Jonathan Andrew Farrimond
email: j.a.farrimond@reading.ac.uk

Marion Simone Mercier

Benjamin J Whalley

Claire M Williams

M Williams

Cannabis sativa and the endogenous cannabinoid system: therapeutic potential for appetite regulation

Keywords: Feeding, Appetite, Phytocannabinoid, Endocannabinoid, Delta-9tetrahydrocannabinol Feeding, appetite, phytocannabinoid, endocannabinoid, 17 ∆ 9 tetrahydrocannabinol

Introduction 1

For ~10,000 years, Cannabis sativa (C. sativa) has been used medicinally and 2 recreationally for its diverse pharmacological actions and psychotropic properties. 3 Typical actions include sedation, analgesia, hypothermia, catalepsy and euphoria which 4 are thought to arise mainly from cannabinoid type 1 receptor (CB 1 R) activation 5 [START_REF] Childers | Cannabis and endogenous cannabinoid systems[END_REF][START_REF] Little | Pharmacology and 10 stereoselectivity of structurally novel cannabinoids in mice[END_REF]. These effects have typically been ascribed to 6 the sixty [START_REF] Howlett | The cannabinoid receptors[END_REF]Pertwee, 2008b) to eighty [START_REF] Izzo | Non-psychotropic 22 plant cannabinoids: new therapeutic opportunities from an ancient herb[END_REF] 7 phytocannabinoids (pCBs) present in C. sativa. In more recent times, C. sativa has been 8

used less widely for medicinal purposes, due in part to the development of other 9 therapeutic agents with better side effect profiles (e.g. [START_REF] Tramer | 20 Cannabinoids for control of chemotherapy induced nausea and vomiting: quantitative 21 systematic[END_REF]. Indeed, 10 cannabinoid side-effects are known to include reduced motor coordination, tachycardia, 11 transient memory loss, disruption to the sense of time, cognitive impairments, anxiety 12 and a disruption of appetite regulation [START_REF] Childers | Cannabis and endogenous cannabinoid systems[END_REF][START_REF] Dewey | Cannabinoid pharmacology[END_REF]Grant et al., 13 2005;[START_REF] Hollister | Health aspects of cannabis[END_REF]Kirkham et al., 2001b;[START_REF] Wilkinson | 19 Medicinal cannabis: is delta9-tetrahydrocannabinol necessary for all its effects?[END_REF] (for a recent 14 review on the safety of C. sativa-based therapy see [START_REF] Wang | Adverse effects of medical cannabinoids: 5 a systematic review[END_REF]. However, the 15 relatively recent identification of the CB 1 R and cannabinoid type 2 (CB 2 R) receptors, [START_REF] Yoshida | 17 Endocannabinoids selectively enhance sweet taste[END_REF] alongside the subsequent characterisation of the endocannabinoid (eCB) system (for a 17 recent review see [START_REF] Maccarrone | Endocannabinoids: Friends and foes of reproduction[END_REF], has driven a resurgence of interest in the potential 18 applications of cannabinoid-based therapy. Cannabinoid therapies have now been 19 investigated for a wide range of neurological and neurodegenerative disorders (Glass, 20 2001; [START_REF] Pryce | 24 Cannabinoids inhibit neurodegeneration in models of multiple sclerosis[END_REF], both in terms of treatment (for a review see [START_REF] Consroe | Brain cannabinoid systems as targets for the therapy of neurological 27 disorders[END_REF]) 21 and/or symptom management [START_REF] Davis | The emerging role of cannabinoid 12 neuromodulators in symptom management[END_REF]. One notable example is in the 22 treatment of multiple sclerosis [START_REF] Pryce | Emerging properties of cannabinoid medicines in 28 management of multiple sclerosis[END_REF] and its symptoms (eg. spasticity), for 23 Spain and Canada by GW Pharmaceuticals. 2 'Cannabinoid' is a well-known term used to describe the compounds isolated 3 from C. Sativa, the pCBs such as ∆ 9 tetrahydrocannabinol (∆ 9 THC), cannabidiol (CBD) 4 and cannabinol (CBN). In addition to these, the term now also encompasses both 5 synthetic exogenous (e.g. SR141716A, a CB 1 R specific antagonist/inverse agonist) and 6 endogenously produced ligands (the eCBs anandamide (AEA) and 2-arachidonyl 7 glycerol (2-AG)) at CB 1 -and CB 2 Rs (for an overview of the cannabinoid receptor 8 (CBR) ligands referred to in this review, see table 1). The development and 9 characterisation of such pharmacological tools has enabled significant progress to be 10 made into understanding the eCB system over the last 20 years (Onaivi et al., 2002). 11 ∆ 9 THC, the main psychoactive component in C. sativa, was first isolated and 12 synthesised in 1964 by Gaoni et al. (Gaoni et al., 1964a). However, it was not until 13 1988 that the first cannabinoid specific G-protein coupled receptor (GPCR; CB 1 R) at 14 which it acts was characterised [START_REF] Devane | Determination 2 and characterization of a cannabinoid receptor in rat brain[END_REF][START_REF] Matsuda | Structure of a 13 cannabinoid receptor and functional expression of the cloned cDNA[END_REF], lending 15 credence to the postulated existence of an eCB system [START_REF] Howlett | The cannabinoid receptor: biochemical, anatomical and behavioral 5 characterization[END_REF]) (for review [START_REF] Yoshida | 17 Endocannabinoids selectively enhance sweet taste[END_REF] see Pertwee, 2005). A series of studies [START_REF] Herkenham | 13 Characterization and localization of cannabinoid receptors in rat brain: a quantitative in 14 vitro autoradiographic study[END_REF][START_REF] Herkenham | Cannabinoid receptor localization in brain[END_REF]) identifying the widespread expression of this receptor in the brain further 18 supported this discovery. These findings were rapidly followed by the isolation and 19 structural determination of the first eCB ligand, AEA, in 1992 [START_REF] Devane | 6 Isolation and structure of a brain constituent that binds to the cannabinoid receptor[END_REF], the 20 identification and characterisation of CB 2 R expressed, on immune cells, in 1993 (Munro 21 et al., 1993), and the identification of a second eCB ligand, 2-AG, in 1995 (Mechoulam 22 et al., 1995b;[START_REF] Sugiura | 2-1 Arachidonoylglycerol: a possible endogenous cannabinoid receptor ligand in brain[END_REF] (See Fig. 1). Since then, significant academic and 23 commercial effort has been invested in characterising and exploiting the eCB system 24 As previously outlined, it is now apparent that the eCB system is physiologically 3 ubiquitous and linked to many pathophysiological states (de Fonseca, 2005), resulting in 4 its identification as a therapeutic target, as well as the publication of numerous reviews 5 regarding the potential benefits of cannabinoid based therapies (e.g. Agrawal et al.,6 2009; [START_REF] Baker | The therapeutic potential of 33 cannabis[END_REF][START_REF] Glass | The role of Cannabinoids in neurodegenerative diseases[END_REF][START_REF] Goutopoulos | From cannabis to cannabinergics: new 34 therapeutic opportunities[END_REF]Makriyannis et al., 7 2005). This is particularly true in the field of body weight regulation and energy balance 8 [START_REF] Yates | Inactivation and Biotransformation of the Endogenous 15 Cannabinoids Anandamide and 2-Arachidonoylglycerol[END_REF], where significant advances have been made in the development of 9 both anti-anorectic (e.g. in hepatitis C [START_REF] Costiniuk | Evaluation of oral cannabinoid-containing 30 medications for the management of interferon and ribavirin-induced anorexia, nausea 31 and weight loss in patients treated for chronic hepatitis C virus[END_REF]; in cancer (Walsh et al., 10 2003); in human immunodeficiency virus (HIV) [START_REF] Woolridge | Cannabis 1 use in HIV for pain and other medical symptoms[END_REF]) and anti -11 obesity [START_REF] Van Gaal | Effects of the 31 cannabinoid-1 receptor blocker rimonabant on weight reduction and cardiovascular risk 32 factors in overweight patients: 1-year experience from the RIO-Europe study[END_REF] cannabinoid-based therapies. However, these advances 12 must be made with care [START_REF] Grant | Cannabis and endocannabinoid modulators: Therapeutic 37 promises and challenges[END_REF], and it is clear that further long-term studies 13 are warranted if the safety profiles of such therapies are to be fully understood (Wang et 14 al., 2008). Indeed, the withdrawal of Acomplia (Rimonabant: SR141716A; see EMA, [START_REF] Kennett | A comparison of the effects 16 of the CB 1 receptor antagonist SR141716A, pre-feeding and changed palatability on 17 the microstructure of ingestive behaviour[END_REF] 2009 for details) from clinical use for the treatment of obesity due to serious cognitive 16 side effects, combined with the cessation of Phase III clinical trials for a second putative 17 anti-obesity drug, Taranabant (MK-0364; Clark, 2009), illustrates the difficulties of 18 manipulating such a complex and ubiquitous system. Nonetheless, it is clear that the 19 eCB system plays an important, if not yet fully understood, role in appetite, obesity and 20 energy balance (for reviews see Cota et al., 2003a;[START_REF] Kirkham | Endocannabinoids and the Neurochemistry of Gluttony[END_REF]. This review will 21 describe the significant progress made, as well as the work still required, in order to 22 fully understand the role played by the eCB system in feeding and appetite regulation. 23 Importantly, by focusing on those studies that have examined pCB effects and how they 24 The endocannabinoid system in the central nervous system 1 Although, CBRs are present in both the peripheral and central nervous systems (CNS), 2 the majority of cannabinoid feeding research to date has focused primarily on centrally-3 mediated feeding mechanisms, a focus that is consequently reflected in this review. A 4 short section outlining the biological components that comprise the eCB system within 5 the CNS is presented hereafter (see also figures 2 and 3). 6

ECBs (AEA and 2-AG) are produced on demand by postsynaptic neurons (Di 7 [START_REF] Marzo | The endocannabinoid system and its 15 therapeutic exploitation[END_REF] in the CNS in response to rises in intracellular [Ca 2+ ] ( Cadas et al., 8 1997; Di [START_REF] Marzo | 18 Formation and inactivation of endogenous cannabinoid anandamide in central neurons[END_REF]. Such increases typically occur following G q -coupled 9 receptor activation which stimulates production of inositol trisphosphate (IP 3 ) by 10 phospholipase C (PLC) leading to a release of Ca 2+ from intracellular stores (for 11 reviews see Berridge, 1995;Berridge et al., 1988;[START_REF] Jr | A model for receptor-regulated calcium entry[END_REF]. Alternatively, 12 ionotropic glutamate receptor activation permits the influx of Ca 2+ to increase eCB 13 production (Freund et al., 2003). 14 AEA synthesis from its precursors, arachidonic acid and N-arachidonoyl 15 phosphatidyl-ethanolamine (NaPE) is an on demand effect. Phosphatidylethanolamine is 16 first catalysed by Ca 2+ -dependent N-acyltransferase [START_REF] Cadas | Occurrence and biosynthesis of endogenous 5 cannabinoid precursor, N-arachidonoyl phosphatidylethanolamine, in rat brain[END_REF] values and so reducing the further pre-synaptic release of neurotransmitter (for review 13 see [START_REF] Mackie | Mechanisms of CB1 receptor signaling: endocannabinoid modulation 39 of synaptic strength[END_REF][START_REF] Südhof | Pharmacology of neurotransmitter release[END_REF]. Indeed, presynaptically it has been conclusively 14 demonstrated that CB 1 R activation has a direct effect on membrane polarisation, for 15 example, in cultured hippocampal neurones [START_REF] Deadwyler | Cannabinoids 32 modulate voltage sensitive potassium A-current in hippocampal neurons via a cAMP-33 dependent process[END_REF]Schweitzer, 2000) 16 and in CA1 pyramidal cells [START_REF] Kirby | Cannabinoid receptor activation in 9 CA1 pyramidal cells in adult rat hippocampus[END_REF]. 17

