UNIVERSITY OF CALIFORNIA, IRVINE
Endocannabinoid signaling in social behavior
DISSERTATION
submitted in partial satisfaction of the requirements for the degree of
DOCTOR OF PHILOSOPHY
in Biomedical Science
by
Don Wei
Dissertation Committee: Professor Daniele Piomelli, Chair Professor Christine Gall Professor James McGaugh Professor Fred Ehlert
2016
© 2016 Don Wei
DEDICATION
To
family and friends
my dear parents
to my love Christine
in recognition of their unconditional support
The lesson from childhood, then, is that if you want to win the war for attention, don’t try to say ‘no’ to the trivial distractions you find on the information smorgasbord; try to say ‘yes’ to the subject that arouses a terrifying longing, and let the terrifying longing crowd out everything else.
David Brooks
--
We must not cease from exploration. At the end of all our exploring will be to arrive where we began and to know the place for the very first time.
TS Eliot
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TABLE OF CONTENTS
Page
LIST OF FIGURES iv
ACKNOWLEDGMENTS v
CURRICULUM VITAE vi
ABSTRACT OF THE DISSERTATION ix
CHAPTER 1: General Introduction 1
CHAPTER 2: Endocannabinoid signaling mediates oxytocin-driven social reward 17
CHAPTER 3: A role for 2-AG in social and high-fat food reward in mice 43
CHAPTER 4: Enhancement of anandamide-mediated endocannabinoid 67 signaling corrects autism-related social impairment
CHAPTER 5: Adolescent cannabinoid exposure persistently impairs social reward 87
CHAPTER 6: Endocannabinoid signaling mediates social protection against pain 93
CHAPTER 7: General Discussion 98
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LIST OF FIGURES
Page
CHAPTER 2: Endocannabinoid signaling mediates oxytocin-driven social reward Figure 2.1: Anandamide signaling at CB1 receptors mediates social reward 21 Figure 2.2: Oxytocin transmission enhances anandamide mobilization 23 in the nucleus accumbens Figure 2.3 3: Anandamide mediates oxytocin-dependent social reward 25 Supplementary Fig. 2.1-2.8 29
CHAPTER 3: A role for 2-AG in social and high-fat food reward in mice Figure 3.1: MGL-Tg mice do not acquire CPPs to high-fat food and social interaction 54 Figure 3.2: MGL-Tg mice show decreased high-fat intake but normal social interaction 55 and social approach behavior Figure 3.3: MGL-Tg mice acquire CPP to cocaine 56 Figure 3.4: Social stimulation increases 2-AG in C57Bl6J mice 58
CHAPTER 4: Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment Figure 4.1: Anandamide activity at CB1 receptors corrects social approach impairment 74 Figure 4.2: URB597 inhibits FAAH and elevates anandamide levels in BTBR mice 76 Figure 4.3: Effects of URB597 in BTBR mice on elevated plus-maze 77 Figure 4.4: FAAH inhibition corrects social approach impairment in fmr1-/- mice 78
CHAPTER 5: Adolescent cannabinoid exposure persistently impairs social reward Figure 5.1: Treatment with cannabinoid agonist in adolescence impairs adult social reward 90
CHAPTER 6: Endocannabinoid signaling in social protection against pain Figure 6.1: Endocannabinoid signaling in social protection against pain in juvenile rats 96
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ACKNOWLEDGMENTS
I am deeply grateful to my mentor and committee chair, Professor Daniele Piomelli. His trusted guidance and faithful support were invaluable to this work. His ability to frame simple observations in important contexts is captivating, and his passion inspiring. I look forward to continuing our scientific discourse.
I would like to thank my committee members, Profs. Christine Gall, James McGaugh, and Fred Ehlert for their kind advice and thoughtful comments. In particular, I will fondly remember the after-hour chats with Dr. Gall and the serendipitous run-ins at the gym with Dr. Ehlert.
I wish to thank members of the Piomelli Laboratory, from the permanent researchers to the visiting scholars. These include, but are not limited to, Prof. Kwang-Mook Jung, Drs. Guillermo Moreno-Sanz, Vidya Narayanaswami, and Nick DiPatrizio, graduate students Agnesa Avanesian and James Lim, as well as lab associate Jennifer Daglian and specialist Fariba Oveisi. I am thankful for their support, friendship, and consultation. I am thankful that they ate the donuts so I didn’t have to.
I wish to thank collaborators and consultants, who played key roles in this work. These include Drs. Olga Peñagarikano, Daniel Geschwind, Gul Dölen, Jacqueline Crawley, and Christine Gall. In particular, thank you to members of the Gall Laboratory, including Carley Karsten, Conor Cox, Kathleen Wang, and Svetlana Kantorovich (Shore). I’ve loved working together, on big projects and small, and hanging out together, for the big and small moments.
I also want to thank all members of the MSTP family at UC Irvine, for their ongoing advice, camaraderie, and support.
Last but not least, I thank the students, technicians, and other scholars who worked directly on these projects, without whom the work obviously would not have been possible. These include, DaYeon Lee, Dr. Dandan Li, Conor Murray, Drake Dinh, Allison Anguren, Kavi Peshawaria, Ernesto Paz, and Jennifer Daglian.
I am grateful for the following sources of funding support: Terri Wang; the Autism Science Foundation Predoctoral Fellowship (to D.W.), National Institute of Health Grant DA012413 (to D.P.), the UC Irvine Medical Scientist Training Program and the UC Irvine Center for Autism Research and Translation.
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CURRICULUM VITAE
Don Wei
CONTACT INFORMATION Email: [email protected] Phone: 916-397-1664 3216 Gillespie Neuroscience Research Facility Univ. California, Irvine 92697
EDUCATION UC Irvine Medical Scientist Training Program 2010 – 2018 Dept. Anatomy & Neurobiology M.D./Ph.D. Candidate Laboratory of Dr. Daniele Piomelli GPA: 4.0/4.0 US Medical Licensing Exam Step I Board Score: 243 (85th percentile) University of Illinois, Urbana-Champaign 2006 – 2010 BS, Psychology, Molecular and Cellular Biology GPA: 3.90/4.00 MCAT: Verbal 11/15, Physical 12/15, Biological 13/15, Writing T/(A-T) (95th percentile) SAT: Math 800/800, Writing 750/800, Verbal 700/800 (98th percentile)
RESEARCH University of Illinois Beckman Institute for Advanced Learning Greenough Laboratory – Undergraduate Researcher, Research Technician 2007 – 2010 Environmental enrichment on callosal myelination Rab3a in synaptic release in a mouse model of Fragile X syndrome U.C. Davis Medical Center M.I.N.D. Institute Noctor Laboratory – Research Associate II Summer 2008 Notch/Numb signaling as cell fate regulators during neurodevelopment Sharp Laboratory – Research Associate I Summer 2007 Golgi-regulated pathways of p53-induced apoptosis
TEACHING UC Irvine MSTP Tutorial Series, Instructor 2012 – 2016 Teach medical students pharmacology, neuroscience TutorNerds, Tutor 2010 – 2016 Guide, coach, and teach high school and college students in subject areas including biology, chemistry, physics, reading, and exams MCAT and DAT, SAT/ACT Kaplan Test Prep, Instructor for MCAT, DAT/OAT 2009 – 2010
AWARDS & HONORS Travel Award for MD-PhD National Conference 2015 UCI Center for Learning and Memory Renee Harwick Graduate Student Award 2015
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Best Poster Presentation, Gordon Conference Cannabinoids in the CNS 2015 Travel Award for NINDS AUPN (Neurology) MD-PhD Clinician-Scientist Course 2015 Travel Award for Molecular Psychiatry / NIMH MD-PhD Conference 2014 Autism Science Foundation Predoctoral Fellowship 2014 Best Poster Presentation, Anatomy & Neurobiology Grad Day 2013, 2014 Dean’s Commendation Honors in Neuroscience, Pharmacology 2011, 2012 Magna Cum Laude, Illinois Latin Honors 2010 School of Liberal Arts and Sciences Excellence Award 2010 University Achievement Full Scholarship 2006 – 2010 Science Olympiad Scholarship (Two National Gold Medals) 2006 – 2010 March of Dimes, Best Youth Group 2009 National Merit Finalist Scholarship 2007 – 2008
PUBLICATIONS & MANUSCRIPTS Lewellyn KJ, Nalbandian A, Gomez A, Wei D, Walker N, Kimonis VE (2015). Administration of CoQ 10 analogue ameliorates dysfunction of the mitochondrial respiratory chain in a mouse model of Angelman syndrome. Neurobiology of disease, 76, 77-86. Wei D, Piomelli D (2015). Cannabinoid-based drugs: potential applications in addiction and other mental disorders. Focus (American Psychiatric Association). Reviewed cannabinoids in mental illnesses and treatment, including aspects of social behavior, PTSD, Tourette’s, psychosis, and addiction. Dotsey EY, Jung KM, Lodola A, Wei D, Mor M, Piomelli D (2015). Control of endocannabinoid signaling through redox regulation of monoacylglycerol lipase activity. Chemistry and Biology. Wei D, Lee D, Karsten CA, Cox CD, Peñagarikano O, Geschwind DH, Gall CM, Piomelli D (2015). Endocannabinoid signaling mediates oxytocin-driven social reward. Proc Natl Acad Sci USA. doi:10.1073/pnas.1509795112 Wei D, Lee D, Dandan L, Daglian J, Jung KM, Piomelli D (in press, 2016). A role for the endocannabinoid 2-arachidonoyl-sn-glycerol for social and high-fat food reward in mice. Psychopharmacology. Wei D, Lee D, Dinh D, Anguren A, Gall C, Piomelli D (in press, 2016). Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment. Cannabis and Cannabinoid Research.
ABSTRACTS/POSTERS Wei D, Murray C, Lee D, Anguren A, Dinh D, Peshawaria K, Jung KM, Piomelli D. Oxytocin-driven endocannabinoid regulation of sociability. Phoenix, AZ: American College of Neuropsychopharmacology, 2014. Abstract/Poster, online. Wei D, Murray C, Lee D, Anguren A, Dinh D, Peshawaria K, Jung KM, Piomelli D. Endocannabinoid regulation of sociability. Washington, D.C.: Society for Neuroscience, 2014. Abstract, online.
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ACKNOWLEDGEMENTS DURING UNDERGRADUATE WORK Stanton, GB, Kohler, SJ, Boklweski, J, Cameron, JL, & Greenough, WT (2014). Cytogenesis in the adult monkey motor cortex: Perivascular NG2 cells are the major adult born cell type. Journal of Comparative Neurology. Performed immunostaining to identify the phenotype of adult-born cells in the motor cortex of macaques. Markham, J. A., Herting, M. M., Luszpak, A. E., Juraska, J. M., & Greenough, W. T. (2009). Myelination of the corpus callosum in male and female rats following complex environment housing during adulthood. Brain research, 1288, 9-17. Performed electron microscopy to determine the effects of environmental enrichment on axonal myelination in the corpus callosum during adulthood. Ran, R., Pan, R., Lu, A., Xu, H., Davis, R. R., & Sharp, F. R. (2007). A novel 165-kDa Golgin protein induced by brain ischemia and phosphorylated by Akt protects against apoptosis. Molecular and Cellular Neuroscience, 36(3), 392-407. Performed Western blotting for quantification of a novel Golgin protein that regulates apoptosis.
PRESENTATIONS Endocannabinoid signaling in social behavior and autism-related social impairment. Irvine, CA: Center for Autism Research and Translation Symposium, 2015 Endocannabinoid regulation of social behavior. Irvine, CA: Anatomy & Neurobiology Grad Day, 2013, 2014; Center for Learning and Memory Renee Harwick Award, 2015 Endocannabinoid regulation of sociability. Irvine, CA: ReMIND Emerging Scientists Symposium, 2014.
SERVICE UC Irvine MSTP Distinguished Lecturer Series, Committee Member, Chair 2012-2014 Invited, hosted, and organized internationally distinguished scientists to lecture and interact with students and faculty, including two Nobel Laureates and many HHMI investigators. UC Irvine Universities Allied for Essential Medicines, Member, President 2010-2012 Lobbied the Univ. of California Regents for a global access licensing policy of UC-patented medicines. Success in 2014 to change policy for equitable licensing in third-world countries. Univ. of Illinois March of Dimes Collegiate Council, Co-Founder 2008-2010 Raised funds and awareness for premature birth and developmental disorders. Activities included organizing penny drives and tours of the NICU (Neonatal Intensive Care Unit).
EMPLOYMENT Univ. of Illinois Carle Foundation Hospital, Emergency Medical Technician 2008-2009 Univ. of Illinois Housing, Resident Advisor 2007-2008 Univ. of Illinois Media Company, Page Designer 2006-2008
MEMBERSHIPS Society for Neuroscience Orange County Medical Association International Society for Autism Research
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ABSTRACT OF THE DISSERTATION
Endocannabinoid signaling in social behavior
By Don Wei
Doctor of Philosophy in Biomedical Science
University of California, Irvine, 2016
Professor Daniele Piomelli, Chair
Many animal species are social, which in many ways is essential for their survival. The neural substrates of social behavior are largely unknown, but studies on marijuana have hinted at a role for endocannabinoid signaling. We investigated this possibility using a combination of pharmacological and genetic approaches, with social place preference and social approach as readouts for social reward and social interest, respectively. The main findings of this work are: (i) oxytocin neurons in the hypothalamus recruits anandamide-mediated endocannabinoid signaling in the nucleus accumbens to regulate social reward; (ii) the endocannabinoid 2-AG is also involved in social reward but with key differences, including being generalizable to other rewards;
(iii) inhibiting enzyme-mediated anandamide hydrolysis to promote its signaling may offer a viable therapeutic approach for autism-related social impairment. These findings provide important insights for larger questions in social neuroscience, including mechanisms of recruiting the endocannabinoid system, social modulation, and social development. They suggest that targeting endocannabinoid signaling may be a worthwhile approach in the treatment of social impairment, which is involved in most mental illnesses.
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Chapter 1
GENERAL INTRODUCTION
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Social behavior
Social behavior is a human hallmark. People spend an enormous amount of time engaging in social interactions. They form loose relationships with acquaintances or strong relationships with friends and family members. Together, these connections form social networks that range from schools and companies to ethnic identities and nationalities.
The social nature of humans has been recognized since Aristotle (Lord, 1984). Its crucial role in human life has been studied in a spectrum of fields in the social sciences. Only recently, however, have biological approaches been used to study the underlying physiology of social behavior.
These approaches have contributed to understanding the neural signaling that may regulate social behavior as well as the impact of social factors on mental and physical health. These lines of research have given rise to the aggregated field of social neuroscience (Cacioppo and Ortigue,
2011).
Firstly, this field has emphasized the importance of social relationships. Social contact is essential for the health of the brain and the body (House et al., 1988). For example, social isolation in critical periods of life wreaks havoc on cognition, emotion and immune responses. Second, social support is a crucial factor for neuropsychiatric and bodily diseases, both in susceptibility and recovery (Cohen and Willis, 1985). Examples abound: abnormal social development predicts addictive behaviors and conduct disorder; social support buffers against not only mental illnesses such as depression but also physical ailments such as chronic pain and recovery from myocardial infarction.
The multitude of health problems associated with social factors also highlights the complexities and fragilities involved in a growing person navigating the social sphere. The nature of social relationships changes significantly over a lifespan, requiring the development of unique social
2 skills. For instance, a child must transition from a strong maternal attachment to mostly peer contact over the course of adolescence, the young adult must transition from singlehood to a state of attachment with a significant other, while the elderly must take on new roles imposed by accrued experience and reduced physical strength. These distinct types of experiences involve a range of cognitive and emotional processes that undoubtedly impact brain development. The impact is likely mutual, as proper development is crucial for ongoing social function. Such mutual interactions must be finely tuned. To date, the neural processes underlying these transitions and the neural systems that regulate social behavior are largely unknown.
Perhaps the best biological account so far has been the study of oxytocin. Oxytocin is a peptide, comprised of nine amino acids, that has long been known for its peripheral hormonal effects in parturition and lactation (Gimpl and Fahrenholz, 2001). More recently, research has elucidated its central effects in affiliative behaviors and social reward. Tom Insel, Larry Young, and colleagues performed seminal studies in prairie voles (Microtus ochrogaster). They compared these rodents, which display monogamous pair bonding, to the genetically similar but polygamous montane voles (Microtus montanus), and found distinct differences in their oxytocin systems
(Insel, 2010). One key difference is that compared to montane voles, prairie voles have increased oxytocin receptors in the nucleus accumbens, a brain region important for reward signaling. In a series of pharmacological and genetic studies, these authors elaborated the crucial roles of oxytocin in social memory and pair-bond formation. Since then, work on oxytocin remains an active area of ongoing investigation. Modern neuroscience tools have been used to examine the circuit dynamics of oxytocin signaling. For example: oxytocin neurons of the paraventricular nucleus of the hypothalamus project to the amygdala to modulate evoked fear (Knobloch et al.,
2012); oxytocin neurons coordinate with serotonergic neurons from the dorsal raphe nucleus to
3 control peer social reward (Dölen et al., 2013); oxytocin in the hippocampus controls signal-to- noise ratio for information processing (Owen et al., 2013); oxytocin receptors are left-lateralized in the auditory cortex to enable maternal retrieval of separated pups (Marlin et al., 2015).
