated orexinergic regulation of many key parts of the reward neurocircuitry, particularly with respect to dopamine. Immunohistochemical staining has shown that projections from orexin neurons are intermingled with dopaminergic fibres in the VTA where it is thought that they interact by volume transmission (Balcita-
Pedicino & Sesack, 2007; Baldo et al., 2003; Fadel & Deutch, 2002). Many tyrosine hydroxylase-positive neurons in the VTA express orexin receptors and can be activated by microinjection of OX-A or OX-B, resulting in increased dopamine in the nucleus accumbens, the PFC, and elsewhere (Korotkova, Sergeeva, Eriksson, Haas, & Brown,
2003; Narita et al., 2006). Dopamine also acts as a feedback mechanism on orexin neurons, disinhibiting them at low concentrations and promoting GABA-mediated inhibition at higher concentrations (Linehan, Trask, Briggs, Rowe, & Hirasawa, 2015). 31
Chapter 1. Introduction
Targeted microinjection studies have implicated orexin signalling in several reward regions in morphine place preference and withdrawal. Microinjection studies have shown that intra-VTA OX-A area can reinstate CPP for morphine (Harris et al.,
2005). As shown in Figure 2, targeted microinjection studies have also implicated the locus coeruleus and the PV. In the LC, SB-334867 pretreatment reduces the morphine withdrawal symptoms precipitated by intra-LC injection of glutamate during the active dark phase, but not the inactive light phase (Hooshmand, Azizi, Javan, & Semnanian,
2017). In the PV, neither SB-334867 nor TCS-OX2-29 reduce acquisition of naloxone- induced morphine withdrawal-associated conditioned place aversion (CPA) but expression of morphine withdrawal-associated CPA is affected by OX2 receptor antagonism (Li et al., 2011). However, the opposite pattern of results is observed in the
Acb, where SB-334867 but not TCS-OX2-29 reduces expression of morphine CPP
(Sadeghzadeh, Namvar, Naghavi, & Haghparast, 2016). This result is surprising because Marcus et al. (2001) reported very little OX1 receptor expression in the Acb and this result has recently been replicated by Ch’ng and Lawrence (2015) who found no evidence of Hcrtr1 mRNA in the nucleus accumbens core (AcbC) or shell. One possible explanation is that these opiate studies used doses (30 – 300 µg) of SB-334867 that may not maintain OX1 selectivity. Other studies have reported effective doses at 3 µg
(Brown et al., 2016) and SB-334867 is only 50-fold more selective for OX1 relative to
OX2 receptors (Porter et al., 2001; Smart et al., 2001). Nonetheless, these studies demonstrate that there is a role for orexinergic signalling in morphine place preference and withdrawal in multiple regions associated with reward signalling.
32
Chapter 1. Introduction
Figure 2. Microinjection studies and opioids. OX-A can reinstate morphine CPP. In the
PV, it is the OX2 but not OX1 receptor which is important for morphine withdrawal conditioned place aversion, but antagonists have produced the opposite effect in the nucleus accumbens for CPP. In the LC, SB-334867 reduces morphine withdrawal during the dark (active) phase, but not the light (inactive) phase. Abbreviations: Amy, amygdala, LV, lateral ventricle. Adapted from Khoo et al. (2017).
33
Chapter 1. Introduction
Similar regions have been implicated in animal models of alcohol self- administration and reinstatement. The VTA and Acb are important in alcohol seeking
(Figure 3), but other regions have also been studied including the PFC and nucleus incertus (NI). OX1 receptor antagonism in the VTA and medial PFC reduce alcohol reinstatement (Brown et al., 2016). Similar effects have been obtained using the dual orexin receptor antagonist, almorexant, which reduces FR3 self-administration when injected into the VTA but not the substantia nigra (Srinivasan et al., 2012).
Interestingly, SB-334867 does not affect c-Fos expression in the VTA during reinstatement of alcohol seeking (Jupp, Krstew, Dezsi, & Lawrence, 2011b), suggesting a dissociation between effects on behaviour and some markers of neural activity. Other studies have implicated the OX2 receptor in FR3 alcohol self-administration, but not reinstatement, an effect which involves the AcbC but not the AcbSh (Brown et al.,
2013). Recently, Kastman et al. (2016) have shown that TCS-OX2-29 injected into the
NI, but not SB-334867, can suppress yohimbine stress-induced reinstatement of alcohol seeking. The NI expresses both OX1 and OX2 receptors (Greco & Shiromani, 2001), is an important region for the integration of stress signals (Ma & Gundlach, 2015) and NI
CRF1 receptors regulate alcohol seeking (Walker et al., 2017). Navarro et al. (2015) have shown that orexin and CRF receptors form heteromers in the VTA and if this were to occur in the NI it would provide a compelling explanation for the convergent results from CRF and orexin receptor antagonist studies. Together, these results implicate several important reward regions in alcohol seeking, but also suggest that for alcohol there may be an interaction with stress at both the anatomical and molecular level which is not activated by alcohol-associated cues alone.
34
Chapter 1. Introduction
Figure 3. Microinjection studies and alcohol seeking. The VTA has been implicated in alcohol self-administration and reinstatement, while the AcbC has been implicated in alcohol self-administration, and the PFC has been implicated in alcohol reinstatement.
Central OX2 receptor antagonism reduces self-administration but not reinstatement.
Adapted from Khoo et al. (2017).
35
Chapter 1. Introduction
Cocaine seeking also relies on an interaction between different reward, arousal, and stress-related signalling pathways (Figure 4). Central administration of OX-A promotes reinstatement of cocaine seeking and the OX-A-induced reinstatement can be blocked by administering CRF or noradrenergic antagonists (Boutrel et al., 2005). Intra-
PV OX-A also precipitates reinstatement of cocaine seeking and this effect can be reduced using the OX2 receptor antagonist TCS-OX2-29, but not the OX1 receptor antagonist SB-334867 (Martin-Fardon & Boutrel, 2012; Matzeu, Kerr, Weiss, &
Martin-Fardon, 2016). These findings are in agreement with James et al. (2011) who found that intra-VTA, but not intra-PV, SB-334867 reduces cue-induced reinstatement of cocaine seeking. The PV is both an important centre for arousal, stress, and reward signalling (Kirouac, 2015; Li et al., 2010) which receives orexinergic innervation from hypothalamus (Kirouac, Parsons, & Li, 2005). Immunohistochemical labelling suggests that hypothalamic orexin and cocaine and amphetamine regulated transcript (CART) fibres project to PV neurons that then go on to project to AcbSh (Parsons, Li, &
Kirouac, 2006) and these fibres are denser than the direct projections of orexin neurons to the AcbSh (Lee & Lee, 2016). Orexinergic regulation of cocaine seeking therefore involves interactions with arousal and stress-related neurocircuitry and transmitter systems.
Cocaine seeking behaviour also involves the CeA and VTA. Intra-amygdala SB-
334867 reduces FR1 cocaine self-administration and yohimbine stress-induced reinstatement in rats given long (6 h) access sessions (Schmeichel, Herman, Roberto, &
Koob, 2017). However, the effect of SB-334867 on self-administration is very small, with a mean difference of approximately 10%. Additionally, Schmeichel et al. (2017) note that the effect did not last for the entire 6 h session which is fairly unsurprising given that SB-334867 has a half-life of approximately 24 min (Porter et al., 2001). 36
Chapter 1. Introduction
While they do not show the data for the intra-CeA test, they do show that 15 – 30 mg/kg i.p. SB-334867 reduces the number of infusions earned during the first hour, but only the 30 mg/kg dose had a significantly lower number of infusions earned after the full 6 h. In the VTA, España et al. (2010) have shown that microinjection of the selective OX1 receptor antagonist SB-334867 reduces PR breakpoint in rats self-administering cocaine, as well as the cocaine-induced increase in Acb dopamine. Conversely, intra-
VTA OX-A increases cocaine PR responding (España, Melchior, Roberts, & Jones,
2011). These effects are consistent with the orexinergic projections to the VTA (Baldo et al., 2003). It is thought that the orexinergic projections to the VTA result in an efflux of dopamine in the prefrontal cortex (Vittoz & Berridge, 2006) and it has more recently been shown that Hcrt-/- mice have reduced dopamine release in the Acb following cocaine administration (Shaw et al., 2017). It is therefore likely that orexinergic input to
VTA dopaminergic neurons contribute to the reinforcing and motivational properties of cocaine.
There is mixed evidence that orexinergic VTA projections regulate some of the stress-related aspects of cocaine seeking. In the VTA, orexin receptors form heterodimers with CRF receptors (Navarro et al., 2015) and Slater, Noches, and Gysling
(2016) have shown that LH orexin neurons also express CRF2 receptors and CRF binding protein which are present at the presynaptic terminals that orexin neurons make in the VTA. However, Wang, You, and Wise (2009) found that the OX1 receptor antagonist SB-408124 could reduce reinstatement precipitated by intra-VTA OX-A, but not footshock. Conversely, intra-VTA microinjection of a CRF antagonist had no effect on OX-A-induced reinstatement of cocaine seeking. While there is anatomical evidence for an interaction between the orexin and CRF systems in the VTA, these antagonist studies suggest that these may not be important for cocaine seeking behaviour. 37
Chapter 1. Introduction
Figure 4. Microinjection studies and cocaine seeking. Microinjection studies have specifically implicated the PV, VTA and Amy. Central or intra-PV administration of
OX-A precipitates cocaine reinstatement. In the PV, OX1 receptor antagonism is ineffective against cue-induced or OX-A-induced reinstatement, but OX2 receptor antagonism is effective against OX-A-induced reinstatement. In the VTA, OX1 receptor antagonism reduces PR breakpoint and cue-induced reinstatement, while OX-A increases PR breakpoint. In the CeA, OX1 receptor antagonism reduces FR1 self- administration for the first hour of 6 h self-administration sessions and also reduces yohimbine stress-induced reinstatement. Adapted from Khoo et al. (2017).
38
Chapter 1. Introduction
Microinjection studies have identified the mesocorticolimbic brain regions involved in natural reward seeking in addition to drug reward seeking. As previously discussed, central administration of the orexin peptides can promote feeding (Asakawa et al., 2002; Sakurai et al., 1998). OX-A and OX-B, when injected into the CeA of
Syrian hamsters also promotes feeding (Alò, Avolio, Mele, Di Vito, & Canonaco, 2015) and OX-A in the VP can enhance the taste reactivity to sucrose thought to express
“liking” (Ho & Berridge, 2013). Thus, orexinergic activity in key reward regions like the VP and amygdala promote appetitive motivation for natural reinforcers.
These subtleties of the orexin/hypocretin system are mirrored in the responses of orexin neurons to various reinforcers. Hamlin et al. (2008) showed that orexin neuron activation, as evidenced by c-Fos expression, was increased during renewal for cocaine.
However, this same increase was not evident for alcohol or sucrose and binge-like alcohol consumption has been shown in mice to reduce OX-A expression in the hypothalamus (Hamlin et al., 2006; Hamlin et al., 2007; Olney, Navarro, & Thiele,
2015). For each of these operant studies, an FR1 schedule was used and there was a relatively short self-administration period of 5 - 10 days. Marchant et al. (2014) found similar null results for orexin neuron activation following context-induced reinstatement of alcohol seeking after punishment-induced abstinence. In their protocol, Marchant et al. (2014) trained rats with 6 FR1 sessions and 6 variable interval 30s (VI30). This was much shorter period of training than used by Moorman, James, Kilroy, and Aston-Jones
(2016), who gave rats a three week period of FR1 training followed by an additional 10 days to transition rats to FR2 and then FR3. Moorman et al. (2016) observed significant correlations between active lever responding and orexin/c-Fos double-labelling in the
DMH and LH during reinstatement. Although these studies vary in multiple aspects, such as the use of intermittent alcohol pre-exposure protocols, reinforcement schedule, 39
Chapter 1. Introduction
length of training, and reinforcer, the differential results suggest a complex interaction between the animal’s reinforcement conditions and the involvement of the orexin/hypocretin system.
Most studies of orexin neuron activation during reinstatement have focussed on contextual stimuli. Contextual stimuli did not result in increased orexin/c-Fos double labelling for alcohol (Hamlin et al., 2007; Marchant et al., 2014), but have been shown to be correlated with orexin neuron activation (Moorman et al., 2016). However, a discriminative olfactory cue for alcohol did produce greater orexin/c-Fos double- labelling during reinstatement when compared to the olfactory cue that was not paired with alcohol (Dayas, McGranahan, Martin-Fardon, & Weiss, 2008). In this paradigm, rats had equal exposure to both discriminative cues and extinction occurred in a third context that lacked either olfactory cue. Similarly, discriminative stimuli result in higher levels of orexin/c-Fos double labelling for rats trained to self-administer cocaine
(Martin-Fardon, Cauvi, Kerr, & Weiss, 2016). Fewer studies have examined cue- induced reinstatement, but Moorman et al. (2016) did not find that there was an orexin/c-Fos correlation in rats following cue-induced reinstatement. Nonetheless, classic orexin antagonist studies have targeted cue-induced reinstatement for reinforcers such as cocaine and alcohol (Lawrence et al., 2006; Smith et al., 2009) and so further investigation of whether discrete reward-associated cues stimulate orexin neuron activation are warranted.
40
Chapter 1. Introduction
Figure 5. Microinjection studies and natural reinforcers. Orexin peptides promote feeding and taste reactivity to sucrose, but null results have been reported for FR3 self- administration following central administration of an OX2 receptor antagonist (TCS-
OX2-29) or for reinstatement of sucrose seeking when an OX1 receptor antagonist (SB-
334867) was injected into the prefrontal cortex. Adapted from Khoo et al. (2017).
41
Chapter 1. Introduction
3. Selecting Orexin Antagonists for Behavioural Studies
Limitations of SB-334867. As noted previously, disorders of appetitive motivation are among the best established examples where an excess of orexin signalling is thought to be important. Indeed, much of the evidence reviewed here, and revealing positive effects of orexin receptor manipulations on appetitive behaviour, rely on the effects of orexin receptor antagonists. Moreover, the vast majority of this positive evidence has relied on use of SB-334867.
The use of SB-334867 as the dominant orexin antagonist has several limitations.
Even though the majority of studies of orexin and appetitive behaviour use SB-334867
(Table 1), it is difficult to dissolve and different groups have used vehicles with varying concentrations of dimethyl sulfoxide (DMSO), hydrochloric acid, 2-hydroxypropyl-β- cyclodextrin (2HPβCD), or combinations of all of these excipients (James et al., 2011;
Moorman et al., 2017; Rusyniak, Zaretsky, Zaretskaia, Durant, & DiMicco, 2012).
Some of these excipients, such as 2HPβCD, are also not very effective at dissolving SB-
334867 (McElhinny Jr et al., 2012), so several studies are likely to have used suspensions rather than solutions for their systemic injections or targeted microinjections. SB-334867 also hydrolyses at rest, with hydrolysis occurring more quickly if the compound is supplied as a hydrochloric acid salt or dissolved in acid
(McElhinny Jr et al., 2012). Its selectivity for the OX1 receptor over the OX2 receptor is only 50-fold and can still result in inhibition of OX2 receptor-mediated calcium responses (Porter et al., 2001; Smart et al., 2001). It also shows some affinity for serotonin receptors 5-HT2B and 5-HT2C, with only 100-fold selectivity for OX1 receptors (Porter et al., 2001). These off-target effects of SB-334867 could also be one explanation for positive effects of the compound in some studies (Jupp et al., 2011a;
Lawrence et al., 2006; Moorman & Aston-Jones, 2009; Richards et al., 2008) but not 42
Chapter 1. Introduction
other others (e.g. Shoblock et al., 2011). It is therefore important to use orexin antagonists other than SB-334867 to ensure that results are as free as possible from confounds.
43
Chapter 1. Introduction
Table 1
Summary of orexin antagonists used in studies of appetitive motivation
Antagonist Studies
SB-334867, selective OX1 Anderson et al. (2014) antagonist Boutrel et al. (2005) Brown et al. (2016) Cason and Aston-Jones (2013b) Cason and Aston-Jones (2013a) España et al. (2010) Harris et al. (2005) Haynes et al. (2002) Hollander et al. (2008) Hollander et al. (2012) Hooshmand et al. (2017) Jupp et al. (2011a) Jupp et al. (2011b) Lawrence et al. (2006) LeSage et al. (2010) Li et al. (2011) Moorman and Aston-Jones (2009) Moorman and Aston-Jones (2010) Moorman et al. (2017) Muschamp et al. (2014) Olney et al. (2015) Plaza-Zabala et al. (2013) Richards et al. (2008) Smith et al. (2009) Smith et al. (2010b) Smith and Aston-Jones (2012) Zhou et al. (2012a) Zhou et al. (2012b)
SB-408124, selective OX1 Shoblock et al. (2011) antagonist Wang et al. (2009)
GSK1059865, selective OX1 Gozzi et al. (2011) antagonist Lopez et al. (2016) Piccoli et al. (2012)
ACT-335827, selective OX1 Steiner, Sciarretta, Pasquali, and Jenck antagonist (2013c)
TCS-OX2-29 (a.k.a. 4PT), selective Brown et al. (2013) OX2 antagonist Plaza-Zabala et al. (2013) Smith et al. (2009)
44
Chapter 1. Introduction
Antagonist Studies
JNJ-10397049, selective OX2 Shoblock et al. (2011) antagonist
LSN2424100, selective OX2 Anderson et al. (2014) antagonist
2-SORA 18, selective OX2 Uslaner et al. (2014) antagonist NBI-80713, selective OX2 Schmeichel et al. (2015) antagonist
Almorexant, dual orexin receptor LeSage et al. (2010) antagonist Srinivasan et al. (2012)
TCS 1102, dual orexin receptor Winrow et al. (2010) antagonist
DORA-22, dual orexin receptor Tannenbaum et al. (2016) antagonist
45
Chapter 1. Introduction
Alternative OX1 receptor antagonists. SB-334867 was the first small molecule
OX1 receptor antagonist, but there are several alternatives available. SB-408124 and
SB-674042 are both small molecule selective OX1 receptor antagonists with good potency and selectivity profiles. SB-408124 has greater affinity than SB-334867 and also approximately 50-fold more selective for OX1 receptors relative to OX2 receptors
(see Table 3 for affinities and/or potencies at each receptor), but SB-674042 has even greater affinity for the OX1 receptor and is 100-fold more selective for OX1 receptors
(Langmead et al., 2004). Thus, assuming equal efficacy, a lower dose or concentration of SB-408124 would be required to obtain a given effect than SB-334867, but the risk of off-target effects at the OX2 receptor is approximately the same. SB-408124 has been reportedly dissolved in artificial cerebrospinal fluid (aCSF; Wang et al., 2009), it seems like this may have been a suspension rather than a solution because its octanol/water partition coefficient (XLogP) is 4.02 and other groups have used cyclodextrins and other excipients in preparing SB-408124 (Shoblock et al., 2011). SB-674042 has not been reported outside of in vitro experiments (Ellis et al., 2006), possibly due to relatively poor drug-like properties (see Table 2) such as an XLogP of 6.81 and a high topological polar surface area (TPSA) of 100.36 Å2. High XLogP and TPSA are associated with reduced solubility and bioavailability (Ertl, Rohde, & Selzer, 2000;
Lipinski, 2004; Lipinski, Lombardo, Dominy, & Feeney, 1997) which may explain why
SB-674042 has been used exclusively in vitro.
46
Chapter 1. Introduction
Table 2 Physicochemical properties associated with drug-like compounds Physicochemical Property Pharmacokinetic Implications (if exceeded)
Lipinski’s Rule of Five
Molecular weight ≤ 500 Poor solubility/permeability/oral availability
Hydrogen bond donors ≤ 5 Poor membrane permeability
Hydrogen bond acceptors ≤ 10 Poor membrane permeability
XLog P ≤ 5 Poor solubility/permeability
Polar surface area* 60 – 70 Å2 Poor CNS penetration
* Polar surface area may be calculated by generating a 3D computer model and summing the 3D surface area or (topologically) from the number and type of polar fragments in the molecular as described by Ertl et al. (2000).
47
Chapter 1. Introduction
Another notable small molecule OX1 receptor antagonist is GSK1059865.
GSK1059865 has a slightly better selectivity profile, with 80-fold selectivity for OX1 receptors over OX2 receptors (Table 3). The results of the binding assay screen performed by CEREP for activity at 113 receptors, transporters, and ion channels found inhibitory effects at κ-opioid receptors with a pKi of 6.5 (Gozzi et al., 2011). While the drug has been used in a few studies on appetitive motivation (Gozzi, Lepore, Vicentini,
Merlo-Pich, & Bifone, 2013; Lopez et al., 2016; Piccoli et al., 2012) and as a control compound while characterising other leads during drug development (Bonaventure et al., 2015), it has not been widely used. GSK1059865 has been reportedly dissolved using combinations of excipients including N-methyl-2-pyrrolidone, Solutol, 2HPβCD,
Tween, DMSO, and sodium lauryl sulfate (Gozzi et al., 2011; Lopez et al., 2016;
Piccoli et al., 2012). The variety and concentrations of these complexing agents, surfactants, and organic solvents suggests that researchers have difficulty working with
GSK1059865 because of poor aqueous solubility, which is consistent with its XLogP of
4.4. Combined with a lack of commercial availability, its superior selectivity profile compared to SB-334867 is not sufficient to make it a practical option for many researchers.
Actelion Pharmaceuticals have recently developed a potent and selective OX1 receptor antagonist, ACT-335827, that presents a credible alternative to SB-334867.