ECB reuptake however remains the subject of much debate. Fatty acid amide 18 hydrolase (FAAH; [START_REF] Cravatt | 5 Molecular characterization of an enzyme that degrades neuromodulatory fatty-acid 6 amides[END_REF] and monoacylglycerol lipase (MAGL; Lambert 19 et al., 2005) are the two main enzymes involved in eCB degradation, and have been 20

known to degrade AEA and 2-AG for some time. However, although a specific 21 transmembrane transporter protein has yet to be identified, it seems that reuptake may 22 be a rapid carrier-mediated transport process (e.g. [START_REF] Gerdeman | Postsynaptic endocannabinoid release is 14 critical to long-term depression in the striatum[END_REF]Ronesi et al., 23 2004) since eCBs within the synaptic cleft are almost immediately inactivated (Di 24 F o r P e e r R e v i e w [START_REF] Marzo | Endocannabinoids and the regulation of their levels in 25 health and disease[END_REF]. 2-AG is typically inactivated by MAGL (Di Marzo, 2008) but also 1 to some extent by FAAH (Blankman et al., 2007), whereas, AEA is thought to be 2 degraded by FAAH alone [START_REF] Vandevoorde | Overview of the chemical families of fatty acid amide hydrolase 40 and monoacylglycerol lipase inhibitors[END_REF]. Four mechanisms are currently 3 proposed to underlie eCB reuptake; a membrane-localised transmembrane carrier 4 protein, FAAH-induced passive diffusion through the lipid bilayer, the creation of 5 caveolae with AEA binding sites on the cell membrane which then internalise AEA 6 and/or 2-AG, and via intracellular sequestration into the cell within a lipid shell (for a 7 recent review see [START_REF] Yates | Inactivation and Biotransformation of the Endogenous 15 Cannabinoids Anandamide and 2-Arachidonoylglycerol[END_REF]. Intracellular FAAH-mediated AEA hydrolysis 8 creates arachidonic acid and ethanolamine [START_REF] Deutsch | The fatty acid amide hydrolase (FAAH)[END_REF] thoroughly review the 9 biochemistry of FAAH) whilst 2-AG undergoes primarily MAGL-mediated hydrolysis 10 into arachidonic acid and glycerol (Blankman et al., 2007). 11

While the mechanisms involved in AEA and 2-AG degradation remain poorly 12 understood, it is clear that FAAH and MAGL blockade could be of therapeutic value. 13 FAAH inhibition, for instance by URB597 [START_REF] Piomelli | 17 Pharmacological profile of the selective FAAH inhibitor KDS-4103 (URB597)[END_REF]; see Vandevoorde,14 2008 for a review of FAAH and MAGL inhibitors), is expected to potentiate AEA 15 effects (and to some extent 2-AG). Such inhibition could, for instance, be expected to 16 have effects on energy homeostasis as AEA and 2-AG administration into the 17 hypothalamic nuclei has been conclusively demonstrated to induce hyperphagia in rats 18 [START_REF] Jamshidi | Anandamide administration into the ventromedial 26 hypothalamus stimulates appetite in rats[END_REF]Kirkham et al., 2001a;[START_REF] Kirkham | Endocannabinoid levels in rat 24 limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: 25 stimulation of eating by 2-arachidonoyl glycerol[END_REF] levels respectively. For instance, some evidence suggests that FAAH inhibition can also 1 decrease 2-AG levels (see Di Marzo et al., 2008 for a review of these data), and MAGL 2 inhibition, by the particularly potent and selective MAGL inhibitor JZL-184, has been 3 found to induce behavioural effects similar to those produced by agonist-induced CB 1 R 4 activation [START_REF] Long | Selective 17 blockade of 2-arachidonoylglycerol hydrolysis produces cannabinoid behavioral effects[END_REF]. The dynamic interplay between these systems must be 5 taken into account when considering new therapeutic approaches if unwanted side-6 effects are to be minimised. 7

The cannabinoid receptors, CB 1 and CB 2 R, are members of the superfamily of 8

GPCRs and are characterised by seven transmembrane helices [START_REF] Turu | Signal transduction of the CB1 cannabinoid receptor[END_REF]. CB 1 R 9 was cloned in 1990 [START_REF] Matsuda | Structure of a 13 cannabinoid receptor and functional expression of the cloned cDNA[END_REF], 26 years after the isolation of ∆ 9 THC. 10 CB 2 Rs were first identified and characterised via polymerised chain reaction 11 experiments in 1993 [START_REF] Munro | Molecular characterization of a peripheral 38 receptor for cannabinoids[END_REF][START_REF] Gong | 30 Cannabinoid CB2 receptors: Immunohistochemical localization in rat brain[END_REF], and were initially thought 12 to be present only in inflammatory/immune cells; Basavarajappa, 2007b; Munro et al., 13 1993), although evidence now shows CB 2 R expression in the brain [START_REF] Gong | 30 Cannabinoid CB2 receptors: Immunohistochemical localization in rat brain[END_REF]14 Onaivi et al., 2006;[START_REF] Van Sickle | Identification and functional characterization of brainstem cannabinoid CB2 37 receptors[END_REF]. Indeed, it is now known that both CB 1 -and 15 CB 2 Rs are widely expressed in the brain [START_REF] Gong | 30 Cannabinoid CB2 receptors: Immunohistochemical localization in rat brain[END_REF]Moldrich et al., 2000) 16 although it is postulated that the majority of the functional eCB effects in the CNS are 17 CB 1 R mediated [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF]. This receptor is one of the most abundant GPCRs in 18 the CNS [START_REF] Herkenham | 13 Characterization and localization of cannabinoid receptors in rat brain: a quantitative in 14 vitro autoradiographic study[END_REF] and its distribution within the mammalian brain 19 reflects the behavioural effects of cannabinoid administration. For instance, CB 1 R levels 20 are particularly high in the cortex, hippocampus, cerebellum and basal ganglia, 21 correlating with the well-known effects of cannabinoids on cognition, memory and 22 motor control respectively. [START_REF] Herkenham | 13 Characterization and localization of cannabinoid receptors in rat brain: a quantitative in 14 vitro autoradiographic study[END_REF][START_REF] Tsou | Immunohistochemical 24 distribution of cannabinoid CB1 receptors in the rat central nervous system[END_REF]; for a detailed 23 review of CB 1 R localisation see [START_REF] Mackie | Distribution of cannabinoid receptors in the central and peripheral 35 nervous system[END_REF]. Although also present in the 24 hypothalamus, CB 1 R levels are lower in this brain region than in those mentioned above 1 [START_REF] Herkenham | 13 Characterization and localization of cannabinoid receptors in rat brain: a quantitative in 14 vitro autoradiographic study[END_REF][START_REF] Tsou | Immunohistochemical 24 distribution of cannabinoid CB1 receptors in the rat central nervous system[END_REF] which is somewhat surprising considering 2 the well-known hypothalamic involvement in feeding-related processes (Elmquist et al., 3 1999). However, a study by Breivogel et al. found that hypothalamic CB 1 Rs are more 4 strongly coupled to G-proteins than those in numerous other brain regions including the 5 cortex and hippocampus (Breivogel et al., 1997), supporting important functional 6 effects of cannabinoids in this brain region, and suggesting that functional gain 7 associated with downstream intracellular signalling cascades following CB 1 R activation 8 may be of crucial importance. 9

While CB 1 -and CB 2 Rs are thought to mediate the majority of cannabinoid 10 effects, both exo-and endo-genous cannabinoids also exert effects via other receptors . 11 In line with this, it has been conclusively demonstrated that the non-psychotropic 12 exogenous cannabinoids (excluding CBN and ∆ 9 tetrahydrocannabivarin; ∆ 9 THCV) 13 exhibit almost no affinity for either CB 1 or CB 2 Rs [START_REF] Petrosino | Endocannabinoid chemical biology: a tool 4 for the development of novel therapies[END_REF] [START_REF] Sanger | Endocannabinoids and the gastrointestinal tract: what are the key 31 questions?[END_REF], is currently a putative third CBR. Upon activation, GPR55 1 suppresses M-type K + currents and stimulates phosphatidylinositol 4,5-bisphosphate 2 (PIP2) -induced Ca 2+ release from intracellular stores [START_REF] Henstridge | The 9 GPR55 ligand L-{alpha}-lysophosphatidylinositol promotes RhoA-dependent Ca2+ 10 signaling and NFAT activation[END_REF]Lauckner 3 et al., 2008). While the status of GPR55 as a CBR has been a topic of fierce academic 4 debate for some years [START_REF] Petitet | GPR55 as a new cannabinoid receptor: still a 1 long way to prove it[END_REF], somewhat more compelling evidence has 5 been presented recently suggesting that it is indeed a CBR [START_REF] Baker | In silico patent searching reveals a 30 new cannabinoid receptor[END_REF] Ryberg (using radioligand binding assays in human embryonic kidney (HEK293) cells; 9 [START_REF] Ryberg | 27 The orphan receptor GPR55 is a novel cannabinoid receptor[END_REF] have shown that CP55,940 (a non-selective CBR agonist), but not 10 WIN55212-2 (a CBR agonist), binds with strong affinity to GPR55; Baker also showed 11 that AEA, 2-AG and ∆ 9 THC bind to this receptor. Moreover, Ryberg presented further 12 evidence in the same manuscript suggesting that GPR55 was G α13 coupled, and that the 13 comparatively late detection of GPR55 as a possible CBR was because the affinity of 14 CP55940 for GPR55 binding was 25 times lower than that for CB 1 R binding. However, 15 in a similar study Lauckner and colleagues [START_REF] Lauckner | The cannabinoid agonist WIN55,212-2 38 increases intracellular calcium via CB1 receptor coupling to Gq/11 G proteins[END_REF] evidence is mounting for GPR55's role in the eCB system, as demonstrated for instance 1 by Kapur and colleagues, who observed AM251 (CB 1 R inverse agonist) and 2 SR141716A (a CB 1 R specific antagonist) binding to GPR55 [START_REF] Kapur | Atypical 1 responsiveness of the orphan receptor GPR55 to cannabinoid ligands[END_REF]. 3