Human studies have been hampered by the difficulty in delivering oxytocin to the brain, but this obstacle was partly removed by the recent development of an intranasal oxytocin spray. Although the pharmacokinetics of intranasal oxytocin is still unclear, its central effects have been widely documented in numerous high-profile publications. For example, intranasal oxytocin impacts one’s trust of others (Kosefield et al., 2005). Intranasal oxytocin is also being tested in ongoing trials for indications including anxiety, autism spectrum disorder, and drug addiction. Drug development is still at an early stage (reviewed in Barrett and Young, 2015), but a first a sign of success on sociability in autism-spectrum patients (caregiver-rated) was reported (Yatawara et al.,
2015).
The effects of marijuana on social behavior
Marijuana has long been used to facilitate social traditions. For example, an Indian decoction called thandai is prepared and consumed slowly, facilitating a social atmosphere amongst family and friends. Below is a passage recounting the use of thandai (from Iversen, 2007):
Preparing thandai is a time-consuming process. A number of dry fruits,
condiments and spices are used in its preparation. Almonds, pistachio, rose petals,
black pepper, aniseed, and cloves are ground on the toothed grinding plate
(silauti); water is added so that a thinly ground paste is obtained. This paste is
dissolved in milk and then bhang is added to the mixture. A few spoons of sugar
or jaggery (boiled brown sugar) are added finally and then the decoction is ready
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for consumption. . . . The preparation of thandai and the social atmosphere it
creates has great significance. Members of the same family, caste or a circle of
friends from the village or the neighbourhood gather in the parlour of a friend.
Different ingredients of the drink are collected and ground on the toothed stone
grinding plate. The whole process takes an hour or so. While preparing the drink,
individuals talk about friends, family members, prices of goods and services and a
host of other problems.
More recent studies of marijuana use in humans are also in line with the assumption that this drug may affect social behavior. In a survey of students who were experienced marijuana users, more than 70 percent said that intoxication makes them want to interact more with others, because the group takes on ‘a much greater sense of unity, or real social relationship’ (Tart,
1970). More than 80 percent reported that they felt more insightful and empathetic of others but they are also less able to play social games, which suggests that marijuana makes users more intuitive and social but hinders their social skills (Tart, 1970). Worth noting, however, is that, a substantial portion of experienced users also preferred to ingest the drug in isolation, perhaps because they like to feel ‘isolated from things around me’ (Tart, 1970). This contrasts with more casual users who typically ingest the drug in a social setting. Experimental studies have also found that marijuana alters social behavior. In a study of aggression, two groups were given a shared task with a frustration stimulus. Compared to placebo, the group given marijuana provided self-rated reports of higher cooperativity and decreased hostility (Salzman, 1976).
Interestingly, the authors also note that marijuana may have been disinhibitory, such that the users were more willing to express their feelings. Similarly, an experimental study in a small- group setting found that active marijuana changed the distribution of social activities by
5 decreasing time spent in verbal exchanges while increasing time spent in co-actation, i.e. engaging in a shared activity, such as playing a game (Foltin, 1988). Together, these studies indicate that active marijuana use can exert powerful effects on social interactions. They also imply that the effects likely depend on the dose, stressful stimuli, and the past experience of the user. Interestingly, the studies note differential effects on the emotional aspects of social experiences (e.g., empathy, calmness, disinhibition) versus the skills needed for social interaction
(e.g., games, co-actation). Thus, the neurobiological consequences of marijuana likely involve a range of dissociable effects throughout the engagement and ongoing development of social interactions.
The endocannabinoid system
The endocannabinoid system, a modulatory neurotransmitter system in the brain, mediates the effects of the psychoactive principle in marijuana, Δ9-tetrahydrocannabinol. Work on the endocannabinoid system in social behavior remains limited and has emerged more recently.
Meanwhile, the more established roles of this system in anxiety, reward, pain, and (to a lesser extent) cognition have hinted at overlapping functions in social behavior.
The endocannabinoid system consists of lipid-derived messengers called ‘endocannabinoids’ whose actions in the brain are mainly, albeit not exclusively, mediated through CB1 cannabinoid receptors. A series of enzymes catalyze the biochemical synthesis and degradation of endocannabinoids to control their signaling activity (for reviews, see Piomelli, 2003; 2014).
These cascades typically start with the release of particular lipid species from phospholipid membranes, involve multiple biochemical pathways, and end with recycling or the directing of products to alternative biochemical signaling cascades. The canonical route for the synthesis of
6 anandamide is thought to involve a calcium-dependent phospholipase D that releases anandamide by hydrolysis of the phospholipid precursor N-arachidonoyl- phosphatidylethanolamine (N-arachidonoyl-PE). Whether and how other enzymes may be involved is unknown, making the molecular mechanisms and localization of anandamide signaling unclear. Anandamide is mainly degraded via intracellular hydrolysis by the enzyme fatty acid amide hydrolase (FAAH). On the other hand, the mechanisms of synthesis for the other endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) are clearer. 2-AG is generated via the hydrolysis of the intracellular second messenger 1,2-diacylglycerol, by diacylglycerol lipase-α
(DGL-α). DGL-α can be coupled to different receptors upstream for the activation of 2-AG by different signaling systems. For example, in glutamatergic neurons, DGL-α forms a complex with the type-1 metabotropic glutamate receptor, mGluR5, which is scaffolded by proteins such as Homer-1a (Jung et al., 2012). The efficiency of coupling in this complex represents a molecular mechanism for signaling regulation. 2-AG is mainly degraded by the serine hydrolase monoacylglycerol lipase (MGL).
The endocannabinoid neurotransmitters possess a unique set of synaptic properties. Both anandamide and 2-AG are unconventional in the sense that they act in a retrograde manner to suppress presynaptic firing (both CB1 receptors and MGL are localized presynaptically). In this regard, they can be thought of as ‘synaptic circuit breakers’ to curb incoming depolarization
(Katona and Freund, 2008). In line with this idea, 2-AG, for example, is generated upon glutamate spillover to the ‘perisynaptic’ region rather than within the post-synaptic density proper – an anatomical feature that allows homeostatic negative feedback. This is not to say, however, that this type of synaptic feedback is the only mode of endocannabinoid signaling.
Another unique property of the system is that endocannabinoids can also act as diffuse local
7 messengers. Anandamide possesses properties of a volume transmitter – diffusing and affecting multiple neighboring cells – whereas 2-AG is thought of more as a point-to-point synaptic transmitter. Accordingly, a third property is that, when the messengers work in concert, their temporal profiles may differ during signaling. For example, when an animal is subjected to acute stress, 2-AG increases within minutes in the periaqueductal grey whereas anandamide rises in the course of an hour to mediate stress-induced analgesia (Hohmann et al., 2005). In other situations, the directionality of changes in 2-AG and anandamide may actually be opposing. For example, non-contingent alcohol exposure increases extracellular 2-AG levels in the nucleus accumbens (as measured by in vivo microdialysis), but reduces anandamide (self-administration increases 2-AG but does not affect anandamide) (Parsons and Hurd, 2015). In the brain, 2-AG is almost 1000-fold more abundant than anandamide. Although this difference could in part be accounted for by structural, intermediary, or otherwise signaling-incompetent pools of 2-AG, the substantial difference nevertheless suggests dichotomous patterns for signaling action. A fourth unique property is that, as lipid mediators, the endocannabinoids are not stored in traditional vesicles but instead ‘demobilized’ (sequestered) in phospholipid membranes at baseline; they become ‘mobilized’ during signaling activity (upon receptor activation) as they are synthesized ‘on-demand’. This means that molecular mechanisms for their recruitment must involve synthetic enzymes for mobilization. This necessity has implications for understanding interactions between systems, from the circuit down to the molecular level. If the synthetic machinery for anandamide is less well known, for example, it becomes difficult to dissect the molecular interactions needed for its recruitment. This can be seen in the contrast between several well-documented cases of 2-AG recruitment by various G-protein-coupled receptors versus only one for anandamide. G-protein-coupled receptors that recruit 2-AG include type-1
8 metabotropic glutamate receptors (Jung et al., 2005), type 1/3 muscarinic acetylcholine receptors
(Kano et al., 2009), and type-1 orexin receptors (Ho et al., 2011). D2 receptors in the dorsal striatum have been shown to recruit anandamide (Giuffrida et al, 1999).
Endocannabinoid signaling in social behavior
The brain distribution of components of the endocannabinoid system is consistent with a role in social behavior. CB1 receptors are highly expressed in associational cortical regions of the frontal lobe and subcortical structures that underpin human social-emotional functioning (Glass et al.,
1997; Seeley et al., 2012; Stanley and Adolphs, 2013). For example, the behavioral variant of frontotemporal dementia, characterized by indifference to social conflict and eccentric social conduct, involves neurodegeneration of regions in salience networks, including the anterior cingulate cortex, frontoinsular cortex, prefrontal cortex, striatum, and thalamus (Seeley et al.,
2012). CB1 receptors are also expressed throughout regions implicated in reward and addiction, including the central and basolateral amygdala, prefrontal cortex, hippocampus, dorsolateral striatum, ventral tegmental area, and, to a lesser extent, the nucleus accumbens (Glass et al.,
1997; Parsons and Hurd, 2015). These regions are considered key parts of the ‘social brain,’ based on imaging and network studies (Stanley and Adolphs, 2013). The regional distribution of the aforementioned endocannabinoid-synthesizing and -degradative enzymes is largely similar to the picture of CB1 depicted here (Suárez et al., 2011; Egertova et al., 2008; Parsons and Hurd,
2015). But it is worth noting that the anatomical distribution of these enzymes has been studied less extensively than that of CB1 receptors. Human PET imaging studies have also validated the distribution of CB1 receptors in, for example, schizophrenia and addiction (Ceccarini et al., 2015;
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Wong et al., 2008). In addition to less documentation, the value of enzyme expression as a proxy for signaling is questionable (despite popularity in the literature), because (i) enzyme activities can be modified post-translationally, (ii) biochemical synthetic routes may be multiple or unclear, particularly for anandamide, and (iii) expression reflects basal rather than stimulated states, which is problematic for the on-demand nature of endocannabinoid mobilization.
Initial animal studies of endocannabinoid signaling in social behavior focused at the level of CB1 receptors. Synthetic cannabinoid agonists tend to decrease direct interactions. Why, however, remains unclear because of the diverse actions of cannabinoids, such as in cognition and stress reactivity. Like marijuana, the broad actions of an agonist expose social effects to modification by a host of factors related to context. One example is the complex effects on aggression – a component of the social repertoire. Consistent with human studies that found that marijuana decreases hostility (Salzman et al., 1976), studies in experimental animals have found that cannabinoid agonists decrease aggression (Cutler et al., 1984; Miczek et al., 1978). But this may not be necessarily prosocial, as agonists can actually increase defensive posturing (Cutler et al.,
1984). Other studies have reported an opposite effect – that agonists actually enhance aggression under certain stressful conditions, in conjunction with flight acts (Dorr and Steinberg, 1976).
Consistently, cannabinoid agonists can be anxiogenic (Genn et al., 2004). Thus, the state of stress may play an important role in modifying the response to a cannabinoid. Indeed, Haller and
-/- colleagues (2004) observed that CB1 mice exhibited less direct social interactions in an unfamiliar environment, but not in a home-cage environment. Regional manipulation of CB1 may also lead to a different outcome than whole-brain manipulation. Overexpression of CB1 in the medial prefrontal cortex reduced interactions and increased withdrawal (Klugmann et al.,
2011). This effect was concomitant with altered emotional reactivity and decreased cognitive
10 flexibility (Klugmann et al., 2011). Together with the previously described human marijuana studies, these results support a role for CB1 in social behavior, but suggest complex interactions between factors such as the affected neural circuits of cognition, stress, and reward, which additionally can be modified by context.
The picture offered by anandamide seems to be, in general, less complex and more prosocial than that provided by cannabinoid receptors. Mice lacking the hydrolytic enzyme of anandamide, fatty acid amide hydrolase (FAAH), which show elevated levels of anandamide, exhibit increased direct social interactions (Cassano et al., 2011). In a model of phencyclidine-induced social withdrawal, which is related to schizophrenia, the FAAH inhibitor URB597 improved the social impairment (Seillier et al., 2013). Trezza and colleagues have investigated the differential roles of
CB1 agonists and anandamide in social play in juvenile rats, a characteristic behavior that peaks in juveniles but wanes in adulthood (2008; 2012). Juvenile peers engage in ‘rough-and-tumble’ play, including pouncing and pinning. Trezza et al. found that a synthetic cannabinoid agonist decreases social play, whereas the FAAH inhibitor URB597 increases social play (2008; 2012).
Furthermore, play increases levels of anandamide in the nucleus accumbens and amygdala
(Trezza et al., 2012). These studies suggest that anandamide may be largely prosocial, in contrast to cannabinoid agonists. However, like studies on CB1, the various neural faculties involved across these studies, from social cognition to reward, raise further questions into how anandamide affects social behavior. In addition, no study to date has reported a clear role for 2-
AG in social behavior.
In a separate line of research, developmental effects of cannabinoids also support their role in social behavior. Developmental exposure to cannabinoid agents has been found to influence the expression of social behavior later in adulthood. Treatment with the cannabinoid agonist, WIN
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55,212-2 (1.2 mg/kg) over 25 days in adolescent rats (postnatal days 15–40) impaired social interactions after the animals developed into adults (Schneider et al., 2005). The authors also conducted lesion experiments to conclude that the medial prefrontal cortex is crucial for the proper development of social functioning and likely involved in this impairment. This type of persistent impairment after adolescent cannabinoid exposure has been confirmed by several other studies (Rubino and Parolaro, 2008). An open question, however, is whether this exposure affects the endocannabinoid system itself or interacts with other systems to lead to long-term impairment. While these are not mutually exclusive, support for the first possibility would also indicate an important role for endocannabinoid signaling in social development. Indeed, it is particularly striking that the expression of CB1 receptors throughout regions of the social brain reaches a peak during adolescence, and decreases into adulthood (Rubino and Parolaro, 2015). A recent study aimed at probing the significance of this increase used a mutagenesis-induced gain- of-function rat model. The study found that overexpressing CB1 receptors prolonged the adolescent behavioral repertoire, including increased impulsivity and social play, into adulthood
(Schneider et al., 2015). That is, the adult mutants’ behavioral profile looked like that of regular adolescent rats. Although the model may have confounders, and endocannabinoid signaling in these mutants requires investigation, the study raises the hypothesis that CB1 receptors are crucial for the proper development of social behavior. They suggest that rather than modulating the development of other social brain systems, endocannabinoid signaling itself mediates the social transition between adolescence and adulthood. Furthermore, these studies highlight the unappreciated, long-term risks of marijuana intake during brain development.
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Scope of the thesis
The studies briefly outlined above, covering from marijuana in humans to components of the endocannabinoid system in rodents, suggest that endocannabinoid signaling in various domains of the social brain plays a role in the development and acute expression of social behavior. In order to establish such a role, however, several knowledge gaps must be addressed: (i) the functions served by endocannabinoid signaling in specific facets of sociality must be isolated, such as cognition, reward, or anxiety; (ii) in this line of thinking, the afferent mechanisms for the recruitment of endocannabinoid signaling must be identified, perhaps within a circuit-based framework to define social behavior; (iii) it must be determined whether impairment in endocannabinoid signaling might be responsible for cardinal social impairments in neuropsychiatric conditions.
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Chapter 2
Endocannabinoid signaling mediates oxytocin-driven social reward
Proceedings of the National Academy of Sciences, 2015
Don Wei1, DaYeon Lee1, Conor D. Cox1, Carley A. Karsten1, Olga Peñagarikano6, Daniel H. Geschwind4,5, Christine M. Gall1, Daniele Piomelli1,2,3
1Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA 92697 2Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697 3Unit of Drug Discovery and Development, Istituto Italiano di Tecnologia, Genova, Italy 4Program in Neurogenetics, Department of Neurology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095, USA. 5Center for Autism Research and Treatment and Center for Neurobehavioral Genetics, Jane and Terry Semel Institute for Neuroscience and Human Behavior, University of California, Los Angeles, Los Angeles, CA 90095, USA. 6Department of Pharmacology, School of Medicine, University of the Basque Country, Barrio Sarriena s/n, Leioa 48940, Spain
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ABSTRACT
Marijuana exerts profound effects on human social behavior, but the neural substrates underlying such effects are unknown. Here we report that social contact increases, whereas isolation decreases, the mobilization of the endogenous marijuana-like neurotransmitter, anandamide, in the mouse nucleus accumbens (NAc), a brain structure that regulates motivated behavior. Pharmacological and genetic experiments show that anandamide mobilization and consequent activation of CB1 cannabinoid receptors are necessary and sufficient to express the rewarding properties of social interactions, assessed using a socially conditioned place preference test. We further show that oxytocin, a neuropeptide that reinforces parental and social bonding, drives anandamide mobilization in the NAc. Pharmacological blockade of oxytocin receptors stops this response while chemogenetic, site-selective activation of oxytocin neurons in the paraventricular nucleus of the hypothalamus stimulates it. Genetic or pharmacological interruption of anandamide degradation offsets the effects of oxytocin receptor blockade on both social place preference and cFos expression in the NAc. The results indicate that anandamide-mediated signaling at CB1 receptors, driven by oxytocin, controls social reward.