ACT-335827 has up to 70-fold selectivity for the OX1 over OX2 receptor, although this selectivity is reduced to 30-fold at high concentrations (Steiner et al., 2013a). Its selectivity for the orexin receptors is also superior to GSK1059865 and SB-334867 because an MDS Pharma screen found only 1 hit at the melatonin MT1 receptor (Steiner et al., 2013a), which has not been shown to have any direct relevance to orexin signalling, unlike κ-opioid receptors (Muschamp et al., 2014). ACT-335827 has high
48
Chapter 1. Introduction
molecular weight (MW) of 518.28 and XLogP of 6.6, which indicate reduced solubility and bioavailability (Lipinski, 2004; Lipinski et al., 1997). Although it has been reported to have no water solubility (Steiner et al., 2013a) and is formulated in polyethylene glycol and methyl cellulose by Actelion scientists (Steiner et al., 2013c), it has also been reportedly administered in a sonicated saline solution/suspension with behavioural effects (Beig, Dampney, & Carrive, 2015a). This suggests that ACT-335827 has properties that are sufficient for use in preclinical animal models, provided that the drug is prepared in a water miscible co-solvent and mixed well if administered as a suspension. Its potency, selectivity, and commercial availability make it a reasonable alternative to SB-334867 that is satisfying both theoretically and practically. Although
ACT-335827 is now commercially available, it has not been widely used in the literature. It has been shown to reduce preference for high fat/sugar foods without affecting overall calorie intake in a rat model of diet-induced obesity (Steiner et al.,
2013c) and to reduce cardiovascular responses to a novel context (Beig et al., 2015a).
OX2 receptor antagonists. The first selective OX2 receptor antagonist, EMPA, was reported nearly a decade after the discovery and characterisation of SB-334867
(Malherbe et al., 2009a). EMPA has over 1000-fold selectivity for OX2 receptors over
OX1 receptors and very high selectivity for the orexins over other receptors. Malherbe et al. (2009a) report that although a CEREP screen initially found that EMPA had activity at the κ-opioid receptor and the hV1a vasopressin receptor, follow up dose-response curves showed that the IC50 was in the micromolar range for both receptors. Although
EMPA has a high TPSA of 101.08 Å2, the polar surface area reported by Malherbe et al.
(2009a) is only 73 Å2, which is only slightly above the recommendations of Kelder,
Grootenhuis, Bayada, Delbressine, and Ploemen (1999) that polar surface area be less than 60 – 70 for optimal brain penetration. Other physicochemical properties are also
49
Chapter 1. Introduction
reasonably favourable, like its XLogP of 3.44, so it is unsurprising that it has favourable ex vivo binding kinetics and can inhibit OX-B-induced hyperlocomotion in mice
(Malherbe et al., 2009a). Other studies have used EMPA in mice and rats to study cardiovascular responses (Beig et al., 2015a; Beig, Horiuchi, Dampney, & Carrive,
2015b) and avoidance learning (Palotai, Telegdy, Ekwerike, & Jászberényi, 2014). Its lower XLogP also allows it to be dissolved more readily and it has been reported to be dissolved in solutions of Tween and as little as 1% DMSO (Beig et al., 2015a; Beig et al., 2015b; Malherbe et al., 2009a; Socała, Szuster-Ciesielska, & Wlaź, 2016). Its main drawback is its high levels of protein binding, with a free fraction of only 10% in rats
(Malherbe et al., 2009a). EMPA is therefore a good candidate drug for preclinical researchers interested in targeting the OX2 receptor because it has negligible off-target effects, has generally favourable physico-chemical properties, and is commercially available.
Johnson & Johnson have also contributed an OX2 receptor antagonist, JNJ-
10397049, that has been used in preclinical studies. The development of JNJ-10397049 was first reported by McAtee et al. (2004) who showed that it had 600-fold selectivity for OX2 receptors over OX1 receptors. However, it has some issues with off-target effects with a CEREP screen reporting inhibition at κ-opioid, neuropeptide Y receptor
Y1, and the neurokinin NK3 receptor (McAtee et al., 2004). These selectivity issues are a distinct disadvantage for what is otherwise a compound with favourable properties.
JNJ-10397049 has a MW of 481.98, TPSA of 59.59 Å2, five hydrogen bond acceptors, and 2 hydrogen bond donors, consistent with Lipinski’s Rule of Five (Lipinski, 2004;
Lipinski et al., 1997). Unfortunately, it has a high XLogP of 5.93 and low aqueous solubility, making it somewhat difficult to work with. Although it has been shown to be brain penetrant and to achieve high OX2 receptor occupancy in rats, JNJ-10397049 is
50
Chapter 1. Introduction
typically dissolved in a solution of 5% Pharmasolve, 15% Solutol and 15% 2HPβCD
(Dugovic et al., 2014; Dugovic et al., 2009; Shoblock et al., 2011) or using fatty acid esters like migliol 812N (Piccoli et al., 2012). The use of these multiple, high MW organic co-solvents and complexing agents would result in a solution that is too viscous for microinjection studies, which requires the use of organic solvents, like the mixture of DMSO, methanol and hydrochloric acid used by Mavanji et al. (2015). The solubility and selectivity problems of JNJ-10397049 suggest that alternatives are likely to be more attractive.
51
Chapter 1. Introduction
Table 3
Synthetic orexin antagonist affinities and potencies
a Antagonist Measure OX1 OX2 Reference
b SB-334867 pKb 7.24 ± 0.04 - Smart et al. (2001)
b b Kb (nM) 27.8 ± 2.6 1704 ± 266 Langmead et al. (2004)
b b SB-408124 Kb (nM) 21.7 ± 2.3 1405 ± 284 Langmead et al. (2004)
b b SB-674042 Kb (nM) 1.1 ± 0.1 129 ± 15 Langmead et al. (2004)
GSK1059865 pKb 8.8 6.9 Gozzi et al. (2011)
ACT-335827 IC50 (nM) 6 417 Steiner et al. (2013a)
b EMPA IC50 (nM) >10 000 8.8 ± 1.7 Malherbe et al. (2009a)
c c JNJ-10397049 pKi 5.3 – 5.8 8 – 8.6 McAtee et al. (2004)
pKi 5.7 8.2 Dugovic et al. (2009)
b b LSN2424100 Ki (nM) 393 ± 47 4.49 ± 1.39 Fitch et al. (2014)
b b Kb (nM) 90.3 ± 17.7 0.44 ± 0.11
2-SORA 18 Ki (nM) 94.5 0.07 Mercer et al. (2013)
IC50 (nM) 292 12
NBI-80713 Ki (nM) 87 2.2 Schmeichel et al. (2015)
b b IPSU pKi 6.34 ± 0.06 7.23 ± 0.04 Betschart et al. (2013)
d d pKb 6.32 – 6.14 8 – 7.68 Callander et al. (2013)
TCS-OX2-29 IC50 (nM) > 10 000 40 Hirose et al. (2003)
52
Chapter 1. Introduction
a Antagonist Measure OX1 OX2 Reference
Almorexant Ki (nM) 2.7 0.19 Winrow et al. (2012)
Kb (nM) 128.4 118.9
b b pKb 8.44 ± 0.06 9.02 ± 0.07 Faedo et al. (2012)
d d pKb 7.8 – 7.75 8.3 – 9.09 Callander et al. (2013)
IC50 (nM) 13 8 Brisbare-Roch et al. (2007)
b b SB-649868 pKi 9.5 ± 0.02 9.4 ± 0.05 Di Fabio et al. (2011)
b b pKb 9.67 ± 0.03 9.64 ± 0.03 Faedo et al. (2012)
d d pKb 9.03 – 9.38 9.52 – 9.82 Callander et al. (2013)
d d Suvorexant pKb 8.39 – 8.73 9 – 9.53 Callander et al. (2013)
Ki (nM) 0.55 0.35 Cox et al. (2010)
IC50 (nM) 50 56
TCS 1102 Ki (nM) 3 0.2 Bergman et al. (2008)
IC50 (nM) 17 4 a Unless otherwise indicated, antagonist affinity and potency is measured against OX-A- induced activity. b Error is reported as standard error of the mean (SEM). c Range represents a 95% confidence interval. d Range represents results after 30 min and results after 4 h.
53
Chapter 1. Introduction
There has recently been a proliferation of new selective OX2 receptor antagonists used in the literature. LSN2424100 was recently reported in the literature by
Eli Lilly with 200-fold selectivity for the OX2 receptor over the OX1 receptor (Fitch et al., 2014). Its physicochemical properties are generally favourable with a MW of 407.11 and TPSA of 74.44, but a high XLogP of 6.82. Although LSN2424100 has been reported to have behavioural effects in rats (Anderson et al., 2014), it is virtually insoluble and has only been reported as a suspension in carboxymethylcellulose and polysorbate (Anderson et al., 2014; Fitch et al., 2014). It also lacks commercial availability and is unlikely to be a widely used OX2 receptor antagonist at this stage.
Merck has also developed a series of 2,5-diarylnicotinamides as selective OX2 receptor antagonists and has tested the most promising, 2-SORA 18, against nicotine reinstatement (Mercer et al., 2013; Uslaner et al., 2014). 2-SORA 18 has over 1000-fold selectivity and 24-fold potency at the OX2 receptor relative to the OX1 receptor (Mercer et al., 2013). However, it has poor selectivity for the orexin receptors, with an MDS
Pharma screen showing inhibition with micromolar IC50’s at dopamine D3 receptors, µ- opioid receptors, adenosine transporters, monoamine transporters, 5-HT1A, 5-HT1B and
5-HT2B serotonin receptors, and the HERG potassium channel (Mercer et al., 2013).
This represents around 200-fold greater potency at the OX2 receptor than at serotonin receptors or adenosine transporters. Its calculated physicochemical properties are a MW of 494, five hydrogen bond acceptors, one hydrogen bond donor, TPSA of 72.28 Å2, and XLogP of 5.32, which are generally favourable but suggest poor water solubility. It also lacks commercial availability.
A third recently used selective OX2 receptor antagonist is NBI-80713.
Schmeichel et al. (2015) report that NBI-80713 (N-[(1R)-2,3-dihydro-1H-inden-1-yl]-2-
{[2-(3,4-dimethoxyphenoxy)ethyl] [(4-fluorophenyl)methyl]amino}acetamide) has 40- 54
Chapter 1. Introduction
fold selectivity for the OX2 over the OX1 receptor. Its drug-like properties can be calculated to be reasonably favourable, with MW of 478.56, six hydrogen bond acceptors, one hydrogen bond donor, TPSA of 60 Å2, and XLogP of 5.38. However, it is not commercially available and there is no report that its selectivity has been screened against libraries using any of the common screening services (e.g. Panlabs/MDS
Pharma or CEREP).
IPSU has recently been reported and used to study sleep, but is currently not commercially available. It was developed by Novartis but has relatively poor selectivity, with only 10 – 40 -fold selectivity for OX2 over OX1 receptors (Table 3) and no details of selectivity relative to other receptors or enzymes (Betschart et al., 2013; Callander et al., 2013). However, it has been shown to have different effects on sleep architecture than suvorexant (Hoyer et al., 2013), and further studies have not been reported in the literature. Its lack of selectivity compared to other OX2 receptor antagonists and lack of commercial availability make it an unattractive option for examining orexin effects.
TCS-OX2-29 has several properties that make it an ideal selective OX2 receptor antagonist for research purposes. It was the first small molecule OX2 receptor antagonist, is potent at the OX2 receptor with an IC50 of 40 nM, has more than 250-fold selectivity for OX2 over the OX1 receptor, and has no off-target effects in a screen of 50 other receptors, ion channels, or transporters (Hirose et al., 2003). Its physicochemical properties are some of the most drug-like of all of the orexin receptor antagonists, with a MW of 397.24, TPSA of 63.69, and XLogP of 2.82. As a result, TCS-OX2-29 has high solubility in water (Hirose et al., 2003) and has been reported to have produced behavioural results in rats following microinjections using aCSF or saline (Brown et al.,
2013; Kastman et al., 2016). It is also commercially available unlike several other OX2 receptor antagonists. TCS-OX2-29 is therefore an ideal preclinical OX2 receptor 55
Chapter 1. Introduction
antagonist because it is readily available and has good pharmacological and physicochemical properties.
Dual orexin receptor antagonists. The first and most widely used dual orexin receptor antagonist is almorexant, developed by Actelion Pharmaceuticals. Almorexant has been shown to have 600-fold selectivity for the orexin receptors relative to any 89 other receptors and enzymes (Brisbare-Roch et al., 2007). As shown in Table 3 it has been characterised in multiple assays and shown to be approximately equally selective for both OX1 and OX2 receptors, with a slight preference for the OX2 receptor. Its physicochemical properties are also generally very favourable, with a TPSA of 50.3, but a very high XLogP of 8.25. Nonetheless, it is orally available and crosses the blood brain barrier and has been shown to have in vivo efficacy in rats and dogs (Brisbare-
Roch et al., 2007). Almorexant was successful enough to advance to clinical trials and early results indicated that it was well-tolerated (Hoch, Hoever, Alessi, Marjason, &
Dingemanse, 2011; Hoever et al., 2013), did not potentiate alcohol-induced impairments (Hoch et al., 2013), and induced sleep (Brisbare-Roch et al., 2007; Hoever et al., 2012). Unfortunately, almorexant was shown to produce mild liver impairment which eventually resulted in termination of its clinical development (Shakeri-Nejad,
Hoch, Hoever, & Dingemanse, 2013). It also does not have widespread commercial availability and groups have either obtained the drug directly (as a gift) from Actelion
Pharmaceuticals (Beig et al., 2015a; Beig et al., 2015b; LeSage et al., 2010) or had it synthesised (Srinivasan et al., 2012). While almorexant would be an excellent dual orexin receptor antagonist for preclinical research, the lack of widespread commercial availability is a barrier.
A dual orexin receptor antagonist from GlaxoSmithKline, SB-649868, also reached clinical trials, but is not commercially available. SB-649868 has high affinity at 56
Chapter 1. Introduction
both the OX1 and OX2 receptors and screening by CEREP found negligible activity at off-target receptors, ion channels, and enzymes (Di Fabio et al., 2011). SB-649868 was reported to have been well tolerated at single doses of 10 – 80 mg or daily 5 – 30 mg doses for 15 days in Phase I clinical trials (Bettica et al., 2012a). In Phase II trials SB-
649868 was reported to increase sleep in men with insomnia, without significantly greater adverse effects than placebo, although there were some inconsistent next-day effects on delayed word recall (Bettica et al., 2012b). Studies of SB-649868 were eventually discontinued over safety concerns (GlaxoSmithKline, 2010).
Merck’s suvorexant, which recently became the first orexin receptor antagonist approved for clinical use, would be another ideal choice for preclinical research.
Similarly to almorexant, suvorexant is approximately equipotent at both the OX1 and
OX2 receptors (Cox et al., 2010). It was also shown to be essentially free of off-target effects in an MDS Pharma screen for 170 receptors, enzymes, and ion channels (Cox et al., 2010). Its physicochemical properties would also be considered very drug-like if examined from first principles because it has 7 hydrogen bond donors, no hydrogen bond acceptors, a MW of 450, TPSA of 80.29 Å2, and XLogP of 4.24. It has been reported to have good thermal and pH-dependent stability, but requires a 20% d-α- tocopheryl polyethylene glycol-100-succinate (Vitamin E-TPGS) solution in order to dissolve (Cox et al., 2010). Results from clinical trials of suvorexant show that it promotes sleep and is generally safe if used nightly over the course of a year (Herring et al., 2012; Ma et al., 2014; Michelson et al., 2014; Sun et al., 2013), however Merck was required to make a lower dose available because the Food and Drug Administration had some safety concerns at the higher doses (Merck, 2013; Rhyne & Anderson, 2015).
While using a clinically approved drug in preclinical research would be ideal,
57
Chapter 1. Introduction
suvorexant is not commercially available so must be synthesised in-house, as Tsuneki et al. (2016) have done.
TCS 1102 is a commercially available dual orexin receptor antagonist with useful properties (Bergman et al., 2008). It was, like suvorexant, developed by Merck, but has a slightly different chemical backbone (Scammell & Winrow, 2011). As shown in Table 3, TCS 1102 has excellent affinity and potency for the OX1 and OX2 receptors.
It has also been shown to have only minimal off-target effects in an MDS Pharma screen at a protanoid monoamine transporter, thromboxane A2, and thyrotropin releasing hormone (Bergman et al., 2008). Its other physicochemical properties are generally quite favourable, with four hydrogen bond acceptors, one hydrogen bond donor, a MW of 470.18, but a high TPSA of 92.5 and XLogP of 4.9. However, the log P reported by Bergman et al. (2008) of 3.4 is lower than the calculated XLogP recorded in
PubChem (CID: 11960895), suggesting that there may be a more favourable profile in practical applications than theoretical calculations suggest. Moreover, Bergman et al.
(2008) have shown that TCS 1102 is brain penetrant and bioavailable in rats. TCS 1102 has been prepared in aqueous solutions with excipients such as polyethylene glycol
(Chen et al., 2014) or 20% Vitamin E-TPGS (Winrow et al., 2010). While the added viscosity from these excipients may make microinjections slightly more difficult, TCS
1102 is a realistic option for dual orexin receptor antagonism given its favourable properties and commercial availability.
4. Aims and Hypotheses
The orexin/hypocretin system has several important functions in arousal, appetite, and reward and the first orexin-based pharmacotherapy recently entered clinical use for insomnia. The orexin/hypocretin system has a pathophysiological role in
58
Chapter 1. Introduction
sleep disorders such as narcolepsy and insomnia, as well as possible roles in PD, cancer, cachexia, and anxiety. The involvement of orexin signalling in drug seeking has been identified in preclinical models for several drugs of abuse, including cocaine, opioids, alcohol, and nicotine. Orexin neurons project widely throughout the brain and their projections to mesocorticolimbic reward circuitry have been implicated in reward seeking behaviours including drug self-administration and relapse-like behaviours. The motivational activator theory is the first coherent account of role of the orexin/hypocretin system in reward seeking and states that the orexin/hypocretin system is involved under conditions where highly motivated behaviours are required. However, many of the studies that inform current understanding of the orexin/hypocretin system should be interpreted with caution because the most commonly used orexin antagonists have important limitations, such as poor stability or selectivity. Many of the compounds available to researchers also have poor solubility, making them difficult to administer especially for in vivo microinjection studies.
Rationale. The motivational activator theory is an attractive explanation for the function of the orexin/hypocretin system in reward and appetitive motivation. It predicts that the orexin/hypocretin system should be involved in appetitive motivation for reinforcers that are high in salience or hedonic value such as drugs or highly palatable foods, when reward seeking is under the control of reward-associated cues, or when rewards are available under a high unit-cost. If this is the case, then orexin antagonists would make effective anti-craving compounds for the treatment of substance use disorders. Indeed, a clinical trial of the dual orexin receptor antagonist, suvorexant, is already recruiting participants in order to examine the effect of orexin antagonism on cocaine cue reactivity (The University of Texas Health Science Center, 2016). Further
59
Chapter 1. Introduction
preclinical evidence will help to establish whether similar trials for legal drugs – alcohol and nicotine – are also warranted and to test the motivational activator theory.
Choice of drug. In order to test the motivational activator theory, TCS 1102 presents the best option. It is potent, selective, brain penetrant, bioavailable in rats, and a dual orexin receptor antagonist like the clinically approved insomnia medication suvorexant. Results obtained using TCS 1102 would therefore have direct relevance to clinical trials, unlike single orexin receptor antagonists,would present evidence that there may or may not be possible additional uses of suvorexant as an addiction pharmacotherapy. Intracerebroventricular administration is used because it delivers the drug directly to the brain bypassing the blood brain barrier, first pass hepatic metabolism, and peripheral side-effects.
Aims. The aims of the present study were therefore to examine the effect of a dual orexin receptor antagonist, TCS 1102, on operant appetitive motivation for alcohol, nicotine, and palatable food.
Hypotheses. It was hypothesised that TCS 1102 would reduce alcohol self- administration and reacquisition, nicotine self-administration and reinstatement, and palatable food self-administration and reinstatement.
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Chapter 2. Methods and Results
Experiment 1. Orexin-A-induced Feeding
The aim of Experiment 1 was to optimise an assay of OX-A-induced feeding against which the dual orexin receptor antagonist, TCS 1102, could be tested. OX-A and OX-B-induced feeding were the first behavioural effects demonstrated for the orexin system (Sakurai et al., 1998). Experiment 1 was therefore designed to replicate these results, but in an operant setting to allow measurement of behaviour to be automated.
Methods
Subjects
Experimentally naive Sprague-Dawley rats at 6 weeks of age weighing 240-280 g were obtained from the Animal Resource Centre (Perth, WA, Australia). Upon arrival rats (n = 16) were housed in groups of four in plastic cages (309 mm wide, 617 mm long, 284 mm high) in ventilated racks with corncob bedding and an alloy mezzanine.
They were maintained on a 12 h: 12 h light/dark cycle (lights on at 07:00) and ad libitum access to food and water prior to behavioural testing, after which they received
22 g/rat/day. Rats were given at least one week to acclimate to the colony room before undergoing behavioural experiments, which were conducted during the light cycle. All procedures were approved by the University of New South Wales Animal Care and
Ethics Committee (ACEC) and conducted in accordance with the National Health and
Medical Research Council’s Australian Code for the Care and Use of Animals for
Scientific Purposes.