Whilst it clearly remains an 'enigmatic' receptor [START_REF] Ross | The enigmatic pharmacology of GPR55[END_REF], research is beginning to 4 focus on possible GPR55 based therapies, with recent studies linking it to 5 neuroinflammation [START_REF] Pietr | Differential 7 changes in GPR55 during microglial cell activation[END_REF], inflammatory and neuropathic pain (Staton et al., 6 2008) and anorexia [START_REF] Ishiguro | 18 Functional polymorphism in the GPR55 gene is associated with anorexia nervosa[END_REF] production and release to some extent, as well as increasing intercellular cyclic 13 adenosine monophosphate (cAMP) accumulation (Ning et al., 2008;[START_REF] Soga | 19 Lysophosphatidylcholine enhances glucose-dependent insulin secretion via an orphan 20 G-protein-coupled receptor[END_REF]. 14 However, despite findings that GPR119 agonism by OEA suppresses feeding in rats 15 [START_REF] Rodriguez De Fonseca | 10 An anorexic lipid mediator regulated by feeding[END_REF], this receptor is unlikely to be involved in centrally 16 mediated eCB feeding effects as it has been found in the gut (Overton et al., 2006;17 Overton et al., 2008). It should nevertheless still be considered when interpreting in vivo 18 feeding study results and could represent a useful target for anti-obesity therapies 19 [START_REF] Hughes | Emerging therapies for metabolic diseases--the focus is on diabetes 15 and obesity[END_REF]. 20 Although, as previously mentioned, this review focuses primarily on central 21 cannabinoid mediated feeding mechanisms, peripheral effects, such as those mediated 22 by peripheral CBRs or GPR119 add considerable complexity when interpreting in vivo 23 feeding data. This is particularly true in light of findings describing peripheral CB 1 R 24 , and a potential modulatory role of these peripheral receptors on feeding 3 behaviour [START_REF] Gomez | A 26 Peripheral Mechanism for CB1 Cannabinoid Receptor-Dependent Modulation of 27 Feeding[END_REF]. Equally, although it is thought that most of the 4 functional eCB effects in the brain are CB 1 R-mediated [START_REF] Piomelli | The molecular logic of endocannabinoid signalling[END_REF], recent work 5 has suggested some functional expression of CB 2 Rs in the brain [START_REF] Gong | 30 Cannabinoid CB2 receptors: Immunohistochemical localization in rat brain[END_REF], 6 particularly at synapses [START_REF] Morgan | Functional CB2 type cannabinoid 32 receptors at CNS synapses[END_REF], and that these may also be involved in 7 feeding behaviours (Onaivi et al., 2008). Furthermore, CBR activation in the gut (as 8 recently reviewed by Izzo et al., 2010) for example, in terms of gut motility, is also 9 likely to have considerable effects on feeding patterns. As such, it is often difficult, 10 when interpreting in vivo experimental results, to exclusively assign cannabinoid effects 11 to either central or peripheral sites of action, or to differentiate between CB 1 -and CB 2 -, 12 or indeed non-CBR-mediated effects. Moreover, ligands which produce well-13 characterised effects at specific CBRs may also exert effects at other receptor sites, 14 thereby creating unexpected and potentially unwanted secondary effects. This is well 15 illustrated by SR141716A (Acomplia/Rimonabant), a well-known selective CB 1 R 16 antagonist/inverse agonist which has recently been found by Kapur et al. to agonise 17 GPR55, and which the authors suggest may have played a part in some of the 'off-18 target' effects that led to its withdrawal as an anti-obesity drug [START_REF] Kapur | Atypical 1 responsiveness of the orphan receptor GPR55 to cannabinoid ligands[END_REF]. 19 There is still much to clarify about how cannabinoids produce their endogenous 20 effects, and how they might interact with each other in vivo (Ben Amar, 2006; Whalley 21 et al., 2004). Furthermore, adding to this complexity is the eCB system's interaction 22 with other systems, in particular the opioid system which itself has been linked to 23 appetite regulation for some time (for an early review of opiates and appetite regulation 24 see [START_REF] Morley | The role of the endogenous opiates as regulators of appetite. 35[END_REF]. Indeed, it is now apparent that both the eCB and opioid 1 systems modulate energy balance, that both systems can modulate food intake 2 independently and that activation or suppression of both systems together can increase 3 or reduce food intake to a greater extent than modulation of either system alone (Gallate 4 et al., 1999; Kirkham et al., 2001c;[START_REF] Solinas | Motivational effects of cannabinoids and opioids on 23 food reinforcement depend on simultaneous activation of cannabinoid and opioid 24 systems[END_REF]. Whilst detailed discussion of 5 such interactions is beyond the scope of this review, they, along with the numerous 6 other complexities outlined above, are important factors to consider when looking at, or 7 indeed conducting and analysing, research on the eCB system and feeding. 8

Cannabinoid-mediated effects upon feeding 1

The last decade has seen considerable advances in our understanding of eCB-mediated 2 control of feeding behaviours. Numerous anecdotal reports exist, and the 'munchies' 3 effect has become a well-accepted physiological response to C. sativa consumption, yet 4 few quantitative reports from this period exist (for a complete review see Kirkham et 5 al., 2001b). While the 'classical' tetrad of cannabinoid effects was defined as decreased 6 spontaneous motor activity, hypothermia, analgesia and catalepsy [START_REF] Little | Pharmacology and 10 stereoselectivity of structurally novel cannabinoids in mice[END_REF], it 7 was only comparatively recently that ∆ 9 THC was demonstrated to induce hyperphagia 8 [START_REF] Williams | Hyperphagia in pre-fed rats following 32 oral ∆ 9 -THC[END_REF], an effect clearly linked to CB 1 R rather than CB 2 R activation 9 (Williams et al., 2002b). Since CB 2 R have only recently been localised to the CNS 10 (Morgan et al., 2009), and have not been found expressed on feeding pathways, little 11 research has considered the possible effects that CB 2 R stimulation may have on feeding. 12 While a limited number of reports which consider CB 2 R effects on feeding do exist (e.g. 13

Onaivi et al., 2008), they have so far presented inconclusive evidence. Furthermore, 14 possible opioid/cannabinoid and cannabinoid/serotonergic interactions have been 15

studied, for example by Williams et al., (2002b). Williams demonstrated a functional 16 relationship between the cannabinoid and opioid systems in the control of appetite, and 17 notably rejected any serotonergic interaction: upon administration of ∆ 9 THC and either 18 SR141716A, SR144528, naloxone or dexfenfluramine, Williams observed that while 19 naloxone could block ∆ 9 THC-mediated feeding increases, dexfenfluramine could not. 20

Unfortunately, a detailed description of such interactions falls beyond the scope of this 21 review, although a recent review of the effects of CB/opioid interactions on the 22 behavioural satiety sequence can be found in [START_REF] Cota | Cannabinoids, opioids and eating 2 behavior: the molecular face of hedonism?[END_REF]. Given the relatively 23 recent success of treatment with ∆ 9 THC-based CB 1 R partial agonists (e.g. dronabinol or 24 Endocannabinoid agonism and its effects on feeding 1 ∆ 9 THC was first shown to exert hyperphagic effects in rats in 1998 (Williams et al., 2 1998). ∆ 9 THC (0.063-2.0mg/kg) administered orally (per ora; p.o.) to pre-satiated (a 3 situation in which eCB tone is reduced by the highly palatable prefeed process), adult, 4 male, Lister hooded rats, at doses >0.5mg/kg significantly increased chow intake 5 relative to control in the first hour following drug administration. Furthermore, no 6 significant differences in food intake between control and any ∆ 9 THC doses were seen 7 over the twenty four hour period following ∆ 9 THC administration. This early study 8 importantly demonstrated that very low concentrations of orally administered ∆ 9 THC 9 could substantially change an animal's motivation to consume; particularly given that a 10 considerable percentage of the administered drug would have undergone first pass 11 metabolism as a result of oral administration. Intriguingly, a later study by Avraham 12 (Avraham et al., 2004) also showed that extremely low ∆ 8 THC doses (a pCB similar to 13 ∆ 9 THC but regarded as more stable; 0.001mg/kg; intraperitoneal; i.p.) increased feeding 14 in mice. These studies suggest that subtle modulation of the eCB system can be 15 achieved without the manifestation of the non-specific behavioural side effects 16 traditionally associated with C. sativa administration [START_REF] Howlett | Cannabinoid physiology and pharmacology: 30 years of progress[END_REF]. The drug 17 administration routes used by Williams (1998; p.o.) and Avraham (i.p.) do, however, 18 limit comparison of these results and prevent specific attribution of the effects to central 19 or peripheral sites. 20

Exogenous AEA administration (0.5, 1.0, 5.0 and 10.0mg/kg; subcutaneous; 21 s.c.) has also been observed to induce hyperphagia in pre-satiated rats (Williams et al., 22 1999). This study demonstrated that AEA administration significantly increased food 23 intake relative to control in the first hour of testing, in a similar fashion to ∆ 9 THC 24 F o r P e e r R e v i e w [START_REF] Williams | Hyperphagia in pre-fed rats following 32 oral ∆ 9 -THC[END_REF], but unlike ∆ 9 THC also induced increases in total food intake. It 1 was proposed that the observed changes in food intake pattern represented an AEA-2 induced modification in the motivation to feed, since AEA administration caused 3 feeding to occur far sooner than under control conditions, suggesting a cannabinoid-4 mediated modulation of normal feeding pathways. Indeed, close inspection of these data 5