Deficits in this signaling mechanism may contribute to social impairment in autism spectrum disorders and might offer an avenue to treat these conditions.
SIGNIFICANCE STATEMENT
We present evidence that an oxytocin-dependent endocannabinoid signal contributes to the regulation of social reward. The results provide insights into the functions of oxytocin, a neuropeptide crucial for social behavior, and its interactions with other modulatory systems that
18 regulate the rewarding properties of social behavior. They further suggest that oxytocin-driven anandamide signaling may be defective in autism spectrum disorders, and that correcting such deficits might offer a strategy to treat these conditions.
INTRODUCTION
Human studies have shown that marijuana heightens the saliency of social interactions (Tart,
1970), enhances interpersonal communication (Tart, 1970), and decreases hostile feelings within small social groups (Salzman et al., 1976). The neural mechanisms underlying these prosocial effects are unclear but are likely to involve activation of CB1 cannabinoid receptors, the main molecular target of marijuana in the human brain (Huestis et al., 2007). Consistent with this idea, CB1 receptors are highly expressed in associational cortical regions of the frontal lobe and subcortical structures that underpin human social-emotional functioning (Glass et al., 1997;
Seeley et al., 2012). Moreover, the receptors and their endogenous lipid-derived ligands – anandamide and 2-arachidonoyl-sn-glycerol (2-AG) (Piomelli, 2014) – have been implicated in the control of social play (Trezza et al., 2012) and social anxiety (Bergamaschi et al., 2014;
Gunduz-Cinar et al., 2013), two crucial aspects of the social experience. Another essential facet of social behavior, the adaptive reinforcement of interactions among members of a group (i.e., the reward of being social), requires the oxytocin-dependent induction of long-term synaptic plasticity at excitatory synapses of the nucleus accumbens (NAc) (Dölen et al., 2013), a key region in the brain reward circuit. Because the endocannabinoid system regulates the reinforcement of various natural stimuli (Fattore et al., 2010) as well as NAc neurotransmission
(Robbe et al., 2002), in the present study we tested the hypothesis that this signaling complex might cooperate with oxytocin to control social reward.
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RESULTS
Anandamide signaling at CB1 receptors mediates social reward
As a first test of the role of anandamide in social reward, we isolated juvenile, group-reared mice for 24 hours and then either returned them to their group or left them in isolation for 3 additional hours (Fig. 2.1A). We then removed and snap-froze their brains, collected micro- punches from various regions of interest (Fig. S2.1) and measured endocannabinoid content by liquid chromatography-mass spectrometry. In contrast to conventional neurotransmitters, which are sequestered in storage vesicles and released by neurosecretion, the endocannabinoids are produced on-demand from membrane phospholipid precursors (Piomelli et al., 2007). For this reason, the tissue levels of these lipid substances provide an accurate estimate of mobilization (i.e. formation minus degradation) during signaling activity (Piomelli, 2014). We found that anandamide levels were substantially elevated in NAc and ventral hippocampus (vHC) of mice that had been returned to their group, when compared to animals left in isolation (Fig. 2.1B). By contrast, no such changes were seen in the amygdala, dorsal striatum, ventral midbrain
(comprising the ventral tegmental area and substantia nigra) (Fig. 2.1B) or other structures included in our survey (dorsal hippocampus, S2 cortex and piriform cortex) (Fig. S2.2).
Moreover, socialization did not change the levels of 2-AG (Fig. 2.1C, Fig. S2.3), an endocannabinoid substance (Mechoulam et al., 1995; Sugiura et al., 1995; Stella et al., 1997) whose roles are often distinct from anandamide’s (Mechoulam et al., 1995); or the levels of oleoylethanolamide (Fig. 2.1D), a bioactive lipid that is structurally related to anandamide but acts through a distinct receptor mechanism (Fu et al., 2003). The results suggest that social contact stimulates anandamide mobilization in NAc and vHC, two regions of the mouse brain
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Fig. 2.1 Anandamide signaling at CB1 receptors mediates social reward. (A) Schematics of the experimental protocol. (B-D) Levels of (B) anandamide (AEA), (C) 2-arachidonoyl-sn-glycerol (2-AG), and (D) oleoylethanolamide (OEA) in nucleus accumbens (NAc), ventral-to-mid hippocampus (vHC), amygdala, dorsal striatum (dStr), and ventral midbrain (vMB) of isolated (Iso) or resocialized (Soc) C57Bl6 mice. (E) Schematics of the social conditioned place preference (sCPP) test. (F,G) sCPP in wild-type (wt) or faah-/- mice treated with vehicle -1 (VEH) or CB1 inverse agonist AM251 (2 mg-kg , intraperitoneal). (H) sCPP in mice treated with FAAH inhibitor URB597 (intraperitoneal). Data are shown as means ± SEM; *P<0.05, compared using Student’s unpaired t test, n = 4-5 (B-D), n = 12-14 (F), Two-way ANOVA with Tukey’s post-hoc test, n = 12-14 (G), or One-way ANOVA with Bonferroni’s post-hoc test, n = 10 (H). that are involved in the control of motivated behavior. Importantly, one of these regions, the
NAc, has been recently implicated in the regulation of social reward by the hypothalamic neuropeptide oxytocin (Kathuria et al., 2003).
Interrupting the activity of fatty acid amide hydrolase (FAAH), the main anandamide-degrading enzyme in the brain, enhances the biological actions of this endocannabinoid neurotransmitter
(Kathuria et al., 2003; Cravatt et al., 2001). Therefore, to explore the function of socially driven anandamide mobilization, we tested juvenile mice lacking the faah gene in a conditioned place preference (CPP) task that specifically evaluates social reward (Dölen et al., 2013) (Fig. 2.1E).
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Compared to their wild-type littermates, faah-/- mice displayed substantially higher levels of social CPP (sCPP) (Fig. 2.1F and G). This phenotypic difference was specific to social context, because CPP for high-fat food or cocaine was unchanged (Fig. S2.4), and was abolished by
-/- administration of the CB1 antagonist AM251, which decreased place preference in both faah and wild-type mice (Fig. 2.1F and G). Furthermore, the two genotypes showed similar performance levels in the three-chambered social approach task, which measures direct social approach behavior rather than social reward (Fig. S2.5).
Confirming FAAH’s role and ruling out developmental compensation as a possible confounder, we noted that single systemic injections of the FAAH inhibitor URB597 (Kathuria et al., 2003;
Cravatt et al., 2001) during the socialization phase of the test (Fig. 2.1E) replicated the prosocial phenotype seen in faah-/- mice in a dose-dependent manner (Fig. 2.1H). Furthermore, as seen with FAAH deletion, blocking FAAH with URB597 did not change performance in the social approach task (Fig. S2.5). Noteworthy, when the sCPP protocol was modified so that mice underwent isolation conditioning first, along with URB597 treatment, the subjects developed normal preference to the social context (Fig. S2.6), suggesting that enhancing anandamide signaling via FAAH inhibition increases social reward rather than decrease isolation aversion.
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Fig. 2.2 Oxytocin transmission enhances anandamide mobilization in the nucleus accumbens (NAc). (A) Anandamide (AEA) levels in the NAc of isolated (Iso) or resocialized (Soc) mice treated with vehicle (VEH) or oxytocin receptor antagonist L-368,899 (5 mg-kg-1, intraperitoneal). (B) AEA levels in the NAc of isolated mice treated with the oxytocin agonist WAY-267464 (5 nmol, intracerebroventricular), with or without the oxytocin receptor antagonist L-368,899 (0.5 nmol, intracerebroventricular). (C,D) Levels of (C) AEA and (D) 2-AG in the NAc of isolated mice with virally directed, oxytocin promoter-restricted expression of DREADD hM3Dq receptors in the paraventricular nucleus of the hypothalamus and were treated with VEH or clozapine-N-oxide (CNO, 5 mg-kg-1, intraperitoneal). Data are shown as means ± SEM; *P<0.05, compared using Two-way ANOVA with Tukey’s post-hoc test, n = 4-5 (A), One-way ANOVA with Bonferroni’s post- hoc test, n = 4-5 (B), or Student’s unpaired t test, n = 4-5 (C,D).
Oxytocin transmission enhances anandamide mobilization in the nucleus accumbens
A primary physiological function of oxytocin is to heighten the saliency of social stimuli (Young and Barrett, 2015). This effect may require the induction of long-term depression at excitatory synapses of the NAc, which receives direct oxytocinergic input from the paraventricular nucleus
(PVN) of the hypothalamus (Dölen et al., 2013). We asked therefore whether socialization- induced oxytocin neurotransmission might regulate anandamide signaling in the NAc, and obtained three sets of results that supported this idea. First, systemic administration of the brain- permeant oxytocin receptor (OTR) antagonist, L-368,899, abolished the rises in anandamide levels elicited in NAc by social contact (Fig. 2.2A). Conversely, intracerebroventricular infusion
23 of the OTR agonist WAY-267464 elevated such levels in the absence of social contact and in an
OTR-dependent manner (Fig. 2.2B). Lastly, and similar to social stimulation, site-selective chemogenetic activation of oxytocin-secreting neurons in the PVN, which we previously showed to increase oxytocin transmission (Peñagarikano et al., 2015), increased anandamide mobilization in the NAc. Administration of clozapine-N-oxide (CNO) in mice engineered to express CNO receptors exclusively in oxytocinergic neurons of the PVN, strongly elevated anandamide content in the NAc, while having no effect on 2-AG (Fig. 2.2C and D). The same interventions produced a similar, but not identical set of responses in the vHC. Intracerebroventricular administration of the OTR agonist WAY-267464 increased anandamide levels (Fig. S2.7A), but oxytocinergic neuron activation with CNO produced only a trend toward increased anandamide mobilization, which was not statistically significant (Fig. S2.7B). Moreover, OTR blockade increased, rather than decreased, anandamide levels in the vHC (Fig. S2.7C). These results suggest that oxytocin transmission tightly controls anandamide mobilization in the NAc, but not the vHC, where its effects appear to be complex and possibly indirect.
Anandamide mediates oxytocin-dependent social reward
If anandamide is a key mediator of the prosocial actions of oxytocin, as our findings seemingly imply, it follows that faah-/- mice, in which anandamide signaling is constitutively hyperactive, should be less sensitive than wild-type mice to the antisocial effects of OTR blockade (Dölen et al., 2013). Confirming this prediction, we found that administration of the OTR antagonist L-
368,899 reduced sCPP in wild-type, but not faah-/- mice (Fig. 3A and B). The pattern of NAc activation, assessed using cFos immunolabeling, also provided indirect support to that
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Fig. 2.3 Anandamide mediates oxytocin-dependent social reward. (A,B) sCPP of wild-type (wt) or faah-/- mice treated with vehicle (VEH) or oxytocin receptor antagonist L-368,899 (5 mg-kg-1, intraperitoneal). (C,D) Representative images and normalized quantification of cFos immunolabeling after isolation or resocialization, following the protocol in Fig. 2.1A; (E-H) cFos levels in socialized faah+/+ mice and faah-/- mice treated with or without the oxytocin receptor antagonist L-368,899 (5 mg-kg-1, intraperitoneal). Scale bar represents 0.1 mm. Dotted line delineates core (inside) and shell (outside). Data are shown as means ± SEM; *P<0.05, compared using Student’s unpaired t test, n = 10-14 (A), n = 3-4 (C,D), n = 4-5 (E- H), or Two-way ANOVA with Tukey’s post-hoc test, n = 10-14 (B). hypothesis. Social contact (Fig. 2.1A) increased the number of cFos-positive cells in the NAc
(Fig. 2.3C and D), and OTR blockade attenuated this effect only in wild-type mice (Fig. 2.3E and F), not in faah-/- mutants (Fig. 2.3G and H). The latter result cannot be attributed to reduced time spent socializing, because faah-/- mice displayed a similar amount of social interactions as did wild-type mice (Fig. S2.5). In contrast to the NAc, social contact did not significantly change the number of cFos-positive cells in the vHC, which was also unaltered by OTR blockade (Fig. S2.8).
DISCUSSION
The present study provides evidence that supports an obligatory role for anandamide in social reward. We show that (i) social contact stimulates anandamide mobilization in a brain region, the NAc, that is crucially involved in the reinforcing properties of natural stimuli (Kelley, 2004), including social reward (12); (ii) this effect is prevented by blockade of the OTR and is mimicked by pharmacological or chemogenetic activation of this receptor; and (iii) heightened anandamide signaling (via FAAH inhibition) enhances social reward and occludes the prosocial
26 effects of oxytocin. cFos expression experiments identify the shell region of the NAc as an important site in which this signaling mechanism might be operational.
In humans, marijuana can either facilitate or impair social interactions and social saliency, possibly depending on dose and context (Tart, 1970; Salzman et al., 1976; Iversen, 2001).
Analogously, in animal models, cannabinoid receptor activation with direct-acting agonist drugs disrupts social interactions, whereas FAAH inhibition enhances them, which is suggestive of a role for anandamide in socialization (Trezza et al., 2010; 2012; Cassano et al., 2011). While important, these data leave unanswered two key questions. The first is whether the anandamide, whose functions in the modulation of stress-coping responses are well recognized (Kathuria et al., 2003, Gunduz-Cinar, 2013; Dölen et al., 2013), might influence social behavior by modulating stress reactivity (Haller et al., 2004). We addressed this question with two complementary sets of experiments. In one, we used a model of socially conditioned place preference that focuses specifically on the acquisition of incentive salience (Panksepp et al.,
2007). Mice were conditioned to social contact with familiar cage-mates for 24 h, an intervention that does not cause stress. When this conditioning procedure was paired with
FAAH inhibition, or faah-/- mice were used instead of wild-type mice, the animals displayed a markedly increased preference for the social context. In separate studies, we evaluated the impact of genetic FAAH deletion or pharmacological FAAH blockade in the social approach task, in which mice are given the option of interacting with a novel conspecific or a novel object for a relatively short period of time (10 min). Socially normal mice spend more time with their conspecific than with the object. We found that neither pharmacological nor genetic FAAH blockade had any effect in this model. Collectively, our findings support the conclusion that
27 anandamide signaling at CB1 receptors specifically regulates the incentive salience of social interactions, and that this effect is independent of anandamide’s ability to modulate anxiety.
A second question addressed by the present work pertains to the neural circuits responsible for recruiting endocannabinoid neurotransmission during socialization. We used convergent experimental approaches to show that OTR activation by endogenously released oxytocin triggers anandamide mobilization in the NAc. This result is consistent with evidence indicating that oxytocin acts as a social reinforcement signal within this limbic region, where it elicits a presynaptic form of long-term depression (LTD) in medium spiny neurons (Dölen et al., 2013).
Thus, a plausible interpretation of our findings is that oxytocin triggers an anandamide-mediated paracrine signal in the NAc, which influences synaptic plasticity through activation of local CB1 receptors. Activation of these receptors is known to induce presynaptic LTD at corticostriatal synapses (Robbe et al., 2002; Gerdeman and Lovinger, 2003).
While providing an economical explanation for our results, the hypothesis formulated above also raises several new questions. Particularly important among them are questions about the roles that other modulatory neurotransmitters may play in regulating the interaction between oxytocin and anandamide. Previous studies point toward serotonin, which is needed for the expression of oxytocin-dependent plasticity in the NAc (Dölen et al., 2013), and dopamine, which has been implicated in striatal anandamide signaling (Giuffrida et al., 1999; Mathur and Lovinger, 2012).
Defining such roles will require, however, further investigation.
In conclusion, our results illuminate a novel mechanism underlying the prosocial actions of oxytocin, and provide unexpected insights on possible neural substrates involved in the social facilitation caused by marijuana. Pharmacological modulation of oxytocin-driven anandamide
28 signaling – by utilizing, for example, FAAH inhibitors – might open new avenues to treat social impairment in autism spectrum disorders.