Drugs and Reagents
Atropine (0.65 mg/mL, as sulphate), Benacillin (150 mg/mL procaine penicillin,
150 mg/mL benzathine, 20 mg/mL procaine hydrochloride) and xylazine (20 mg/mL) 61
Chapter 2. Methods and Results
were obtained from Troy Laboratories (Smithfield, NSW, Australia). Carprofen (50 mg/mL), Marcain® (5 mg/mL bupivacaine hydrochloride anhydrous, 8 mg/mL sodium chloride) and 0.9% w v-1 saline were obtained from Pfizer (West Ryde, NSW,
Australia). DBL® Cephazolin (100 mg/mL) was obtained from Hospira Australia
(Mulgrave, VIC, Australia). Ketamine (100 mg/mL) was obtained from Ceva Animal
Health (Glenorie, NSW, Australia). Riodine™ (10% w v-1 povidone iodine) and
Riotane™ (0.5% chlorhexidine, 70% ethanol in water v v-1) was obtained from ORION
Laboratories (Balcatta, WA, Australia). Sodium pentobarbitone (325 mg/mL) was obtained from Virbac Animal Health (Milperra, NSW, Australia) and diluted with bupivacaine to reduce peritoneal irritation upon injection. OX-A (Batch No:
13D/179299; MW: 3561.12) was obtained from Tocris Bioscience (Bristol, United
Kingdom). OX-A was dissolved at a concentration of 0.5 µg/µL in 0.9% saline and stored at -20C until use.
Surgery
Rats were anaesthetised using a stock solution of ketamine and xylazine (1 mL ketamine, 0.5 mL xylazine and 5.1 mL saline; 6.5 mL/kg) via intra-peritoneal (i.p.) injection. Local anaesthetic (bupivacaine) was also applied. Anaesthesia was confirmed by the absence of reflex response to a paw pinch. The non-steroidal anti-inflammatory drug (NSAID) carprofen (5 mg/kg, s.c.) and atropine (1 mg/kg, i.p.) were administered preoperatively for pain relief and to prevent adverse reactions to anaesthesia respectively. After induction of anaesthesia, the rat’s head was shaved, swabbed with iodine and placed in a stereotaxic frame (David Kopf Instruments, Tujunga, CA,
U.S.A.). The sterility of tools and implants was maintained by immersion in 70% ethanol/0.5% chlorhexidine. Guide cannulae (26 ga; PlasticsOne, Roanoke, VA, U.S.A.) were implanted unilaterally targeting the right lateral ventricle (Brown et al., 2013)
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Chapter 2. Methods and Results
using the following coordinates (from Bregma) based on Paxinos and Watson (1998): -
0.8 mm anteroposterior (A/P), +1.5 mm mediolateral (M/L) and -4 mm dorsoventral
(D/V). Although the cannula implant was unilateral, previous studies have used unilateral implants because the ventricles are connected and so the solution diffuses through both sides of the brain (Brown et al., 2013). The guide cannula was secured to the skull using dental acrylic (Vertex Dental, Zeist, The Netherlands). Prophylactic
Benacillin (0.3 mL, i.p.) and cephazolin (0.15 mL, s.c.) were administered to prevent infection. Rats were given 7 days recovery before behavioural experiments during which time they were monitored and weighed daily using the monitoring sheet approved by the ACEC.
Behaviour
Operant self-administration was conducted in eight identical operant chambers
(Med Associates, St. Albans, VT, U.S.A.) housed in sound and light attenuation boxes equipped with fans to mask external noise. Each chamber was 24 cm long, 30 cm wide and 21 cm high, with a Perspex door, rear wall and ceiling and stainless steel end walls.
On the left end wall was a houselight and the centre of the right end wall had a recessed magazine connected to a pellet dispenser which delivered 45 mg food pellets (#F0165,
BioServ, Flemington, NJ, U.S.A.). To the right of the magazine was a retractable lever that was initially used as the manipulanda to activate delivery of food pellets.
Rats were first given six daily 1 h training sessions on an FR1 schedule whereby each lever press resulted in the delivery of a food pellet reward. Lever presses made while the pellet dispenser was operating were recorded but did not trigger the delivery of an additional food pellet. However, once the pellet dispenser finished operating (in 1 s), the next lever press was able to produce pellet delivery. However, following preliminary testing where rats did not respond to OX-A microinjections, the task was
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Chapter 2. Methods and Results
changed to a variable interval-20 s (VI-20) schedule that rewarded magazine entries and the session length was extended to 2 h. Rats were then retrained for an additional 7 days. This change was made to combine the seeking and consummatory components of feeding and to make the behavioural assay more similar to the free feeding experiments that have shown strong orexin-mediated effects in the past (Sakurai et al., 1998) while providing the unbiased, automated data collection afforded using an operant paradigm.
For four days prior to their first test day, rats were habituated to the microinjection procedure by having their obturators removed and replaced and gently restrained as they would be for microinjections before their operant session. 5 µL of
OX-A or its vehicle were delivered by a Chemyx F400 syringe pump (Chemyx Inc.,
Houston, TX, U.S.A.) via a 10 µL Hamilton syringe (Hamilton Company, Reno, NV,
U.S.A.) connected by polyethylene tubing to a 33 ga injector (PlasticsOne). The tubing was loaded with sterile saline and 0.5 – 1 µL of air was drawn up before 6-8 µL of solution. The microinjection was delivered over 2 min, with injectors left in place for an additional 30 s to allow the solution to circulate to both sides of the brain before being withdrawn. After receiving the microinjection, rats were returned to their home-cage for
10 min to allow the drug to circulate throughout the brain, which is typically done in
ICV studies (Brown et al., 2013). This time is necessary because it can take a few minutes for ICV-mediated effects to occur, such as angiotensin II-mediated rapid dipsogenesis (McKinley et al., 2003). Delivery of the injection was verified by checking for the movement of the air bubble in the tubing. Any excess solution was also expelled from the line which also verified that there was no blockage. The tubing and injector were flushed with 80% ethanol and reloaded with sterile saline between each microinjection.
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Chapter 2. Methods and Results
Rats were initially tested in two rounds of microinjections, initially comparing vehicle and 5 µg OX-A and then comparing vehicle and 2.5 µg OX-A. At least one day of normal self-administration was allowed between each test. Testing order within each round was counterbalanced.
Histology
After the conclusion of behavioural testing, rats were anaesthetised with an overdose of sodium pentobarbitone diluted with a local anaesthetic solution
(bupivacaine). Cresyl violet (25 µL) was injected into the cannula to verify accurate microinjection delivery. The larger volume of cresyl violet compared to microinjections ensured that it was more readily visible in the ventricles and able to spread to both sides of the brain post-mortem. Rats were decapitated and brains were removed and cut coronally with a razor blade. Rats which did not have ink present in the ventricles were excluded.
Data Analysis
Data were analysed using paired t-tests in SPSS 22.0 (IBM, New York, NY,
U.S.A.) and are presented as means ± SEM. 5 rats were excluded from final analyses because of misplaced cannulae or illness. One additional rat was excluded between the 5
µg OX-A and 2.5 µg OX-A tests due to a blocked cannula.
Results
Rats were initially tested for lever responding after 6 days of self-administration training. However, OX-A did not increase the number of lever responses (M = 60.6,
SEM = 11.1 compared to M = 70.9, SEM = 15.8 after saline) so rats were retrained on a
VI-20 magazine entry task to model free feeding. After 7 days of retraining on the VI-
20 task, rats made M = 733.8, SEM = 219 magazine entries to earn M = 59.4, SEM =
10.4 food pellets or approximately 2.7 g of food. However, 5 µg OX-A did not 65
Chapter 2. Methods and Results
significantly alter magazine entries, t(10) = 0.487, p = 0.637, or the number of rewards earned, t(10) = -2.14, p = 0.058 (Figure 6). In contrast, 2.5 µg OX-A significantly increased the number of magazine entries, t(9) = -2.49, p = 0.034 and number of rewards earned, t(9) = -5.21, p = 0.001. While there have been studies which have associated orexin signalling with anxiety (Alò et al., 2016), no additional or unusual behaviour was observed in the present experiment.
Discussion
The results of this experiment showed that 2.5µg, but not 5µg of OX-A administered ICV was able to significantly increase magazine entries and food rewards earned. This suggests a possible inverted U shaped dose response function so that the optimal dose of OX-A that stimulates feeding is a moderate one, and additional OX-A has no effect on feeding behaviour. For present purposes, these results indicate that
TCS 1102 should be tested against a dose of 2.5µg of OX-A.
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Chapter 2. Methods and Results
Figure 6. The effect of orexin-A on operant free-feeding. (a) The number of magazine entries made was not significantly altered by ICV 5µg OX-A (n = 11), (b) but it was significantly increased by 2.5µg of OX-A (n = 10). (c) Similarly, the number of rewards earned was not significantly altered by 5µg OX-A, (d) but it was significantly increased by 2.5µg OX-A. Data are means ± SEM. * p < 0.05.
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Chapter 2. Methods and Results
Experiment 2. TCS 1102 and Orexin-A-induced Feeding
TCS 1102 is a relatively new drug that has been made commercially available to research scientists (Bergman et al., 2008). As such, it is not as commonly used as the first small molecule orexin receptor antagonist, SB-334867 (Smart et al., 2001). But even though SB-334867 has been used for many years, it is not without its difficulties
(McElhinny Jr et al., 2012; see also Chapter 1 [Introduction]). Therefore, it is important to validate the efficacy of TCS 1102 against OX-A. The aim of Experiment 2 was to determine if TCS 1102 could antagonise OX-A-induced feeding.
Methods
Experimentally naive Sprague-Dawley rats at 6 weeks of age weighing 240-280 g were obtained from the Animal Resource Centre (Perth, WA, Australia). Upon arrival rats (n = 16) were housed in groups of four in plastic cages (309 mm wide, 617 mm long, 284 mm high) in ventilated racks with corncob bedding and an alloy mezzanine.
Rats experienced a 12 h: 12 h light/dark cycle (lights on at 07:00) and ad libitum access to food and water. Rats were given a week to acclimate to the colony room, handled for a week and then surgically implanted with chronic indwelling guide cannula targeting the lateral ventricles as previously described for Experiment 1. After a week of recovery they were food restricted to 22 g/rat/day.
Drugs
TCS 1102 (Batch No: 2A/155485; MW: 475.09) was obtained from Tocris
Bioscience (Bristol, United Kingdom). The vehicle solution was 20% Vitamin E-TPGS
(Lot #BCBM7443V; Sigma-Aldrich, Castle Hill, NSW, Australia) in 0.9% saline. TCS
1102 was stored at room temperature according to the manufacturer’s instructions until it was dissolved and frozen at -20 °C in aliquots.
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Chapter 2. Methods and Results
Behaviour
Rats were then given 7 daily 2 h VI-20 free feeding sessions, where magazine entries produced delivery of 45 mg food pellets to the magazine as per Experiment 1.
They were habituated to microinjection procedures as previously described and tested for feeding behaviour following a single microinjection of saline or double microinjections of OX-A followed by either 20% Vitamin E-TPGS/0.9% saline vehicle or TCS 1102. On the single saline microinjection day, rats received 5 µL of saline and were returned to their home-cage for 10 min before being placed in the operant chamber. On the double microinjection days, rats received 2.5 µg of OX-A in 5 µL and were returned to their home-cage for 5 min. They then received the 30 µg of TCS 1102 in 20% Vitamin E-TPGS vehicle or vehicle alone and were returned to their home-cage for another 10 min before being placed in the operant chamber for behavioural testing.
At least one day of normal food self-administration was given between test days to ensure there were no residual effects carried over from one test to the next.
Histology and Data Analysis
At the end of behavioural testing rats were euthanased with an overdose of sodium pentobarbitone, injected with ink and dissected as previously described. One rat was excluded because of a misplaced cannula. Data analysis was performed in SPSS
22.0 using a repeated measures ANOVA and Helmert contrasts. Helmert contrasts were used for factors with more than two levels because they are a coherent and statistically powerful type of orthogonal contrast that compares the control condition to all others and then compares between treatment conditions (Bird, 2004).
Results
After 10 days of food self-administration rats made a mean of 582 ± 20 magazine entries, resulting in 57 ± 1.07 pellets delivered or 2.5 g of food. There was a 69
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significant main effect of test, F(2, 28) = 4.85, p = 0.016. Helmert contrasts showed that there was no difference between the saline test and two OX-A test days, F(1, 14) = 2.62, p = 0.127, however there was a significant difference in the number of magazine entries on the OX-A/Vitamin E-TPGS and OX-A/TCS 1102 test days, F(1, 14) = 7.92, p =
0.014 (Figure 7). For the number of rewards earned, there was a significant main effect of test, F(2, 28) = 14.7, p < 0.001, and Helmert contrasts showed a significant difference between the saline test and two OX-A test days, F(1, 14) = 18.54, p = 0.01, as well as between the OX-A/Vitamin E-TPGS and OX-A/TCS 1102 test days, F(1, 14) = 8.99, p
= 0.01.
Discussion
These results show that OX-A-induced feeding is significantly attenuated by
TCS 1102. This validates the use of TCS 1102 as a dual orexin receptor antagonist. It demonstrates that TCS 1102 administered ICV is able to reach brain regions that are relevant for appetitive behaviours and that its effects persist long enough to be meaningful in behavioural assays. TCS 1102 has been reported to have a short half-life of just 18 min, but this is not that much different to that of SB-334867, which has a half-life of 24 min (Bergman et al., 2008; Porter et al., 2001). Sakurai et al. (1998) showed that ICV administration of OX-A or OX-B increases feeding behaviour over 2-
4 hours in freely fed rats. While the lack of a double microinjection vehicle control
(saline/TPGS) could potentially confound the interpretation of the OX-A effect,
Experiment 1 demonstrated that at this dose the OX-A is effective in increasing feeding.
This experiment also did not include a vehicle/TCS 1102 to directly control for non- specific effects of TCS 1102 on arousal, but given the lack of locomotor effects in
Experiments 5, 7, and 8, such effects are unlikely. It can therefore be concluded that
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TCS 1102 is able to counteract orexinergic activity over the course of a standard 1 h operant behavioural session.
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Chapter 2. Methods and Results
Figure 7. Effect of the dual orexin receptor antagonist, TCS 1102, on orexin-A-induced feeding. (a) 30 µg TCS 1102 (ICV) significantly reduced the number of magazine entries made following 2.5µg OX-A administration compared to a 20% Vitamin E-
TPGS vehicle. (b) OX-A administration produced a significant increase in the number of rewards earned, which was attenuated by 30 µg TCS 1102. n = 15. Data are means ±
SEM. * p < 0.05 compared to OX-A/TPGS; # p < 0.05 compared to both OX-A testing conditions.
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Experiment 3. Palatable Food Self-Administration and Reinstatement
The orexin system has been shown to be involved in the seeking of natural rewards, but results have been mixed. For example, Borgland et al. (2009) showed that the OX1 receptor antagonist, SB-334867, had effects on PR responding for chocolate pellets. Srinivasan et al. (2012) have shown that the dual orexin receptor antagonist, almorexant, reduces 5% sucrose seeking. However, it has also been reported that SB-
334867 does not alter responding for SuperSac, a mixture of 3% glucose and 0.125% saccharin (Martin-Fardon & Weiss, 2014b). The aim of Experiment 3 was therefore to test the dual orexin receptor antagonist, TCS 1102, on operant self-administration and reinstatement of palatable food pellets. It also examined whether motivational state – hungry versus sated – determined the impact of TCS 1102 by comparing the effects of the antagonist in hungry and sated rats.
Methods
Experimentally naive Sprague-Dawley rats at 6 weeks of age weighing 240-280 g were obtained from the Animal Resource Centre (Perth, WA, Australia). Upon arrival rats (n = 24) were housed in groups of four in plastic cages (309 mm wide, 617 mm long, 284 mm high) in ventilated racks with corncob bedding and an alloy mezzanine.
Rats experienced a 12 h: 12 h light/dark cycle (lights on at 07:00) and ad libitum access to food and water. Rats were given a week to acclimate to the colony room, handled for a week and then surgically implanted with chronic indwelling guide cannula targeting the lateral ventricles using the same procedures as described for Experiment 1.
Behaviour
Rats were trained for operant self-administration of 45 mg palatable food pellets
(18.1% protein, 23.4% fat, 44.2% carbohydrate, #F06162, BioServ, Frenchtown, NJ,
U.S.A.) in 8 identical operant chambers (Med Associates, St Albans, VT, U.S.A.) 73
Chapter 2. Methods and Results
situated in sound attenuating boxes with a fan to mask external noise. Each chamber had a houselight on one side and on the other side there were two retractable levers. For half of the boxes the active lever was the left lever and for the other half the active lever was the right lever. A cue light was situated above each lever and a magazine was situated between the two levers.
Before the commencement of self-administration training, half of the rats were placed on a food restriction schedule (20g/rat/day) beginning two days before behavioural testing (hungry) while the rest of the rats remained on an ad libitum supply of food (sated). Rats then received two days of 1 hr habituation sessions. During these sessions the levers remained retracted and the houselight was on.
Self-Administration
Rats then received 10 days of 1 hr self-administration training. The levers were extended and responses on the active lever resulted in a delivery of a 45 mg palatable food pellet to the magazine. The cue light above the active lever was illuminated for 3 s and the houselight was also switched off for the rest of the timeout period, which was a total of 23 s. Responses made during the timeout period, or on the inactive nosepoke, were counted but had no programmed consequences.
Rats were then tested for self-administration following doses of 1, 3, 10 and 30
µg TCS 1102 in 20% Vitamin E-TPGS/0.9% saline vehicle following microinjection procedures as described for Experiment 1 using a within-subjects design. A day of standard self-administration was interleaved between each test day.
Extinction and Reinstatement
Following the completion self-administration testing, rats underwent 9 days of extinction training. The extinction context was identical to the self-administration context because the houselight and fan were on, but responses on the levers produced no
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programmed consequences. Rats then received a microinjection of vehicle or 30 µg
TCS 1102 10 min before a cue/prime reinstatement session. During this session, the magazine was primed with 3 palatable food pellets and active lever presses produced a cue presentation, but no additional food pellet deliveries.
Histology and Data Analysis
Microinjection site validation was performed as previously described. There was a reduction in the number of rats between the extinction tests and reinstatement tests due to damaged and blocked cannula. Data were analysed using a repeated measured
ANOVA or mixed-design ANOVA in SPSS. Data are presented as means ± SEM.
Results
Self-Administration
After 10 days of acquisition, all rats showed a strong preference for responding on the active lever, M = 120.8, SEM = 12.9, relative to the inactive lever, M = 3.52,
SEM = 0.85. Rats earned a high number of food pellets, M = 56, SEM = 5.1, corresponding to approximately 2.5 g of palatable food which magazine checks after each session showed were all consumed. There was no difference between sated and hungry rats in terms of active lever presses, t(21) = 1.03, p = 0.315, or the number of rewards earned, t(21) = 1.21, p = 0.241. ICV administration of TCS 1102 had no effect on self-administration (Figure 8). For sated rats, there was no main effect of dose, F(4,
44) = 0.61, p = 0.66, or dose x lever interaction, F(4, 44) = 0.616, p = 0.65. However, rats showed significant discrimination for the active lever over the inactive lever, F(1,
11) = 65.96, p < 0.001. There was also no effect of TCS 1102 on magazine entries, F(4,
44) = 1.01, p = 0.414, or rewards earned, F(4, 44) = 1.116, p = 0.361. Similarly, for hungry rats, there was no main effect of dose, F(4, 36) = 0.349, p = 0.843, or dose x lever interaction, F(4, 36) = 0.38, p = 0.82. Hungry rats also showed significant 75
Chapter 2. Methods and Results
discrimination for the active lever over the inactive lever, F(1, 9) = 53.76, p < 0.001.
There was also no effect of TCS 1102 on magazine entries, F(4, 36) = 0.436, p = 0.782, or rewards earned, F(4, 36) = 0.505, p = 0.732.
Cue/Prime Reinstatement
After 9 days of extinction training rats reduced their active lever presses, M =
17, SEM = 2.84. In the reinstatement session, the food-associated cues were once again available and the magazine was primed with three food pellets at the start of the session.
Rats in the sated group did not show a robust reinstatement response because although there was a session x lever interaction, F(1, 9) = 15.16, p = 0.004, there was no main effect of session, F(1, 9) = 3.196, p = 0.107 (Figure 9A). Further, TCS 1102 had no effect because there was no session x lever x dose interaction, F(1, 9) = 3.53, p = 0.093, lever x dose interaction, F(1, 9) = 0.617, p = 0.452, or session x dose interaction, F(1, 9)
= 2.256, p = 0.167. There was also no effect of the reinstatement session on the number of magazine entries, F(1, 9) = 1.439, p = 0.261, and TCS 1102 had no effect as evidenced by the lack of a session x dose interaction, F(1, 9) = 0.141, p = 0.716.
Rats in the hungry group, in contrast, showed a relatively strong reinstatement
(Figure 9B), with a significant session x lever interaction, F(1, 5) = 56.68, p = 0.001, and main effect of session, F(1, 5) = 20.6, p = 0.006. However, there was no effect of
TCS 1102 because there was no session x lever x dose interaction, F(1, 5) = 0.012, p =
0.918, or lever x dose interaction, F(1, 5) = 1.433, p = 0.285, or session x dose interaction, F(1, 5) = 1.69, p = 0.25. Magazine entries also showed a significant increase consistent with a reinstatement response. There was a significant main effect of session,
F(1, 5) = 52.9, p = 0.001, but no session x dose interaction, F(1, 5) = 0.044, p = 0.842.