shows that while vehicle-treated animals consumed only small amounts of food in the 6 first hour of testing, they consumed ten-fold more during the third hour. In slightly later 7 work, Williams et al. (2002a) used two groups of twelve, male, adult, Lister-hooded rats 8 which received ∆ 9 THC (0.5, 1.0 or 2.0mg/kg; s.c.) or AEA (0.0, 1.0, 5.0 or 10.0mg/kg; 9 s.c.) and video recordings of post-administration activity to investigate feeding 10 behaviours. It became clear that administration of both ∆ 9 THC and AEA significantly 11 decrease the latency to feeding onset, increase the duration of intake and the number of 12 meals, but that only AEA increased total intake. Alongside the well-reported increases 13 in short term chow intake associated with ∆ 9 THC, these data suggest that ∆ 9 THC and 14 AEA administration induce their effects by subtly different, and yet to be elucidated 15 pathways since two distinct changes to feeding behaviours were observed. [START_REF] Yoshida | 17 Endocannabinoids selectively enhance sweet taste[END_REF] Like ∆ 9 THC, extremely low doses of AEA (0.001mg/kg; i.p.) have also been 17 demonstrated to induce significant increases in food intake in female mice in 18 experiments which used a 40% diet restriction protocol [START_REF] Hao | Low dose anandamide affects 1 food intake, cognitive function, neurotransmitter and corticosterone levels in diet-2 restricted mice[END_REF]. It is 19 interesting that in a situation when eCB tone would already be increased due to the diet 20 restriction paradigm used, further slight increases in eCB tone caused by extremely low 21 doses of AEA induced changes in feeding patterns. Furthermore, it is also interesting 22 that when Hao and colleagues administered doses of 0.7 and 4.0mg/kg AEA (also i.p.) 23 in the same set of experiments no significant feeding effects were observed, even 24 paradigms are almost complete opposites. Indeed, Williams used a prefeed paradigm 4 followed by s.c. injections in male rats and recorded food intake over a period of six 5 hours whereas Hao used a diet restricted paradigm followed by i.p. injections in female 6 mice and recorded daily food intake over a period of one week. 7 AEA feeding effects were localised to CB 1 R sites by Williams using a pre-8 satiated paradigm in 1999. Rats received AEA (0.5, 1.0, 5.0 and 10.0mg/kg; s.c.) and, in 9 a subsequent trial, AEA (1.0mg/kg; s.c.) plus SR141716A (0.1, 0.5 and 1.0mg/kg; s.c.; 10 [START_REF] Williams | Anandamide induces overeating: mediation by 23 central cannabinoid CB 1 receptors[END_REF]. This demonstrated that AEA significantly increased food intake 11 relative to control (in a similar fashion to ∆ 9 THC; [START_REF] Williams | Hyperphagia in pre-fed rats following 32 oral ∆ 9 -THC[END_REF] and that the 12 effect could be blocked by SR141716A, thus confirming a CB 1 R-mediated mechanism 13 of action for the observed feeding effects. In 2002 the hyperphagia induced by ∆ 9 THC 14 administration was also localised to CB 1 R sites (Williams et al., 2002b). Williams first 15 induced hyperphagia by administering ∆ 9 THC (1.0mg/kg; s.c.) and in further trials 16 attenuated this effect with the coadministration of SR141716A (0.1, 0.5 and 1.0mg/kg; 17 s.c.) thereby demonstrating CB 1 R involvement in this effect. CB 2 R-mediation was then 18 discounted as coadministration of ∆ 9 THC (1.0mg/kg; s.c.) and SR144528 (0, 0.05, 0. and WIN55,212 (10.0µg/kg) increased food intake, but that i.c.v. SR141716A 2 administration (0.1, 0.4, 2.0 or 10.0µg/kg) did not reduce feeding. In contrast, whilst i.p. 3 administration of AEA (0.1, 1.0 and 10.0mg/kg) and WIN55,212 (0.4, 2.0 and 4 10.0mg/kg) similarly increased food intake, i.p. administration of SR141716A (0.3, 1.0 5 and 3.0mg/kg) reduced feeding. Furthermore, the authors demonstrated that the changes 6 in feeding patterns they observed were unaffected by a CB 2 R antagonist (SR144528; 7 3.0mg/kg; i.p.), principally rejecting the hypothesis that these peripheral effects were in 8 fact CB 2 R-as opposed to CB 1 R-mediated. SR141716A's failure to reduce feeding after 9 i.c.v. but not i.p. administration led Gomez to suggest that CB 1 Rs must be present in the 10 periphery and that these peripheral CB 1 Rs can affect feeding. Gomez supports this 11 assertion by showing that intestinal AEA levels increase after food deprivation, 12 suggesting that intestinal endocannabinergic tone plays a role in energy balance. 13 However, the experimental paradigm used by Gomez did not account for the 14 concentrations of SR141716A, AEA and WIN55,212 that would be present in the CNS 15 following i.p. injection or for the considerably different dose ranges used. As such, 16 while this evidence is suggestive of a peripheral CB 1 R-mediated role in energy balance, 17 further experiments are needed to confirm this. 18 It has been well described that upon administration of an exogenous cannabinoid 19 agonist endocannabinergic tone will be increased and that alterations to feeding patterns 20 can then be observed. Furthermore, it has been demonstrated that naturally occurring 21 eCB concentrations fluctuate in various feeding states. Alongside evidence gathered 22 using exogenous cannabinoid administration to affect feeding patterns, this elegantly 23 demonstrates a link between how the natural eCB system modulates feeding patterns 24 and how artificial alterations to its state modulate the same patterns. AEA and 2-AG 1 levels in the rat limbic forebrain, hypothalamus and cerebellum were quantified in three 2 feeding states; fasted, feeding and satiated [START_REF] Kirkham | Endocannabinoid levels in rat 24 limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: 25 stimulation of eating by 2-arachidonoyl glycerol[END_REF]. Four groups of 3 between eight and ten male rats were either: 1) given ad libitum access to food and 4 sacrificed during a period of low spontaneous feeding; 2) fed with a wet mash for 15 5 minutes and then sacrificed during feed consumption; 3) fed with a wet mash and 6 sacrificed once they stopped eating or 4) given a 20% maintenance diet for 24hrs and 7 sacrificed at the start of their red light cycle the following day. Brain tissue was then 8 extracted and AEA and 2-AG levels were assessed in the limbic forebrain, 9 hypothalamus and cerebellum. AEA and 2-AG levels were significantly elevated by 10 food deprivation in the limbic forebrain while 2-AG concentration was significantly 11 reduced in the hypothalamus during the feeding state but significantly increased during 12 the deprived state. Since 2-AG levels were increased in the limbic forebrain and 13 hypothalamus, and AEA levels were significantly increased in the limbic forebrain, it 14 appears that elevated eCB levels in important reward-related brain areas during food 15 deprivation play a role in motivating animals towards food. The reduction of 2-AG 16 levels in the hypothalamus during feeding suggests that 2-AG is actively suppressed 17 during feeding to facilitate satiation. Such data suggest subtly differing roles for AEA 18 and 2-AG in feeding cycles. 19 Further experiments published in the same manuscript revealed that 2-AG (i.c.v. 20 into the nucleus accumbens at 0.5 and 2.0µg) could significantly increase food intake 21 over a one hour period while coadministration of SR141716A (0.5mg/kg; s.c. plus 22 0.5µg 2-AG; i.c.v.) could almost fully attenuate this effect, thereby demonstrating CB 1 R 23 mediation. The increases in food intake seen in [START_REF] Kirkham | Endocannabinoid levels in rat 24 limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: 25 stimulation of eating by 2-arachidonoyl glycerol[END_REF] the similar studies performed by the same group following ∆ 9 THC administration 1 (Williams et al., 2002b;[START_REF] Williams | Hyperphagia in pre-fed rats following 32 oral ∆ 9 -THC[END_REF], however are considerably different from 2 a feeding study which administered AEA [START_REF] Williams | Anandamide induces overeating: mediation by 23 central cannabinoid CB 1 receptors[END_REF]. Indeed, AEA 3 (1.0mg/kg; s.c.) effects were observed over four hours and during the first hour 4 significantly increased intake by a factor of approximately two, as well as significantly 5 elevating food intake over the entire four hour period. In contrast, 2-AG (0.5 and 6 1.0µg/kg; i.c.v.) significantly increased food intake by factors of nine and seven, 7 respectively, over a one hour period. However, it is important to note that in this 8 instance 2-AG administration was i.c.v. and AEA administration was s.c., and it is 9 possible that AEA-mediated feeding increases would have been larger had 10 administration been i.c.v. Given the strong anatomical connections between the nucleus 11 accumbens shell and the hypothalamus [START_REF] Stratford | Evidence of a Functional Relationship between the 31 Nucleus Accumbens Shell and Lateral Hypothalamus Subserving the Control of 32 Feeding Behavior[END_REF] and the importance of the 12 hypothalamus in the integration of feeding pathways [START_REF] Maccarrone | The 32 Endocannabinoid System and Its Relevance for Nutrition[END_REF] these 13 data represent a demonstration that modulation of this pathway can have profound 14 feeding effects. [START_REF] Kennett | A comparison of the effects 16 of the CB 1 receptor antagonist SR141716A, pre-feeding and changed palatability on 17 the microstructure of ingestive behaviour[END_REF] As considerable differences in eCB levels in the limbic forebrain were seen 16 during various feeding states in [START_REF] Kirkham | Endocannabinoid levels in rat 24 limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: 25 stimulation of eating by 2-arachidonoyl glycerol[END_REF] and because the limbic forebrain 17 is traditionally associated with reward processing, possible interactions between the 18 eCB and opioid systems were then considered by Williams and Kirkham (Williams et 19 al., 2002b). It was demonstrated that ∆ 9 THC-induced hyperphagia could be attenuated 20 by the opioid antagonist naloxone. This proves a link between opioid food reward 21 processing and the eCB system. These experiments are also exciting in terms of putative 22 anti-obesity treatments. Indeed, it has been reported that in obese humans, eCB levels 23 are elevated by up to 52% [START_REF] Engeli | 35 Activation of the peripheral endocannabinoid system in human obesity[END_REF]; as such experiments which artificially 24 elevate eCB activity using food-restriction paradigms are particularly valuable since 1 they more accurately model the human obese condition. Moreover, since in both the 2 food deprived and obese states eCB levels are increased, this further supports the theory 3 that eCB dysfunction plays an important part in human obesity. Further studies which 4 directly compared AEA and 2-AG-induced feeding effects (both centrally and 5 peripherally administered) would be extremely useful. It would be equally compelling 6 for further work to consider fully the putative effects of peripheral cannabinoid 7 receptors on feeding behaviours, for example in terms of gut motility. 8

Given the previously discussed alterations to eCB concentrations in the limbic 9 forebrain, an area traditionally associated with reward processing and well documented 10 opioid/cannabinoid interactions, it is clear that the changes to behavioural patterns 11 observed during cannabinoid administration are due to changes in reward levels. 12 Therefore, cannabinoid agonist effects on the consumption of different types of 13 rewarding ingesta, such as sweet versus fatty foods, was addressed in a study by Koch 14 (2001). Here, ∆ 9 THC (0.0, 0.5, 1.0 and 2.5mg/kg; i.p.) was administered to rats 15 receiving either control (chow), high fat (HF) or high fat plus sugar (HFS) diets and 16 food intake was recorded over 24 hours post-injection. After the first hour, 0.5 and 17 1.0mg/kg ∆ 9 THC significantly increased food intake versus control within the HF diet 18 group only, while doses of 2.5mg/kg had no significant effects on intake in any group. 19 However, overall food intake during the first hour was significantly greater in the HF 20 group than in either the control chow or HFS groups. During the second hour of testing, 21 the 0.5 and 1.0mg/kg ∆ 9 THC doses significantly increased intake versus control in all 22 three groups with 1.0mg/kg causing the largest increases. This dose also induced a 23 significantly greater intake increase in the HF (but not in the HFS) than in the control 24 chow group, although overall food intake across all doses was significantly greater in 1 both the HF and HFS groups, relative to the control chow group. Finally in the fourth 2 hour, unlike previous feeding studies employing ∆ 9 THC which showed effects lasting 3 fewer than four hours, ∆ 9 THC doses of 1.0mg/kg still induced significant increases in 4 consumption compared to vehicle in the HF and HFS diet groups. 5

The increases in intake seen at lower dose levels by Koch appear comparable 6 (though reduced) to those previously reported [START_REF] Williams | Hyperphagia in pre-fed rats following 32 oral ∆ 9 -THC[END_REF] and support a 7 cannabinoid-mediated stimulation of intake [START_REF] Williams | Anandamide induces overeating: mediation by 23 central cannabinoid CB 1 receptors[END_REF]Williams et al., 8 1998), although the non-significant effects of 2.5mg/kg ∆ 9 THC remain unexplained, 9 particularly as no changes to motor coordination or the appearance of tetrad effects were 10 reported for this dose. Therefore, it is clear from these results that eCB system 11 stimulation induces greater increases in the intake of highly fatty food-types (HF) than 12 other, less calorific or more palatable, diets (HFS). Previous work by Arnone et al. 13 (1997) and Simiand et al. (1998) (this work is fully discussed in 'Endocannabinoid 14 antagonists and their effects on feeding') would suggest that the HFS intake should also [START_REF] Kennett | A comparison of the effects 16 of the CB 1 receptor antagonist SR141716A, pre-feeding and changed palatability on 17 the microstructure of ingestive behaviour[END_REF] have been increased by ∆ 9 THC administration. Indeed, ∆ 9 THC administration induced 16 similar intake increases in the HFS and HF diet groups but only after the fourth and 17 twenty-fourth hours of testing. These data could suggest that the eCB system is 18 involved more strongly in increasing the motivation to consume high fat foods and that 19 this effect manifests more quickly than eCB increases due to other 'bland' food types. 20 From the perspective of evolution, an increased motivation to consume high fat foods is 21 obvious and as such it is reasonable to suggest from this data that the eCB system is 22 intimately involved in this process. 23 In a later study specifically designed to look at cannabinoid modulation of 1 palatability, [START_REF] Higgs | Cannabinoid influences on palatability: 20 microstructural analysis of sucrose drinking after ∆ 9 -tetrahydrocannabinol, anandamide, 21 2-arachidonoyl glycerol and SR141716[END_REF] tested the effects of various cannabinoid receptor 2 ligands on consumption of a 10% sucrose solution in rats. In this test ∆ 9 THC or AEA 3 (0.5, 1.0 or 3.0mg/kg in both cases; i.p.) but not 2-AG (0.2, 0.5 and 2.0mg/kg; i.p.) 4 significantly increased sucrose solution consumption. By analysing lick patterns using 5 the mathematical model of ingestive control proposed by [START_REF] Davis | A model for the control of ingestion[END_REF], this effect 6 was ascribed to a cannabinoid-induced increase in perceived palatability. The lack of 7 significant 2-AG-induced effect is surprising, since both 2-AG and AEA are partial 8 agonists at CB 1 R sites (although it has been suggested that 2-AG is the main ligand 9 associated with CB 2 R sites; [START_REF] Sugiura | 38 Evidence that the cannabinoid CB1 receptor is a 2-arachidonoylglycerol receptor[END_REF][START_REF] Sugiura | 2-Arachidonoylglycerol and the cannabinoid receptors[END_REF], and because of 10 the significant effects observed by [START_REF] Kirkham | Endocannabinoid levels in rat 24 limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: 25 stimulation of eating by 2-arachidonoyl glycerol[END_REF]. Higgs suggests that different 11 speeds at which the exogenous and endogenous ligands act, coupled with the probable 12 lack of 2-AG brain penetration (due to the route of administration), can explain the 13 reduced effects of AEA compared to ∆ 9 THC and could, to some extent, shed light on 14 the limited effects of 2-AG. Administration of SR141716A alone was also tested (0.5, 15 1.0 and 3.0mg/kg; i.p.) by Higgs, and found to elicit a dose-dependent decrease in 16 consumption, thus linking the observed effects to modulation of endogenous CB 1 R 17 activation. Yet, it could be argued that the dose-dependent decreases in consumption 18 induced by SR141716A seen here were caused by a reduction in eCB tone of which 2-19 AG plays a part, and as such it remains impossible to discount any effects of 2-AG 20 acting at CB 2 R sites on this process. This finding demonstrates that the eCB system 21 must have an active effect upon appetite and feeding since CB [START_REF] Williams | Anandamide induces overeating: mediation by 23 central cannabinoid CB 1 receptors[END_REF] it can be suggested that eCB system 1 stimulation upregulates the palatability of high calorie, low palatability foods and 2 conversely, reducing endocannabinergic tone diminishes perceived palatability. This 3 could explain why in Koch's work the HF diet intake was increased above HFS diet as 4 it was both palatable and calorie-rich. 5