SUPPLEMENTARY FIGURES
Supplementary Fig. 2.1 Pictures of punches of tissue used for LC/MS-based lipid analysis. Each region starts with a picture of the coronal section before the punch was taken, and then progresses through what the punch looks like as sections were cut toward the posterior of the brain. Arrows indicate the (A) nucleus accumbens (NAc), (B) dorsal striatum (dStr), (C) S2 cortex, (D) dorsal hippocampus (dHC) and (E) ventral-to-mid hippocampus (vHC). Arrowheads indicate (C) the amygdala (D) the piriform cortex. (F) Punches of the ventral midbrain, which likely includes the ventral tegmental area and the substantia nigra. 29
Supplementary Fig. 2.2 Survey of anandamide levels during socialization. (A) Levels of anandamide (AEA) in isolated (Iso) or resocialized (Soc) C57Bl6 mice in the dorsal hippocampus (dHC), S2 cortex and piriform (Pir) cortex. The experimental protocol followed the schematic in Fig. 2.1A. Data are shown as means ± SEM; n = 4-6.
Supplementary Fig. 2.3 Survey of 2-arachidonoyl-sn-glycerol levels during socialization. (A) Levels of 2-arachidonoyl-sn-glycerol (2-AG) in isolated (Iso) or resocialized (Soc) C57Bl6 mice in the ventral-to-mid hippocampus (vHC), amygdala, dorsal striatum (dStr), dorsal hippocampus (dHC), S2 cortex and piriform (Pir) cortex. The experimental protocol followed the schematic in Fig. 2.1A. Data are shown as means ± SEM; n = 5-6.
30
Supplementary Fig. 2.4 High-fat food or cocaine CPP is not overtly altered in faah-/- mice. (A) Schematic of the experimental paradigm, (B) high-fat chamber times and (C) high-fat chamber preference. (D) Schematic of the experimental paradigm, (E) cocaine chamber times and (F) cocaine chamber preference. Data are shown as means ± SEM; n = 8-10.
31
Supplementary Fig. 2.5 Anandamide enhancement does not change social approach performance. (A) Schematic of the experimental paradigm. (B-C) Chamber and sniffing times of (B) wildtype (wt) or faah-/- mice, or (C) C57Bl6J mice treated with vehicle (VEH) or FAAH inhibitor URB597 (1 mg-kg- 1, intraperitoneal). Data are shown as means ± SEM; n = 8-10.
Supplementary Fig. 2.6 Anandamide enhancement by the FAAH inhibitor URB597 administered during isolation in an inverted sCPP does not alter development of preference to the social environment. (A) Schematic of the experimental paradigm, in which isolation conditioning precedes socialization and is paired with URB597, in contrast to the normal sCPP procedure according to Fig. 2.1E. (B,C) Social chamber time and social preference of C57Bl6J mice treated with vehicle (VEH) or FAAH inhibitor URB597 (1 mg-kg-1, intraperitoneal) during isolate conditioning. Data are shown as means ± SEM; n = 10-12.
32
Supplementary Fig. 2.7 Effects of oxytocin on anandamide mobilization in the ventral hippocampus (vHC). (A) Levels of anandamide (AEA) in the vHC of isolated mice treated with the oxytocin agonist WAY-267464 (5 nmol, intracerebroventricular), with or without the oxytocin receptor antagonist L-368,899 (0.5 nmol, intracerebroventricular). (B) Levels of AEA in the vHC of isolated mice with virally directed, oxytocin promoter-restricted expression of DREADD hM3Dq receptors in the paraventricular nucleus of the hypothalamus and were treated with VEH or clozapine-N-oxide (CNO, 5 mg-kg-1, intraperitoneal). (C) Levels of AEA in the NAc of isolated (Iso) or resocialized (Soc) mice treated with vehicle (VEH) or oxytocin receptor antagonist L-368,899 (5 mg-kg-1, intraperitoneal). Data are shown as means ± SEM; *P<0.05, compared using One-way ANOVA with Bonferroni’s post-hoc test, n = 4-5 (A), or Student’s unpaired t test, n = 4-5 (B-C).
Supplementary Fig. 2.8 Effects of oxytocin and anandamide on cFos expression in the ventral hippocampus. (A) Normalized quantification of cFos immunolabeling after isolation or resocialization, following the protocol in Fig. 3.1a; (B) cFos levels in socialized faah+/+ mice and faah-/- mice treated with or without the oxytocin receptor antagonist L-368,899 (5 mg-kg-1, intraperitoneal). Data are shown as means ± SEM; *P<0.05, compared using Student’s unpaired t test, n = 3 (A), n = 4- 5 (B). 33
MATERIALS AND METHODS
Animals. We used juvenile male mice (4-8 weeks) with C57Bl6J background. They were weaned at P21 and group-reared socially in cages of 4-5 animals each. Testing was done during the light cycle (on at 0630 h and off at 1830 h). All procedures met the National Institute of Health guidelines for the care and use of laboratory animals and were approved by the Institutional
Animal Care and Use Committee at University of California, Irvine.
Drug preparation and treatment. We dissolved URB597 (synthesized in the lab) and AM251
(Cayman Chemicals, Ann Arbor, MI) in a vehicle of saline/propylene glycol/Tween-80 (90/5/5, v/v). L-368,899 (Tocris, Ellisville, MI) was dissolved in saline for intraperitoneal (i.p.) injections or in DMSO for intracerebroventricular (i.c.v.) injections. WAY-267464 (Tocris) was dissolved in DMSO. Clozapine-N-oxide (CNO, Tocris, Ellisville, MI) and cocaine (Sigma-Aldrich, St.
Louis, MO) were dissolved in saline. For lipid analyses, L-368,899 was administered 0.5 h before the start of socialization (3.5 h before sacrifice), WAY-267464 was administered by i.c.v. injection to 24-h isolated animals 0.5 h prior to sacrifice, and CNO to 24-h isolated animals 1 h before sacrifice. For the socially conditioned place preference (sCPP) test, animals were habituated to injections for 3 days leading up to the experiment. URB597, L-368,899 and
AM251 were administered twice a day during social conditioning (AM/PM, i.p.). Balancing vehicle treatments were given during isolation conditioning (AM/PM). For the social approach task, URB597 was administered i.p. 3 h before starting the test.
Intracerebroventricular (i.c.v.) drug infusions. We anesthetized mice with ketamine-xylazine and stereotaxically implanted a 22-gauge guide cannula (3.1 mm in length, Plastics One,
34
Wallingford, CT) positioned 1 mm above the right lateral ventricle at coordinates from bregma:
AP -0.2, ML -1.0 and DV -1.3 mm (Franklin and Paxinos, 3rd Ed.). Animals were allowed to recover 10 days after surgery, during which they were maintained in social housing (groups of 2-
3). Infusions were made in wake animals through a 33-gauge infusion cannula (Plastics One) that extended 1 mm beyond the end of the guide cannula. The injector was connected to a 10-
µL Hamilton syringe by PE-20 polyethylene tubing. The syringe was driven by an automated pump (Harvard Apparatus, Holliston, MA) at a rate of 0.66 µL/min to provide a total infusion volume of 0.5 µL. Cannula placements were verified histologically.
Recombinant adeno-associated (AAV) DREADD virus. We used a serotype 2 AAV vector that was previously constructed and validated to express a modified muscarinic receptor (hM3Dq) and the fluorescent protein mCherry (University of Pennsylvania Viral Core) (Peñagarikano et al., 2015). The hM3Dq receptor is exclusively activated by the otherwise inert compound clozapine-N-oxide. Expression of the virus is restricted by the oxytocin promoter, which directs oxytocin cell-specific expression of hM3Dq receptors and mCherry. mCherry expression in the paraventricular nucleus (PVN) was verified using a Leica 6000B epifluorescence microscope.
Viral infusions. We bilaterally injected the AAV2-oxt-hM3Dq-mCherry construct into the
PVN using the following coordinates (Peñagarikano et al., 2015): AP -0.70, ML ±0.30 and DV -
5.20 mm. We used an adaptor with 33-G needle (Hamilton) connected via polyethylene tubing to a 10-µL Hamilton syringe driven by an automated pump (Harvard Apparatus, Holliston,
MA). We waited for 5 min prior to infusion in order for tissue to seal around the needle, infused a total volume of 0.5 µL (~10^10 titer genome/mL) over 5 min and waited 10 min after infusion
35 before removing the needle. Experiments were conducted 3 weeks after viral injections to allow for recovery and adequate expression. During this period, animals were maintained in social housing (groups of 2-3).
Brain micropunches. Whole brains were collected and flash-frozen in isopentane at -50 to -
60 °C. Frozen brains were maintained in liquid nitrogen on the day of sacrifice until they were transferred to storage at -80 °C. To take micropunches of brain tissue, we first transferred frozen brains to -20°C in a cryostat and waited 1 h for brains to attain local temperature. We then cut to the desired coronal depth and collected micropunches from bilateral regions of interest using a
1x1.5-mm puncher (Kopf Instruments, Tujunga, CA). The micropunches weighed approximately 1.75 mg. A reference micropunch was taken to normalize each punch to the brain’s weight. Bilateral punches were combined for lipid analyses.
Lipid analyses. Procedures were described previously (Astarita and Piomelli, 2009). Briefly, tissue samples were homogenized in methanol containing internal standards for H2-anandamide
2 2 2 2 2 (H -AEA), H -oleoylethanolamide (H -OEA) and H8-2-arachidonoyl-sn-glycerol ( H8-2-AG)
(Cayman Chemicals, Ann Arbor, MI). Lipids were separated by a modified Folch-Pi method using chloroform/methanol/water (2:1:1) and open-bed silica column chromatography. For
LC/MS analyses, we used an 1100 liquid chromatography system coupled to a 1946D-mass spectrometer detector equipped with an electrospray ionization (ESI) interface (Agilent
Technologies, Palo Alto, CA). The column was a ZORBAX Eclipse XDB-C18 (2.1x100 mm,
1.8 µm, Agilent Technologies). We used a gradient elution method as follows: solvent A consisted of water with 0.1% formic acid, and Solvent B consisted of acetonitrile with 0.1%
36 formic acid. The separation method used a flow rate of 0.3 mL/min. The gradient was 65% B for
15 min, then increased to 100% B in 1 min and kept at 100% B for 14 min. The column temperature was 15°C. Under these conditions, Na+ adducts of anandamide/H2-anandamide had retention times (Rt) of 6.9/6.8 min and m/z of 348/352, OEA/H2-OEA had Rt 12.7/12.6 min
2 and m/z 326/330, and 2-AG/ H8-2-AG had Rt 12.4/12.0 min and m/z 401/409. An isotope- dilution method was used for quantification (Giuffrida et al., 2000).
Perfusion and immunohistochemistry. Ketamine-xylazine anesthetized mice were perfused through the heart left ventricle, first with ice-cold saline solution and then with a fixation solution containing 4% paraformaldehyde (PFA) in 0.1 M phosphate buffered saline (PBS, pH
7.4). Brains were collected, post-fixed for 1.5 h and cryoprotected using 30% sucrose in PBS.
Coronal sections were cut using a microtome (30 µm thickness) and mounted on Superfrost plus slides (Fisher, Houston, TX). For cFos immunostaining, sections were washed in 0.1 M glycine solution (in 0.1 M PBS) to quench excess PFA. Sections were incubated for 1 h in blocking solution (10% normal swine serum plus 0.3% Triton-X in 0.1 M PBS). Washed sections were incubated for 48 h at 4°C with anti-cFos antibody (1:500, ab7963 from Abcam, Cambridge,
MA). After washing with 0.1M PBS to remove unbound primary antibody, sections were then incubated for 1 h at room temperature with donkey anti-rabbit IgG conjugated to Alexa Fluor
594. Slides were cover-slipped with Vectashield plus DAPI (Vector Labs, Burlingame, CA).
Images were captured using a 10x objective (Plan Apo, NA 0.4) on a Leica 6000B epifluorescence microscope with PCO Scientific CMOS camera and Metamorph acquisition software.
37 cFos quantification. Image montages were stitched together using FIJI (Preibisch et al., 2009).
Variability in background fluorescence was standardized by subtracting a gaussian-blurred image of each image from itself. Objects of cellular size and shape were then detected using Python 2.7 and FIJI. Brain regions were traced by hand in FIJI using an atlas reference (Franklin and
Paxinos, 3rd Ed.), and resulting coordinates were used to restrict cell counts. Because immunostaining varies across animals and experiments, values were normalized as a ratio to the dorsal striatum (dStr) of the same animal. The dStr was selected as an internal control because it did not vary across compared groups (Student’s t test, n=4-5).
Socially conditioned place preference (sCPP). Following previously described procedures (Dölen et al., 2013), mice were placed in an opaque acrylic box (27.3 x 27.3 cm), divided into two chambers by a clear acrylic wall with a small opening. In the box, a 30-min pre-conditioning test was used to establish baseline non-preference to two types of autoclaved, novel bedding (Alpha
Dry, PharmaServ, Framingham, MA; and Kaytee Soft Granules, Petco, Irvine, CA), which differed in texture and shade (white vs. dark brown). Individual mice with strong preference for either type of bedding were excluded – typically, those that spent more than 1.5x time on one bedding over the other. The next day, animals were assigned to a social cage with cage-mates to be conditioned to one type of novel bedding for 24 h, then moved to an isolated cage with the other type of bedding for 24 h. Bedding assignments were counterbalanced for an unbiased design. Animals were then tested alone for 30 min in the two-chambered box to determine post- conditioning preference for either type of bedding. Fresh bedding was used at each step and chambers were thoroughly cleaned between trials with SCOE 10X odor eliminator (BioFOG,
Alpharetta, GA) to avoid olfactory confounders. Volumes of bedding were measured to be
38 consistent – 300 mL in each side of the two-chambered box and 550 mL in the home-cage.
Animals from the same cage (and thus with familiar odors) were run concurrently in four adjacent, opaque CPP boxes. Scoring of chamber time and locomotion were automated using a validated image analysis script in ImageJ – the static background image was subtracted, moving objects of mouse shape and size were thresholded out and frames were counted in a position- restricted manner.
Three-chambered social approach task. Test mice were habituated to an empty three-chambered acrylic box (40.6 x 21.6 cm), as previously described (Silverman et al., 2010). Habituation consisted of a 10-min trial in the center chamber with doors closed, and then a 10-min trial in all chambers with doors open. Then, during the 10-min testing phase, subjects were offered a choice between a novel object and a novel mouse in opposing side-chambers. The novel object was an empty inverted pencil cup and the novel social stimulus mouse was a sex, age and weight- matched 129/SvImJ mouse. These mice were used because they are relatively inert, and they were trained to prevent aggressive or abnormal behaviors. Weighted cups were placed on top of the pencil cups to prevent climbing. Low lighting was used – all chambers were measured to be 5 lux. The apparatus was thoroughly cleaned with SCOE 10X odor eliminator (BioFOG) between trials to preclude olfactory confounders. Object/mouse side placement was counterbalanced between trials. Chamber time scoring was automated as in social CPP. Sniffing time was scored by trained assistants who were unaware of treatment conditions. Subjects with outlying inactivity or side preference were excluded.
39
Cocaine and high-fat diet CPP. These paradigms were largely similar to social CPP, including unbiased and counterbalanced design, cleaning and habituation, exclusion criteria, and scoring, except for the following key differences which followed established methods (Dölen et al., 2013;
Perello et al., 2010). Mice were conditioned and tested in a two-chambered opaque acrylic box
(31.5 x 15 cm) with a small opening. Pre- and post-conditioning tests allowed free access to both chambers and each had durations of 15 min (cocaine) and 20 min (high-fat). For conditioning, animals underwent 30-min sessions alternating each day between saline/cocaine (8 sessions total,
4 each) or standard chow pellet/high-fat pellet (12 sessions total, 6 each). The two chambers offered conditioning environments that differed in floor texture and wall pattern – sparse metal bars on the floor and solid black walls vs. dense-wire-mesh floors and striped walls. Sparse metal bars allowed for paw access to the smooth acrylic floor, whereas dense-wire mesh did not. For high-fat diet CPP, animals were given one pellet of standard chow and an isocaloric amount of high-fat food (Research Diets, Inc., New Brunswick, NJ). As high-fat pellets have a different color and consistency, they were also given to home cages the day before pre-conditioning to prevent neophobia.
Statistical analyses. Results are expressed as means ± SEM. Significance was determined using two-tailed Student's t-test, One-way or Two-way analysis of variance (ANOVA) with Tukey’s post-hoc test and differences were considered significant if P<0.05. Analyses were conducted using GraphPad Prism (GraphPad Software).