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Chapter 2. Methods and Results
Discussion
These results show no effect of TCS 1102 on palatable food self-administration or reinstatement in rats that were hungry and rats that were sated. However, a robust reinstatement effect was not observed for rats in the sated group. Rats in the hungry group did show a robust reinstatement effect, despite the smaller group size due to attrition in terms of damaged or blocked cannula between the self-administration tests and the reinstatement test. While it may seem surprising that rats in both the hungry and sated groups had similar levels of active lever responding and rewards received, this may actually be due to a ceiling effect. The timeout for each pellet was 23 s, which gives a theoretical maximum of 156 pellets earned during the session. However, rats did not respond perfectly, with approximately 50% of their responses being made during the timeout period, and they would also need time to collect and consume their pellets.
Earning 50 – 60 pellets may be the maximum that is practical for a rat in this assay to collect. It is possible that placing rats on a more restrictive feeding schedule would increase the number of pellets earned. However, the mild food restriction used in this experiment was sufficient to produce differences in reinstatement, suggesting that the food restriction schedule used was adequate for promoting learning and motivation.
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Figure 8. Effect of TCS 1102 on palatable food self-administration in sated and hungry rats. There was no effect of 1, 3, 10 or 30 µg TCS 1102 (ICV) on the number of lever presses in (a) sated (n = 12) or (b) hungry rats (n = 10). Additionally, there was no effect on the number of rewards earned by (d) sated or (e) hungry rats. (g) TCS 1102 also did not show any effect on the number of magazine entries made by sated rats or
(h) hungry rats. Data are means ± SEM.
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Figure 9. Effect of ICV TCS 1102 on cue and primed reinstatement for palatable food.
(a) Rats in the sated group did not show a robust reinstatement effect in the number of lever presses made (Vehicle n = 5, TCS30 n = 6). (b) Rats in the hungry group showed reinstatement of the number of lever presses but no effect of 30 µg TCS 1102 (Vehicle n = 4, TCS30 n = 3). Similar effects were observed for magazine entries as (c) rats in the sated group did not show reinstatement and (d) rats in the hungry group showed reinstatement but no effect of TCS 1102. Data are means ± SEM.
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Experiment 4. Varied Unit Costs for Palatable Food
The motivational activator theory (see Chapter 1) posits that the orexin system contributes to behaviour under conditions of high unit-cost (Mahler et al., 2014). As reviewed previously, the impacts of orexin receptor antagonists are seen more frequently under higher, rather than lower, ratio reinforcement schedules (e.g., FR5 v
FR1). One explanation for why there was no observed effect of TCS 1102 on palatable food self-administration or reinstatement in Experiment 3 is the low unit-cost of the behavioural assay used. In order to test this possibility, the unit cost of each reward was varied using different fixed-ratios and PR. The result was also replicated at the highest fixed unit-cost tested using single orexin receptor antagonists which have all been reported to have had behavioural effects, including SB-334867 (Borgland et al., 2009;
Lawrence et al., 2006), ACT-335827 (Steiner et al., 2013a; Steiner et al., 2013c) and
TCS-OX2-29 (Brown et al., 2013).
Methods
Experimentally naive Sprague-Dawley rats (n = 16) were obtained, housed, handled and implanted with a guide cannula targeting the lateral ventricles as previously described for Experiments 1-3. The behavioural protocol was also identical to
Experiment 3 in the acquisition phase. Rats were first food restricted to 20 g/rat/day and then given 2 days of habituation and 10 days of 1 h self-administration sessions. Rats were then tested following microinjections of 30 µg TCS1103 or a 20% Vitamin E-
TPGS/0.9% saline vehicle. They were first tested for self-administration under an FR1 schedule. They were then given 3 days of FR3 self-administration training, followed by
5 days of FR5 self-administration training and tested for self-administration under FR5 conditions. Rats then received an additional 5 days of FR10 self-administration training
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and were tested under FR10 conditions. All tests were conducted using a counterbalanced within-subjects design and at least 1 day of self-administration between tests. Two rats were excluded because they lost their headmounts before the completion of behavioural testing.
Progressive Ratio
Following the completion of FR10 testing, rats were given a single PR training session. In the PR session, the number of active lever presses required for each pellet delivery was given by the function 5e(reward number × 0.2) – 5 (Richardson & Roberts, 1996) and the breakpoint was the highest number of lever presses that was performed in order to obtain a reward. Rats were then tested in counterbalanced order following microinjections of TCS 1102 or its vehicle with a day of FR10 self-administration in between the two test days. FR10 days were used as a baseline because PR sessions provide extinction-like conditions, especially towards the end of the session where the required ratio is too high.
Antagonist Comparison
After completion of the PR test, rats were returned to the FR10 schedule of self- administration for another day. They were then tested for FR10 self-administration following ICV administration of single orexin receptor antagonists. Each antagonist was tested separately because they each required different vehicle solutions. TCS-OX2-29
(Batch No: 2; MW: 488.02; Tocris Bioscience, Bristol, United Kingdom) was tested first at a dose of 100 µg in 5 µL of 0.9% saline. ACT-335827 (Batch No: 1A/151172;
MW: 518.64; Tocris Bioscience) was then tested at a dose of 150 µg in 5 µL 20%
Vitamin E-TPGS/0.9% saline. Due to the poor solubility of ACT-335827 in aqueous solutions (Steiner et al., 2013a), ACT-335827 was vortexed immediately before each microinjection. Finally, SB-334867 (Batch No: 11A/175721; MW: 332.83; Tocris
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Bioscience) was tested at a dose of 30 µg in 2 µL of 100% DMSO (Sigma-Aldrich,
Castle Hill, NSW, Australia). These doses where chosen based on previous studies where ACT-335827 or SB-334867 have been administered systemically (Beig et al.,
2015a; Lawrence et al., 2006; Steiner et al., 2013c) or where SB-334867 or TCS-OX2-
29 have been microinjected (Brown et al., 2013; Brown et al., 2016). Although these doses are not all specifically ICV doses, they provide a guide to selecting an ICV dose.
For example, SB-334867 has been administered to rats at systemic doses of 30 mg/kg
(Lawrence et al., 2006; Smith et al., 2009), 16 µg ICV (Karasawa, Yakabi, Wang, &
Taché, 2014), and targeted microinjections have used 3 µg/side (Brown et al., 2016).
Rounding to the nearest half-log step yields the ratio 10 mg/kg systemic:10 µg ICV:1
µg/side intra-parenchymal. ACT-335827 is approximately as potent (IC50 = 6 nM) at the OX1 receptor as TCS 1102 (IC50 = 17 nM). However, in vitro results may vary significantly, as is the case for almorexant which has pKb values of 6.9 (Winrow et al.,
2012), 7.8 (Callander et al., 2013), and 8.4 (Faedo et al., 2012), variation of nearly two orders of magnitude. The doses were therefore based on previous in vivo studies which have administered 100 – 300 mg/kg of ACT-335827 (Beig et al., 2015a; Steiner et al.,
2013a).
Histology and Data Analysis
Histology was performed as previously described for Experiment 1. Lever response data were analysed using a two-way repeated measures ANOVA with lever and treatment as factors. Magazine entries, rewards and breakpoint were analysed using separate paired t-tests in SPSS. Data are presented as means ± SEM.
Results
After 10 days of self-administration training, rats acquired responding on the active lever, M = 137.2, SEM = 37.5. Few responses were made on the inactive lever, 82
Chapter 2. Methods and Results
M = 3.43, SEM = 0.72. The number of rewards earned, M = 53.8, SEM = 7.6, corresponded to approximately 2.4 g of palatable food, a comparable amount to all previous experiments.
FR1 Self-Administration
TCS 1102 had no effect on FR1 self-administration (Figure 10). Two-way repeated measures ANOVA showed a main effect of lever, F(1, 13) = 61.5, p < 0.001, but no treatment x lever interaction, F(1, 13) = 0.352, p = 0.563, or main effect of treatment, F(1, 13) = 0.379, p = 0.549. Similarly there was no effect on magazine entries, t(13) = 0.026, p = 0.98, or the number of rewards earned, t(13) = -0.325, p =
0.751.
FR5 Self-Administration
TCS 1102 had no effect on FR5 self-administration. Two-way repeated measures ANOVA showed a main effect of lever, F(1, 13) = 88.95, p < 0.001, but no treatment x lever interaction, F(1, 13) = 2.16, p = 0.166, or main effect of treatment,
F(1, 13) = 2.02, p = 0.179. Similarly there was no effect on magazine entries, t(13) = -
1.03, p = 0.323, or the number of rewards earned, t(13) = -0.628, p = 0.541.
FR10 Self-Administration
TCS 1102 had no effect on FR10 self-administration. Two-way repeated measures ANOVA showed a main effect of lever, F(1, 12) = 57.38, p < 0.001, but no treatment x lever interaction, F(1, 12) = 3.71, p = 0.078, or main effect of treatment,
F(1, 12) = 4.05, p = 0.067. Similarly there was no effect on magazine entries, t(12) = -
1.37, p = 0.2, or the number of rewards earned, t(12) = -1.32, p = 0.21. One rat was temporarily ill for the FR10 test and was not tested on FR10 self-administration, but recovered and was included in subsequent tests.
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Progressive Ratio
TCS 1102 had no effect on PR responding (Figure 11). There was no effect on breakpoint, t(13) = -0.739, p = 0.473. Two-way repeated measures ANOVA showed a main effect of lever, F(1, 13) = 74.4, p < 0.001, but no treatment x lever interaction, F(1,
13) = 1.66, p = 0.22, or main effect of treatment, F(1, 13) = 1.1, p = 0.314. Similarly there was no effect on magazine entries, t(13) = -0.719, p = 0.485, or the number of rewards earned, t(13) = -0.493, p = 0.63.
Antagonist Comparison
The OX2 receptor antagonist, TCS-OX2-29, had no effect on FR10 self- administration (Figure 12). Two-way repeated measures ANOVA showed a main effect of lever, F(1, 13) = 62.1, p < 0.001, but no treatment x lever interaction, F(1, 13) =
1.814, p = 0.201, or main effect of treatment, F(1, 13) = 1.829, p = 0.199. Similarly there was no effect on magazine entries, t(13) = -1.962, p = 0.072, or the number of rewards earned, t(13) = -1.473, p = 0.165.
The OX1 receptor antagonist, ACT-335827, had no effect on FR10 self- administration. Two-way repeated measures ANOVA showed a main effect of lever,
F(1, 13) = 56.43, p < 0.001, but no treatment x lever interaction, F(1, 13) = 2.77, p =
0.12, or main effect of treatment, F(1, 13) = 2.76, p = 0.12. Similarly there was no effect on magazine entries, t(13) = 0.882, p = 0.394, or the number of rewards earned, t(13) =
1.263, p = 0.229. The OX1 receptor antagonist, SB-334867, also had no effect on FR10 self-administration. Two-way repeated measures ANOVA showed a main effect of lever, F(1, 13) = 78.95, p < 0.001, but no treatment x lever interaction, F(1, 13) = 0.011, p = 0.918, or main effect of treatment, F(1, 13) = 0.016, p = 0.901. Similarly there was no effect on magazine entries, t(13) = 1.396, p = 0.186, or the number of rewards
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earned, t(13) = 0.19, p = 0.852. There were no differences in the timecourses of responding (data not shown).
Discussion
This series of experiments found that there was no effect of the dual orexin receptor antagonist, TCS 1102, on self-administration of palatable food under FR1,
FR5, FR10 or PR schedules. Additionally, no effect of the selective OX1 receptor antagonists, SB-334867 and ACT-335827, or the selective OX2 receptor antagonist,
TCS-OX2-29, was observed on FR10 self-administration. This was surprising given that these single orexin receptor antagonists have been used widely in the literature, at these or similar doses, and been shown to be effective in reducing appetitive motivation for different reinforcers (Beig et al., 2015a; Brown et al., 2013; Cason & Aston-Jones,
2013a, 2013b; Kastman et al., 2016). These drugs have usually been shown to be effective when given systemically and TCS-OX2-29 has been shown to be effective when administered ICV using nearly identical procedures (Brown et al., 2013). For SB-
334867 and ACT-335827, it is plausible that there is an issue with the route of administration, but this is not the case for TCS-OX2-29. Alternatively, there may be differences in the motivational properties of the reinforcer used in the present study. It may be that the reinforcing effects of a food pellet high in fat and sugar are mediated by multiple different neural systems and that this provides a level of redundancy that protects against orexin antagonism. It may also be the case that the associative learning processes that control responding for palatable food are not orexin-mediated. It is therefore necessary to test whether orexin neurons are activated in a situation where food-associated cues drive responding, such as during cue-induced reinstatement.
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Figure 10. TCS 1102 does not affect palatable food self-administration at multiple unit costs. (a-c) At FR1, ICV administration of 30 µg TCS 1102 had no effect on lever responding, rewards earned or magazine entries (n = 14). (d-f) At FR5, TCS 1102 had no effect on lever responding, rewards earned or magazine entries (n = 14). (g-i) At
FR10, TCS 1102 had no effect on lever responding, rewards earned or magazine entries
(n = 13). Data are means ± SEM.
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Figure 11. TCS 1102 had no effect on progressive ratio responding. (a) ICV administration of 30 µg TCS 1102 had no effect on lever responding, (b) magazine entries, or (c) rewards and breakpoint during PR testing. n = 14. Data are means ± SEM.
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Figure 12. Comparison between single orexin receptor antagonists on FR10 self- administration. (a-c) ICV administration of 100 µg of the OX2 receptor antagonist,
TCS-OX2-29, had no effect on lever responding, rewards earned, or magazine entries.
(d-f) 150 µg of the OX1 receptor antagonist, ACT-335827, had no effect on lever responding, rewards earned, or magazine entries. (g-i) 30 µg of the OX1 receptor antagonist, SB-334867, had no effect on lever responding, rewards earned, or magazine entries. n = 14. Data are means ± SEM.
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Experiment 5. Neuronal Activation During Reinstatement
Experiments 3-4 have failed to yield robust evidence for an effect of the orexin receptor antagonist TCS 1102 on various forms of palatable food motivated behaviours.
This could suggest that the orexin system is not heavily involved in the palatable food assays used here. Previous studies have shown activation of hypothalamic orexin neurons, as revealed by expression of the c-Fos protein, correlates with alcohol-seeking behaviour during renewal (Hamlin et al., 2007). Additionally, SB-334867 has been shown to reduce neuronal activity, as measured by c-Fos expression, in the prelimbic and nucleus accumbens core during reinstatement (Jupp et al., 2011b). The motivational activator theory of orexins posits that the orexins are involved in appetitive behaviour when this behaviour is under the control of cues (Mahler et al., 2014) and would therefore predict that orexin neurons should be involved during cue-induced reinstatement. The present study therefore examined the activation of orexin neurons during cue-induced reinstatement of palatable food using orexin and c-fos immunohistochemistry.
Methods
Subjects
Experimentally naive male Sprague-Dawley rats (n = 20) were obtained from the Animal Resources Centre (Perth, WA, Australia) at 6 weeks of age weighing 240-
280g and housed in groups of four in plastic tubs (24 cm high × 64 cm long × 40 cm wide) with wire tops, corncob bedding and environmental enrichment (aspen blocks for chewing, plastic tunnels and cardboard) under a reverse 12 h: 12h light/dark cycle
(lights off at 06:00) with ad libitum food and water prior to behavioural testing. Rats were given one week to acclimate to the colony room and then handled daily for 5 days
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before undergoing surgery and behavioural testing. Two days prior to commencement of self-administration training, rats were placed on a mild food restriction schedule (20-
22g/rat/day).
Acquisition and Extinction
Rats were trained for operant self-administration of palatable food in 8 identical operant chambers (Med Associates, St Albans, VT, U.S.A.) which were 24 cm long,
30.5 cm wide and 29 cm tall and situated in a sound attenuating box. Each chamber had a houselight and two nosepoke holes which flanked a magazine. For half of the chambers, the left nosepoke was active and for the other half the right nosepoke was active. Additionally, these chambers were equipped with four evenly spaced photobeam sensors to monitor locomotor activity during operant sessions. Before the commencement of self-administration training, rats were placed on a food restriction schedule (20g/rat/day) for two days. Rats then received two days of 1 hr habituation sessions. During these sessions the nosepokes were covered and the houselight was on.
Rats then received 10 days of 1 hr self-administration training. The nosepokes were uncovered and responses on the active nosepoke resulted in a delivery of a 45 mg pellet of palatable food (18.1% protein, 23.4% fat, 44.2% carbohydrate; #F06162,
Bioserv, Frenchtown, NJ, U.S.A.) to the magazine. Illumination of the nosepoke cue light and the houselight was switched off during reward delivery and for the rest of the timeout period, which was a total of 23 s. Responses made during the timeout period, or on the inactive nosepoke, were counted but had no programmed consequences.
During this acquisition phase, training was conducted 5 days per week and rats were considered to have acquired if they achieved at least 30 pellet deliveries during the
60 min session.
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Rats then underwent 9 consecutive days of extinction training. During extinction sessions, the houselight and fan were on for the 1 hr session but nosepokes had no consequences.
Behavioural Testing and Perfusion
Rats were tested and perfused over two consecutive days and randomly allocated to home-cage, extinction or cue-induced reinstatement groups. Rats which were not perfused on day 1 underwent a 10th day of extinction and were tested and perfused on day 2. On test days, rats in the home-cage group were left in their home-cage. Rats in the extinction group experienced an extinction session. Rats in the cue-induced reinstatement groups experienced a session identical to acquisition, except no pellets were available. For the rats in the extinction and cue-induced reinstatement groups, they were returned to their home-cage for 90 min before being transcardially perfused.
Perfusion
Rats were deeply anaesthetised using an overdose of sodium pentobarbitone
(108.3 mg/mL) combined with the local anaesthetic bupivacaine (1.7 mg/mL) to reduce discomfort associated with the high pH of sodium pentobarbitone (Ambrose, Wadham,
& Morton, 2000; Svendsen, Kok, & Lauritzenã, 2007). Once reflexes were no longer evident via tail and paw-pinch tests, the abdomen and chest cavity were opened to reveal the heart. A catheter needle was then inserted into the left ventricle, and the right atrium was cut (to allow blood to escape into the cavity). 200 mL of a 0.1 M phosphate buffer solution was then pumped into the left ventricle at a rate of 50 mL/min. This was followed by 200 mL of 4% paraformaldehyde, 0.1 M phosphate buffer solution. Brains were then dissected and left for at least one hour in paraformaldehyde solution, before being removed and placed in 20% sucrose overnight.
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Cryosectioning and Immunohistochemistry
Brains were removed from solution, embedded in O.C.T. Compound
(TissueTek®, ProSciTech, Thuringowa Central, QLD, Australia) at -18 °C and sectioned at 40 µm in a cryostat (CM1950, Leica Biosystems, North Ryde, NSW,
Australia). Sections were collected in a 1:4 series and stored in a 0.1% sodium azide,
0.1 M phosphate buffered saline solution at 4 °C until immunohistochemical staining.
Immunohistochemistry for c-Fos and OX-A was conducted. Brains were washed for 30 min in 0.1M phosphate buffer, 50% ethanol, 50% ethanol/1% hydrogen peroxide and 0.1M phosphate buffer/5% normal horse serum until they were incubated for 72 h in a 0.1M phosphate buffered saline/1% sodium azide solution containing 2% normal horse serum, 0.2% triton-X, rabbit anti-c-Fos antibody diluted 1/2000 and goat anti-OX-
A antibody diluted 1/2000 (Santa Cruz Biotechnology, Dallas, TX, U.S.A.) at room temperature. Brains were then washed 3 x 20 min in 0.1M phosphate buffer and incubated in 0.1M phosphate buffer, 2% normal horse serum, 0.2% triton-X and biotinylated donkey anti-rabbit (diluted 1/2000; Jackson ImmunoResearch Laboratories,
West Grove, PA, USA) overnight. Brains were then washed 3 x 20 min in 0.1M phosphate buffer) and incubated for 2 h in 0.1M phosphate buffer, 2% normal horse serum, 0.2% triton-X and ABC solution (Vectastain®, Vector Labs, Burlingame, CA,
USA). Then brains were washed, 2 x 0.1M phosphate buffer, 1 x 20 min in 0.1M sodium acetate (pH = 6) and incubated for 15 min in diaminobenzidine (0.2% D- glucose, 0.04% ammonium chloride and 2% nickel sulfate hexahydrate). The reaction was catalysed by glucose oxidase for 8 min before brains were washed in 0.1M sodium acetate, 0.1M phosphate buffer and then incubated overnight in a secondary antibody solution which contained biotinylated donkey anti-sheep (diluted 1/2000; Jackson
ImmunoResearch Laboratories) instead of the biotinylated donkey-anti-rabbit. The 92
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DAB reaction was performed again, but this time without the nickel sulfate hexahydrate to produce a brown reaction product. Brains were then returned to sodium azide before mounting on 4% gelatinised slides. Six sections of each rat were imaged at 20x magnification on an Olympus BX50 microscope (Olympus Life Science, Tokyo, Japan).
Counting of neurons that showed OX-A immunoreactivity (brown) and both
OX-A and c-Fos immunoreactivity (brown and black) was performed manually while blind to data and group allocations using the count function in Adobe Photoshop CC
2014 (Adobe Systems Incorporated, San Jose, CA, U.S.A.). As described by Hamlin et al. (2007), the boundaries of the medial hypothalamus were taken to be between the third ventricle and the medial edge of the mammillothalamic tract, the perifornical hypothalamus then extended to approximately half the width of the fornix past the lateral edge of the fornix. Anything lateral of this boundary was counted as part of the lateral hypothalamus.