In a very recent study Yoshida and colleagues (2010) administered AEA and 2-6 AG to both wild type and CB 1 R knock-out mice. They observed that AEA and 2-AG 7 preferentially increased taste responses to sweet rather than salty, sour, bitter or umami 8 (a Japanese term for a meaty or savoury taste) flavours in the chorda tympani nerve, 9 fungiform taste buds and during a short-term lick response test. Furthermore, they did 10 not observe this effect in their knock-out mice, hence linking this effect to CB 1 R. Given 11 their i.p. route of administration, the authors have suggested a peripheral effect, 12 alongside the well described central effect, of eCBs on taste. In comparison with the 13 work of [START_REF] Higgs | Cannabinoid influences on palatability: 20 microstructural analysis of sucrose drinking after ∆ 9 -tetrahydrocannabinol, anandamide, 21 2-arachidonoyl glycerol and SR141716[END_REF] who found 2-AG to have little effect on palatability in a 14 progressive lick-based paradigm, and considering that both studies used i.p. 15 administration and observed peripheral taste responses, it is not immediately apparent 16 why different eCBs are identified as the principal modulators of taste. 17

In earlier taste reactivity studies, Gallate et al. (1999) presented rats with various 18 concentrations of 'beer' (<0.5%, 2.7% or 4.0% ethanol solutions) or an 8.6% sugar 19 solution, and their motivation to consume these substances was measured using a lick-20 based progressive ratio paradigm. In order to assess CB 1 R involvement, animals were 21 treated with either vehicle, the CB 1 R agonist CP55,940 (10.0, 30.0 or 50.0µg/kg; i.p.), 22 CP55,940 (30.0µg/kg; i.p.) plus SR141716A (1.5mg/kg; i.p.), or naloxone (2.5mg/kg; 23 i.p.) plus CP55,940 (30.0µg/kg; i.p.). This drug regime therefore also examined 24 reducing beer solution intake to levels that did not differ from vehicle treatments. 5 Furthermore, CP55,940 plus naloxone administration significantly reduced beer intake 6 to approximately 50% of vehicle intake, confirming a strong interaction between CB 1 7 and opioid receptors in the control of alcohol consumption. This study demonstrated 8 that CB 1 R activation could induce significant increases in palatable solution intake 9 when compared to vehicle-treatments, and that this effect was attenuated by CB 1 R 10 antagonists; further supporting the theory of CB 1 R involvement in perceived 11 demonstrated that the eCB system mediated the perceived palatability of sweet and 14 bitter tastes. In trials using adult, male Sprague-Dawley rats, ∆ 9 THC (0.5mg/kg; i.p.), 15 ∆ 9 THC (0.5mg/kg; i.p.) plus SR141716A (2.5mg/kg; i.p.) or AM251 (1.0mg/kg; i.p.), or 16 AM251 (1.0mg/kg; i.p.) alone were administered via intraoral cannulae and the 17 orofacial reactions to intraoral administration of either sucrose (2, 10 and 32%), or 18 quinine (0.01 or 0.05%) solutions were recorded. ∆ 9 THC increased the perceived 19 palatability of the sucrose solution, an effect attenuated in animals treated with ∆ 9 THC 20 plus SR141716A [START_REF] Jarrett | Effect of [Delta]9-tetrahydrocannabinol 29 on sucrose palatability as measured by the taste reactivity test[END_REF]. In the later study, ∆ 9 THC reduced aversiveness 21 to 0.05% quinine solution, an effect that was blocked by coadministration of AM251 22 [START_REF] Jarrett | Effect of [Delta]9-tetrahydrocannabinol 33 on quinine palatability and AM251 on sucrose and quinine palatability using the taste 34 reactivity test[END_REF]. The authors suggest that their results indicate that increases in 23 CB 1 R responsiveness could be involved in a general upregulation of the ingesta 24 In further experiments during the same two studies, Jarrett and colleagues 1 also demonstrated that SR141716A administration alone does not reduce perceived 2 palatability [START_REF] Jarrett | Effect of [Delta]9-tetrahydrocannabinol 29 on sucrose palatability as measured by the taste reactivity test[END_REF] but AM251 administration enhances aversiveness 3 [START_REF] Jarrett | Effect of [Delta]9-tetrahydrocannabinol 33 on quinine palatability and AM251 on sucrose and quinine palatability using the taste 34 reactivity test[END_REF]. This may suggest that the increased palatability of sweet food can 4 activate the eCB system and upregulate eCB tone. However, in the case of bitter tasting 5 food, when eCB tone would not be increased by the low palatability of the food, CB 1 R 6 antagonism can then reduce the perceived palatability of the food further. 7

Further supporting this palatability hypothesis, it has recently been demonstrated 8 that, under a reinforced learning paradigm which included food type (normal lab chow, 9 chocolate pellets or fat-enriched pellets) selection as an experimental variable, mice will 10 select the sugar rich, highly-palatable food type or fatty foods over standard food after 11 ∆ 9 THC (1.0mg/kg; i.p.) administration [START_REF] Barbano | Delta-9-36 tetrahydrocannabinol enhances food reinforcement in a mouse operant conflict test[END_REF]. Since, the chocolate-12 enhanced pellets used in this study had the same calorific content as the standard food 13 pellets also employed while the fat enriched pellets contained approximately 66% more 14 calories per gram but no added chocolate, it can be observed that ∆ 9 THC administration 15 increased the palatability of fatty foods to a level comparable to that of sweet foods. [START_REF] Yoshida | 17 Endocannabinoids selectively enhance sweet taste[END_REF] The conclusion that the eCB system exerts a level of control over palatability 17 reward is well supported. It has been shown that increased eCB tone increases the 18 palatability of sweet liquids (Gallate et al., 1999; [START_REF] Higgs | Cannabinoid influences on palatability: 20 microstructural analysis of sucrose drinking after ∆ 9 -tetrahydrocannabinol, anandamide, 21 2-arachidonoyl glycerol and SR141716[END_REF], bland fatty foods 19 (Koch, 2001) or even aversive, bitter liquids [START_REF] Jarrett | Effect of [Delta]9-tetrahydrocannabinol 29 on sucrose palatability as measured by the taste reactivity test[END_REF][START_REF] Jarrett | Effect of [Delta]9-tetrahydrocannabinol 33 on quinine palatability and AM251 on sucrose and quinine palatability using the taste 34 reactivity test[END_REF]. 20 Indeed, eCB levels change naturally reflecting the current feeding state, and it is this 21 basal tone that can be altered with the application of CB 1 R ligands. Furthermore, the 22 meal pattern changes induced by stimulation of the eCB system are also well 23 understood. ∆ 9 THC administration has been demonstrated to reduce the latency to 24 feeding onset in periods of reduced endocannabinergic tone and has also been shown to 1 increase meal sizes. If we consider appetitive behaviours as those that regulate the 2 latencies to meal onset and consummatory behaviours as those that regulate the size of 3 any given meal, what becomes clear from these data is that modulation of the eCB 4 system can affect consummatory and/or appetitive behaviours and that these different 5 behavioural changes can be manipulated individually. For example, in the work of 6 Williams et al. (2002a), a distinct separation was seen between the AEA and ∆ 9 THC 7 groups in terms of feeding behaviour. In this instance, AEA administration produced 8 effects comparable to ∆ 9 THC: AEA significantly reduced the latency to feeding onset, 9 whilst also increasing the number of eating bouts. AEA administration also increased 10 meal duration and the duration of the longest bout versus control conditions. However, 11 unlike ∆ 9 THC, AEA induced significant increases in total intake over the test period. 12 Given these data it can be clearly seen that the changes to feeding patterns (either in 13 gross terms or in terms of palatability levels) are due to changes to reward processing. 14 Indeed, the decreased latency to feeding onset and the increased size and length of 15 meals thereafter coupled with increased lick responses can all be linked to increased 16 reward. 17 Endocannabinoid antagonism and its effect on feeding 1 Alongside the well documented hyperphagic effects of ∆ 9 THC administration there is 2 comprehensive complimentary evidence which details the effects of reducing eCB tone 3 on feeding behaviours. Indeed the use of CB 1 R antagonist-based studies represents an 4 elegant way to study the effects of basal eCB tone on physiological feeding 5 mechanisms. This is particularly true as many CBR antagonists are highly selective. In 6 1997 Arnone [START_REF] Arnone | 19 Selective inhibition of sucrose and ethanol intake by SR141716, an antagonist of central 20 cannabinoid (CB1) receptors[END_REF] and colleagues performed a comprehensive set of 7 experiments which revealed the changes to feeding behaviours induced by CB 1 R 8 blockade by the administration of SR141716A (0.0, 0.1, 0.3, 1.0 or 3.0mg/kg: i.p.). In 9 experiments which studied either intake of sucrose pellets, intake of a 5% sucrose 10 solution or a 10% ethanol solution, Arnone demonstrated that eCB tone plays an 11 important role in the perceived appetitive values of both alcohol and sugar. Firstly, [START_REF] Matsuda | Structure of a 13 cannabinoid receptor and functional expression of the cloned cDNA[END_REF] Arnone and colleagues measured the weight of food consumed in 30min open field tests 13 where food-restricted male, Wistar rats (a situation in which one would expect eCB tone 14 to be increased by the food restriction [START_REF] Kirkham | Endocannabinoid levels in rat 24 limbic forebrain and hypothalamus in relation to fasting, feeding and satiation: 25 stimulation of eating by 2-arachidonoyl glycerol[END_REF]) had access to either 15 sucrose pellets and standard lab chow or standard lab chow alone after SR141716A 16 administration. The quantity of sucrose pellets consumed by rats was reduced from 17 3.1±0.4g under control conditions to 1.0±0.4g after the 3.0mg/kg SR141716A dose in a 18 dose-dependent fashion; furthermore, the two highest doses of SR141716A induced 19 significant reductions when compared to control intakes. Standard chow intake was 20 unaffected. However, when Arnone repeatedly administered 3.0mg/kg SR141716A in 21 mice with a predisposition to alcohol consumption over four, six hour test periods in 22 one day, SR141716A induced a suppression of ethanol intake. Secondly, rats (male, 23 Sprague-Dawley) were given access to a 5% sucrose solution for a period of four hours 24 given access to water for the same test period. Sucrose solution intake was significantly 2 reduced by the two highest doses of SR141716A and a significant dose-dependent 3 reduction in sucrose solution intake was apparent. Water intake was unaffected by 4 SR141716A administration at any dose. Thirdly, Arnone tested the effect SR141716A 5 had on ethanol consumption in male C57BL6 mice; once again administration of either 6 1.0 or 3.0mg/kg SR141716A induced significant reductions in consumption versus 7 control-treatment and a significant dose-dependent effect was apparent for the entire 8 dose range. Water-only tests were used as a control, as per the 5% sucrose solution test, 9 and no effect of SR141716A was apparent. These three experiments demonstrate that 10 the eCB system plays an active role in the control of the appetitive value of sweet and 11 alcoholic ingesta. Indeed, such experiments prove that the eCB system must have a 12 constantly active basal tone as antagonists applied to an inactive system would induce 13 no behavioural changes alone. Furthermore, given the selectivity of SR141716A we can 14 be confident that this effect is localised to CB 1 R sites. [START_REF] Kennett | A comparison of the effects 16 of the CB 1 receptor antagonist SR141716A, pre-feeding and changed palatability on 17 the microstructure of ingestive behaviour[END_REF] Reductions in eCB tone induced by the administration of SR141716A (0.0, 1.0 16 and 3.0mg/kg; p.o.) were extended to marmosets in 1998 by Simiand (Simiand et al., 17 1998). Marmosets were habituated to a high-sugar mash (HSM; 33% sugar, 67% milk 18 and cereal) and after habituation administered SR141716A twice a week; their food 19 (either HSM or standard diet) intake was then observed for a period of six hours. 20 Administration of the 3.0mg/kg dose induced significant reductions in HSM intake 21 versus control-treatments after one, three and six hours while the 1.0mg/kg dose only 22 reduced HSM intake versus control-treatments during hours one and three. Notably 23 standard diet intake was unaffected by SR141716A administration with the exception of 24 a significant increase in intake seen during hour six. These data demonstrate that 1 SR141716A can preferentially reduce intake of sweet, highly palatable food, versus 2 bland food. This suggests that eCB tone is responsible for increasing the appetitive 3 values of high-sugar foods under normal physiological conditions. Indeed, as previously 4 discussed, such work has been supported by later experiments which showed 5 SR141716A administration could reduce the reward associated with sucrose solutions 6 [START_REF] Higgs | Cannabinoid influences on palatability: 20 microstructural analysis of sucrose drinking after ∆ 9 -tetrahydrocannabinol, anandamide, 21 2-arachidonoyl glycerol and SR141716[END_REF]. Since [START_REF] Arnone | 19 Selective inhibition of sucrose and ethanol intake by SR141716, an antagonist of central 20 cannabinoid (CB1) receptors[END_REF] and [START_REF] Simiand | SR 141716, a CB1 cannabinoid 6 receptor antagonist, selectively reduces sweet food intake in marmoset[END_REF], together, 7 used two strains of rats, one strain of mouse and one type of primate and observed 8 similar results, it can be suggested that the effects of the eCB system are similar across 9 species. However, the work of Arnone and Simiand did not consider a high fat diet 10 alternative to the high-sugar foods they used, and they only antagonised the eCB system 11 over a shorter period of time. It is worthwhile comparing the works of Arnone and 12Simiand to that of Williams and colleagues. Indeed, while Williams observed CB 1 R 13 stimulation to increase feeding, and that this effect could be blocked by 14 coadministration of SR141716A, Arnone and Simiand demonstrated a basal 15 endocannabinergic tone which when blocked with SR141716A alone could reduce 16 feeding. Furthermore, as discussed, the eCB system has been demonstrated to alter its 17 activity level dependent on feeding state (e.g. deprived of food versus eating; Kirkham 18 et al., 2002). Taken together, these studies demonstrate that eCB tone is a powerful 19 effector of perceived hunger and that feeding reduces endocannabinergic tone in the 20 same way as blocking CB 1 R sites in the CNS. 21 or fatty (10% intralipid) solutions and analysed the lick-based response in a way similar 3 to [START_REF] Higgs | Cannabinoid influences on palatability: 20 microstructural analysis of sucrose drinking after ∆ 9 -tetrahydrocannabinol, anandamide, 21 2-arachidonoyl glycerol and SR141716[END_REF]. Appealingly, this paradigm also compared the hypophagic 4 SR141716A effects with those induced by behavioural manipulations designed to 5 reduce either motivation to feed (pre-feeding) or food palatability (addition of quinine to 6 the intralipid solution and reduction of sucrose concentration to 5%) which, combined 7 with the lick-based microstructure analysis, allowed for more detailed interpretation of 8 the results. Supporting previous findings, this study demonstrated that SR141716A 9 treatment reduced both sucrose and intralipid solution intake, and microstructure 10 analysis revealed that these reductions were due to decreased perceived palatability of 11 ingesta. However, whilst these significant reductions were induced by both 1.0mg/kg 12 and 3.0mg/kg SR141716A for the intralipid solution, only 3.0mg/kg SR141716A 13 significantly reduced sucrose solution consumption. Furthermore, reduced lick duration 14 of intralipid solution observed after drug administration led the authors to suggest that 15 the SR141716A-induced reduction in consumption of this solution was a result of a 16 decrease in motivation to feed, as well as an effect on perceived palatability. In contrast, 17 reductions in sucrose solution intake were less pronounced than those seen for the 18 intralipid solution, suggesting that eCBS may be more strongly implicated in 19 consumption of highly calorific than sweet, palatable food-types. Taken alone, this work 20 suggests that the eCB system can play its role in energy homeostasis via modulation of 21 the motivation to consume fatty foods specifically; this long term effect may be due to 22 changes in meal patterning over the shorter term, particularly with respect to the control 23 of fat intake. Indeed, the work of Thornton-Jones further supports the findings of 24 and strengthens the link between CB 1 R activation and the control of feeding. 2