40
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Mechoulam R, et al. (1995) Identification of an endogenous 2-monoglyceride, present in canine gut, that binds to cannabinoid receptors. Biochem Pharmacol 50(1):83–90. Panksepp JB, Lahvis GP (2007) Social reward among juvenile mice. Genes, Brain and Behavior 6:661–671. Peñagarikano O et al. (2015) Exogenous and evoked oxytocin restores social behavior in the Cntnap2 mouse model of autism. Science Translational Medicine 7:271ra8–271ra8. Perello M et al. (2010) Ghrelin Increases the Rewarding Value of High-Fat Diet in an Orexin- Dependent Manner. Biological Psychiatry 67:880–886. Piomelli D (2014) More surprises lying ahead. The endocannabinoids keep us guessing. Neuropharmacology 76:228–234. Piomelli D (2014) More surprises lying ahead. The endocannabinoids keep us guessing. Neuropharmacology 76:228–234. Piomelli D, Astarita G, Rapaka R (2007) A neuroscientist's guide to lipidomics. Nat Rev Neurosci 8(10):743–754. Preibisch S, Saalfeld S, Tomancak P (2009) Globally optimal stitching of tiled 3D microscopic image acquisitions. Bioinformatics 25:1463–1465. Robbe D, Kopf M, Remaury A, Bockaert J, Manzoni OJ (2002) Endogenous cannabinoids mediate long-term synaptic depression in the nucleus accumbens. Proc Natl Acad Sci USA 99:8384–8388. Salzman C, Kochansky GE, Van der Kolk BA, Shader RI (1977) The Effect of Marijuana on Small Group Process. The American journal of drug and alcohol abuse 4:251–255. Salzman C, Van der Kolk BA, Shader RI (1976) Marijuana and hostility in a small-group setting. AJP 133:1029–1033. Seeley WW, Zhou J, Kim E-J (2012) Frontotemporal dementia: what can the behavioral variant teach us about human brain organization? Neuroscientist 18:373–385. Silverman JL, Yang M, Lord C, Crawley JN (2010) Behavioural phenotyping assays for mouse models of autism. Nat Rev Neurosci 11:490–502. Stella N, Schweitzer P, Piomelli D (1997) A second endogenous cannabinoid that modulates long-term potentiation. Nature 388(6644):773–778. Sugiura T, et al. (1995) 2-Arachidonoylgylcerol: A Possible Endogenous Cannabinoid Receptor Ligand in Brain. Biochemical and Biophysical Research Communications 215(1):89–97. Tart CT (1970) Marijuana intoxication: Common experiences. Nature. Trezza V et al. (2012) Endocannabinoids in Amygdala and Nucleus Accumbens Mediate Social Play Reward in Adolescent Rats. The Journal of neuroscience 32:14899–14908. Trezza V, Baarendse PJJ, Vanderschuren LJMJ (2010) The pleasures of play: pharmacological insights into social reward mechanisms. Trends in Pharmacological Sciences 31:463–469. Young LJ, Barrett CE (2015) Can oxytocin treat autism?: We are still at an early stage of assessing oxytocin-based therapy for autism spectrum disorders. Science (New York, NY) 347:825.
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Chapter 3
A role for the endocannabinoid 2-arachidonoyl-sn- glycerol for social and high-fat food reward in mice
Submitted
Don Wei1, DaYeon Lee1, Dandan Li1,4, Jennifer Daglian1, Kwang- Mook Jung1, Daniele Piomelli1,2,3
1Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA 92697 2Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697 3Unit of Drug Discovery and Development, Italian Institute of Technology, Genova, Italy 4Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
43
ABSTRACT
Rationale
The endocannabinoid system is an important modulator of brain reward signaling. Investigations have focused on cannabinoid (CB1) receptors, because dissection of specific contributions of individual endocannabinoids has been limited by the available toolset. While we recently showed the important role of the endocannabinoid anandamide in regulating social reward, in part because of these limitations, the role of the endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) in social reward remains unknown.
Objectives
To study the role of 2-AG in natural reward, we used a transgenic mouse model in which forebrain 2-AG levels are selectively reduced without overt compensation in other elements of the endocannabinoid system. We complemented behavioral analysis of these mice with measurements of brain 2-AG levels.
Methods
Forebrain overexpression of the 2-AG-hydrolyzing enzyme monoacylglycerol lipase (MGL) in
MGL-Tg mice reduces 2-AG levels by 50-75%. We tested these mice in conditioned place preference (CPP) paradigms for high-fat food, social contact and cocaine. Additionally, we measured 2-AG by isotope-dilution liquid chromatography/mass spectrometry (LC/MS).
44
Results
MGL-Tg mice are impaired in developing CPP for high-fat food and social interaction, but do develop CPP for cocaine. Furthermore, compared to isolated mice, levels of 2-AG in socially stimulated wild-type mice are 83% higher in the nucleus accumbens and 40% higher in the ventral hippocampus, but unchanged in the medial prefrontal cortex.
Conclusions
The results suggest that reducing 2-AG-mediated endocannabinoid signaling impairs social and high-fat food reward, and that social stimulation mobilizes 2-AG in key brain regions for reward. The time course of this response differentiates 2-AG from anandamide, whose role in mediating social reward was previously documented.
45
INTRODUCTION
The use of marijuana is reinforced through activation of the mesolimbic reward circuit (Gardner,
2005). In a related but distinct modulatory process, the neurotransmitter system mediating the effects of marijuana in the brain – the endocannabinoid system – also facilitates the reward of other stimuli, such as food or drugs of abuse (Solinas et al., 2008; Maldonado et al., 2006). The endocannabinoid system has three main components: (i) two lipid-derived local messengers – 2- arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA), (ii) enzymes and transporters that mediate their formation and elimination, and (iii) receptors that are activated by endocannabinoids and regulate neuronal activity (Piomelli, 2014; Mechoulam and Parker, 2013).
Genetic and pharmacological studies have unveiled key roles of the CB1 receptor in the modulation of reward signaling. Less is known about the functions served by individual endocannabinoid messengers. In particular, an emerging question is whether endocannabinoids might also regulate the reward of social interactions. We recently demonstrated that anandamide regulates social reward via cooperative signaling with oxytocin, a neuropeptide that is crucial for social bonding (Wei et al., 2015). The role of 2-AG remains unknown, however.
One way to assess the specific contribution of individual endocannabinoids is to manipulate the enzymes responsible for their formation and deactivation. For example, pharmacological inhibition or genetic deletion of the enzyme that hydrolyzes anandamide, fatty acid amide hydrolase (FAAH), markedly increases anandamide activity at CB1 cannabinoid receptors
(Cravatt et al., 2001, Kathuria et al., 2003). Analogous strategies exist for 2-AG. Indeed, MGL-/- mice, in which the 2-AG-hydrolyzing enzyme monoacylglycerol lipase (MGL) is deleted, and
46 thus 2-AG levels are elevated, as well as DGL-α-/- mice, in which the 2-AG-synthesizing enzyme diacylglycerol lipase (DGL-α) is deleted, and thus 2-AG levels are very low (Gao et al.,
2010; Chanda et al., 2010). However, the effects of these radical modifications are often difficult to interpret because of the emergence of profound compensatory changes in the brain, such as desensitization of CB1 receptors and elevation in anandamide and arachidonic acid levels (Gao et al., 2010; Chanda et al., 2010; Schlosburg et al., 2010).
We have recently generated a novel transgenic mouse model – MGL-Tg mice – which selectively overexpress MGL in forebrain neurons under the control of the CaMKIIα promoter
(Jung et al., 2012). These mutant mice display a forebrain-selective accrual in MGL hydrolyzing activity and a 50-75% decrement in 2-AG content. This reduction in 2-AG is not accompanied by overt changes in levels of other endocannabinoid-related lipids (anandamide, arachidonic acid), cannabinoid receptors, or other endocannabinoid-related proteins (Jung et al., 2012).
To investigate the role of 2-AG in reward-related behaviors, we tested MGL-Tg mice in conditioned place preference (CPP) paradigms for high-fat food, social, or cocaine stimuli.
Based on a rich theoretical framework, CPP assesses the rewarding value of test stimuli by pairing them with neutral environments (Bardo et al., 2000). Because less is known about endocannabinoid signaling and social behavior, we also investigated the effects of social interaction on 2-AG signaling in reward-related regions of the brain. We hypothesized that
MGL-Tg mice are deficient in reward signaling and that rewarding social stimuli drive 2-AG signaling in normal mice.
47
MATERIALS AND METHODS
Animals
Male mice (6-10 weeks) were used. They were weaned at postnatal day 21 and group-reared. All testing was conducted during the light cycle (on at 0630 h and off at 1830 h). All procedures met the National Institute of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at University of California,
Irvine.
Socially conditioned place preference (sCPP). Procedures were previously described (Wei et al.,
2015). Briefly, mice were placed in a two-chambered acrylic box (27.3 x 27.3 cm). A 30-min pre- conditioning session was used to establish baseline neutral preference to two types of autoclaved, novel bedding (Alpha Dry, PharmaServ, Framingham, MA; and Kaytee Soft Granules, Petco,
Irvine, CA). These differed in texture and shade (white vs. dark brown). Individual mice with strong baseline preference for either type of bedding were excluded – typically, those that spent more than 1.5x time on one bedding over the other. The next day, animals were randomly assigned to a social cage with cage-mates to be conditioned to one type of novel bedding for 24 h
(in an unbiased, counterbalanced manner), then moved to an isolated cage with the other type of bedding for 24 h. On the next day, animals were tested alone for 30 min in the two-chambered box to determine post-conditioning preference for either type of bedding. Bedding volumes were
300 mL in each side of the two-chambered box and 550 mL in the home-cage. Familiar animals from the same cage were tested concurrently in four adjacent, opaque CPP boxes. Between trials, boxes were thoroughly cleaned with SCOE 10X odor eliminator (BioFOG, Alpharetta, GA).
Scoring was automated using a validated image analysis script in ImageJ (Wei et al., 2015).
48
Cocaine and high-fat diet CPP. Procedures were previously described (Wei et al., 2015). Briefly, these paradigms were similar to social CPP, including unbiased and counterbalanced design, cleaning and habituation, exclusion criteria, and scoring, except for the following main differences, which followed reported methods (Bardo et al., 2000; Perello et al., 2010). Mice were conditioned and tested in a two-chambered acrylic box (31.5 x 15 cm). Pre- and post- conditioning tests allowed free access to both chambers and each had durations of 15 min
(cocaine) and 20 min (high-fat). For conditioning, animals underwent 30-min sessions alternating each day between saline/cocaine (8 sessions total, 4 each) or standard chow pellet/high-fat pellet (12 sessions total, 6 each). The two chambers offered conditioning environments that differed in floor texture and wall pattern – sparse metal bars on the floor and solid black walls vs. dense-wire-mesh floors and striped walls. Sparse metal bars allowed for paw access to the smooth acrylic floor, whereas dense-wire mesh did not. For high-fat diet CPP, animals were given one pellet of standard chow and an isocaloric amount of high-fat food
(Research Diets, Inc., New Brunswick, NJ). As high-fat pellets have a different color and consistency, they were also given to home cages the day before pre-conditioning to prevent neophobia.
High-fat food intake
Intake of high-fat pellets (Research Diets, Inc., New Brunswick, NJ) was recorded in free- feeding mice using an automated monitoring system (Scipro, New York, NY, USA), as described previously (Gaetani et al., 2003). Food intake was measured for two days, and the average of intake was normalized to the body weight at the start of feeding.
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Social interaction test
The test was conducted according to established methods (File and Seth, 2003). To mimic the conditions of the social CPP task, mice were first isolated for 30 min and tested in dim-light conditions (5 lux). Pairs of mice were tested in an open field arena (50 x 50 x 40 cm) for 5 min.
Scoring for social interaction time included behaviors such as sniffing, following, grooming, mounting and crawling over or under. Passive interaction, in which mice were in close proximity but without these interactions, was not included in the scoring.
Three-chambered social approach task
The procedure was previously described (Wei et al., 2015), which was based on an established protocol (McFarlane et al., 2008; Silverman et al., 2012). Briefly, test mice were first habituated to an empty three-chambered acrylic box (40.6 x 21.6 cm), including to the center chamber for
10 min, and then to all chambers for 10 additional min. Mice were then tested for 10 min.
Subjects were offered a choice between a novel object and a novel mouse in opposing side- chambers. The novel object was an empty inverted pencil cup and the novel social stimulus mouse was a sex, age and weight-matched 129/SvImJ mouse. These mice were used because they are relatively inert. They were trained to prevent erratic or aggressive behaviors, such as biting the cup. Weighted cups were placed on top of the pencil cups to prevent climbing. Low lighting
(5 lux) was used. The apparatus was thoroughly cleaned with SCOE 10X odor eliminator
(BioFOG) between trials to preclude olfactory confounders. Object/mouse side placement was counterbalanced between trials. Chamber time scoring was automated using image analysis.
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Sniffing time was scored by trained assistants who were unaware of treatment conditions.
Outliers in inactivity or side preference were excluded.
Brain micropunches. The procedure was previously described (Wei et al., 2015). Briefly, whole brains were collected and flash-frozen in isopentane at -50 to -60 °C. Frozen brains were transferred to -20°C in a cryostat and kept for 1 h to attain local temperature. The brain was then cut to the desired coronal depth and micropunches from bilateral regions of interest were collected using a 1x1.5-mm puncher (Kopf Instruments, Tujunga, CA). The micropunches weighed approximately 1.75 mg. A reference micropunch was taken to normalize each punch to the brain’s weight. Bilateral punches were combined for lipid analyses.
Lipid analyses
Procedures were previously described (Astarita and Piomelli, 2009; Wei et al., 2015). Briefly, tissue samples were homogenized in methanol containing internal standards for H2-anandamide
2 2 2 2 2 (H -AEA), H -oleoylethanolamide (H -OEA) and H8-2-arachidonoyl-sn-glycerol ( H8-2-AG)
(Cayman Chemicals, Ann Arbor, MI). Lipids were separated by a modified Folch-Pi method using chloroform/methanol/water (2:1:1) and open-bed silica column chromatography. For
LC/MS analyses, we used an 1100 liquid chromatography system coupled to a 1946D-mass spectrometer detector equipped with an electrospray ionization (ESI) interface (Agilent
Technologies, Palo Alto, CA). The column was a ZORBAX Eclipse XDB-C18 (2.1x100 mm,
1.8 µm, Agilent Technologies). We used a gradient elution method as follows: solvent A consisted of water with 0.1% formic acid, and Solvent B consisted of acetonitrile with 0.1% formic acid. The separation method used a flow rate of 0.3 mL/min. The gradient was 65% B for
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15 min, then increased to 100% B in 1 min and kept at 100% B for 14 min. The column temperature was 15°C. Under these conditions, Na+ adducts of anandamide/H2-anandamide had retention times (Rt) of 6.9/6.8 min and m/z of 348/352, OEA/H2-OEA had Rt 12.7/12.6 min
2 and m/z 326/330, and 2-AG/ H8-2-AG had Rt 12.4/12.0 min and m/z 401/409. An isotope- dilution method was used for quantification.
Statistical analyses. Results are expressed as means ± SEM. Significance was determined by two- tailed Student's t-test, One-way or Two-way analysis of variance (ANOVA) with Tukey’s post- hoc test. Differences were considered significant if P<0.05. Analyses were conducted using
GraphPad Prism (GraphPad Software).
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RESULTS
MGL-Tg mice do not acquire CPP for high-fat food or social interaction
MGL-Tg mice eat less chow (either standard or high-fat) than do their wild-type littermates
(Jung et al., 2012). The food intake phenotype, however, does not dissociate the effects of 2-AG signaling on metabolic and reinforcement processes. Furthermore, altered feeding can be interpreted as either decreased or increased reward to the food stimulus. To isolate the effects of reduced 2-AG signaling on reward, we tested MGL-Tg mice and their wild-type (WT) littermates (C57Bl6J) in a CPP task for high-fat food. In a standard CPP box, mice were conditioned for 30-min sessions to either standard chow or isocaloric high-fat food for 6 sessions each, alternating over 12 days total (Perello et al., 2010). In WT mice, we found that this conditioning protocol was sufficient to elicit a preference for the high-fat-paired chamber during post-conditioning testing. Animals spent 137 seconds more in the high-fat chamber compared to the standard-chow chamber (Fig. 3.1a-d). In contrast, MGL-Tg mice did not develop a preference for either chamber (Fig. 3.1a-d). This result suggests that 2-AG signaling is involved in conditioned reward processes of high-fat food.
We then asked whether this role for 2-AG signaling could translate to the reward produced by social interaction (Panksepp et al., 2007; Pearson et al., 2012; Dölen et al., 2013). We conditioned mice for 24 h with cage-mates in their home-cage to one type of bedding, then we conditioned them for 24 h isolated to another bedding (Dölen et al., 2013). In the post- conditioning test in a standard CPP box, we found that this conditioning was sufficient to elicit a preference in WT mice for the social bedding (Fig. 3.1e-h). In contrast, MGL-Tg mice did not develop a preference for either bedding (Fig. 3.1e-h). Together with the high-fat-food CPP
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Fig. 3.1 MGL-Tg mice do not acquire CPPs to high-fat food and social interaction. (a) Schematic of conditioned place preference (CPP) task for high-fat food. (b) Time spent in high- fat chamber. (c) High-fat preference as a ratio of time spent in high-fat chamber during post- conditioning trial to pre-conditioning trial. (d) High-fat preference as a difference in time spent in high-fat chamber between post-conditioning and pre-conditioning trials. (e) Schematic of CPP task for social interaction. (f) Time spent in social chamber. (g) Social preference as a ratio of time spent in social chamber during post-conditioning trial to pre-conditioning trials. (h) Social preference as a difference in time spent in social chamber between post-conditioning and pre- conditioning trials. Results are expressed as mean ± SEM; n=10-12 per group. Students t test, comparing pre- to post-conditioning (b,f), or WT to Tg (c-d, g-h). *P < 0.05, **P < 0.01. results, these results suggest that 2-AG signaling may underlie aspects of reward processes common to both natural stimuli.