Data Analysis
Data were analysed using mixed-design ANOVA or one-way ANOVA in SPSS as required. As previously described, Helmert contrasts were used to examine whether there were differences between groups. Data are means ± SEM.
Results
After 10 days of acquisition rats showed robust acquisition, with M = 306.9,
SEM = 30.5 active nosepokes and M = 7.05, SEM = 1.68 inactive nosepokes. This resulted in earning M = 120, SEM = 5.99 rewards or 5.4 g of palatable food. After 9 days of extinction, the number of active nosepokes had decreased to M = 9.9, SEM =
1.08 and the number of inactive nosepokes was M = 2.95, SEM = 0.57. Upon test, rats showed a robust reinstatement effect (Figure 13A). A mixed-design ANOVA showed
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that there was a significant manipulanda x session x group interaction, F(1, 12) = 5.527, p = 0.037, session x group interaction, F(1, 12) = 4.83, p = 0.048, manipulanda x session interaction, F(1, 12) = 7.61, p = 0.017, manipulanda x group interaction, F(1,
12) = 4.822, p = 0.048, and main effect of manipulanda, F(1, 12) = 24.3, p < 0.001.
Reinstatement also persisted throughout the session because there was no difference in active nosepoke responding during the first 30 min, M = 19.3, SEM = 5.58, and during the last 30 min, M = 16.1, SEM = 6.09, t(6) = 0.583, p = 0.581. Cell counting results showed no overall differences in the total number of orexin neurons between groups in the lateral hypothalamus, F(2, 19) = 0.147, p = 0.865, perifornical hypothalamus, F(2,
19) = 1.055, p = 0.37, or medial hypothalamus, F(2, 19) = 1.069, p = 0.0365. Helmert contrasts showed that in the lateral hypothalamus there was no difference between the home-cage control group and the context-exposed groups (extinction and reinstatement), t(17) = -0.192, p = 0.85, or between the extinction and reinstatement groups, t(17) = -0.567, p = 0.578. In the perifornical hypothalamus, there was a significant difference between the home-cage control and the context-exposed groups, t(17) = -2.256, p = 0.038, but no significant difference between the extinction and reinstatement groups, t(17) = -1.279, p = 0.218. In the medial hypothalamus there was also a difference between the home-cage control and the context-exposed groups, t(17)
= -3.28, p = 0.004, but not between the extinction and reinstatement groups, t(17) = -
0.392, p = 0.7. Representative photomicrographs for each group, selected from rats that showed a number of double-labelled cells close to the mean, are shown in Figure 14.
Discussion
These results show that hypothalamic orexin neurons in the perifornical and medial hypothalamus are activated during exposure to an appetitive context, but that there is no additional activation during cue-induced reinstatement. These results suggest 94
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that in this behavioural assay the presentation of the palatable food-associated cues was able to precipitate reinstatement without recruiting additional orexin neuron activation over and above that recruited by the appetitive context, at least when this activation is assessed via expression of the c-Fos protein. One caveat with this experiment is that if strong neuronal activity occurred only at the beginning of the session rather than throughout the session, that perfusion would have taken place past the peak levels of c-
Fos expression. In hypothalamic brain regions, it takes 3 – 4 h for c-Fos levels to return to baseline so although it may be a suboptimal time point much of the c-Fos would still be present at this time (Giovannelli, Shiromani, Jirikowski, & Bloom, 1990, 1992;
Herrera & Robertson, 1996). However, in the present study reinstatement was observed throughout the hour-long session because there was no significant difference between active nosepoke responding during the first 30 min relative to the last 30 min.
Reinstatement-related neuronal activation was probably also occurring throughout the session. The difference in levels of activation between the home-cage control group and the context-exposed groups may therefore be due to general arousal rather than a specific cue-driven response.
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Figure 13. Effect of cue-induced reinstatement for palatable food on orexin neuron activation. (a) In a between-subjects design, rats showed a robust cue-induced reinstatement effect. (b) In the lateral hypothalamus, there was no difference number of orexin and c-Fos double-labelled neurons. (c) In the perifornical hypothalamus, there was a difference between the home-cage control group and the context-exposed groups
(extinction and reinstatement) in orexin/cFos double-labelled cells. (d) In the medial hypothalamus, there was also a difference between the home-cage control group and the context-exposed groups, but no difference between the extinction and reinstatement groups. There were no differences in the total number of orexin neurons in any region.
Home n = 6, Extinction (EXT) n = 7, Reinstatement (CIR) n = 7. * p < 0.05 compared to Home. Data are means ± SEM.
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Figure 14. Representative photomicrographs of orexin-A and c-Fos immunoreactivity.
(a) Home-cage control group. Arrows point to brown OX-A-IR cells. (b) Extinction group. Arrows point to brown and black OX-A/c-Fos-IR double-labelled cells. (c)
Reinstatement group. In all cases the left division is the lateral hypothalamus, the middle division is the perifornical hypothalamus and the right division is the medial hypothalamus. Scale bar = 500 µm. 97
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Experiment 6. Beer Self-Administration and Reacquisition
The results from Experiments 3 and 4 indicate that dual orexin receptor antagonism has no effect on self-administration or reinstatement of palatable food seeking. However, the orexin system has previously been reported to be able to distinguish between different reinforcers (Borgland et al., 2009; Brown et al., 2013;
Khoo & Brown, 2014). In particular, operant alcohol self-administration has been shown to respond to both OX1 and OX2 receptor antagonism (Brown et al., 2013;
Lawrence et al., 2006; Shoblock et al., 2011). Therefore it is possible that the beer model (McGregor & Gallate, 2004) which has previously been used to obtain robust levels of operant self-administration (Perry & McNally, 2013) as well as recruitment of c-Fos expression in orexin neurons (Hamlin et al., 2007) that may be sensitive to orexin antagonism.
Methods
Subjects
Experimentally naive male Long Evans rats (n = 32) were obtained from the
Florey Institute of Neuroscience and Mental Health (Melbourne, VIC, Australia). Upon arrival, rats were housed in groups of three or four in plastic cages (309 mm wide, 617 mm long, 284 mm high) in ventilated racks with corncob bedding and an alloy mezzanine. Rats experienced a 12 h: 12 h light/dark cycle (lights on at 07:00) and ad libitum access to food and water prior to behavioural testing, after which point they received 1 hr of daily access to maintain them at 85% of their free-feeding bodyweight.
Rats were given at least one week to acclimate to the colony room before undergoing behavioural experiments, which were conducted during the light cycle. All procedures were approved by the University of New South Wales Animal Care and Ethics
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Committee and conducted in accordance with the National Health and Medical
Research Council’s Australian Code for the Care and Use of Animals for Scientific
Purposes.
Drugs
TCS 1102 (Batch No: 2A/155485; MW: 475.09) was obtained from Tocris
Bioscience (Bristol, United Kingdom). The vehicle solution was (2-hydroxypropyl)-β- cyclodextrin obtained from Sigma-Aldrich (Castle Hill, NSW, Australia) and diluted with saline to a final concentration of 20% 2HPβCD. Beer (Coopers Birell Ultra Light) was obtained from Coopers (Regency Park, SA, Australia), decarbonated by stirring overnight and made up to a 4% v v-1 alcoholic beer solution by adding absolute ethanol.
In these experiments, it was expected that doses of 10 µg TCS 1102 or less would be sufficient and so they were administered in a 20% 2HPβCD vehicle which was less viscous than the 20% Vitamin E-TPGS solution.
Surgery
Rats were deeply anaesthetised and implanted with guide cannulae targeting the lateral ventricles as described for Experiment 1.
Behavioural Apparatus
Operant self-administration training was conducted, as previously described by
Hamlin et al. (2007) in 8 identical operant chambers (Med Associates, St. Albans, VT,
U.S.A.) housed in sound attenuation boxes equipped with fans to mask external noise.
Each chamber was 24 cm long, 30 cm wide and 21 cm high, with a Perspex door, rear wall and ceiling and stainless steel end walls. On the left end wall was a houselight and the centre of the right end wall had a recessed magazine connected to a syringe pump
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and flanked on both sides by nosepoke holes. Responding on the left (active) nosepoke activated the syringe pump for 6 s to deliver of 0.6 mL of alcoholic beer solution to the magazine via plastic tubing (PE100). The availability of beer was signalled by illumination of the active nosepoke hole light. All sessions were conducted with the houselight off.
Operant Self-Administration Training
Rats were placed on food and water restriction beginning two days before operant training, with 1 hr of food and water access in the evening. Rats were then given two days of magazine training to habituate them to beer, where both nosepoke holes were inactive and 10 deliveries of beer were given at random intervals. On each day, rats were given two 20 min magazine training sessions, for a total of four magazine training sessions. This was followed by seven days of acquisition training where beer was only delivered following an active nosepoke during the 60 min session. Nosepokes made during the 24 s timeout period (which included the 6 s of syringe pump operation) were counted, but did not result in any additional reinforcement. Nosepokes made on the inactive nosepoke are counted, but did not have any programmed consequences.
Self-Administration Testing
After 7 days of self-administration training, rats (n = 16) were given microinjections of TCS 1102 before a self-administration session, using the same procedures as Experiments 1 - 4. Rats received microinjections of vehicle, 1, 3 and 10
µg TCS 1102 prior to operant self-administration sessions in counterbalanced order because doses as low as 10 – 15 mg/kg have been reported to have behavioural effects in other studies (Bergman et al., 2008; Chen et al., 2014; Winrow et al., 2010). Between
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each testing day, rats were allowed 2 - 3 days of normal self-administration until they returned to baseline.
Reacquisition
A separate cohort of rats (n = 16) was used to examine whether a dose of 10 µg
TCS 1102 would reduce reacquisition after a period of extinction. Following 7 days of self-administration as described above, rats were given 5 days of extinction training which were run under identical conditions to self-administration sessions, except no syringe was in the syringe pump so no beer was delivered. Rats were randomly assigned to receive a microinjection of vehicle or TCS 1102 before a reacquisition session, where beer was available (identical conditions to self-administration). This was chosen because rates of responding during reacquisition are high and animals express high levels of motivation to respond for alcoholic beer during reacquisition (Willcocks &
McNally, 2011).
Self-Administration Re-Test
After the reacquisition test was complete, rats were retrained under normal self- administration conditions. Following the null results observed for doses up to 10 µg of
TCS 1102, a higher dose of 30 µg TCS 1102 was tested on self-administration. Rats were given vehicle and 30 µg TCS 1102 in counterbalanced order, with one day of normal self-administration in between test days to allow them to return to baseline.
Histology and Data Analysis
Injection site validation was performed as previously described for Experiment
1. Rats that did not complete the experiments due to illness, lost headmounts or inaccurately delivered injections were excluded from analysis (n = 6). Statistical
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analysis was performed using repeated measures ANOVA or mixed-design ANOVA in
SPSS. Greenhouse-Geisser sphericity adjustments were applied to degrees of freedom as required. Data are presented as means ± SEM.
Adverse Events
An adverse event report was filed with the Animal Care and Ethics Committee during this experiment. One rat which had received a microinjection of 10 µg TCS
1102, been tested for self-administration and then provided ad-libitum food and water overnight was found dead the next day. A necropsy was performed and found that the rat appeared to have been in good health, with no sign of infection or inflammation near the cannula implant. The cause of death was determined to be due to a haematoma near the cerebellum.
Results
Self-Administration
After 7 days of operant self-administration, rats acquired mean responding of M
= 63.1, SEM = 12.4 active nosepokes, showing discrimination against the inactive nosepoke where only M = 1.8, SEM = 0.31 responses were made. 10 rats completed the experiment and received accurate microinjections. As shown in Figure 15, there was no effect on beer self-administration. No significant interaction was observed between the treatment and the manipulanda, F(3, 27) = 0.239, p = 0.87. Additionally, no main effect of treatment was observed, F(3, 27) = 0.241, p = 0.87. Rats only showed significant discrimination between the active and inactive manipulanda, F(1, 9) = 32.7, p < 0.001.
There was no significant difference in the number of rewards earned, F(3, 27) = 0.528, p
= 0.67, or the number of magazine entries, F(1.51, 13.57) = 2.76, p = 0.109.
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Reacquisition
After 7 days of operant self-administration, rats acquired mean responding of M
= 49, SEM = 13.2 active nosepokes, showing discrimination against the inactive nosepoke where only M = 1.75, SEM = 0.47 responses were made. After 5 days of extinction training, rats made M = 9.4, SEM = 2.5 active nosepokes and M = 27.9, SEM
= 7.47 inactive nosepokes. 14 rats completed the experiment and received accurate microinjections (n = 7 per group). However, there was no effect on beer reacquisition
(Figure 16). A mixed-design ANOVA with the manipulanda and session as within subjects factors and treatment as a between subjects factor showed that although there was significant discrimination for the active nosepoke, F(1, 12) = 43.4, p < 0.001, there was no significant session x manipulanda x treatment interaction, F(1, 12) = 0.727, p =
0.411, session x treatment interaction, F(1, 12) = 0.103, p = 0.754, or manipulanda x treatment interaction, F(1, 12) = 0.073, p = 0.792. However, the reacquisition effect was significantly stronger on the active nosepoke, F(1, 12) = 20.47, p = 0.001. While the number of rewards/cue presentations earned was significantly increased in the reacquisition session, F(1, 12) = 94.2, p < 0.001, there was no session x treatment interaction, F(1, 12) = 0.851, p = 0.375. Similarly, the number of magazine entries was increased in the reinstatement session, F(1, 12) = 12.2, p = 0.004, but there was no session x treatment interaction, F(1, 12) = 0.53, p = 0.48.
Self-Administration Re-Test
In order to test whether a higher dose of TCS 1102 would reduce self- administration, rats which had completed Experiment 2 were given 30 µg TCS 1102 or vehicle in counterbalanced order (Figure 15D-F). Two additional rats did not complete this experiment due to illness. Although there was significant discrimination between
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the manipulanda, F(1, 11) = 69.2, p < 0.001, there was no significant treatment x manipulanda interaction, F(1, 11) = 0.626, p = 0.45, or main effect of treatment, F(1,
11) = 0.789, p = 0.39. Additionally, there was no difference in the number of rewards earned, t(11) = 1.16, p = 0.271, or the number of magazine entries, t(11) = 0.597, p =
0.562.
Discussion
This experiment found that TCS 1102 had no effect on operant self- administration of alcoholic beer or on reacquisition after extinction. This finding is surprising because multiple laboratories have reported effects of different orexin receptor antagonists on alcohol self-administration and reinstatement. This includes the use of both OX1 receptor antagonists (Jupp et al., 2011a; Lawrence et al., 2006;
Moorman & Aston-Jones, 2009), OX2 receptor antagonists (Brown et al., 2013;
Shoblock et al., 2011), and dual orexin receptor antagonism (Srinivasan et al., 2012).
However, no previous study has specifically examined the use of orexin receptor antagonists on beer self-administration which may differ as a reinforcer from solutions of ethanol in water or because of differences in the training protocol.
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Figure 15. The effect of TCS 1102 on 4% alcohol beer self-administration. (a-c) There was no effect of doses up to 10 µg of TCS 1102 (ICV) on nosepoke responding for beer, the number of rewards earned or magazine entries made (n = 10). (d-f) In a follow-up experiment performed after reacquisition of beer seeking, rats were tested for self- administration following microinjections of vehicle or 30 µg of TCS 1102. There was also no effect on nosepoke responding, rewards earned or magazine entries (n = 12).
Data are means ± SEM.
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Figure 16. Effect of ICV TCS 1102 on reacquisition of beer. (a) There was robust reacquisition effect on total active nosepoke responding for beer, but no effect of 10 µg
TCS 1102. (b-c) Reacquisition was also observed for the number of cue presentations earned (which were rewarded during reacquisition but not extinction sessions) and the number of magazine entries made, but TCS 1102 had no effect. Vehicle n = 7, TCS10 n
= 7. Data are means ± SEM. 106
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Experiment 7. Nicotine Self-Administration and Reinstatement
Thus far there is little evidence from these studies to implicate the orexin system in various aspects of motivation for instrumentally earned reinforcers, with the only effects reported on food intake. The latter is consistently observed in the literature but the lack of effect of TCS 1102 against instrumental responding for palatable food was unexpected. These null effects might be attributable to the reinforcers (food, beer) used.
The orexin system has also previously been shown to be important in various aspects of nicotine self-administration and behaviour. Notably, operant nicotine self- administration is reduced by the OX1 receptor antagonist, SB-334867, and nicotine alters expression of Hcrtr1 mRNA that encodes for the OX1 receptor (Hollander et al.,
2008; LeSage et al., 2010). Studies in mice have found effects of SB-334867 on signs of withdrawal and cue-induced reinstatement of operant intravenous self-administration
(Plaza-Zabala, Flores, Maldonado, & Berrendero, 2012; Plaza-Zabala et al., 2013).
Uslaner et al. (2014) have also found that an OX2 antagonist can reduce cue induced reinstatement. Therefore, Experiment 7 aimed to examine the effect of the dual orexin receptor antagonist, TCS 1102, on operant nicotine self-administration and reinstatement.
Methods
Subjects
Experimentally naive male Sprague-Dawley rats (n = 48) were obtained from the Animal Resources Centre (Perth, WA, Australia) at 6 weeks of age weighing 240-
280g. Rats for Experiment 7 (n = 16) were housed in groups of four in plastic tubs (24 cm high × 64 cm long × 40 cm wide) with wire tops, corncob bedding and environmental enrichment (aspen blocks for chewing, plastic tunnels and cardboard) 107
Chapter 2. Methods and Results
under a reverse 12 h: 12h light/dark cycle (lights off at 06:00) with ad libitum food and water prior to behavioural testing. Rats were given one week to acclimate to the colony room and then handled daily for 5 days before undergoing surgery and behavioural testing. Two days prior to commencement of self-administration training, rats were placed on a mild food restriction schedule (20-22g/rat/day).
Drugs
Drugs used were as previously described for Experiments 1-5. Heparin was obtained from Hospira Australia (Mulgrave, VIC, Australia). Isoflurane was obtained from Delvet (Seven Hills, NSW, Australia).
Surgery
A single surgery was performed to implant both the chronically indwelling jugular vein catheters as previously described by Macnamara et al. (2016) and a single guide cannula targeting the lateral ventricle. Briefly, rats were deeply anaesthetized using 3% isoflurane and given a pre-emptive analgesic injection of carprofen (5 mg/kg, s.c.). The catheter was implanted such that it passed from a back mount to the right jugular vein in the neck and then it was secured and flushed with 0.2 mL of 100 mg/mL cephazolin. Rats remained under anaesthesia while being transferred to a stereotaxic.
Local anaesthetic (bupivacaine) was applied to the head and a guide cannula (26 ga;
PlasticsOne, Roanoke, VA, U.S.A.) was implanted targeting the lateral ventricles using the following coordinates (from Bregma): -0.8 mm anteroposterior (A/P), +1.5 mm mediolateral (M/L) and -4 mm dorsoventral (D/V). Rats were given 2 days of post- operative carprofen (5 mg/kg, s.c.) and 7 days recovery before behavioural experiments.
The catheter was flushed daily with heparinised (150 IU/mL) cephazolin (0.2 mL, 100 mg/mL) until the end of behavioural testing.
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Acquisition
Rats were trained for operant self-administration of nicotine in 8 identical operant chambers (Med Associates, St Albans, VT, U.S.A.) which were 24 cm long,
30.5 cm wide and 29 cm tall and situated in a sound attenuating box. Each chamber had a houselight and two nosepoke holes which flanked a magazine. For half of the chambers, the left nosepoke was active and for the other half the right nosepoke was active. Additionally, these chambers was equipped with four evenly spaced photobeam sensors to monitor locomotor activity during operant sessions. The infusion line was fed through a spring connector and a weighted fluid swivel assembly (Instech Laboratories,
Pylmout Meeting, PA, U.S.A.) to the syringe pump which was situated outside the sound attenuating box.
Rats received two days of 1 h habituation sessions in the self-administration chambers. During these sessions the nosepokes were covered, the houselight was on, and rats were not connected to the infusion line.
Rats then received daily 1 h sessions of nicotine self-administration. The nosepokes were uncovered and responses on the active nosepoke resulted in a 3 s infusion of nicotine (30 µg/kg/infusion), illumination of the nosepoke cue light (3 s) and the houselight was switched off during a 20 s time-out period. Responses made during the timeout period, or on the inactive nosepoke, were counted but had no programmed consequences.
During this acquisition phase, training was conducted 5 days per week and rats were considered to have acquired if they achieved at least 6 infusions of nicotine during the 60 min session and at least 2:1 discrimination for the active nosepoke. After the commencement of drug testing, all sessions took place on consecutive days.
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Self-Administration Testing
Once rats had experienced 10 acquisition sessions and were at stable responding
(<30% variation in the number of infusions earned per day), they were given a series of microinjections to test the effects of TCS 1102 on nicotine self-administration.
Procedures for habituation to microinjections were as described for Experiment 1.