Using a different experimental approach, a recent study by [START_REF] Mathes | Cannabinoid-1 receptor antagonists 9 reduce caloric intake by decreasing palatable diet selection in a novel dessert protocol in 10 female rats[END_REF] 3 also addressed these issues, and provided further support for eCB system involvement 4 in the specific consumption of highly fatty and/or sweet foods. A 'dessert protocol' was 5 employed, whereby rats were given ad libitum access to a wet mash diet made from 6 standard laboratory chow in addition to simultaneous but time limited access to a 7 'dessert' diet high in fat and sugar. Vitally, this methodology gave rats a choice between 8 two different diets, allowing within-group analysis of diet selection. eCB system 9 involvement in the consumption of these two different food-types was assessed by 10 administration of either of the CB 1 R antagonists, SR141716A (1.0mg/kg; i.p.) or 11 AM251 (0.3, 1.0 or 3.0mg/kg; i.p.) 30min prior to dessert access. Both compounds 12 significantly reduced caloric intake over a 24 hour period, but this effect was due to a 13 significant decrease in the consumption of the high fat and sugar diet only, with no 14 effect on the consumption of the standard chow. When considered alongside Arnone et 15 motor coordination, which may have been the cause for its effects on feeding 5 behaviours, PSNCBAM-1, AM4113 and MK-5596 have been demonstrated to reduce 6 short term intake and reduce body weight in animal models without apparent side 7 effects. Further work using these novel agents would benefit from the inclusion of meal 8 patterning analysis as this would allow the study of appetitive and/or consummatory 9 behaviours, as well as effects on perceived palatability; such an analysis may shed more 10 light on eCB mediation of feeding behaviours. Non-∆ 9 THC phytocannabinoid effects on feeding 1

Very recently it has been suggested that non-∆ 9 THC pCBs can also modulate rodent 2 feeding patterns (Farrimond et al., 2010a;Farrimond et al., 2010b) and induce different 3 changes than ∆ 9 THC, without any apparent non-specific behavioural effects. Before 4 2009 there had been a limited number of studies which demonstrated non-∆ 9 THC pCB-5 mediated feeding effects. Unfortunately these studies were either unrepeated or 6 contradictory. [START_REF] Sofia | Comparative effects of various naturally occurring 15 cannabinoids on food, sucrose and water consumption by rats[END_REF] reported that the CB 1 R agonist CBN reduced 7 intake in rats, an effect yet to be repeated, and that CBD also reduced feeding in rats. 8 However, [START_REF] Wiley | 15 CB1 cannabinoid receptor-mediated modulation of food intake in mice[END_REF] reported that CBD administration had no effect on food 9 intake in mice, a result repeated by [START_REF] Riedel | 37 Synthetic and plant-derived cannabinoid receptor antagonists show hypophagic 38 properties in fasted and non-fasted mice[END_REF] who observed only a small, non-10 significant reduction in food intake and weight gain, also in mice. It should be noted 11 that the dose ranges used by Sofia, Knobloch and Wiley are considerably higher than 12 those used by Farrimond and colleagues and, given the well described bi-phasic dose 13 effects of CB 1 R activation [START_REF] Glick | Increased and decreased eating following THC 23 administration[END_REF] on feeding, this may explain the 14 differences in observed effect patterns. Work in our laboratory has demonstrated that a 15 range of pCBs may have significant effects on feeding patterns. Thirty-six pre-satiated, 16 male, Lister-hooded rats received a purified, C. sativa-derived ∆ 9 THC (hereafter 17 purified ∆ 9 THC), a synthetic ∆ 9 THC and a high-∆ 9 THC standardised botanical drug 18 substance (BDS), similar in composition to that used in Sativex and containing a typical 19 array of non-∆ 9 THC pCBs (high-∆ 9 THC BDS; all treatments were dose matched for 20 ∆ 9 THC content; 0.00, 0.34, 0.67, 1.34 and 2.68mg/kg; p.o.; Farrimond et al., 2010a). 21 Synthetic and purified ∆ 9 THC administration induced classical ∆ 9 THC hyperphagia in 22 this study: the latency to the first meal was reduced and both the first hour intake and 23 first meal duration were increased versus vehicle treatments. Intriguingly, the effects of 24 the high-∆ 9 THC BDS administered showed significantly reduced hyperphagia in 1 comparison. These results suggested that the combination of pCBs (and, potentially, 2 non-cannabinoid components) in the high-∆ 9 THC BDS attenuated the hyperphagic 3 effects of ∆ 9 THC. Given the constituents of the high-∆ 9 THC BDS, (∆ 9 THC; 67.0%, 4 Cannabigerol (CBG); 1.7%, Cannabichromene (CBC); 1.6%, CBN; 1.5%, ∆ 9 THCV; 5 0.9%, ∆ 9 tetrahydrocannbinolic acid (∆ 9 THCA); 0.3%, CBD; 0.3% and Cannabitriol 6 (CBO); 0.2%) it is possible that the pCBs present could have affected feeding 7 synergistically, or that at least one pCB modulated the ∆ 9 THC-induced hyperphagia. 8 Indeed, the high-∆ 9 THC BDS used in Farrimond et al., (2010a) contained known CB 1 R 9 antagonists (∆ 9 THCV, [START_REF] Dennis | Effects of ∆ 9 -tetrahydrocannabivarin on 36 [35S] GTPγS binding in mouse brain cerebellum and piriform cortex membranes[END_REF][START_REF] Ma | The phytocannabinoid ∆ 9 -25 tetrahydrocannabivarin modulates inhibitory neurotransmission in the cerebellum[END_REF][START_REF] Thomas | 12 Evidence that the plant cannabinoid ∆ 9 -tetrahydrocannabivarin is a cannabinoid CB 1 13 and CB 2 receptor antagonist[END_REF] in 10 addition to modulators of Ca 2+ homeostasis (CBD; [START_REF] Ryan | Cannabidiol Targets 24 Mitochondria to Regulate Intracellular Ca2+ Levels[END_REF] and CBN (Rao et 11 al., 2006); which could modulate eCB levels. However, these results do not preclude the 12 possibility that non-cannabinoid material also present in the high-∆ 9 THC BDS also 13 contributed to the observed behavioural effects. 14

Consistent with these results we have also demonstrated that non-∆ 9 THC pCBs 15 can affect feeding when co-administered with sub-effective concentrations of ∆ 9 THC 16 (Farrimond et al., 2010b; please also see [START_REF] Riedel | 37 Synthetic and plant-derived cannabinoid receptor antagonists show hypophagic 38 properties in fasted and non-fasted mice[END_REF] for non-∆ 9 THC pCB-17 induced hypophagia fully discussed below). Ten, pre-satiated, adult, male, Lister-18 hooded rats received a low-∆ 9 THC standardised BDS (low-∆ 9 THC BDS; 0.0, 0.5, 1.0, 19 2.0 and 4.0mg/kg; p.o.) containing only 6.9% ∆ 9 THC, alongside 14.2% other pCBs. 20 Conversely, at these concentrations, rather than having no effect on feeding (as it might 21 be expected, given the use of a prefeed paradigm with an extremely low dose of ∆ 9 THC 22 and pCBs that have previously been shown to reduce ∆ 9 THC-mediated hyperphagia), 23 the low-∆ 9 THC BDS significantly increased chow intake during the first hour, by 24 significantly reducing the latency to the first contact with food for the highest doses 1 versus vehicle-treatments. These reductions in meal one latency mirror those previously 2 demonstrated for considerably higher concentrations of pure ∆ 9 THC (Farrimond et al., 3 2010a; [START_REF] Williams | Hyperphagia in pre-fed rats following 32 oral ∆ 9 -THC[END_REF]. Furthermore, while the latency to the first meal was 4 significantly reduced, the size and duration of this meal was unaffected. Therefore, we 5 suggested that the ratio of non-∆ 9 THC pCBs present in the low-∆ 9 THC extract 6 significantly increased appetitive (but not consummatory behaviours), since the 7 concentrations of ∆ 9 THC present were between two-and ten-fold lower than those 8 previously demonstrated to induce hyperphagia (Farrimond et al., 2010b). 9