MGL-Tg mice show decreased intake of high-fat food but normal social interactions
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Fig. 3.2 MGL-Tg mice show decreased high-fat intake but normal social interaction and social approach behavior. (a) High-fat food intake. The average of two days was normalized to the starting weight. (b) Social interaction time in free reciprocal interactions in open field. (c- d) Social approach in a three-chambered apparatus, in which a novel stimulus mouse is contained in a pencil cup (object) in one side chamber and the opposite side chamber contains only the same empty object. Results are expressed as mean ± SEM; n=4-5 per group (a,b), n=8 per group (c,d). Students t test, comparing WT to Tg (a,b), or object to mouse time (c,d). *P < 0.05, **P < 0.01.
The lack of CPP can be attributed to impairments in the generation and processing of the reward, the consolidation of the memory for the reward, or a combination of these processes. To evaluate whether high-fat food stimuli are generated and processed properly, we measured initial intake of high-fat pellets over 2 days. MGL-Tg mice show a 16% reduction in normalized intake compared to WT littermates over this period (Fig. 3.2a). The combined phenotype of MGL-Tg mice showing a lack of CPP and decreased intake suggests that 2-AG plays a role in the generation and processing of high-fat food reward. Strict interpretation of these results, however, may be complicated by the role of 2-AG in energy metabolism (Jung et al., 2012).
For the same reason, we also examined the direct social activity and the social approach interest of MGL-Tg mice using the social interaction test (File and Seth, 2003) and the three- chambered social approach test (McFarlane et al., 2008; Silverman et al., 2012), respectively.
These tests differ in two key ways: (i) the social interaction test evaluates interactions that are reciprocal and direct, whereas the social approach test measures approach activity to a stimulus
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Fig. 3.3 MGL-Tg mice acquire CPP to cocaine. (a) Schematic of conditioned place preference (CPP) task for cocaine (5 mg-kg-1, intraperitoneal). (b) Time spent in cocaine chamber. (c) Cocaine preference as a ratio of time spent in cocaine chamber during post-conditioning trial to pre-conditioning trial. (d) Cocaine preference as a difference in time spent in cocaine chamber between post-conditioning and pre-conditioning trials. Results are expressed as mean ± SEM; n=10 per group. Students t test, comparing pre- to post-conditioning (b), or WT to Tg (c,d). *P < 0.05. mouse that is sequestered in an inverted wire cup; (ii) the social interaction test uses familiar cage-mates, whereas the social approach test uses a novel mouse as a stimulus. In the social interaction test, we observed that MGL-Tg mice trend toward less interaction time, but this result was not significant (Fig. 3.2b). In the social approach test, we found that both MGL-Tg and WT mice preferred the social chamber over the object chamber and sniffed the stimulus mouse more than the object (Fig. 3.2c,d). MGL-Tg mice were similar to WT mice in the amount of time spent in the social chamber and sniffing the stimulus mouse (Fig. 3.2c,d). These results suggest that MGL-Tg mice show similar levels of social interaction and social interest as
WT mice, regardless of the familiarity or the reciprocity of the social stimulus. Therefore, in contrast to high-fat food, the impaired CPP of MGL-Tg mice to social interactions may represent a problem of consolidation rather than reward processing.
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MGL-Tg mice acquire CPP to cocaine
It has been postulated that drugs of abuse ‘hijack’ natural reward circuits (Gardner, 2005; Fattore et al., 2010). We therefore tested MGL-Tg mice in cocaine CPP to see if the drug might recruit
2-AG signaling. In a standard CPP box, we conditioned mice to either saline or cocaine (5 mg- kg-1, intraperitoneal) in four 30-min sessions, alternating over a total of 8 days (Bardo et al.,
2000). Both WT and MGL-Tg mice developed a strong post-conditioning preference for the cocaine chamber (Fig. 3.3a-d). This result suggests that the conditioned effects of cocaine do not depend on 2-AG signaling. Furthermore, it indicates that MGL-Tg mice are cognitively able to perform CPP in general.
Social stimulation increases 2-AG
Previous work has linked feeding status and social contact with brain endocannabinoid levels (Di
Marzo et al., 2001; Kirkham et al., 2002; Wei et al., 2015). In particular, we have recently shown that social contact increases anandamide mobilization in the NAc, and that this response plays an important role in social reward (Wei et al., 2015). If 2-AG is also involved in social-reward processes, as our previous results suggest, social stimulation should increase 2-AG levels. We isolated juvenile C57Bl6J mice for 24 h, and then returned half to their cage-mates while keeping the other half isolated for 6 h (Fig. 3.4a). We collected and snap-froze their brains, took micro-punches of tissue, and measured lipid content using liquid chromatography-mass spectrometry (LC/MS). We found that, compared to mice that remained isolated, socially stimulated mice showed an 83% increase in 2-AG levels in the nucleus accumbens (NAc) and a
40% increase in the ventral hippocampus (vHC), but did not show a change in 2-AG levels in the medial prefrontal cortex (mPFC) (Fig. 3.4b-d). This suggests that social interactions cause a
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Fig. 3.4 Social stimulation increases 2-AG in C57Bl6J mice. (a) Schematic of social stimulation. (b-d) 2-AG levels in the nucleus accumbens (NAc), ventral-to-mid hippocampus (vHC), and medial prefrontal cortex (mPFC). (e) Levels of anandamide (AEA) in the NAc. (f) Schematic of isolation. (g-i) 2-AG levels in the NAc, vHC, mPFC. (j) Levels of anandamide (AEA) in the NAc. Results are expressed as mean ± SEM; n=4-6 per group. Students t test. *P < 0.05, **P < 0.01. ns = not significant.
mobilization of 2-AG signaling. Levels of the endocannabinoid anandamide, in contrast, were unchanged in the NAc after this 6-h social stimulation (Fig. 3.4e). It is important to note, however, that we previously observed an opposite pattern for a 3-h social stimulation, which elicited an increase in levels of anandamide but not 2-AG (Wei et al., 2015).
It is possible that social contact elevates 2-AG levels due to an increase in general activity.
Therefore, we compared 24-h-isolated animals to control animals that were similarly handled but remained social (Fig. 3.4f). In this comparison, isolation had no effect on levels of 2-AG in
58 the NAc, but decreased 2-AG by 55% in the vHC and by 50% in the mPFC (Fig. 3.4g-i). This profile of 2-AG change was not the opposite of that elicited by social stimulation – that is, NAc
2-AG increased during stimulation but did not change during isolation, and mPFC did not change during stimulation but decreased during isolation. In contrast, the pattern of 2-AG levels was opposite in the vHC – 2-AG increased during stimulation and decreased during isolation.
The magnitudes of these opposing effects in the vHC were relatively comparable (40% increase and 55% decrease). Isolation also did not alter levels of anandamide in the NAc (Fig. 3.4j).
We interpret these results to mean that social stimulation induces 2-AG signaling in the NAc that is distinct from what is active during baseline social activity, i.e. a ‘social 2-AG tone.’ Other contributions in the mPFC and vHC remain to be determined. Nevertheless, because the NAc is a key region for brain reward, the results suggest that 2-AG likely contributes to the reward of social interactions. These results support the previous finding that MGL-Tg are deficient in social CPP.
DISCUSSION
It has been postulated that the signaling system underlying reward for social interactions overlaps with those involved in the control of other natural rewards (Panksepp et al., 1997). The available literature emphasizes the important role of the endocannabinoid system in motivated behaviors
(Fattore et al., 2005; Maldonado et al., 2006; Solinas et al., 2008; Parsons and Hurd, 2015), raising the theoretical possibility of an endocannabinoid substrate that is common to different natural rewards. Our study provides evidence in support of this possibility through complementary behavioral analysis of a unique model of selective 2-AG reduction with
59 biochemical analysis of 2-AG mobilization in brains regions important for reward. Our main findings are that 2-AG reduction impairs CPP for social interactions, and that social stimulation increases 2-AG levels in the NAc. These results clearly identify 2-AG as a reward signal for social interaction. Additionally, we found that 2-AG reduction impairs CPP to high-fat food.
Collectively, our results suggest that 2-AG is involved in regulating the incentive salience of two essential aspects of behavior, feeding and social contact.
A limited number of studies have addressed the involvement of endocannabinoid signaling in the control of social behavior. Genetic CB1 deletion and administration of CB1 agonists alter social interactions. The effect of CB1 agonists can be bidirectional, depending on dosage and thus likely the competing circuits involved (Haller et al., 2004; Trezza and Vanderschuren, 2008).
Furthermore, increasing anandamide activity via genetic deletion or inhibition of its hydrolytic enzyme, FAAH, increases direct social interactions in mice and social play in rats (Cassano et al.,
2011; Trezza et al., 2012). Recently, we demonstrated that anandamide-mediated endocannabinoid signaling is important in the control of social reward (Wei et al., 2015). Social contact mobilizes anandamide in an oxytocin receptor-dependent manner. Consistently, chemogenetic activation of oxytocin neurons in the hypothalamus increases anandamide mobilization in the NAc. Pharmacological and genetic enhancement of anandamide activity (i.e. with FAAH inhibitors or genetic FAAH deletion) increases social CPP and offsets the effects of oxytocin blockade. These results suggest that a cooperative oxytocin-driven anandamide signal regulates social reward (Wei et al., 2015).
Thus, the distinct effects of CB1 agonists and FAAH inhibitors on social behavior present a complex picture with two knowledge gaps: (i) whether 2-AG-mediated activation of CB1 receptors also participates in social behaviors and (ii) if so, the type of interaction and associated
60 neural circuits that are regulated. The present study addresses these gaps by (i) exploiting a new transgenic mouse to reduce endogenous 2-AG signaling without overt compensation, (ii) using
CPP to specifically model social reward rather than interaction in general, and (iii) measuring socially mobilized 2-AG levels in brain areas that are part of the reward circuit.
At the same time, our study raises an important question for future studies. The temporal profiles of socially stimulated 2-AG versus anandamide are distinct. In the NAc, a 3-h social stimulation increases the levels of anandamide, but not 2-AG (Wei et al., 2015), whereas a 6-h stimulation elevates levels of 2-AG, but not anandamide (Fig. 3.4b,e). Distinct temporal profiles of endocannabinoid mobilization have been demonstrated in other situations – for example, in response to drugs of abuse (Solinas et al., 2008) as well as in stress-induced analgesia mediated by the periaqueductal grey (Hohmann et al., 2005) – and thus are likely to be functionally meaningful. One plausible explanation is that anandamide is involved in the initial saliency of a social encounter whereas 2-AG is involved in consolidating information from prolonged social contact. This hypothesis is supported by two pieces of evidence. First, oxytocin is thought of as a saliency signal that is more proximal to the initial processing of social sensory information (Insel,
2010; Young and Barrett, 2015). Oxytocin tightly drives anandamide formation in the NAc, whereas 2-AG production is driven by prolonged social contact but not oxytocin (Wei et al.,
2015; Fig. 3.4b,c). Secondly, anandamide is more specific to social reward whereas 2-AG is more generalizable to other rewards, as elevating anandamide increases CPP for social interactions but not for high-fat food or cocaine (Wei et al., 2015), whereas reducing 2-AG activity reduces CPP to both social interactions and high-fat food (Fig. 3.1).
An extensive literature also supports an important role of CB1 signaling in food reward. CB1 receptor antagonism and genetic deletion suppress not only food intake, but also CPP and self-
61 administration (Arnone et al., 1997; Sanchis-Segura et al., 2004; Solinas and Goldberg, 2005). In contrast, information is sparse regarding the individual endocannabinoid transmitters that might be involved. Available studies have found that systemic administration of exogenous anandamide increases food intake (Williams and Kirkham, 1999), and 2-AG administration into the NAc increases feeding (Kirkham et al., 2002). Exogenous administration, however, does not simulate physiological conditions. The intake phenotype may also equivocate the metabolic and motivational aspects of endocannabinoid signaling. Our study addresses these concerns by specifically manipulating 2-AG signaling and using a model of CPP to represent the rewarding value of high-fat food.
A lack of CPP encompasses different impairments in aspects of reward signaling, such as the processing of initial sensory cues, integration and recruitment of limbic regions, and the consolidation of the memory for the reward. In order to specify the component in which 2-AG may play a larger role, we also measured the intake of high-fat food and the social interactions of
MGL-Tg mice. We found that MGL-Tg mice show less intake of high-fat food but appear normal in direct, reciprocal social interactions as well as social approach. In light of the lack of
CPP for both high-fat and social stimuli in MGL-Tg mice, these results may point to a dichotomous role for 2-AG – perhaps in the processing of high-fat reward versus the consolidation of social reward. These speculations are consistent with the aforementioned feeding literature and prolonged action of 2-AG, as compared to anandamide, after social stimulation. In line with this thinking, the phenotype of a lack of CPP across high-fat and social stimuli, but present CPP for cocaine, must be taken to represent a qualitative rather than quantitative difference. That is, reducing 2-AG impairs different processes for the reward signaling of these stimuli, rather than different amounts of the same process. Perhaps because
62 cocaine bypasses 2-AG-regulated signaling and is more dopamine-dependent, CPP develops regardless of 2-AG signaling. Nevertheless, whether 2-AG influences dopamine in MGL-Tg mice and whether dopamine is the ultimate effector of all these rewards remains to be determined. Indeed, the magnitude of cocaine CPP as a function of dosage does not vary in a graded manner, but develops in an all-or-none fashion, starting at the minimum dose range that we used (5 mg-kg-1, i.p.) (Brabant et al., 2005). Additional investigation into these possibilities is needed.
While MGL-Tg mice represent a technological advance to address the aforementioned knowledge gaps, phenotypic results also come with potential weaknesses. First, forebrain 2-AG reduction may alter general cognition so that mice are unable to perform the CPP task.
However, our finding that MGL-Tg mice develop normal cocaine CPP argues against this possibility. Second, improper development or socialization may render MGL-Tg abnormal. But this is unlikely because MGL overexpression is controlled by the CAMKII promoter, which lacks developmental activity, and MGL-Tg mice do not show overt abnormalities in tests of general health (Jung et al., 2012). Normal levels of social interaction and social approach also speak to proper socialization. It remains true that our results do not completely exclude these possibilities. More nuanced forms of cognition or social interaction may not be detected in our tests.
In conclusion, our study identifies a common endocannabinoid substrate – 2-AG – in the regulation of two natural rewards, feeding and social contact. This identification provides a basis to motivate further investigation of the circuitry and physiology regulated by 2-AG signaling in natural reward, the dichotomous roles of anandamide and 2-AG in different social contexts, as well as how such signaling may be exploited in addiction.
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Models of Autism. Science Translational Medicine 4:131ra51–131ra51. doi: 10.1126/scitranslmed.3003501 Solinas M, Goldberg SR (2005) Motivational effects of cannabinoids and opioids on food reinforcement depend on simultaneous activation of cannabinoid and opioid systems. Neuropsychopharmacology 30:2035–2045. doi: 10.1038/sj.npp.1300720 Solinas M, Goldberg SR, Piomelli D (2008) The endocannabinoid system in brain reward processes. British Journal of Pharmacology 154:369–383. doi: 10.1038/bjp.2008.130 Trezza V, Vanderschuren LJ (2008) Bidirectional cannabinoid modulation of social behavior in adolescent rats. Psychopharmacology 197:217–227. doi: 10.1007/s00213-007-1025-3 Wei D, Lee D, Cox CD, et al (2015) Endocannabinoid signaling mediates oxytocin-driven social reward. Proc Natl Acad Sci USA 201509795. doi: 10.1073/pnas.1509795112 Williams CM, Kirkham TC (1999) Anandamide induces overeating: mediation by central cannabinoid (CB1) receptors. Young LJ, Barrett CE (2015) Can oxytocin treat autism?: We are still at an early stage of assessing oxytocin-based therapy for autism spectrum disorders. Science (New York, NY) 347:825.