They then received microinjections of vehicle (20% (2-hydroxypropyl)-β- cyclodextrin, 0.5% saline), 1µg, 3µg or 10µg TCS 1102. These doses were chosen because systemic administration of as little as 10 - 15 mg/kg TCS 1102 reduced nicotine-induced reinstatement of food seeking in rats (Bergman et al., 2008; Chen et al., 2014; Winrow et al., 2010). TCS 1102 was administered 10 min before the operant session, using a within-subjects design. Two days of normal self-administration elapsed between test days to allow for washout and a return to baseline responding. The 10 µg
TCS 1102 test was then repeated using a 20% Vitamin E-TPGS/0.9% saline vehicle to ensure that there was no difference between vehicle solutions.
Extinction and Reinstatement
Following the completion of self-administration testing, rats were placed on normal self-administration for four days, to ensure that they had returned to stable baseline responding. At this point they had received a total of 29 self-administration days and extinction training commenced.
During extinction sessions, the houselight and fan were on for the 1 h session but nosepokes had no consequences. Rats received at least 6 daily extinction sessions until they met extinction criteria which was active nosepoke responding of ≤30% of responding on the last day of self-administration for two consecutive days. Once extinction criteria was met they were tested for reinstatement. For each reinstatement 110
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test, rats were randomly assigned (between-subjects) to receive a microinjection of either Vitamin E-TPGS vehicle or 10 µg TCS 1102 10 min prior to the session.
Cue-induced reinstatement was precipitated by reinstating the response- contingent availability of the visual cues. The program was identical to the program used in training except that if a rat made no active nosepokes within the first 10 min a non-contingent cue presentation was delivered. Although rats were attached to the tethers, they did not receive infusions.
Rats then underwent a second phase of re-extinction until they met the extinction criteria again, and then were tested for nicotine-primed reinstatement.
Nicotine primed reinstatement was precipitated by an injection of nicotine immediately before being placed in the chamber (0.3 mg/kg, 1 mL/kg, s.c.) and carried out under extinction conditions (houselight on, but all nosepokes were inactive). This procedure was then repeated for cue/nicotine compound reinstatement which involved a session identical to cue-induced reinstatement with a priming injection of nicotine (0.3 mg/kg, 1 mL/kg, s.c.) immediately before the session.
Histology
Once rats had completed all experiments, they were euthanized using a 0.2 mL
IV infusion of 325 mg/mL sodium pentobarbitone via their catheters, therefore simultaneously verifying catheter patency. To check the accuracy of the microinjections, cresyl violet was injected via guide cannulae and brains were dissected as previously described for Experiment 1.
The injection site of one animal, whose guide cannula became blocked before the cue/nicotine-primed reinstatement test, was validated by sectioning in a CM1950
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cryostat (Leica Microsystems, North Ryde, NSW, Australia) and staining with cresyl violet to check the cannula track entered the ventricles.
Data Analysis
Statistical analysis was performed using SPSS. Repeated measures
ANOVA was used for the self-administration data and mixed-design ANOVA for the reinstatement tests. Greenhouse-Geisser sphericity adjustments were applied to degrees of freedom as required. As described for previous experiments, Helmert contrasts were performed for factors with more than two levels. Data are presented as means ± SEM.
Results
Nicotine Self-Administration
After 10 days of self-administration, 13 rats met inclusion criteria. They made a mean of 21.9 ± 2.5 active nosepokes and 7.85 ± 1.68 inactive nosepokes, corresponding to 15.5 ± 1.44 infusions of 30 µg/kg nicotine during the operant session. There was a significant increase in active nosepoke responding over the course of training, F(9, 108)
= 3.41, p = 0.001, but not for the inactive nosepoke, F(4.52, 54.3) = 2.1, p = 0.086, indicating that rats rapidly increased responding on the active nose-poke across training session.
Across self-administration, there was no effect of TCS 1102 across the doses tested on responding for nicotine overall (Figure 17A). A repeated measures ANOVA showed that there was no dose x manipulanda interaction, F(3, 36) = 0.968, p = 0.418, or main effect of dose, F(3, 36) = 1.323, p = 0.282. However rats retained their significant preference for the active nosepoke, F(1, 12) = 24.99, p < 0.001. For active nosepoke responding there was no significant dose x time interaction, F(9, 108) = 0.601,
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p = 0.793, main effect of dose, F(3, 36) = 1.248, p = 0.307, or main effect of time, F(3,
36) = 0.356, p = 0.785, indicating there was no effect of TCS 1102 on the timecourse of responding during the session (Figure 17B).
There was also no effect on locomotor activity during the operant session, overall, F(3, 36) = 0.397, p = 0.756, as shown in Figure 17C. Repeated measures
ANOVA showed that while rats varied their activity over time, F(3, 36) = 5.907, p =
0.002, there was no dose x time interaction, F(3.65, 43.76) = 0.51, p = 0.712, or main effect of dose, F(1.87, 22.47) = 0.139, p = 0.858. There was therefore no effect of TCS
1102 on activity over the course of the session (Figure 17D).
This result was then replicated (Figure 18) using 10 µg TCS 1102 and a 20%
Vitamin E-TPGS/0.9% saline vehicle to ensure that this was not due to any artefacts caused by using an alternate vehicle solution. Repeated measures ANOVA showed that while rats still preferred the active nosepoke, F(1, 12) = 33.38, p < 0.001, there was still no dose x manipulanda interaction, F(1, 12) = 0.31, p = 0.588, or main effect of dose,
F(1, 12) = 0.02, p = 0.891. There was also no significant effect on the timecourse of active nosepoke responding, as evidenced by a lack of a dose x time interaction, F(3,
36) = 1.05, p = 0.383, or main effect of dose, F(1, 12) = 0.105, p = 0.752. Responding also did not significantly differ across time, F(3, 36) = 0.474, p = 0.702. Similarly, there was no significant difference the overall amount of activity, t(12) = 0.224, p = 0.826, or on the timecourse of activity as evidenced by the lack of a significant dose x time interaction, F(3, 36) = 0.515, p = 0.674, main effect of dose, F(1, 12) = 0.05, p = 0.826, or main effect of time, F(3, 36) = 2.5, p = 0.075.
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Figure 17. Effect of ICV TCS 1102 on nicotine self-administration. (a) Doses of up to
10 µg TCS 1102 had no effect on the overall number of nosepokes made. (b) There was no change to the timecourse of active nosepoke responding. (c) TCS 1102 had no effect on locomotor activity, as measured by photobeams within the operant chamber. (d) TCS
1102 had no effect on the timecourse of locomotor activity over the course of the session. n = 13. Data are means ± SEM.
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Figure 18. Effect of ICV TCS 1102 on nicotine self-administration, using a Vitamin E-
TPGS vehicle. (a) To ensure there were no differences between the 2HPβCD and
Vitamin E-TPGS vehicles, the 10 µg TCS 1102 dose was repeated. There was no effect on self-administration. (b) Similarly, there was no effect on the timecourse of responding. (c-d) There was also no effect on the overall amount of locomotor activity, or its timecourse, as measured by photobeams in the operant chamber. n = 13. Data are means ± SEM.
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Nicotine Reinstatement
As shown in Figure 19, rats showed a robust cue-induced reinstatement of responding on the active nosepoke. There was a significant manipulanda x session interaction, F(1, 11) = 9.69, p = 0.01, and main effect of session, F(1, 11) = 8.263, p =
0.015. Rats significantly preferred the active nosepoke, F(1, 11) = 21.32, p = 0.001.
However, there was no effect of treatment as shown by the lack of a significant manipulanda x session x treatment interaction, F(1, 11) = 0.005, p = 0.946, session x treatment interaction, F(1, 11) = 0.036, p = 0.852, or main effect of treatment, F(1, 11) =
0.405, p = 0.538. The timecourse of active nosepoke responding was similarly unaffected. Mixed-design ANOVA showed no session x time x treatment interaction,
F(3, 33) = 0.137, p = 0.938, session x treatment interaction, F(1, 11) = 0.018, p = 0.895, or time x treatment interaction, F(3, 33) = 0.124, p = 0.945.
Rats showed nicotine-primed reinstatement but there was no effect of TCS 1102.
Mixed-design ANOVA showed significant main effects of manipulanda, F(1, 11) =
6.447, p = 0.028, and session, F(1, 11) = 13.065, p = 0.004. However, there was no manipulanda x session interaction, F(1, 11) = 3.485, p = 0.089. There was no manipulanda x session x treatment interaction, F(1, 11) = 0.04, p = 0.845, session x treatment interaction, F(1, 11) = 0.323, p = 0.581, or main effect of treatment, F(1, 11) =
0.367, p = 0.557.The timecourse of active nosepoke responding was similarly unaffected. Mixed-design ANOVA showed no session x time x treatment interaction,
F(3, 33) = 0.202, p = 0.894, session x treatment interaction, F(1, 11) = 0.042, p = 0.842, or time x treatment interaction, F(3, 33) = 0.608, p = 0.614.
Rats showed a robust cue/prime compound reinstatement and there was evidence for a transient effect of TCS 1102. Mixed-design ANOVA showed a significant manipulanda x session interaction, F(1, 10) = 10.989, p = 0.008, and main 116
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effects of manipulanda, F(1, 10) = 13.32, p = 0.004, and session, F(1, 10) = 12.77, p =
0.005, indicating that rats reinstated responding. There was no significant manipulanda x session x treatment interaction, F(1, 10) = 0.747, p = 0.408, session x treatment interaction, F(1, 10) = 1.05, p = 0.329, or main effect of treatment, F(1, 10) = 1.27, p =
0.286. The timecourse of the session was mostly unaffected. Mixed-design ANOVA showed no session x time x treatment interaction, F(3, 30) = 0.77, p = 0.52, session x treatment interaction, F(1, 10) = 0.326, p = 0.581, or time x treatment interaction, F(3,
30) = 0.303, p = 0.823. However, there was a significant difference between the first 15 min of the session and the remainder, F(1, 10) = 4.993, p = 0.049. Post-hoc testing showed that during the first 15 min there was a significant difference between the vehicle and TCS 1102 treatment groups (p = 0.039).
Discussion
These results show that TCS 1102 has a small and transient effect on nicotine- seeking during cue/prime compound reinstatement, but that it does not affect operant self-administration, cue-induced reinstatement or nicotine-primed reinstatement. The transient nature of the effect during the first 15 min of cue/prime reinstatement is consistent with the short half-life of TCS 1102 (Bergman et al., 2008). However, several studies have reported effects of systemic TCS 1102 which have persisted for a full hour or more (Bergman et al., 2008; Chen et al., 2014; Winrow et al., 2010). It may be that there are differences in the distribution of the drug when administered systemically compared to when administered ICV. The lack of observed effects on other forms of reinstatement is somewhat inconsistent with previous reports that have used different orexin receptor antagonists to produce behavioural effects (Hollander et al., 2008;
LeSage et al., 2010; Uslaner et al., 2014). Patterns of responding also appear odd, with responding escalating in the last 15 min of cue-induced reinstatement and over the 117
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course of the cue/prime compound reinstatement session. Most of the animals had made responses within the first 3 – 4 min of the session, so in most cases the escalation cannot be explained by the rat being exposed late to the cue and reinstating at the end of the session. Perhaps the late escalation reflects the animal’s frustration at the lack of reward which can drive vigorous responding (Amsel, 1992; Amsel & Roussel, 1952). The effect of TCS 1102 on cue/prime compound reinstatement also occurred after a lengthy period of training and several other operant and reinstatement tests. This effect is consistent with the motivational activator theory because it predicts that the orexin/hypocretin system will be involved when responding is higher and driven by cues. Recently, it has been argued that future studies of orexin in addiction should examine whether there is a motivational shift between short periods of access and alternative long or intermittent access paradigms (James et al., 2017). Therefore, we next attempted to examine this effect in a group of rats given short term access to nicotine.
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Figure 19. Effect of ICV TCS 1102 on reinstatement of nicotine seeking. (a-b) Rats showed cue-induced reinstatement, but 10 µg TCS 1102 had no effect on responding or the timecourse of active responding. Vehicle n = 6, TCS10 n = 7. (c-d) Rats showed nicotine-primed reinstatement, but there was no effect of TCS 1102 on overall responding or the timecourse of active nosepoke responding. Vehicle n = 7, TCS10 n =
6. (e) Rats showed cue/prime compound reinstatement but there was no effect on overall responding, but (f) there was a significant difference in the first 15 min. n = 6 per group.
* p < 0.05 for vehicle vs TCS10. Data are means ± SEM. 119
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Experiment 8. Cue/Prime Compound Reinstatement and FR5
The previous experiment found an effect of TCS 1102 on cue/prime compound reinstatement of nicotine seeking. The present experiment was therefore designed to examine if this effect would be observed with a shorter period of nicotine self- administration and a within-subjects design. A higher dose of TCS 1102 was also tested to examine whether the dose used in the previous experiment may have been suboptimal. After reinstatement testing, rats were retrained and tested on an FR5 schedule in order to examine whether TCS 1102 would affect self-administration at a higher unit-cost.
Methods
Male Sprague-Dawley rats (n = 16) underwent surgery, 15 days of acquisition and extinction in a manner identical to Experiment 7. Testing for cue/nicotine compound reinstatement procedures were also identical, except rats were tested following microinjection of vehicle, 10 µg or 30 µg TCS 1102 using a within-subjects design. As for Experiment 7, there were at least two re-extinction sessions between reinstatement tests to allow rats to meet extinction criteria.
After the conclusion of reinstatement testing, rats were retrained to enable FR5 testing. Rats were given 5 days of normal nicotine self-administration at FR1, 3 days at
FR2, 3 days at FR3, and 7 days at FR5. Rats were then tested in counterbalanced order following microinjection of vehicle and 30 µg TCS 1102. Histology and data analysis were performed as described for Experiment 7.
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Results
14 rats met inclusion criteria, which was to earn at least 6 infusions of nicotine during the session. After 15 days of nicotine self-administration, rats made a 26.4 ± 4.17 active nosepokes, 5.64 ± 1.43 inactive nosepokes and received 17.6 ± 2.57 infusions of nicotine. Over the course of acquisition there was a significant day x nosepoke interaction, F(14, 182) = 7.76, p < 0.001, with the number of responses increasing over time, F(14, 182) = 9.97, p < 0.001, and more for the active nosepoke than inactive, F(1,
13) = 65.1, p < 0.001.
A repeated measures ANOVA comparing the final extinction session and reinstatement tests following vehicle, 10, and 30 µg TCS1103 showed that there was a significant manipulanda x session interaction, F(1.65, 21.4) = 4.549, p = 0.028, and main effects of session, F(1.88, 24.4) = 3.71, p = 0.041, and manipulanda, F(1, 13) =
8.533, p = 0.012 (Figure 20A). Helmert contrast comparing the final extinction session to the reinstatement sessions revealed that there was a significant interaction with the manipulanda, F(1, 13) = 10.6, p = 0.006), and a significant difference between the final extinction session and each of the reinstatement tests, F(1, 13) = 9.04, p = 0.02, indicating a robust reinstatement effect. However, the Helmert contrast comparing the vehicle and TCS 1102 reinstatement tests showed no effect, F(1, 13) = 0.57, p = 0.474, or interaction with manipulanda, F(1, 13) = 1.732, p = 0.211. Similarly, the Helmert contrast comparing the 10 and 30 µg TCS 1102 showed no effect, F(1, 13) = 0.262, p =
0.617, or interaction with manipulanda, F(1, 13) = 0.235, p = 0.636.
As shown in Figure 20B, there was no significant difference in the timecourse of responding between sessions, as shown by a lack of session x time interaction, F(3.23,
31.9) = 1.915, p = 0.138, main effect of time, F(1.37, 17.8) = 2.926, p = 0.095, although
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there was a significant main effect of session, F(1.77, 23.1) = 4.135, p = 0.033. Helmert contrasts showed that there was no difference between the vehicle and TCS 1102 tests,
F(1, 13) = 1.037, p = 0.327, and there were no significant interactions with time either in the first 15 min, F(1, 13) = 3.08, p = 0.103, the second 15 min, F(1, 13) = 1.93, p =
0.188, or third 15 min, F(1, 13) = 0.404, p = 0.536, compared to the rest of the session.
After retraining on normal self-administration, only n = 4 rats met acquisition criteria of earning at least 6 infusions during the session. As shown in Figure 21A, there was no significant treatment x manipulanda interaction, F(1, 3) = 0.192, p = 0.691 or main effect of treatment, F(1, 3) = 0.407, p = 0.569. However, rats continued to show a preference for the active nosepoke over the inactive nosepoke, F(1, 3) = 20.2, p = 0.021.
Similarly, there was no effect on the timecourse of active nosepoke responding (Figure
21B), as shown by a lack of treatment x time interaction, F(1, 3) = 1.274, p = 0.341, or main effect of treatment, F(1, 3) = 0.319, p = 0.611. Figure 21C shows that there was no effect on locomotor behaviour measured by photobeam sensors in the operant chamber, t(3) = 0.036, p = 0.973. There was also no effect on the timecourse of locomotor behaviour (Figure 21D), as there was no treatment x time interaction, F(1, 3) = 2.382, p
= 0.137, or main effect of treatment, F(1, 3) = 0.001, p = 0.973.
Discussion
The results from this experiment show that there was no effect of TCS 1102 on cue/prime compound reinstatement after a short period of access. Rats also appeared to follow a similar pattern of responding to Experiment 7 by escalating over the course of the session (although this was not statistically significant). These results suggest that the effect observed in Experiment 7 may have been specific to conditions of a chronic period of nicotine access. Additionally, there was no effect of TCS 1102 on FR5 self-
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administration. However, the number of rats that maintained self-administration on an
FR5 schedule was very low.
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Figure 20. Effect of ICV TCS 1102 on cue/prime compound reinstatement of nicotine seeking. (a) Rats showed a robust reinstatement effect on the active nosepoke in a within-subjects series of reinstatement tests. However, there was no effect of 10 or 30
µg TCS 1102 on nosepokes during cue/prime reinstatement overall. (b) There was also no effect of TCS 1102 on the timecourse of active nosepoke responding. n = 14. Data are means ± SEM.
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Figure 21. Effect of ICV TCS 1102 on FR5 self-administration. (a) 30 µg TCS 1102 had no effect on FR5 self-administrion or (b) the timecourse of active nosepoke responding. (c) Similarly, there was no effect on the number of beam breaks recorded from photobeam sensors in the operant chamber. (d) There was also no effect on the timecourse of locomotor behaviour. n = 4. Data are means ± SEM.
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Chapter 3. General Discussion
The aim of this thesis was to examine the role of the orexin/hypocretin system in palatable food seeking, alcoholic beer seeking, and nicotine seeking. TCS 1102, a potent and selective dual orexin receptor antagonist, was administered prior to tests of palatable food self-administration and reinstatement, alcohol beer self-administration and reacquisition, and nicotine self-administration and reinstatement. This was done because it has previously been shown that the orexin/hypocretin system is involved in regulating appetitive motivation for food and drugs, with the recently developed motivational activator theory predicting that it is most involved when highly motivated behaviour is required, such when there is a highly salient reinforcer, the reinforcer is available under a high unit-cost, or responding is driven by reward-associated cues.
Additionally, many of the previous preclinical studies only used an antagonist selective for one of the orexin receptors. The recent clinical approval of the dual orexin receptor antagonist suvorexant presents an opportunity to examine whether dual orexin receptor antagonists may be a useful pharmacotherapy for addiction. Since clinical trials of suvorexant for cocaine are already underway (The University of Texas Health Science
Center, 2016), the potential for a dual orexin receptor antagonist to inhibit self- administration or relapse-like behaviour for palatable food, alcoholic beer, and nicotine was examined. These results therefore provide the first experimental examination of the predictions of the motivational activator theory and have important implications for the potential of orexin-based pharmacotherapies for addiction.
The results show that TCS 1102 effectively reduces OX-A-induced feeding.
However, TCS 1102 has no effect on FR1, FR5, FR10, or PR self-administration or cue/prime compound reinstatement of palatable food seeking. There was no effect on
FR1 self-administration or reacquisition of 4% alcoholic beer. Additionally, there was 126
Chapter 3. General Discussion
no effect on FR1 or FR5 nicotine self-administration, cue-induced reinstatement, or nicotine-primed reinstatement. There was a small, transient effect on cue/prime compound reinstatement of nicotine seeking, this was observed after a chronic, but not short, period of self-administration. Moreover, OX-A and c-Fos immunohistochemistry indicated that the operant context is associated with an increase in orexin neuron activation that was not specific for reward-associated cues.
This discussion considers the effects of TCS 1102 on palatable food, alcoholic beer, and nicotine seeking. The results from each reinforcer are considered in terms of the motivational activator theory’s predictions with respect to unit cost, reward salience, and cue-driven behaviour. Additionally, the impact of the pharmacodynamics and pharmacokinetics of TCS 1102 and other orexin receptor antagonists are considered.
Palatable Food
The lack of an effect of TCS 1102 on palatable food self-administration and reinstatement is consistent with some previous studies but not others. McGregor, Wu,
Barber, Ramanathan, and Siegel (2011) showed that Hcrt-/- mice acquire food self- administration just as quickly as wild-type mice, suggesting that orexin signalling may not be necessary for FR operant food seeking behaviour. However, McGregor et al.
(2011) also found that the Hcrt-/- mice were selectively impaired in PR performance during the light phase. The present study is not consistent with this result or the results of Borgland et al. (2009) who found SB-334867 reduced PR responding for high fat pellets. Of course it is likely that the depressive effect on the orexin/hypocretin system of the dual orexin antagonism using TCS 1102 in rats, during the light phase, is not as compete or profound a total hypocretin knockout in mice. For Hcrt-/- mice, there is never any orexin signalling, which could affect the animal’s development or result in
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adaptations in other neural pathways. However, for rats given a dual orexin receptor antagonist, orexin signalling is part of their normal neural functioning and its suppression produces an altered internal state which is presumably physiologically and psychologically different to their internal state at rest.