When these two studies are considered together (Farrimond et al 2010a and 10 2010b), a number of suggestions can be made: firstly, since the observed effects of the 11 non-∆ 9 THC components of the extracts administered reduced hyperphagia when 12 presented in one ratio and increased hyperphagia when presented in a different ratio, it 13 is highly unlikely that the plant matter also present was responsible for these effects, 14

given that its content did not change between the two tests. Secondly, it is possible that 15 the pCBs present in both studies did not act in isolation such that more than one 16 compound induced the observed effects. Thirdly, these effects could involve non-CBR 17 dependent pathways since several of the non-∆ 9 THC pCBs present are known to act via 18 mechanisms only partially connected to CBR (see [START_REF] Izzo | Non-psychotropic 22 plant cannabinoids: new therapeutic opportunities from an ancient herb[END_REF] for review). 19 Finally, these results give further credence to the already well supported therapeutic 20 potential of non-∆ 9 THC pCBs for the treatment of energy balance disorders. Indeed, 21 non-∆ 9 THC pCB-based treatment may prove more tolerable than ∆ 9 THC treatment 22 alone (e.g. Nabilone) due to the absence of psychotropic side effects associated with 23 non-∆ 9 THC pCBs. 24 (Riedel et al., 4 2009). Indeed, given the difficulties associated with the use of the highly specific and 5 potent CB 1 R antagonist/inverse agonist SR141716A in the clinic, the use of ∆ 9 THCV, 6 as a neutral antagonist, to reduce food intake may be of therapeutic value. Riedel et al. 7 (2009) demonstrated that purified ∆ 9 THCV (3.0, 10.0 and 30.0mg/kg; i.p.) significantly 8 reduced chow intake over a period 12 hours and induced weight loss in male C57BL6 9 mice. Subsequent treatment with extremely low ∆ 9 THCV BDS doses (which contained 10 between 0.1 and 0.3mg/kg ∆ 9 THC) failed to induce changes to feeding patters; however 11 the ∆ 9 THC quantities presented were within the range previously observed to affect 12 meal patterning in rats (Farrimond et al., 2010). Unfortunately, meal microstructure was 13 not analysed in this study. In a further experiment, Riedel hypothesised that if CBD 14 (10.0mg/kg; i.p.) were to be co-administered with these very low ∆ 9 THCV BDS doses, 15 the effects of the present ∆ 9 THC would be antagonised, and the hypophagic properties 16 previously induced by purified ∆ 9 THCV would therefore emerge. However, the 17 evidence for in vivo antagonism of ∆ 9 THC by CBD has so far only been demonstrated 18 in a memory task (Fadda et al., 2004), and Riedel observed no significant effects on 19 feeding or weight change following CBD co-administration. The authors suggest that 20 this lack of effect may also be attributed to other pCBs, present in his extract, or simply 21 because the concentrations of ∆ 9 THCV administered were too low. However, Riedel 22 recorded all feeding data during the light phase of the rats' day during which time little 23 feeding typically occurs. Furthermore, it is also possible that the experimental paradigm 24 used was not optimised for the observation of hypophagia. For instance, introducing 1 food-deprivation prior to dosing could accentuate possible differences between control 2 and conditions measurements, thereby rendering the experimental paradigm more 3 sensitive to ∆ 9 THCV-mediated reductions in feeding. While, Riedel has confirmed that 4 purified ∆ 9 THCV can reduce food intake in mice, further experiments which use more 5 sensitive paradigms and analyse meal microstructure after ∆ 9 THCV BDS administration 6 may prove useful in fully understanding Riedel's data. 7

The work discussed throughout this manuscript demonstrates that some pCBs 8 can alter appetitive and consummatory behaviours separately (e.g. [START_REF] Higgs | Cannabinoid influences on palatability: 20 microstructural analysis of sucrose drinking after ∆ 9 -tetrahydrocannabinol, anandamide, 21 2-arachidonoyl glycerol and SR141716[END_REF]. 9

As such if it were possible to use at least one non-∆ 9 THC pCB to modulate 10 consummatory behaviours (to increase or decrease the quantity consumed during a 11 natural eating event) rather than inducing immediate short term post-administration 12 hunger (the 'munchies', a colloquial term meaning increased hunger normally due to 13 smoking cannabis recreationally) it may be possible to produce a C. sativa-based 14 medicine which could control absolute intake quantity. Even though the mechanisms by 15 which the eCB system modulates palatability and therefore diet selection remain 16 unclear, direct effects on diet selection caused by cannabinoid administration have also 17 been demonstrated. Given the presented data which suggests that increased eCB tone 18 leads to increased selection of fatty foods over sweet foods, it is a possibility that non-19 psychotropic pCBs could also be used to modulate diet selection i.e. to reduce fatty food 20 palatability in obesity. Indeed, putative anti-obesity treatments using pCBs are of great 21 interest. Equally, while some pCBs have been shown to induce significant reductions in 22 food intake and in some cases reduce body weight (e.g. ∆ 9 THCV), little research is 23 A considerable quantity of characterisation work has been performed on ∆ 9 THC-2 mediated feeding effects, but the specific roles of other pCBs remain less well 3 understood. Work in our laboratory and others suggests that non-∆ 9 THC pCBs can have 4 profound effects on feeding behaviours (Farrimond et al., 2010a;Farrimond et al., 5 2010b;[START_REF] Riedel | 37 Synthetic and plant-derived cannabinoid receptor antagonists show hypophagic 38 properties in fasted and non-fasted mice[END_REF][START_REF] Sofia | Comparative effects of various naturally occurring 15 cannabinoids on food, sucrose and water consumption by rats[END_REF][START_REF] Wiley | 15 CB1 cannabinoid receptor-mediated modulation of food intake in mice[END_REF]. However, these are 6 not yet well characterised. As such considerable further work is required to assess the 7 effects of both purified single pCBs as well as combinations of numerous pCBs. With 8 the recent UK approval of the ∆ 9 THC CBD combination in Sativex, it can be clearly 9 seen that the use of pCBs in the clinic is gaining public acceptance that regulatory 10 bodies are willing to accept plant-derived medicines alongside their synthetic 11 counterparts. Indeed, non-∆ 9 THC pCBs may represent an untapped clinical potential for 12 a number of feeding disorders. Such a need is particularly important given the non-13 specific behavioural side effects associated with the use of ∆ 9 THC and its analogues in 14 the clinic. Given the encouraging results demonstrating non-∆ 9 THC pCB-mediated 15 modulation of feeding, it is tempting to suggest that these compounds may provide at 16 least the basis for novel therapies for the treatment of feeding disorders. 17 DAG respectively and released into the synaptic cleft from the postsynapse (red). Upon 6 neurotransmitter binding to both metabotropic Glutamate and Acetylcholine receptors G 7 subunits disassociate and stimulate an increase in PLC-β levels. This leads to an 8 upregulation of intracellular IP 3 which gates calcium channels on the endoplasmic 9 reticulum thus increasing intracellular Ca 2+ . Furthermore, the release of G αi/o subunits 10 upregulates the production of cAMP and therefore allows further Ca 2+ entry into the cell 11 via VGCCs. Equally upon activation of iGluR channels calcium is allowed into the cell. 12 Once released into the extracellular space the endocannabinoids bind to CB 1 R sites on 13 the presynapse (green) causing the presynaptic cell to move closer to its resting 14 membrane potential principally by closing VGCCs and opening VGPCs thus reducing 15 the available intracellular Ca 2+ required to facilitate further neurotransmitter release. 16

  -based compound (Sativex®) has recently been licensed in the U.K., 1

  reviews see Basavarajappa, 2007a; Basavarajappa, 2007b; Bisogno et al., 1 2005; Freund et al., 2003). 2

into NaPE 17 (

 17 figure 3; Ohno-Shosaku et al., 2001; Piomelli et al., 2000; Wilson et al., 2001) that are 8

  g. in the vas deferens: Pertwee et al., 1996; in the heart, blood vessels and 1 bladder: Howlett, 2002; the liver and fatty tissue: Cota et al., 2003b; Osei-Hyiaman et 2 al., 2005)

  (1999) demonstrated AEA effects at 1.0 and 5.0mg/kg (s.c.). 1 However, as the authors note, it is particularly difficult to compareWilliams et al. 2 (1999) andWilliams et al. (2002a) to[START_REF] Hao | Low dose anandamide affects 1 food intake, cognitive function, neurotransmitter and corticosterone levels in diet-2 restricted mice[END_REF] since the experimental 3

1 , 19 0

 119 .5 and 1.0mg/kg; s.c.) failed to reduce the hyperphagia caused by ∆ 9 THC 20 administration. 21Although[START_REF] Williams | Anandamide induces overeating: mediation by 23 central cannabinoid CB 1 receptors[END_REF] localised AEA-mediated effects on feeding to 22 CB 1 R, an effect which one can attribute to CNS receptors, evidence which links 23 peripheral mechanisms to these effects also exists. Gomez and colleagues (Gomez et al., 24 that intracerebroventricular (i.c.v.) AEA administration (10.0µg/kg) 1

  1 R blockade alone, 22 without co-administration of a cannabinoid agonist (c.f. Gallate et al., 1999; Williams et 23 al., 1999), significantly reduces consumption. Considering the works of Koch (2001), 24 Gallate et al. (1999) and

  the cannabinoid and opioid systems. In agreement with 1 previous research, CP55,940-treated animals exhibited a significant trend whereby 2 increasing dose led to increases in both beer and sucrose solution consumption. 3 SR141716A-induced CB 1 R blockade during CP55,940 treatment attenuated this effect, 4

  2007 Jarrett et al. (Jarrett et al., 2005; Jarrett et al., 2007) 13

  food and water. As a control experiment another group of rats was 1

∆

  9 THC-induced effects on high fat versus high sugar foods were well described 22 by Koch in 2001. However, only recently were antagonist studies used to facilitate the 23 understanding of the role of CB 1 R alone in the selection and consumption of highly 24 foods. In 2007, a study by Thornton-Jones et al. (2007) examined the 1 effects of SR141716A-induced CB 1 R blockade on consumption of sweet (10% sucrose) 2

  Arnone et al. (1997), Simiand et al. (1998) and Koch (2001), as previously discussed, 1

  al.(1997),Koch (2001),[START_REF] Simiand | SR 141716, a CB1 cannabinoid 6 receptor antagonist, selectively reduces sweet food intake in marmoset[END_REF] and Thornton-Jones et al.(2007), these[START_REF] Yoshida | 17 Endocannabinoids selectively enhance sweet taste[END_REF] findings suggest that CB 1 R-mediation of intake not only modulates intake of fatty food 17 more that sugary food, but does so in a preferential fashion. Unfortunately, although 18 Mathes et al., in the earlier stages of this study, compared the consumption of the high 19 fat and sugar diet with another diet high in sugar only, the effects of CB 1 R blockade on 20 this sugar-only diet were not examined. 21While the use of the previously discussed antagonists has been shown to reduce 22 feeding in a number of models, there is considerable on-going research looking for 23 novel compounds which perform similar actions. Such research is performed to further 24 the eCB system and its ligands, whilst also searching for new anti-1 obesity drugs(for examples please see PSNCBAM-1, Horswill et al., 2007; AM4113, 2 Sink et al., 2007; O-2050, Gardner et al., 2006; MK55-96, Yan et al., 2010; and MK-3 0364, Fong et al., 2007). While, O-2050 was found to induce significant reductions in 4

  mechanism underlying the findings reported by Farrimond et 1 al., (2010a and b) remains unknown, the naturally occurring pCB and CB 1 R neutral 2 antagonist ∆ 9 THCV, which was also present in the extracts employed in Farrimond et 3 al., has been considered alone and shown to exert hypophagic actions

Figure 1 ; 1 Figure 2 ;

 112 Figure 1; the development of cannabinoid pharmacology 1964 -2010. 1

Figure 3 ;

 3 Figure 3; simplified diagram illustrating retrograde signalling by eCBs in the CNS. 17

Following

  vesicular neurotransmitter release from the presynaptic cell (green), 18 neurotransmitters bind to receptor proteins (black) on the postsynaptic cell (red) 19 stimulating eCB production and release from the postsynaptic cell. eCBs then diffuse 20 back across the synaptic cleft and bind to CBRs (blue) on the presynaptic cell. This 21 predominately leads to a suppression of [Ca + ] influx but also an increase in [K + ] efflux, 22 thus hyperpolarising the presynaptic cell and inhibiting further neurotransmitter release.23

F

Figure 1 ;Figure 2 ;Figure 3 ;

 123 Figure 1; the development of cannabinoid pharmacology 1964 -2010. 117x78mm (600 x 600 DPI)

  ). Critical Enzymes Involved in Endocannabinoid Metabolism. Babbs AJ, Doel SM, Fyfe MCT, Gardner LS, Griffin G, et al. (2006). 1 Deorphanization of a G protein-coupled receptor for oleoylethanolamide and its use in 2 the discovery of small-molecule hypophagic agents. Cell Metabol 3(3): 167-175.