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Chapter 4
Enhancement of anandamide-mediated endocannabinoid signaling corrects autism-related social impairment
Submitted
Don Wei1, Drake Dinh1, DaYeon Lee1, Dandan Li1,4, Allison Anguren1, Guillermo Moreno-Sanz1, Christine M. Gall1, Daniele Piomelli1,2,3
1Department of Anatomy and Neurobiology, University of California, Irvine, Irvine, CA 92697 2Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697 3Unit of Drug Discovery and Development, Italian Institute of Technology, Genova, Italy 4Department of Ophthalmology, the Fourth Affiliated Hospital of Harbin Medical University, Harbin, Heilongjiang, China
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ABSTRACT
We recently uncovered a signaling mechanism by which the endocannabinoid anandamide mediates the action of oxytocin, a neuropeptide that is crucial for social behavior, to control social reward. Oxytocin signaling has been implicated in autism spectrum disorder (ASD) and social reward is a key aspect of social functioning that is thought to be disrupted in ASD.
Therefore, as a proof of principle for the core component of ASD – social impairment – we tested an endocannabinoid-enhancing compound on two widely studied mouse models of ASD, the BTBR and fmr1-/- (model of Fragile X Syndrome) mice, using the established three- chambered social approach test. We specifically increased the activity of anandamide by administering a selective inhibitor of fatty acid amide hydrolase (FAAH), the hydrolytic enzyme for anandamide. Remarkably, we found that FAAH blockade completely reversed the social impairment in both mouse models. CB1 receptor blockade prevented the prosocial action of
FAAH inhibition in BTBR mice. These results were likely independent of effects on anxiety, as
FAAH inhibition did not alter the performance of BTBR mice in the elevated plus maze. The results suggest that increasing anandamide activity at CB1 receptors improves ASD-related social impairment, and identify FAAH as a novel therapeutic target for ASD.
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INTRODUCTION
A core feature across autism spectrum disorder (ASD) is impairment in social functioning1,2.
People with ASD restrict themselves to repetitive behaviors and show deficits in social reciprocity and communication1. The underlying basis for social impairment in ASD is unknown and no pharmacological treatment is available. One theory – the social motivation theory – posits that the psychopathology of ASD is rooted in a decreased desire to be social3,4.
The neural substrates of normal social behavior are only now beginning to emerge5. Perhaps the best account so far has come from the study of oxytocin. This neuropeptide is crucial in many aspects of social behavior, including affiliation6 and reward7. Investigations are ongoing into the contributions of the oxytocin system to ASD and oxytocin-based therapies for ASD8. Recent reports suggest that early treatment with oxytocin may be useful for improving social behavior in animal models9 as well as in human patients10. Nevertheless, identifying the key neural systems underlying social behavior and understanding how they interact with oxytocin remains an enormous challenge5.
One candidate is the endogenous cannabinoid (‘endocannabinoid’) system, a modulatory neurotransmitter system that may play an important role in social behavior. This signaling complex consists of lipid-derived messengers called ‘endocannabinoids’ whose actions in the brain are mainly, albeit not exclusively, mediated through CB1 cannabinoid receptors. A series of enzymes catalyze endocannabinoid synthesis and degradation to control the activity of these substances11,12. Fatty acid amide hydrolase (FAAH) catalyzes the intracellular hydrolysis of the endocannabinoid anandamide. In an effort aimed at probing anandamide function in social behavior, we found that genetic removal of FAAH in mice increases direct social interactions13, while Trezza et al. noted that pharmacological FAAH inhibition promotes social play in juvenile
69 rats14. More recently, we identified a signaling mechanism by which oxytocin drives anandamide-
15 mediated signaling at CB1 receptors to control the rewarding properties of social interactions .
Social contact among male mice stimulates the mobilization of anandamide in the nucleus accumbens (NAc), a key brain region for reward signaling, in an oxytocin-dependent manner.
Conversely, in isolated male mice, specifically stimulating oxytocin neurons in the paraventricular nucleus of the hypothalamus drives anandamide mobilization in the NAc. Consequent activation of CB1 receptors is necessary and sufficient to regulate social reward. Anandamide enhancement offsets the effects of oxytocin receptor blockade on social reward and NAc activity15. In addition, human studies also support the notion that the endocannabinoid system affects social behavior.
Acutely intoxicated marijuana users enjoy interacting more and feel more connected and
16,17 empathetic . Chakrabarti and Baron-Cohen reported that a polymorphism in the CB1 cannabinoid receptor gene modulates social gaze18.
Based on those studies, we hypothesized that enhancement of anandamide-mediated signaling at
CB1 receptors might offer a therapeutic strategy for ASD-related social impairment. As a first test of this idea, we examined the effects of a well-characterized FAAH inhibitor, the compound
URB59719, in two established mouse models of ASD: (i) BTBR mice, an inbred strain discovered through the Mouse Phenotype Project, which shows pronounced deficits in social approach, reciprocal social interactions, and juvenile play20,21, and (ii) fmr1-/- mutant mice – a model of Fragile X Syndrome, the most common monogenetic cause of ASD, which features persistent social deficits22-24. We show that pharmacological FAAH inhibition substantially improves social behavior in BTBR and fmr1-/- mice, and that this effect is independent of anxiety modulation.
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MATERIALS AND METHODS
Animals. We used male mice (8-10 weeks) bred at UC Irvine. The mice were weaned at P21 and group-reared in cages of 4-5 animals. Testing was done during the light cycle (on at 0630 h and off at 1830 h). Naïve mice were used for each behavioral experiment. All procedures met the
National Institute of Health guidelines for the care and use of laboratory animals and were approved by the Institutional Animal Care and Use Committee at University of California,
Irvine.
Drug preparation and treatment. URB597 (synthesized in the lab) and AM251 (Cayman
Chemicals, Ann Arbor, MI) were dissolved in a vehicle of saline/propylene glycol/Tween-80
(90/5/5, v/v). URB597, and AM251 were administered i.p. 3 h before starting the behavioral testing28. Drugs were administered at 4 µL / g weight of the animal.
FAAH assay. Procedures were described previously25. Briefly, reactions were conducted with tissue homogenate dissolved in 0.5 mL Tris buffer containing [3H]-anandamide at 37°C for 30 min. Reactions were stopped with 1 mL of chloroform/methanol (1:1,v/v). Samples were centrifuged at 3000xg for 10 min at 4oC. Radioactivity in the aqueous layer was measured by liquid scintillation counting.
Lipid analyses. Procedures were described previously26. Briefly, tissue samples were homogenized in methanol containing internal standards for H2-anandamide, H2-oleoylethanolamide (H2-
2 2 OEA) and H8-2-arachidonoyl-sn-glycerol ( H8-2-AG) (Cayman Chemicals, Ann Arbor, MI).
Lipids were separated by a modified Folch-Pi method using chloroform/methanol/water (2:1:1)
71 and open-bed silica column chromatography. For LC/MS analyses, we used an 1100 liquid chromatography system coupled to a 1946D-mass spectrometer detector equipped with an electrospray ionization (ESI) interface (Agilent Technologies, Palo Alto, CA). The column was a ZORBAX Eclipse XDB-C18 (2.1x100 mm, 1.8 µm, Agilent Technologies). We used a gradient elution method as follows: solvent A consisted of water with 0.1% formic acid, and
Solvent B consisted of acetonitrile with 0.1% formic acid. The separation method used a flow rate of 0.3 mL/min. The gradient was 65% B for 15 min, then increased to 100% B in 1 min and kept at 100% B for 14 min. The column temperature was 15°C. Under these conditions, Na+ adducts of anandamide/H2-anandamide had retention times (Rt) of 6.9/6.8 min and m/z of
2 2 348/352, OEA/H -OEA had Rt 12.7/12.6 min and m/z 326/330, and 2-AG/ H8-2-AG had Rt
12.4/12.0 min and m/z 401/409. An isotope-dilution method was used for quantification.
Three-chambered social approach task. Previously established methods were followed21. Test mice were habituated to an empty three-chambered acrylic box (40.6 x 21.6 cm). Habituation included a 10-min session in the center chamber with doors closed, and then a 10-min session in all chambers with doors open. Test mice were then tested in a 10-min session. Subjects were offered a choice between a novel object and a novel mouse in opposing side-chambers. The novel object was an empty inverted pencil cup and the novel social stimulus mouse was a sex, age and weight-matched 129/SvImJ mouse. These mice were selected because they are relatively inert, and they were trained to prevent abnormal behaviors, such as biting the cup. Weighted cups were placed on top of the pencil cups to prevent climbing. Low lighting was used – all chambers were measured to be 5 lux before testing. The apparatus was thoroughly cleaned with SCOE 10X odor eliminator (BioFOG) between trials to preclude olfactory confounders. Object/mouse side
72 placement was counterbalanced between trials. Chamber time scoring was automated using image analysis in ImageJ. Sniffing time was scored by trained assistants who were unaware of treatment conditions. Excluded were subjects with outlying inactivity.
Elevated plus-maze. The procedure was based on previously published methods27. The maze was made of black Plexiglass and consisted of two open arms (30 x 5 cm) and closed arms (30 x 5 cm, with 15-cm side panels on closed arms). The arms extended from a center square (5 x 5 cm). The maze was mounted on a Plexiglas base and raised 39 cm above the ground. Lighting consisted of two 40W incandescent bulbs, each hanging at a height of 1 m above the open arms of the test apparatus. The floor of the apparatus was cleaned with a SCOE 10X odor eliminator (BioFOG) between trials. The animals were placed in the center square and allowed to freely explore for 5 min. The amount of time spent in each arm and the number of entries into each arm were quantified by Ethovision 3.1 video tracking system (Noldus, Netherlands).
Statistical analyses. Results are expressed as means ± SEM. Significance was determined by two- tailed Student's t-test, One-way or Two-way analysis of variance (ANOVA) with Tukey’s post- hoc test. Differences were considered significant if P<0.05. Analyses were conducted using
GraphPad Prism (GraphPad Software).
73
RESULTS
74
Figure 4.1 Anandamide activity at CB1 receptors corrects social approach impairment in BTBR mice. (a-b) Time spent in chamber (a) or sniffing (b) by C57Bl6J mice in the three-chambered -1 social approach assay after treatment with the CB1 inverse agonist AM251 (2 mg-kg , i.p.) or the FAAH inhibitor URB597 (1 mg-kg-1, i.p.). (c-d) Chamber and sniffing times of BTBR mice treated with AM251 (2 mg-kg-1, i.p.), URB597 (i.p.), or AM251 and URB597 in concert. (e-f) Social index, calculated as (social chamber time – object chamber time) / (total side- chamber time). Results are expressed as mean ± SEM. (a-f) n=12-16. (a-d) Students t test, comparing object to social, or VEH to URB597; (e-f) One-way ANOVA with Tukey’s post- hoc test, comparing to VEH. *P < 0.05, **P < 0.01, ***P < 0.001.
FAAH inhibition corrects social impairment in BTBR mice
28 We used the CB1-receptor inverse agonist, AM251, or the FAAH inhibitor, URB597 , to probe the role of anandamide in social behavior of young adult mice. Social activity was evaluated in the widely used social approach test20,21. The mice were placed in a dimly lit three-chambered apparatus, and given a choice between (i) a novel, inert mouse restrained by an inverted pencil cup in one chamber or (ii) a novel object (empty pencil cup) in the opposite chamber20. We first evaluated maximally effective doses of AM251 (2 mg-kg-1, i.p.)29 or URB597 (1 mg-kg-1, i.p.)28 in socially normal C57Bl6J mice, and found that neither drug altered the two outcome measures of the test, namely the time spent in the social chamber and the time spent sniffing the target mouse (Fig. 4.1a,b).
In contrast to C57Bl6J mice, BTBR mice show no social preference in the test (Fig. 4.1c-d) 20,21.
However, administration of URB597 (0.3 or 1 mg-kg-1, i.p., 3 h before testing) significantly increased the time BTBR mice spent in the social chamber and sniffing, to levels that were comparable to those displayed by control, socially normal C57Bl6J mice (Fig. 4.1c,d). The effect of FAAH inhibition on social approach depended on CB1 receptors, and thus presumably on anandamide accumulation, because it was prevented by concomitant administration of AM251
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Figure 4.2 URB597 inhibits FAAH and elevates anandamide levels in the forebrain of BTBR mice. (a) FAAH enzymatic activity, (b) anandamide (AEA) levels, and (c) 2-arachidonoyl-sn- glycerol (2-AG) levels after treatment with URB597 (1 mg-kg-1, i.p.).
(2 mg-kg-1, i.p.) (Fig. 4.1c,d). The results on time spent in the social chamber are summarized as an index (Fig. 4.1e,f).
Using an enzymatic activity assay and liquid chromatography-mass spectrometry (LC/MS), we confirmed that URB597 inhibited FAAH and substantially increased the levels of anandamide in the forebrain of BTBR mice (Fig. 4.2a,b), without affecting levels of the other endocannabinoid 2-arachidonoyl-sn-glycerol (2-AG) (Fig. 4.2c). Together, the results suggest that elevated anandamide activity at CB1 receptors corrects social-approach behavior in BTBR mice, whereas it does not alter social approach in control C57Bl6J mice.
FAAH inhibition does not alter performance of BTBR mice in the elevated plus-maze
The endocannabinoids are important modulators of stress reactivity in humans and experimental animals19,30. In rodent experiments, this modulation depends on the adverseness of environmental conditions, such as the intensity of ambient lighting13,31,32. To test whether the prosocial actions of FAAH inhibition in BTBR mice might be due to a general reduction in anxiety, we asked whether URB597 exerted anxiolytic-like effects in the elevated plus-maze test
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Figure 4.3 Effects of URB597 in BTBR mice on elevated plus-maze under dim lighting conditions. (a-c) Open arm time, entries, and total entries in elevated plus-maze after treatment with URB597 (1 mg-kg-1, i.p.). Results are expressed as mean ± SEM. n=10 per group. Student’s t test. under the same dim lighting conditions used for the social approach test (5 lux). We found that
URB597 (1 mg-kg-1, i.p.) administered to BTBR mice did not alter the time spent in the open arms, or the number of open-arm entries (Fig. 4.3a-c), suggesting that the prosocial effect of this compound cannot be attributed to reduced anxiety.
FAAH inhibition corrects social impairment in fmr1-/- mice
Interpretation of data obtained with BTBR mice is limited by the possibly polygenetic contributions to the phenotype of this inbred strain. Therefore, we asked whether the prosocial effect of anandamide is translatable to a monogenetic model of ASD-related social impairment.
Fmr1-/- mutant mice bred on an FVB/NJ background have been reported to exhibit a deficit in social approach 22. In our hands, however, this deficit was not statistically significant (Fig.
4.4a,b). Nevertheless, we found that acute administration of URB597 (0.3 mg-kg-1, i.p.), which did not alter the time spent in the social chamber or sniffing in wild-type fmr1+/+ (FVB/NJ) mice
77
(Fig. 4.4a-c), increased the time spent by fmr1-/- mice in the social chamber and the time spent sniffing,
Figure 4.4 Increasing anandamide activity via FAAH inhibition corrects social approach impairment in fmr1-/- mice. (a-b) Chamber and sniffing times of wild-type (FVB/NJ) or fmr1-/- mice in the three-chambered social approach assay after treatment with the FAAH inhibitor URB597 (0.3 mg-kg-1, i.p.). (c) Social index, calculated as (social chamber time – object chamber time) / (total side-chamber time). Results are expressed as mean ± SEM. n = 12-14 per group. (a-b) Students unpaired t test, comparing object to social, (c) Two-way ANOVA with Tukey’s post-hoc test. *P < 0.05, **P < 0.01, ***P < 0.001.
78 to levels to those displayed by control mice (Fig. 4.4a-c). These results suggest that the prosocial action of increased anandamide activity (via FAAH inhibition) is generalizable across at least two distinct models of ASD-related social impairment, without affecting socially normal animals. We did not test fmr-/- mice in the elevated plus maze test because these mice display an innate preference for the open arms of the elevated plus-maze27, which might confound data interpretation.
DISCUSSION
For better or worse, desperate parents are treating their autistic children with various forms of marijuana. Successes have been reported in high-profile anecdotes publicized by social media.
This societal experiment highlights the lack of scientific knowledge regarding the therapeutic utility and safety of cannabinoid agents in ASDs. The present study provides an initial test of the idea that enhancing anandamide-mediated endocannabinoid signaling might help to alleviate social impairment in ASD. We show that inhibition of the anandamide-deactivating enzyme
FAAH corrects social impairment in two distinct ASD-related models – BTBR and fmr1-/- mice.
We confirm that FAAH inhibition is an appropriate strategy to elevate levels of anandamide, and thus anandamide signaling, in BTBR mice. Furthermore, we show that the prosocial action of FAAH inhibition is independent of reducing anxiety in BTBR mice. Together, the results put forth FAAH as a novel therapeutic target for ASD-related social impairment.