Additionally, the palatable food reinforcer used in the present study may have different motivational properties to the reinforcers used in other studies. Different reinforcers may have different motivational properties due to their physiological and psychological effects. Hernandez and Hoebel (1988) showed that rats experience a 37% increase in accumbal dopamine following a rewarded operant response for food, but that accumbal dopamine is 5-fold higher following an acute microinjection of cocaine. Other studies have found that accumbal dopamine can be increased up to 800% of baseline levels following experimenter-administered cocaine (Bradberry & Roth, 1989; Czoty,
Justice Jr, & Howell, 2000; Hurd, Weiss, Koob, And, & Ungerstedt, 1989; Pettit &
Justice Jr, 1989), but that animals will self-administer cocaine to maintain dopamine levels around 350% of baseline (Pettit & Justice Jr, 1989). Regardless, while food evokes a dopamine response that is of a lesser magnitude than that evoked by cocaine, rats actually prefer saccharin solutions to intravenously self-administered cocaine
(Cantin et al., 2010). This occurs despite cocaine eliciting greater responding during a
PR test, where rats earn twice as many cocaine rewards as they do saccharin rewards
(Cantin et al., 2010). So while rats show a greater dopamine response and PR motivation for cocaine, they prefer sweet rewards indicating that drug and non-drug rewards have different motivational and physiological properties. As Shaw et al. (2017) have shown, Hcrt-/- mice have reduced accumbal dopamine responses to cocaine injections and do not acquire CPP, unlike wild-type mice. This suggests that there may be an important dopaminergic component to orexin-based regulation of appetitive 128
Chapter 3. General Discussion
motivation which could be influencing whether orexin manipulations are effective in altering motivation for non-drug reinforcers like palatable food.
The palatable food results observed in the present study are also inconsistent with predictions from the motivational activator theory. Recall that this theory states that orexins are involved in appetitive motivation when highly motivated behaviours are required, such as when there is a high unit-cost (as in PR studies), the reinforcer is highly salient, or reward-seeking is cue-driven (Mahler et al., 2014; Mahler et al.,
2012). Experiment 4 systematically manipulated the unit cost of palatable food across
FR1, FR5, FR10, and PR conditions and assessed the effects of several doses of TCS
1102. The motivational activator theory would predict that at higher unit costs, such as
FR10 or PR, there should be an effect of orexin antagonism (Mahler et al., 2014).
Previous studies using cocaine have shown, using behavioural economics approaches, that higher unit costs are sensitive to orexin antagonism (Bentzley & Aston-Jones, 2015;
España et al., 2010). In these studies, the unit cost was manipulated by progressively decreasing the amount of cocaine delivered per infusion while holding constant the operant reward conditions. But while orexin antagonism decreased an animal’s willingness to work for cocaine at higher unit costs, there was no such evidence here. Of course, it is possible that these discrepant results were due to differences in the unit cost manipulation (reducing the amount of reinforcer per fixed response versus maintaining the amount of reinforcer and increasing the response requirement), but it is not practical to reduce the amount of reinforcer delivered when using a food pellet as it is for a cocaine infusion. Regardless, increasing the unit cost by increasing the number of operant responses required should involve the orexin/hypocretin system because previous studies have shown effects of orexin antagonism in PR studies (Mahler et al.,
2014; Mahler et al., 2012). 129
Chapter 3. General Discussion
One explanation for this lack of an effect is that unit cost alone is not the determining factor of the orexin/hypocretin system’s involvement. There may be a qualitative difference between receiving a reward of decreasing value at a regular interval or after a given number of responses versus receiving a reward of the same value at increasing intervals. Although the unit cost of the reward is the same, the animal might learn something qualitatively different. Conditions of non-reward when a reward is expected are aversive to the animal, prompting rats to defecate and urinate
(Amsel, 1992). When rewards are merely downshifted, as in the negative contrast paradigm, animals also reduce responding below levels that they would reach if they had been originally trained for the lower reward, indicating that the downshift is aversive (Mitchell & Flaherty, 2005) which can be attenuated with benzodiazepines
(Phelps, Mitchell, Nutt, Marston, & Robinson, 2015). Reward magnitude is represented by a subset of amygdala neurons, in the PFC, Acb, and subthalamic nucleus (Bermudez
& Schultz, 2010; Fiallos et al., 2017; Lardeux, Pernaud, Paleressompoulle, & Baunez,
2009; van Duuren, Lankelma, & Pennartz, 2008, 2010) and varying the reward magnitude may be subject to anchoring effects (Smith, Peterson, & Kirkpatrick, 2016).
Rescorla and Wagner (1972) would predict that varying reward magnitude in a behavioural economics assay would induce negative prediction error and extinction as the value of the outcome gradually decreased. There is some support for this in the work of Itzhak and Anderson (2012) who extinguished cocaine CPP using descending doses of cocaine, suggesting that decreasing the reward magnitude does, in fact, cause extinction. On the other hand, accumbal dopamine responses during a PR session do not decrease over the course of a PR session (Nicola & Deadwyler, 2000), as might be expected if the increasing ratios were represented as negative prediction errors (Schultz,
Dayan, & Montague, 1997). Nonetheless, both the behavioural economics task and the
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PR task require positively motivated behaviours and motivation for cocaine in both tasks is sensitive to the addition of histamine to the cocaine solution (Freeman,
McMaster, Roma, & Woolverton, 2014). The differences between the observed effects in the present study and the behavioural economics studies may be because the effect of
SB-334867 on cocaine demand curves is simply not very large and involves an increasing demand elasticity mostly in a small number of animals (Bentzley & Aston-
Jones, 2015).
These results suggest that there is a complex interaction between the nature of the reinforcer, the motivational state of the animal, and the conditions of the operant task that determine the involvement of the orexin/hypocretin system. The palatable food reinforcer used here evoked very high levels of responding from rats and self- administration behaviour was rapidly acquired in all animals. Additionally, effects of orexin antagonism on animals that were food restricted have been observed for sucrose and saccharin under FR1 self-administration conditions (Cason & Aston-Jones, 2013a,
2013b). In addition to testing TCS 1102, the selective OX1 receptor antagonists SB-
334867 and ACT-335827, and the selective OX2 receptor antagonist TCS-OX2-29 were also tested here with no effect on FR10 palatable food self-administration. While activation of either OX1 or OX2 receptors tends to drive appetitive behaviour, as demonstrated by the effects of OX-A and OX-B on feeding (Sakurai et al., 1998), the antagonism of both receptors receptor may produce different effects to antagonism of a single orexin receptor (Dugovic et al., 2009), but this was not the case in the present study. The rats in both the present study and in the previous studies by Cason and
Aston-Jones (2013a, 2013b) were tested using the same drug (SB-334867) and the same motivational state. Moreover, the use of FR10 conditions at test should have made it more likely that an effect would be observed given the predictions of the motivational 131
Chapter 3. General Discussion
activator theory. The lack of an observed effect of these different orexin receptor antagonists suggests that orexinergic involvement in appetitive motivation is not simply determined by highly motivated behaviour for physiologically or psychologically salient rewards.
The importance attributed to orexins for cue-driven reward seeking would have predicted an effect of TCS 1102 on the cue/prime reinstatement test. Saccharin and sucrose pellet seeking is reduced by SB-334867 after FR1 training in food restricted rats
(Cason & Aston-Jones, 2013a, 2013b) and studies with cocaine have shown cue-driven reward seeking is particularly vulnerable to orexin antagonism (Bentzley & Aston-
Jones, 2015; Mahler & Aston-Jones, 2012; Smith et al., 2009). However, several studies have not reported significant effects of orexin antagonists on reinstatement for natural reinforcers such as a sucrose solution (Brown et al., 2016) and sweetened condensed milk (Martin-Fardon & Weiss, 2014a). The observed null effects of TCS 1102 here on palatable food reinstatement are therefore partially consistent with previous findings, but not necessarily entirely consistent with the motivational activator theory.
It is unlikely that these results and discrepancies are due to the hedonic value of the palatable food pellets used. Here, both hungry and sated rats consumed an average of 50-60 pellets, or 2.5 g in total of the palatable food pellets, which was comparable to the average number of 45 mg saccharin pellets consumed by rats used by Cason and
Aston-Jones (2013a). Although it was less than the number of sucrose pellets consumed by food restricted rats in Cason and Aston-Jones (2013b), a 45% kcal from fat (23.4% fat content) formulation is sufficient to cause rats to become obese (Brown et al., 2015).
Additionally, obese-prone rats earn more pellets on FR5 and PR schedules and, after prolonged exposure to the diet, neurons in their nucleus accumbens core have electrophysiological properties, such as higher AMPA/NMDA ratios, that are often 132
Chapter 3. General Discussion
characteristic of animals in drug self-administration paradigms (Brown et al., 2015;
Scofield et al., 2016; Shen, Moussawi, Zhou, Toda, & Kalivas, 2011). The palatable food pellets used in the present study can therefore be considered to have high hedonic value that should be sufficient to produce high levels of motivation in the animals.
A variety of single orexin antagonists were used to target FR10 palatable food self-administration, with consistent results. SB-334867 is the same drug as used by the vast majority of previous studies, including those targeting natural reinforcers (Borgland et al., 2009; Cason & Aston-Jones, 2013a, 2013b; Martin-Fardon & Weiss, 2014a).
ACT-335827 has also been shown to be a highly potent and selective OX1 antagonist with behavioural effects reported in rats at systemic doses of 100 mg/kg (Beig et al.,
2015a; Steiner et al., 2013a). The selective OX2 receptor antagonist, TCS-OX2-29, has also been shown to be effective when centrally administered at the dose used (Brown et al., 2013). Additionally, the vehicle solution and pretreatment protocol for ICV microinjections was identical to Brown et al. (2013), who gave rats 10 min in their home cage to allow the drug to circulate but performed microinjections over 15 s with
10 s for diffusion rather than 2 min with 30 s for diffusion before removing the injector.
The consistent lack of effect of any of these orexin antagonists suggests that the null result is unlikely to be due to insufficient doses, inadequate penetration of drug to key brain regions, or functional antagonism between the two orexin receptors.
Rather, the lack of observed effects of TCS 1102 on palatable food seeking suggests that dual orexin antagonists may lack effects on normal motivation and everyday functions. Current anti-craving pharmacotherapies, such as naloxone, also reduce motivation for sucrose (Shoblock et al., 2011). This presents a therapeutic disadvantage because motivated behaviours are normal and serve functions that are usually adaptive. Although it has been suggested that orexins may have a role in obesity 133
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(Cason et al., 2010), given that exogenous orexin peptide does not result in weight gain
(Ida et al., 1999; Yamanaka et al., 1999), it is unlikely that an orexin antagonist would be an effective anti-obesity medication. The present observed null results for palatable food are therefore a potential positive for the use of orexin antagonists in other therapeutic contexts.
Neuroanatomy
The present study found that in Orexin/c-Fos double-labelling was higher in rats given an extinction or reinstatement session, than for the home-cage control rats who were not given an operant session at all, but there was no difference between the extinction group and the reinstatement group. The finding that there was no specific activation of the orexin/hypocretin system during reinstatement for palatable food suggests that the orexin/hypocretin system may provide general arousal, but not be specifically responding to reward-associated cues. Previous studies have examined orexin neuron activation during relapse-like behaviours and found similar effects. For renewal of alcohol seeking, activation of orexin neurons as measured by expression of c-Fos did not significantly differ between ABA and ABB tests (Hamlin et al., 2007), although there was a correlation between active nosepoke responding and orexin/c-Fos double-labelling. Null results have also been reported for sucrose (Hamlin et al., 2006) and only correlations have been reported in some hypothalamic subregions in other studies (Moorman et al., 2016). Few studies have examined between-group effects which makes it difficult to draw conclusions about whether the reported relationships between orexin neuron activation and reinstatement have any causal role. Hamlin et al.
(2008) found that there was an increase in the LH but not PeF or DMH during ABA renewal testing for cocaine seeking and Martin-Fardon et al. (2016) found increases in the LH, PeF, and DMH during cocaine reinstatement. Orexin/c-Fos correlations were 134
Chapter 3. General Discussion
reported by Harris et al. (2005), but these were with CPP scores for morphine, food, and cocaine.
It is therefore relatively unsurprising that neither a between-group difference nor a correlation was found in this study. Results from previous studies may have been reported as correlations because it is possible to obtain a significant correlation even if the between subjects data are non-significant (Hamlin et al., 2007). This may be because the orexin/hypocretin system provides a general arousal signal rather than a specific response to reward-associated cues. The correlation between orexin neuron activation and an operant response may be mediated by this arousal rather than cue or context- induced reward seeking. It has previously been shown that arousal to reward-associated cues can occur independently of a pharmacologically suppressed orexin/hypocretin system (Tannenbaum et al., 2016). Moreover, hypocretin knockout mice show normal
PR responding during the active dark phase (McGregor et al., 2011), which would suggest that orexins during a rodent’s active dark phase are not necessary for motivated behaviours. The neuroanatomical results are therefore consistent with a role for orexins in general arousal, but without a response specific to reward-associated cues.
One way in which the present result is surprising is in the activated subregions, which were the PeF and DMH, but not the LH. This is in contrast to previous studies that have found correlations between c-Fos expression in LH orexin neurons and CPP scores (Harris et al., 2005) and ABA renewal for cocaine (Hamlin et al., 2008).
However, not all studies have shown orexin neuron activation specifically in the LH.
Dayas et al. (2008) showed increases in the LH/PeF and DMH and Moorman et al.
(2016) showed context-induced reinstatement was associated with greater activation in the DMH and LH. Martin-Fardon et al. (2016) have also found increases in the LH,
PeF, and DMH during cocaine reinstatement. 135
Chapter 3. General Discussion
It has previously been argued that there is a dichotomy between LH and
PeF/DMH orexin neurons (Harris & Aston-Jones, 2006). It has been argued that that LH orexin neurons are specifically involved in reward learning because of their correlation with CPP (Harris et al., 2005; Harris, Wimmer, Randall-Thompson, & Aston-Jones,
2007). PeF and DMH orexin neurons are supposedly involved in arousal because they vary diurnally while LH neurons do not (Estabrooke et al., 2001). Unpredictable chronic mild stress also results in selective activation of PeF and DMH orexin neurons (Nollet et al., 2011). However, the findings that both lateral and PeF/DMH subregions are activated during reinstatement (Martin-Fardon et al., 2016; Moorman et al., 2016) are not consistent with this notion. This notion does suggest that during extinction and reinstatement of palatable food seeking there is a general arousal response to the operant chamber, but not a specific cue-elicited response. However, this does not explain why reinstatement in the present study is different to reinstatement in previous studies. It is unclear why reinstatement for palatable food would activate the arousal-related
PeF/DMH neurons, but be insensitive to TCS 1102, while reinstatement for drugs activates the reward-related LH neurons. Further studies are required to establish what these differences are and why they produce differential patterns of orexin neuron activation.
Alcoholic Beer
It was surprising that TCS 1102 had no effect on alcoholic beer self- administration or reacquisition because several previous studies have reported effects.
Reductions in alcohol self-administration have been observed following systemic OX1 receptor antagonism (Lawrence et al., 2006; Moorman & Aston-Jones, 2009; Richards et al., 2008), systemic or central OX2 receptor antagonism (Brown et al., 2013;
Shoblock et al., 2011), and dual orexin receptor antagonism in the VTA (Srinivasan et 136
Chapter 3. General Discussion
al., 2012). It was therefore hypothesised that central dual orexin antagonism would be effective at reducing alcoholic beer self-administration. Given that dual orexin receptor antagonism is more effective than single orexin receptor antagonism at promoting sleep
(Morairty et al., 2012), a centrally administered dual orexin receptor antagonist might be expected to produce an even greater reduction than antagonism of either orexin receptor alone. However, the results of the present study suggest orexin antagonism does not affect self-administration or reacquisition of alcoholic beer.
There are several differences between the beer model used in the present study and the behavioural protocols of previous studies. Firstly, the alcoholic beer reinforcer is of a lower concentration (4% v/v) than that used in other studies, which vary from
10% to 20% (Lawrence et al., 2006; Moorman & Aston-Jones, 2009; Moorman et al.,
2016). Secondly, the beer model does not use a sucrose fade or pre-exposure phase and provides rats with alcohol in a decarbonated beer solution rather than water. The beer model has previously been shown to produce higher levels of alcohol consumption than equivalent concentrations of alcohol in water, elevated blood alcohol concentrations, and binge-like patterns of consumption that are sensitive to the same pharmacotherapies as human alcohol consumption (Hargreaves, Monds, Gunasekaran, Dawson, &
McGregor, 2009; Hargreaves, Wang, Lawrence, & McGregor, 2011; McGregor &
Gallate, 2004; McGregor, Saharov, Hunt, & Topple, 1999). A key advantage of the beer model is that since no sucrose fade or pre-exposure protocol is required, rats do not associate their operant response with anything other than the alcoholic beer reward, and they have very little experience with non-contingent deliveries (outside of magazine training). This ensures that the animal experiences no confusion about the identity of the reinforcer and that the associated contextual and discrete cues become associated only with the reinforcer that will be used during testing. 137
Chapter 3. General Discussion
Other relevant differences in the beer model include the reinforcement ratio and motivational state of the animals. In previous studies, rats have been tested at FR3 with ad libitum access to food and water (Brown et al., 2013; Brown et al., 2016; Jupp et al.,
2011a; Lawrence et al., 2006; Moorman & Aston-Jones, 2009; Moorman et al., 2017;
Richards et al., 2008). Rats were also generally singly housed, although it is likely that
Lawrence et al. (2006) pair or triple-housed their rats because that was the practice of the laboratory when animals had not undergone surgery (Brown et al., 2013; Brown et al., 2016). In the present study, rats were quad-housed and testing was performed on an
FR1 schedule. Rats were also food and water restricted which should, if anything, have increased the likelihood that orexin antagonism would be effective (Cason & Aston-
Jones, 2013b). However, patterns of orexinergic regulation seem to vary between reinforcers, so it is difficult to say which, if any, of these factors may have contributed to the different results obtained in the present study relative to previous studies.
Another key difference is the use of a reacquisition test instead of a reinstatement or context-induced renewal test. In the reacquisition test, active nosepokes are reinforced because conditions are identical to acquisition. In contrast, during reinstatement or renewal, operant responses are not reinforced. The reacquisition test was chosen because relapse in humans is usually reinforced and a model of relapse that involves reinforcement may better model what happens in this context. The disadvantage of this test is that the presence of the reinforcer may have been confounding and evoked neural responses more similar to a regular self-administration session. However, given that OX1 receptor antagonism reduces both self-administration and reinstatement and OX2 receptor antagonism reduces self-administration, a relapse test that incorporated aspects of self-administration should be more, not less, likely to be vulnerable to dual orexin receptor antagonism. It is therefore surprising that TCS 1102 138
Chapter 3. General Discussion
had no effect, but it may be indicative that there are other differences in the psychological and neural processes that are involved in the reinforced reacquisition of an operant response compared to the non-reinforced reinstatement of an extinguished operant response. Although the two different tests were not directly compared and such a comparison is beyond the scope of the present study, it may be a consideration for future studies.
Nicotine
The finding that TCS 1102 affected nicotine-seeking in rats given chronic but not short-term periods of access to nicotine is consistent with the literature. Moreover, the effects of TCS 1102 were only observed during the compound cue+prime reinstatement procedure. These results are in accordance with the motivational activator theory because it would seem reasonable to assume that the highest level of motivational activation in the experiments reported here would have been in rats with prolonged self-administration of nicotine and tested under the compound cue+prime reinstatement condition. Recently, it has been argued that the difference between standard short access protocols and alternative longer or intermittent access protocols may result in an orexin-mediated motivational shift (James et al., 2017). The finding that TCS 1102 reduces nicotine-seeking in rats given chronic but not short-term periods of access is therefore consistent with the motivational activator theory.
However, the effect of TCS 1102 against compound reinstatement was transient, being significant only in the first 15 min of the cue/prime compound reinstatement session. This transient effect is specific for nicotine and no similar transient effects were observed for palatable food or beer (data not shown). This may be because the half-life of TCS 1102 is quite short, at approximately 20 min, but it is comparable to the 24 min
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half-life of SB-334867 (Porter et al., 2001) and behavioural effects for TCS 1102 have been reported following systemic doses as low as 10 - 15 mg/kg and these persist throughout tests that are up to 180 min in length (Bergman et al., 2008; Chen et al.,
2014; Winrow et al., 2010). Several studies have shown that the degree of addiction-like behaviours that an animal exhibits, such as seeking despite adverse consequences, increases with chronic access (Ahmed, 2012; Deroche-Gamonet, Belin, & Piazza,
2004). For nicotine, it has been shown that rats given chronic access will engage in longer bouts of operant responding to earn more infusions (Clemens, Lay, & Holmes,
2015). These behavioural changes are accompanied by neurobiological changes as well, such as an upregulation of nicotinic ACh receptors (Govind, Walsh, & Green,
2012). The orexin/hypocretin system also becomes more excitable as it is exposed to stimulant drugs (Rao et al., 2013; Yeoh et al., 2012). These neuroplastic changes may alter the degree to which the orexin/hypocretin system is involved in nicotine-seeking, which would explain why we found an effect on nicotine-seeking after chronic access, but not short-term access.