		50 57 58
	1 2 3 4 1 2 3 1 2 Overton HA, 3 Protein Peptide Lett 14(3): 237. Basavarajappa BS (2007b). Neuropharmacology of the Endocannabinoid Signalling Fadda P, Robinson L, Fratta W, Pertwee R, Riedel G (2004). Differential effects of THC-or CBD-rich cannabis extracts on working memory in rats. Neuropharmacology 47(8): 1170-1179. Naidu P, Kinsey S, Guo T, Cravatt B, Lichtman A (2010). Regulation of Inflammatory Pain by Inhibition of Fatty Acid Amide Hydrolase. J Pharmacol Exp Ther 334(1): 182. 3 4 Ning Y, Neill KO, Lan H, Pang L, Shan LX, Hawes BE, et al. (2008). Endogenous and 4 4
	5 5 5 5	System -Molecular Mechanisms, Biological Actions and Synaptic Plasticity. Curr synthetic agonists of GPR119 differ in signalling pathways and their effects on insulin Overton HA, Fyfe MCT, Reynet C (2008). GPR119, a novel G protein-coupled receptor
	6 6 6 6	Neuropharmacol 5(2): 81-97. Farrimond J, Hill A, Whalley B, Williams C (2010a). Cannabis constituents modulate secretion in MIN6c4 insulinoma cells. Brit J Pharmacol 155(7): 1056-1065. target for the treatment of type 2 diabetes and obesity. Brit J Pharmacol 153(S1): S76-
	7 8 7 7 7 8 8 8	Ben-Zvi Z, Mechoulam R, Burstein S (1970). Identification through synthesis of an 9-tetrahydrocannabinol-induced hyperphagia in rats. Psychopharmacology 210(1): 97-106. S81. Ohno-Shosaku T, Maejima T, Kano M (2001). Endogenous cannabinoids mediate
	9 9 9 9	active .DELTA.1(6)-tetrahydrocannabinol metabolite. J Am Chem Soc 92(11): 3468-retrograde signals from depolarized postsynaptic neurons to presynaptic terminals. Oz M (2006). Receptor-independent effects of endocannabinoids on ion channels. Curr
	10 10 10 10	3469. Farrimond JA, Whalley BJ, Williams CM (2010b). A low ∆9tetrahydrocannabinol Neuron 29(3): 729-738. Pharm Des 12(2): 227-239.
	11 11 11 11	cannabis extract induces hyperphagia in rats. Behav pharmacol in press.
	12 13 14 15 16 17 18 19 20 21 12 13 14 15 16 17 18 19 20 21 22 12 13 14 15 12 13 14 15 16 17 18 16 17 18 19 20 21 22 19 20 21	Ben Amar M (2006). Cannabinoids in medicine: A review of their therapeutic potential. J Ethnopharmacol 105(1-2): 1-25. Berridge M (1995). Capacitative calcium entry. Biochem J 312(Pt 1): 1. (1988). Inositol trisphosphate and calcium signaling. Cold Spring Harbor Symposia on Quantitative Biology. Cold Spring Harbor Laboratory Press. p 927. Bisogno T, Ligresti A, Di Marzo V (2005). The endocannabinoid signalling system: Biochemical aspects. Pharmacol Biochem Be 81(2): 224-238. F o r P e e r F o r P e e r F o r P e e r F o r P e e r Felder CC, Joyce KE, Briley EM, Mansouri J, Mackie K, Blond O, et al. (1995). Comparison of the pharmacology and signal transduction of the human cannabinoid CB1 and CB2 receptors. Mol Pharmacol 48(3): 443-450. Fong TM, Guan X-M, Marsh DJ, Shen C-P, Stribling DS, Rosko KM, et al. (2007). Antiobesity Efficacy of a Novel Cannabinoid-1 Receptor Inverse Agonist, N-[(1S,2S)-3-(4-Chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-{[5-(trifluoromethyl)pyridin-2-yl]oxy}propanamide (MK-0364), in Rodents. J Pharmacol Exp Ther 321(3): 1013-1022. F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r Okamoto K, Wang J, Morishita J, Ueda N (2009a). Biosynthetic Pathways of the Endocannabinoid Anadamide. In: Lambert DM (ed)^(eds). Cannabinoids in Nature and Medicine, edn. Zurich and Weinheim: VHCA and WILEY-VCH. p^pp 267-320. Okamoto Y, Morishita J, Tsuboi K, Tonai T, Ueda N (2004). Molecular characterization Pacheco M, Childers S, Arnold R, Casiano F, Ward S (1991). Aminoalkylindoles: actions on specific G-protein-linked receptors. J Pharmacol Exp Ther 257(1): 170. F o Pertwee R (2001). Cannabinoid receptor ligands. Tocris Rev 16: 1-8. r of a phospholipase D generating anandamide and its congeners. J Biol Chem 279(7): 5298-5305. Pertwee R, Griffin G, Fernando S, Li X, Hill A, Makriyannis A (1995). AM630, a P competitive cannabinoid receptor antagonist. Life Sci 56(23-24): 1949-1955. e Okamoto Y, Tsuboi K, Ueda N (2009b). Chapter 1 Enzymatic Formation of Anandamide. In: Gerald L (ed)^(eds). Vitamins & Hormones, edn, Vol. Volume 81: Academic Press. p^pp 1-24. e Pertwee R, Joe-Adigwe G, Hawksworth G (1996). Further evidence for the presence of r cannabinoid CB1 receptors in mouse vas deferens. Eur J Pharmacol 296(2): 169-172.
	22 23 24 25 26 27 28 29 30 31 23 24 25 26 27 28 29 30 23 24 25 26 27 28 29 30 22 23 24 25 26 27 28 29 31 31 30 32 31 32 32 33 33 32	Blankman JL, Simon GM, Cravatt BF (2007). A comprehensive profile of brain enzymes that hydrolyze the endocannabinoid 2-arachidonoylglycerol. Chem Biol 14(12): 1347-1356. Bortolato M, Mangieri R, Fu J, Kim J, Arguello O, Duranti A, et al. (2007). Antidepressant-like activity of the fatty acid amide hydrolase inhibitor URB597 in a rat model of chronic mild stress. Biol Psychiat 62(10): 1103-1110. Breivogel CS, Sim LJ, Childers SR (1997). Regional differences in cannabinoid receptor/G-protein coupling in rat brain. J Pharmacol Exp Ther 282(3): 1632-1642. R e v i e w R e v i e w R e v i e w R e v i e w Fredriksson R, Höglund PJ, Gloriam DEI, Lagerström MC, Schiöth HB (2003). Seven evolutionarily conserved human rhodopsin G protein-coupled receptors lacking close relatives. FEBS Letters 554(3): 381-388. Freund TF, Katona I, Piomelli D (2003). Role of Endogenous Cannabinoids in Synaptic Signaling. Physiol. Rev. 83(3): 1017-1066. Gaetani S, Fu J, Cassano T, Dipasquale P, Romano A, Righetti L, et al. (2010). The Fat-Induced Satiety Factor Oleoylethanolamide Suppresses Feeding through Central R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w R e v i e w Onaivi E, Carpio O, Ishiguro H, Schanz N, Uhl G, Benno R (2008). Behavioral effects of CB2 cannabinoid receptor activation and its influence on food and alcohol consumption. Ann. NY Acad. Sci 1139: 426-433. Onaivi E, Ishiguro H, Gong J, Patel S, Perchuk A, Meozzi P, et al. (2006). Discovery of the Presence and Functional Expression of Cannabinoid CB2 Receptors in Brain. Ann. N.Y. Acad. Sci.(1074): 514-536. R Pertwee RG (2008a). The diverse CB1 and CB2 receptor pharmacology of three plant cannabinoids: ∆ 9 -tetrahydrocannabinol, cannabidiol and ∆ 9 -tetrahydrocannabivarin. Brit e J Pharmacol 153(2): 199-215. v i Pertwee RG (2008b). The diverse CB 1 and CB 2 receptor pharmacology of three plant e cannabinoids: ∆ 9 -tetrahydrocannabinol, cannabidiol and ∆ 9 -tetrahydrocannabivarin. Brit J Pharmacol 153(2): 199-215. w Release of Oxytocin. J Neurosci 30(24): 8096. Onaivi E, Leonard C, Ishiguro H, Zhang P, Lin Z, Akinshola B, et al. (2002). Pertwee RG (2007). GPR55: a new member of the cannabinoid receptor clan? Brit J Endocannabinoids and cannabinoid receptor genetics. Progress in Neurobiol 66: 307-Pharmacol 152(7): 984-986.
	33 34 35 34 34 33 35 35 34 36 36 35	Brown A, J. , Wise A (2001). Identification of modulators of GPR55 activity: GlaxoSmithKline. Gallate JE, Saharov T, Mallet PE, McGregor IS (1999). Increased motivation for beer in rats following administration of a cannabinoid CB1 receptor agonist. Eur J Pharmacol 344. 370(3): 233-240. Pertwee RG (1999). Pharmacology of cannabinoid receptor ligands. Curr Med Chem Osei-Hyiaman D, DePetrillo M, Pacher P, Liu J, Radaeva S, Batkai S, et al. (2005). 6(8): 635-664.
	36 37 37 38 37 36 38 37 39 39 38 38 40 39	Brown AJ (2007). Novel cannabinoid receptors. Brit J Pharmacol 152(5): 567-575. Gaoni Y, Mechoulam R (1966). Cannabichromene, a new active principle in hashish. Chemical Communications (London) 1966(1): 20-21. Endocannabinoid activation at hepatic CB1 receptors stimulates fatty acid synthesis and contributes to diet-induced obesity. J Clin Invest 115(5): 1298-1305. Pertwee RG (2005). The therapeutic potential of drugs that target cannabinoid receptors or modulate the tissue levels or actions of endocannabinoids. Aaps J 7(3): E625-654.

Table 1 ;

 1 endocannabinergic tone effecter ligands. A brief overview of the CBR ligands 1 discussed throughout this review, giving further background to their discovery and 2 where appropriate, their non-feeding effects. 3 Abbreviations: 2-AG, 2-archidonlyglycerol; ∆ 8 THC, ∆ 8 tetrahydrocannbinol; ∆ 9 THC, 4 ∆ 9 tetrahydrocannbivarin; AEA, anandamide; CB 1 R, cannabinoid type 1 receptor; CB 2 R, 6 cannabinoid type 2 receptor; CBR, cannabinoid receptor; CNS, central nervous system; 7 FAAH, fatty acid amide hydrolase; MAGL, monoacylglycerol lipase; OEA, n-8 oleoylethanolamide; P-gp, p-glycoprotein; VR 1 , transient receptor potential vanniliod 1; 9 TRPA 1 , transient receptor potential cation channel, subfamily A, member 1. 10

						66
	5	∆ 9 tetrahydrocannabinol;	∆ 9 THCA,	∆ 9 tetrahydrocannbinolic	acid;	∆ 9 THCV,
		o r
			P
			e
			e r
				R
				e
				v i e
				w

11 Page 66 of 69 http://mc.manuscriptcentral.com/ptr

	Phytotherapy Research
	F o
	r
	P
	e e r
	R e v i e w

http://mc.manuscriptcentral.com/ptr Phytotherapy Research

http://mc.manuscriptcentral.com/ptr

Acknowledgements

This research was supported in part by the University of Reading Research Endowment 22

Trust Fund (to JAF). ://mc.manuscriptcentral.com/ptr