Intriguingly, these results suggest that cues alone are insufficient to provoke orexinergic regulation of nicotine-seeking. In contrast with Uslaner et al. (2014), who showed effects of OX2 antagonism on cue-induced but not nicotine-primed reinstatement, I found that TCS 1102 affected neither cue-induced nor nicotine-primed reinstatement and only transiently reduced cue/prime compound reinstatement. This is surprising, given that one of the functions of chronic access is to make rats susceptible to cue-induced reinstatement (Clemens et al., 2015), but is consistent with the other effects reported in this thesis. However, the rats in the studies by Uslaner et al. (2014) had also undergone extensive prior training prior to cue-induced reinstatement including a sequence of steps from FR1 food pre-training, FR1 to FR2 and FR5 nicotine 140
Chapter 3. General Discussion
responding and then a period of PR training before being tested for reinstatement. Rats in the present study had only undergone training on FR1 nicotine self-administration and testing of self-administration behaviour. The different training histories between the two studies makes it difficult to draw strong conclusions because rats which have been trained under multiple schedules are likely to assign a different level of salience and motivational strength to the nicotine and its associated cues.
The present study has also found no effect of the dual orexin receptor antagonist on operant nicotine self-administration. This is somewhat inconsistent with previous studies, which have found effects of OX1 receptor antagonism on nicotine self- administration (Hollander et al., 2008; LeSage et al., 2010). TCS 1102 was used because of its high potency and selectivity for the orexin receptors. As previously reported, TCS 1102 reduces OX-A-induced Ca2+ at 17 and 4 nM concentrations for the
OX1 and OX2 receptors respectively (Bergman et al., 2008). TCS 1102 is highly selective for the orexin receptors, with only three hits in µM range reported on an MDS
Pharma screen for a monamine transporter, thromboxane A2 and thyrotropin releasing hormone. The doses used here of 10-30µg administered intracerebroventricularly should avoid hepatic metabolism and produce brain-concentrations equivalent to those achieved by effective systemic doses. Indeed, Experiment 2 showed that central TCS
1102 reduces OX-A-induced feeding, demonstrating that TCS 1102 is acting in the brain to block orexin signalling. It is therefore unlikely that the lack of observed effects on self-administration and other types of reinstatement are due to off-target non- orexinergic effects, potency, half-life or vehicle solution.
However, it is possible that the differential pharmacokinetics of TCS 1102 and other orexin receptor antagonists are relevant. Although there are no published reports on the antagonist mechanism of TCS 1102, it was an early precursor to suvorexant 141
Chapter 3. General Discussion
which has been characterised as a competitive antagonist (Mould, Brown, Marshall, &
Langmead, 2014). In contrast, it has been shown that SB-334867 is a non-competitive antagonist (Malherbe, Borroni, Pinard, Wettstein, & Knoflach, 2009b) and almorexant may also be a non-competitive antagonist (Malherbe et al., 2009b; Mould et al., 2014).
However, it is unclear whether this non-competitive binding is due to irreversible antagonism or allosteric modulation (Mould et al., 2014). It is possible that the differences in results between studies have been affected by the different mechanisms by which these antagonists act, especially if non-competitive antagonism may mean that commonly used orexin antagonists like almorexant and SB-334867 are biasing the intracellular signalling cascades triggered by the endogenous orexin peptides differently to TCS 1102. This remains an important issue to be explored.
Another possible explanation for these discrepancies in the role of the orexin/hypocretin system in nicotine self-administration is the self-administration protocols used. All of the previously discussed operant nicotine intravenous self- administration studies in rats have used an extensive food pre-training protocol and an
FR5 schedule (Hollander et al., 2008; LeSage et al., 2010; Uslaner et al., 2014).
However, here rats never associated their operant responses with obtaining food and only ever associated the context of the operant chamber with nicotine rewards. It has now been shown that rats prefer sweet rewards to nicotine, even after a chronic period of access (Huynh, Fam, Ahmed, & Clemens, 2015), consistent with findings comparing preferences for natural reinforcers to drug reinforcers like cocaine and heroin (Madsen
& Ahmed, 2015). By pre-training rats with food and using an FR5 schedule, it is possible that previous studies have enhanced the salience which animals assign to the context and cues in the operant chamber. Moreover, when nicotine is used to precipitate reinstatement of operant responding for food-associated cues TCS 1102 can attenuate 142
Chapter 3. General Discussion
reinstatement (Winrow et al., 2010). Instead of the orexin/hypocretin system being involved in simply regulating nicotine seeking, it may be involved in situations where there has been some ambiguity about the presence and identity of the reinforcer.
Switching between food and nicotine would produce a change in the reward magnitude and identity, which produces a dopamine response and greater activation in the nucleus accumbens (Collins et al., 2016; Veldhuizen, Douglas, Aschenbrenner, Gitelman, &
Small, 2011). It is therefore plausible that changing the reinforcer from food to nicotine alters the animal’s expectations and the altered prediction error then enhances the motivational qualities of nicotine, facilitating an eventual role for orexin. Although this is speculative, small differences in experimental protocol can have significant impacts on subsequent results.
Implications
Interpretation of the results of the present study is complicated by a general lack of positive effects, but it still has some implications for the role of the orexins in appetitive motivation and the motivational activator theory. There is an extensive history of single orexin receptor antagonists administered both systemically or targeting specific brain regions exerting effects on appetitive motivation for a variety of reinforcers (Borgland et al., 2009; Brown et al., 2013; Cason & Aston-Jones, 2013a,
2013b; Harris et al., 2005; Lawrence et al., 2006; Moorman & Aston-Jones, 2009;
Smith & Aston-Jones, 2012; Smith et al., 2009). One possible interpretation is that the
ICV TCS 1102 manipulation was simply ineffective because the target orexin receptors are too far from the ventricles for the drug to diffuse to. However, previous studies using ICV TCS-OX2-29 have shown effects on alcohol self-administration (Brown et al., 2013) and the present studies used an ICV microinjection protocol that was identical except for a longer microinjection time of 2 min with 30 s diffusion. ICV microinjection 143
Chapter 3. General Discussion
actually presents several advantages over systemic drug administration because the drug is delivered directly to the brain, bypassing plasma protein binding, hepatic metabolism, the blood brain barrier, and avoiding peripheral side effects. These advantages are especially important for a drug that has a short half-life, like TCS 1102, although this has not prevented SB-334867 from having effects following systemic administration despite its 24 min half-life (Porter et al., 2001). The ICV route of administration and short half-life also did not prevent TCS 1102 from reducing OX-A-induced feeding behaviour, an effect which persisted over a 2 h session.
The small and transient effect of TCS 1102 on nicotine seeking behaviour in
Experiment 7 was only present after chronic access to nicotine, but not the short-term period of access in Experiment 8. While caution should be applied in interpreting small and transient effects, it also suggests that length of access could be a factor. Indeed,
James et al. (2017) argue that protocols that produce greater levels of compulsivity may involve greater orexinergic recruitment. Rats in the present studies often had a shorter period of self-administration training, for example, rats in Experiment 6 only had 7 days of acquisition, while rats in studies of orexin and alcohol have had more than 25 days of self-administration training, not including a period of sucrose fading (Brown et al.,
2013). In order to investigate if this is the case, it would be important to replicate previous studies exactly, except for the use of ICV TCS 1102. This would confirm that the reason for the present null results is the difference in the behavioural protocol being used and not the drug or route of administration. Another method would be to replicate the previous studies, but to administer single and dual orexin antagonists systemically, to rule out the route of administration interpretation.
The motivational activator theory remains, at present, the best present explanation of the general function of the orexin system, but results from the present 144
Chapter 3. General Discussion
study suggest that it requires further refinement. The orexins have previously been shown to be involved in many different appetitive behaviours, using both agonists to promote motivation for food or sweet pellets (Sakurai et al., 1998; Thorpe, Cleary,
Levine, & Kotz, 2005) and antagonists to suppress motivation for a variety of drugs of abuse (Brown et al., 2013; Harris et al., 2005; Hollander et al., 2008; Lawrence et al.,
2006; Plaza-Zabala et al., 2013; Smith & Aston-Jones, 2012; Smith et al., 2009; Smith et al., 2010b). However, given the variance that is observed between different reinforcers, even reinforcers that are as similar as sucrose and saccharin (Cason &
Aston-Jones, 2013a, 2013b), it seems that the orexins are playing multiple specific roles in motivation. At times, it appears that orexin neurons may respond to contextual or discriminative cues, but they may not be sufficiently activated by discrete cues to express c-Fos (Moorman et al., 2016). There may also be preconditions before the orexin system will become involved, such as length of training or an animal’s general motivational state or stress level which may predispose an animal toward compulsive behaviour (James et al., 2017). In some circumstances, a single orexin receptor may also be involved, as has previously been shown for alcohol where OX2 receptor antagonism does not affect cue-induced reinstatement, but OX1 receptor antagonism does (Brown et al., 2013; Lawrence et al., 2006). Therefore, a multiplicity of roles for the orexin system is likely, with the exact effects that the orexin system exerts on a behaviour under control of various preconditions which are a partially described by the motivational activator theory, such as unit cost, highly salient rewards, and cue driven reward seeking, but may also include other reinforcer properties, length of training, and the type of cue which is driving behaviour.
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Illness and Adverse Events
Orexin receptor antagonists have previously failed to gain clinical approval due to adverse events or side effects that were discovered during clinical trials (Cruz et al.,
2014b). In the present study, a small number of animals became ill during experiments and there was one adverse event. It is expected that a small number of animals may become ill during microinjection experiments because microinjections deliver drugs directly to the brain and testing involves extra stress for the animals. No pattern was detectable among the small number of animals that became ill during these experiments.
It is therefore difficult to draw conclusions about any potential adverse effects from these incidents without conducting additional experiments explicitly designed to detect whether dual orexin receptor antagonists cause adverse effects.
Limitations
This thesis used intracerebroventricular microinjections of orexin receptor antagonists which have several limitations. Perhaps the most important is that it is difficult to know if the drug is reaching the most important target regions for reward seeking. While Experiment 2 provided a positive control demonstrating behavioural effects of centrally administered TCS 1102 against centrally administered OX-A, it is not known which region(s) TCS 1102 is working in to exert these effects. Theoretically, the ventricles should transport the drug throughout the brain but the unique properties of each compound, such as its solubility, MW, TPSA, and half-life, will determine how far it diffuses from the ventricles. An additional positive control, using a drug known to disrupt self-administration or reinstatement would have provided additional evidence that the ICV manipulations were working as required for the purpose of testing self- administration or reinstatement. Moreover, previous studies using both central and
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intraparenchymal (or intracerebroventricular and region-specific) administration of
TCS-OX2-29 have found that both types of microinjections can produce behavioural effects (Brown et al., 2013). For SB-334867, central microinjections of as little as 6 µg have produced behavioural effects (Mediavilla, Cabello, & Risco, 2011), despite SB-
334867’s high TPSA of 92.94 which should reduce its brain penetrance (Kelder et al.,
1999). As a small-molecule that is soluble in organic solvents and has been shown to be brain penetrant when systemically administered (Bergman et al., 2008), TCS 1102 should be able to diffuse to key regions to exert its effects. Bergman et al. (2008) showed that 30 min after a 100 mg/kg injection in rats, there was a brain concentration of 2370 nM and a CSF concentration of 43 nM, the latter being nearly three times greater than its IC50 of 17 nM at the OX1 receptor and 10 times greater than its IC50 of 4 nM at the OX2 receptor. As Smith, Di, and Kerns (2010a) have argued, the CSF concentration approximates the free fraction of drug in the brain and is close to what receptors in the brain would be exposed to. Although the high brain/CSF concentration suggests TCS 1102 is subject to significant non-specific binding, systemic injections of as little as 10 mg/kg have been shown to be effective on models of fear in rats (Chen et al., 2014) and 15 mg/kg was shown to be effective for nicotine-induced reinstatement of food seeking (Winrow et al., 2010). These studies demonstrate that TCS 1102 does indeed cross the blood brain barrier and circulates in the brain at concentrations sufficient to produce behavioural effects. TCS 1102 should therefore be able to access key brain regions when injected intracerebroventricularly. However, as there were few effects of centrally administered TCS 1102 in the results reported here, it is not known if its low CSF/brain concentration could be affecting its distribution and efficacy when microinjected into the ventricles.
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There are also other limitations of microinjection studies that should be acknowledged. While TCS 1102 has been screened for off-target effects, there may be off-target effects that were not detected during selectivity screening. Additionally, as a relatively new compound, it is possible that TCS 1102 has other stability or toxicological issues that are currently unknown. These may become clear in time, as they did for SB-334867 (McElhinny Jr et al., 2012). Vehicle solutions for microinjections may also contain components that have some activity. The Vitamin E-
TPGS component of the vehicle solution for TCS 1102 is known to inhibit P- glycoprotein and is more viscous than water (Guo, Luo, Tan, Otieno, & Zhang, 2013), but this should not have had any effects on appetitive motivation. Importantly, the
Vitamin E-TPGS vehicle did not affect OX-A-induced feeding or prevent TCS 1102 from attenuating the behaviour, but potential activity of excipients is still important to consider.
There were also differences in the strains, the operant manipulanda, and food/water restriction schedules used throughout these experiments which was due to both practical and theoretical considerations. Long Evans rats from Monash Animal
Services have historically been used in the McNally laboratory for alcoholic beer studies (Hamlin et al., 2006; Hamlin et al., 2008; Hamlin et al., 2007; Marchant,
Furlong, & McNally, 2010; Millan, Furlong, & McNally, 2010; Perry & McNally,
2013), but they were unavailable for the present series of experiments. Long Evans rats are a good model animal for alcohol studies because they can be trained to drink solutions of up to 20% ethanol without sucrose fading (Simms, Bito-Onon, Chatterjee,
& Bartlett, 2010) and so Long Evans rats from the Florey Institute of Neuroscience and
Mental Health were used. However, in the Clemens lab where Experiments 5, 7, and 8 were conducted has historically used Sprague-Dawley rats from Animal Resources 148
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Centre (Castino, Cornish, & Clemens, 2015; Macnamara et al., 2016; Motbey et al.,
2013). It has previously been shown that Long Evans and Sprague-Dawley rats are broadly similar in exploration, anxiety, and learning, but have some differences in social behaviour (Ku, Weir, Silverman, Berman, & Bauman, 2016). Therefore, it is unlikely that strain differences present a significant confound. Similarly, it is unlikely that the different operant manipulanda (nosepokes and levers) made a significant difference.
Rats will naturally explore with their vibrissae and noses, making the acquisition of a nosepoke response potentially easier than a lever press. The differences in manipulanda between experiments were because nosepokes have been used for alcoholic beer and nicotine studies in the McNally and Clemens laboratories (Macnamara et al., 2016;
Prasad & McNally, 2014), but levers are used for studies involving food pellets in the
McNally laboratory (Jean-Richard-dit-Bressel & McNally, 2016; Sengupta, Winters,
Bagley, & McNally, 2016; Yau & McNally, 2015). However, rats in Experiments 1 – 4 showed rapid acquisition of lever pressing for palatable food and similar levels of self- administration to rats in Experiment 5 who made nosepokes for palatable food. It is therefore possible, but unlikely, that differences in strain and manipulanda may have had an impact on the results of the present study.
Food and water restriction schedules were also different between experiments.
Importantly, these restriction schedules were applied to rats in a group-housed situation.
The 20 g/rat/day and 1 h/day to 85% of free feeding bodyweight schedules were based on established practice within the laboratory for nicotine studies and alcoholic beer studies respectively (Macnamara et al., 2016; Prasad & McNally, 2014). The 22 g/rat/day restriction schedule was chosen because it was the least restrictive schedule that would leave the rats in a hungry motivational state before their palatable food session. Rats on the 20-22 g/day restriction schedules all continued to gain weight at 149
Chapter 3. General Discussion
similar rates throughout the experiment, suggesting approximately equal food consumption. Rats in the 1 h/day condition all ate and drank to satiety within their allotted time and, after an initial decrease, resumed weight gain during the experiment.
While individual variation in food and/or water consumption in the home cage may have enhanced the variability in food responding to obscure small effects of treatment, the welfare benefit of group housing for the animals, along with the low clinical relevance of a small preclinical effect meant that single housing of animals to control for food and/or water consumption was not justifiable.
Orexin neurons also express multiple other neurotransmitters, such as glutamate, neurotensin, and dynorphin (Furutani et al., 2013; Henny et al., 2010; Muschamp et al.,
2014; Rosin et al., 2003). Orexin neuron activation may therefore not necessarily mean that there is orexin peptide release. Recent work on the hypothalamus has identified glutamate and GABA neurons in the lateral hypothalamus that are distinct from orexin and MCH neurons which also regulate feeding and reward independently of orexin neurons (Jennings, Rizzi, Stamatakis, Ung, & Stuber, 2013; Jennings et al., 2015;
Stuber & Wise, 2016). Moreover, orexin neurons themselves have been shown to have inhibitory effects on MCH neurons which require GABAA receptors (Apergis-Schoute et al., 2015). Glutamate release has also been demonstrated to produce different temporal effects and to require a lower level of stimulation than orexin release from orexin neurons (Schöne, Apergis-Schoute, Sakurai, Adamantidis, & Burdakov, 2014).
Studies that examine c-Fos expression in OX-A immunoreactive cells are therefore not necessarily showing that a neuron was releasing orexin peptides during a behaviour.
Antagonism of orexinergic activity may also leave the non-orexinergic components of orexin neuron signals intact. Caution must be exercised in drawing conclusions from these studies that should be limited to the activity of the orexin neuron which may or 150
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may not involve orexin peptide or orexinergic signalling that may be dissociable from cotransmitters expressed by orexin neurons.
Finally, it is worth noting that there is increased recognition of the role of non- orexin LH neurons in reinstatement, feeding, and reward. For example, ventral striatal control over feeding and reward depends on LH GABA neurons, not LH orexin neurons, and LH orexin neurons receive very few direct inputs from ventral striatum
(Jennings et al., 2015; O’Connor et al., 2015). This finding is especially important when evaluating the role(s) of orexin in reinstatement of drug seeking models because past research, using CPP for morphine (Harris et al., 2005) and context-induced reinstatement of alcohol seeking after extinction or punishment, has shown that LH is causal to reinstatement (Marchant, Hamlin, & McNally, 2009; Marchant &
Kaganovsky, 2015) and is associated with recruitment of ventral striatal inputs to LH
(Marchant et al., 2009). In other words, multiple LH neuronal subtypes may contribute to reinstatement.
Conclusions
The present study has shown, for the first time, that the dual orexin receptor antagonist, TCS 1102, attenuates OX-A-induced feeding but has little efficacy when applied to appetitive motivation for palatable food, beer, and nicotine. Orexin neurons were recruited in the PeF and DMH during both extinction and cue-induced reinstatement for palatable food, but with no significant difference between extinction or reinstatement tests. It also tested several predictions of the motivational activator theory with mixed support for its predictions. There was only a small, transient effect on cue/nicotine compound reinstatement after chronic, but not short term, access to nicotine. These findings suggest that the orexin/hypocretin system may change its role
151
Chapter 3. General Discussion
in regulating appetitive motivation for a reinforcer over time with the role of the orexin/hypocretin system in regulating nicotine seeking increasing as the amount of experience the animal has with self-administering the drug increases. While this finding might be consistent with the motivational activator theory, which posits that the orexin/hypocretin system responds to reward-associated cues, several of the findings of the present study are not. Indeed, in no other test of relapse-like behaviour was there any effect of orexin antagonism despite several previous studies implicating the orexins in reinstatement of drug reinforcers such as alcohol, nicotine, and cocaine and, in certain circumstances, for non-drug reinforcers. There was no effect of TCS 1102 on reinstatement of nicotine seeking induced by cues alone or reinstatement of palatable food seeking. Neuroanatomical evidence also showed that although orexin neurons were recruited by testing, there was no specific activation of orexin neurons in response to cues during reinstatement because there was difference between the extinction and reinstatement groups and the home-cage control, but no difference between the extinction group and the reinstatement group.
It appears that orexin signalling is not necessarily required when a reward is highly salient or available under a high unit-cost. There was no effect of TCS 1102 on
FR1 responding for alcoholic beer, nicotine, or palatable food and no effect of SB-
334867, ACT-335827, or TCS-OX2-29 on FR10 self-administration of palatable food.
A small number of animals also self-administered nicotine under FR5 conditions, but
TCS 1102 has no effect on these high responders. These rewards are all plausibly highly salient, especially for animals that are motivated to obtain rewards because of varying degrees of food and water restriction. It is therefore not simply the case that orexins are involved where reinforcers are highly salient or unit-costs are high. The orexin/hypocretin system may provide motivational activation in some circumstances, 152
Chapter 3. General Discussion
but not under most of the conditions examined in this thesis. Thus, while the motivational activator theory may explain orexinergic regulation of cocaine seeking
(Mahler et al., 2014), it may not apply as well to other reinforcers or experimental conditions such as those studied here.
Finally, it appears that, due to the large number of instances where TCS 1102 failed to affect appetitive motivation for food and drugs, dual orexin antagonists are unlikely to be effective pharmacotherapies for addiction to alcohol, nicotine, or palatable food. However, while these results do not support targeting the orexin/hypocretin system for addiction pharmacotherapy, there are still questions regarding whether the orexin/hypocretin system provides general arousal during reinstatement or is specifically activated by cues. Further studies are also required to investigate which combinations of reinforcers and training conditions elicit orexinergic regulation in order to establish the exact role of this neuropeptide system in appetitive motivation.
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