Separate lines of previous research suggest the general notion that abnormal endocannabinoid signaling might contribute to ASD. First, endocannabinoids play important roles in neurodevelopment, which is also affected by exogenous cannabinoids33. Second, ASD-related alterations in synaptic signaling have been linked to the endocannabinoid system. For example,
79 mutations in neuroligins, a family of ASD-linked synaptic adhesion proteins, impair tonic endocannabinoid signaling34. In a related example, we found that deletion of FMRP, the mRNA-trafficking protein missing in Fragile X Syndrome, impairs the formation of a key signaling complex that links metabotropic glutamate receptor-5 (mGluR5) and the 2-AG- synthesizing enzyme diacylglycerol lipase-α (DGL-α)27. Third, ASD-related insults disturb resting endocannabinoid levels or endocannabinoid system components. For instance, we found that chronic isolation increases 2-AG in the prefrontal cortex, without affecting anandamide, and increases both 2-AG and anandamide in the piriform cortex35. Furthermore, developmental treatment with valproic acid reduces cerebellar mRNA levels of DGL-α and reduces hippocampal mRNA levels of the 2-AG-hydrolyzing enzyme monoacylglycerol lipase (MGL)36.
Importantly, however, these lines of research have not addressed whether deficient endocannabinoid signaling contributes to the core component of ASD – social impairment.
A limited literature has hinted at this possibility indirectly by suggesting a role for endocannabinoid signaling in normal social behaviors. Genetic removal of CB1 receptors alters social interactions in mice in a context-dependent manner32, which may be related to social
37,38 39 anxiety and/or cognition . CB1 agonists impair social play in rats . In contrast, genetic removal of FAAH in mice increases social interactions13, and FAAH inhibition promotes social play in rats14. Thus, the bidirectional modulation of social behavior likely depends on the dose and the identity of the affected circuits. We recently identified a signaling mechanism in male mice by which oxytocin drives anandamide-mediated endocannabinoid signaling to control social reward15. In addition, human studies have found that marijuana may enhance sociability16,17 and a
18 polymorphism in the CB1 cannabinoid receptor gene modulates social gaze .
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Given that endocannabinoid signaling has been linked to ASD and might play a role in normal social behavior, we focused the present investigation on the possible role of endocannabinoid signaling in the social impairment component of ASD. We found that that social impairment is corrected in two distinct mouse models by increasing anandamide activity through FAAH inhibition.
This correction raises the immediate question of whether increasing anandamide activity in these mice is prosocial per se or simply anxiolytic. This question is particularly important in light of the known roles of endocannabinoids in stress modulation19,30,40,41. In our case, we found that the prosocial action of anandamide is unlikely to be due to general anxiolysis, because FAAH inhibition did not alter BTBR performance in the elevated plus-maze when tested in the dim lighting conditions of the social approach test. This result is in line with previous reports showing that FAAH inhibition results in anxiolytic-like effects only under ‘high-light’ conditions42. While this phenotype does not exclude more nuanced forms of social anxiety, it does support the ideathat the modulation of stress reactivity and social behavior can be dissociable. In line with this notion, CB1 overexpression in the medial prefrontal cortex alters social interactions without overtly changing the anxiety-related phenotype43.
In contrast to socially impaired mice, parallel experiments in normal mice indicated that increasing anandamide does not alter social approach. One explanation could be technical – namely, that the social approach test has a ceiling effect or is unable to capture more subtle qualities of social interaction44. Indeed, the task is typically used as a screening tool rather than a continuous-scale measure of sociability. Another explanation could be biological – that signaling systems in a healthy brain are able to compensate for endocannabinoid enhancement in a way that socially impaired brains cannot. These explanations are in line with previous reports of
81 different social situations in which faah-/- mice demonstrated increased direct reciprocal interactions13, as well as URB597-treated juvenile rats engaging in more social play14. Therefore, expanded investigation is warranted into how anandamide contributes to different social contexts and the qualities of social interactions. Nevertheless, our set of results suggests that the prosocial action of FAAH blockade is selective for social impairment in certain contexts, which may be therapeutically advantageous for the spectral nature of ASD.
Based on our results and the available literature, we can reasonably speculate on two possible scenarios improved by anandamide signaling that may underlie social impairment in BTBR and fmr1-/- mice. First, oxytocin-driven anandamide activity in the nucleus accumbens, which we previously demonstrated to be important for social reward15, may be impaired in these mice.
Consistent with this idea, BTBR mice were found to have abnormal oxytocin expression in the hypothalamus45. BTBR mice were also reported to be deficient in conditioned place preference to social interactions46. However, because social conditioned place preference is a relatively new construct, and the learning impairments in these mice make interpretation problematic, further support from the literature is lacking. A second possible scenario is that anandamide might correct an imbalance of excitatory and inhibitory neurotransmission in the cortex, which has been postulated to underlie ASD47. Enhancing GABAergic activity in BTBR mice ameliorates their social impairment, and negative allosteric GABA modulation in C57Bl6J mice recapitulates social impairment48. This suggests that a loss of balance between inhibitory and excitatory activity might contribute to social impairment. A simplified view of this result orients us to interpret our findings as indicating that anandamide could modulate such balance. This view is consistent with the presence of CB1 receptors on presynaptic terminals of both glutamatergic projection neurons and GABAergic interneurons11.
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In conclusion, the present study provides new insights into the role of endocannabinoid signaling in social behavior and validates FAAH as a novel therapeutic target for the social impairment of
ASD.
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Chapter 5
Adolescent cannabinoid exposure persistently impairs social reward
Unpublished preliminary data
87
INTRODUCTION
The use of marijuana, the most widely consumed illicit drug in the world, and synthetic cannabinoid drugs (e.g., Spice, K2) typically starts in adolescence. Its usage pattern is striking.
According to the 2014 NIDA survey, Monitoring the Future, past-month usage of marijuana was at 6.5% among 8th graders, 16.6% among 10th graders, and 21.2% among 12th graders (NIDA,
2014). The percentage of high school seniors who think marijuana is harmful has declined from
52.4% five years ago to only 36.4% today, indicating a possible prelude to increasing usage in the coming years (NIDA, 2014). In addition to recreational use, the accelerating prevalence of cannabinoids as a medicine, e.g. in child epilepsy and autism, is exposing growing numbers of young people to the drug. These usage trends are especially concerning within a rapidly changing social sphere, consisting of technological advancements in marijuana cultivation and cannabinoid synthesis, widespread cultural acceptance, as well as novel politics and economies.
Long-term effects are likely as a consequence of the roles of the endocannabinoid system in the developing brain. This signaling system has three main components: (i) two lipid-derived local messengers – 2-arachidonoyl-sn-glycerol (2-AG) and anandamide (AEA), (ii) enzymes and transporters that mediate their formation and elimination, and (iii) cannabinoid receptors that are activated by endocannabinoids and regulate neuronal activity (Piomelli, 2014; Mechoulam and Parker, 2013). Endocannabinoid activity regulates processes such as neuronal genesis, migration, and differentiation, as well as in synaptic pruning and glial cell formation (Fernández-
Ruiz et al., 2000; Rubino and Parolaro, 2015). With regard to the adolescent period, developmental fluctuations have been reported on the expression levels of CB1 cannabinoid receptors in corticostriatal areas and, to a lesser extent, levels of endocannabinoids. For example,
88 levels of CB1 receptors increased in the cortex and striatum during this period (Schneider et al.,
2015), while 2-AG is reduced in these regions and anandamide continuously increases in the prefrontal cortex (Ellgren et al., 2008).
These lines of evidence each emphasize the vulnerability of the developing brain to consequences of early cannabinoid exposure. However, they also highlight the knowledge gap regarding the molecular mechanisms responsible for these consequences, especially with regard to brain reward systems (Volkow et al., 2014; Parsons and Hurd, 2015; Hurd et al., 2014). In fact, it remains unknown whether disrupted endocannabinoid signaling is responsible for impairment. We hypothesized that non-physiological activation of cannabinoid receptors during adolescence persistently impairs the endocannabinoid regulation of social reward in early adulthood.
RESULTS
We first administered a synthetic cannabinoid receptor agonist, WIN55212-2 (WIN, 1.2 mg- kg-1, intraperitoneal) (Schneider, 2005) for 2 weeks to adolescent mice at postnatal day (PND)
30-43. We chose the WIN compound at this dose because it models the effects of marijuana on cannabinoid receptors over other bioactive constituents. After a two-week washout period, at
PND58, we found that whereas vehicle-treated mice developed a social place preference, WIN- treated mice developed no such preference (Fig. 5.1A-D). This result suggests that improper cannabinoid receptor activation during adolescence impairs the later expression of social reward in adulthood.
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Figure 5.1 | Treatment with the cannabinoid agonist WIN-55212-2 in adolescence impairs social reward in early adulthood. (A-D) Socially conditioned place preference of early-adult mice (PND 58) that had been treated either with vehicle or WIN-55212-2 during adolescence (PND 30-43, 1.2 mg-kg-1, i.p., daily). (E-F) Socially stimulated levels of anandamide (AEA) (for schematic of stimulation, see Fig. 2.1A) of early-adult mice (PND58) that had been previously treated with either vehicle or WIN (same protocol). Data are shown as means ± SEM; *P<0.05, compared using Student’s unpaired t test, n = 14-15. Wei et al., unpublished data.
It is reasonable to expect that socially stimulated anandamide mobilization, which we previously found to be important in social reward (Wei et al., 2015), would also be disrupted by non- physiological activation of cannabinoid receptors in early life. Indeed, we found that after a two- week washout, at PND58, mice previously treated with WIN had increased levels of anandamide in the nucleus accumbens (NAc), medial prefrontal cortex (mPFC), and ventral hippocampus
(vHC) (Fig. 5.1E-G). Thus, socially stimulated anandamide signaling in these regions may no longer occur properly following WIN treatment.
90
DISCUSSION
These preliminary results require additional confirmation. Nevertheless, the results are a proof in principle that early, non-physiological activation of cannabinoid receptors can persistently impair social reward, and that this impairment is due to an underlying disruption in anandamide signaling.
Future investigation should be aimed at: (i) the critical window that leads to persistent impairment; (ii) other persistent molecular changes, such as 2-AG levels and CB1 surface expression; and (iii) whether oxytocin-stimulated anandamide signaling is also impaired. These investigations would provide a more complete picture of the molecular changes responsible for persistent impairment. They would be relevant to the long-term effects of early cannabinoid exposure on mental illness and addiction.
91
REFERENCES
Ellgren, M., Spano, S. M., & Hurd, Y. L. (2007). Adolescent cannabis exposure alters opiate intake and opioid limbic neuronal populations in adult rats. Neuropsychopharmacology, 32(3), 607–615. http://doi.org/10.1038/sj.npp.1301127 Fernández-Ruiz, J., Berrendero, F., Hernández, M. L., & Ramos, J. A. (2000). The endogenous cannabinoid system and brain development. Trends in Neurosciences, 23(1), 14–20. http://doi.org/10.1016/S0166-2236(99)01491-5 Hurd, Y. L., Michaelides, M., Miller, M. L., & Jutras Aswad, D. (2014). Trajectory of adolescent cannabis use on addiction vulnerability. Neuropharmacology, 76 Pt B, 416–424. http://doi.org/10.1016/j.neuropharm.2013.07.028 Mechoulam, R., & Parker, L. A. (2013). The Endocannabinoid System and the Brain. Annual Review of Psychology, 64(1), 21–47. http://doi.org/10.1146/annurev-psych-113011- 143739 NIDA Monitoring the Future Study (2014, December).
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Chapter 6
Endocannabinoid signaling in social protection against pain
Unpublished preliminary data
93
INTRODUCTION
Social interactions and social support protect against physical pain. Such protection exerts powerful effects in adaptive as well as pathological contexts, with profound consequences for pain and pain-related health outcomes (George et al., 1989; Gil et al., 1987). Basic neural mechanisms linking a social signal to an analgesic one, however, are poorly defined. We recently identified a mechanism through which social stimulation mobilizes the endocannabinoid neurotransmitter anandamide, which in turn enhances the saliency of the social interaction (Wei et al., 2015). As endocannabinoid signaling is known for its analgesic effects (Guindon and Hohmann, 2009) independently of social factors, we therefore tested the idea that social stimulation might also recruit the endocannabinoid system as a mediator of analgesia. We targeted a key area for descending pain modulation, the periaqueductal grey (Guidon and Hohmann, 2009).
RESULTS
We used juvenile rats in order to analyze subdivisions of the periaqueductal grey and because of previous investigation of opioids in separation distress (Panksepp et al., 1980). These models, however, have not been extensively replicated. In preliminary experiments, we isolated juvenile rats for 24 h, then re-socialized half while keeping the other half isolated for 3 additional hours.
This social stimulation protocol is consistent with that which elicits an increase in anandamide in the mouse nucleus accumbens, ventral hippocampus, and medial prefrontal cortex (Wei et al.,
2015; unpublished data – Chapter 4 Fig. 4.1F). We found that acute social stimulation in this manner increases levels of anandamide in the ventral periaqueductal grey (PAG), but not the dorsal PAG (Fig. 6.1A-F). In a survey of other regions, we found that the cingulate and insular cortices trended toward an increase, but these results were not significant, and the nucleus
94 accumbens did not show a change in anandamide (data not shown). Paralleling this phenomenon, we used the hot-plate test to assess for acute pain response. Animals were placed inside a large beaker which had been heated to 55 degrees on a hot plate, and their latency to withdrawal or lick their hindpaw was measured. We found that social stimulation increased the threshold for paw withdrawal (Fig. 6.1H). The CB1 cannabinoid receptor antagonist AM251 (1 mg-kg-1, i.p.) prevented such an effect. These results suggest that social stimulation induces anandamide-mediated endocannabinoid signaling (i.e., via CB1 receptors) in order to exert social protection against pain.
DISCUSSION
These preliminary experiments illustrate the concept that socially mobilized endocannabinoid signaling is recruited to buffer against physical pain. Further investigation should be directed toward: (i) establishing the stimulation protocol, as the profile of endocannabinoid changes between mice and rats are clearly different – it is possible that rats may be more sensitive to the rewarding effects of play and thus require a shorter stimulation; (ii) this would be needed to understand whether endocannabinoids modulate an affective component of pain through reward signaling, or actually modulates descending pain at the level of the periaqueductal grey; (iii) whether isolation and social stimulation are qualitatively different or simply opposite effects; (iv) the confounding effects of stress on pain; and (v) whether endocannabinoid signaling elicited here is also oxytocin-stimulated and, if so, the responsible circuitry.
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Figure 6.1 | Endocannabinoid signaling in social protection against pain in juvenile rats. (A-F) Socially stimulated levels of anandamide (AEA), oleoylethanolamide (OEA), and 2-AG in the ventral and dorsal periaqueductal grey (PAG). Juvenile rats were isolated for 24 h and then half were re-socialized for 3 additional hours. (H) Latency on the hot-plate test after the same -1 stimulation, with or without treatment with the CB1 antagonist AM251 (1 mg-kg , i.p., given with socialization). Data are shown as means ± SEM; *P<0.05, compared using Student’s unpaired t test, n = 4-6 per group (A-F), 10-12 per group (H). Wei et al., unpublished data.
96
REFERENCES
George, L. K., Blazer, D. G., Hughes, D. C., & Fowler, N. (1989). Social support and the outcome of major depression. The British Journal of Psychiatry : the Journal of Mental Science, 154(4), 478–485. http://doi.org/10.1192/bjp.154.4.478 Gil, K. M., Keefe, F. J., Crisson, J. E., & Van Dalfsen, P. J. (1987). Social support and pain behavior. Pain. Guindon, J., & Hohmann, A. G. (2009). The Endocannabinoid System and Pain. CNS & Neurological Disorders-Drug Targets (Formerly Current Drug Targets-CNS & Neurological Disorders), 8(6), 403–421. Panksepp, J. (1980). Brief social isolation, pain responsivity, and morphine analgesia in young rats. Psychopharmacology, 72(1), 111-112. Wei, D., Lee, D., Cox, C. D., Karsten, C. A., Peñagarikano, O., Geschwind, D. H., et al. (2015). Endocannabinoid signaling mediates oxytocin-driven social reward. Proceedings of the National Academy of Sciences of the United States of America, 201509795. http://doi.org/10.1073/pnas.1509795112
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Chapter 7
GENERAL DISCUSSION
98
The available literature has implicated the endocannabinoid system in the control of social behavior. Studies include those of marijuana in humans, rodent play and anxiety in a social context, and cannabinoid exposure during development. They raise several questions regarding the signaling dynamics of endocannabinoids, their recruitment, and the nature of the social experiences that are affected. The need to address these questions is highlighted by the current state of knowledge and treatment. Social factors are crucial determinants and social impairments are core features of mental illness, but underlying neural substrates remain largely unknown.
Cannabinoids are being used recreationally, including by adolescents, and medicinally, including for autism spectrum disorder, but scientific understanding of their impact is lacking.
The main findings of my work are: (i) oxytocin neurons in the hypothalamus recruits anandamide-mediated endocannabinoid signaling in the NAc to regulate social reward; (ii) 2-
AG is also involved in social reward, but with three key differences – 2-AG may be generalizable to other natural rewards, its social mobilization is independent from oxytocin, and its temporal profile is distinct from anandamide; (iii) inhibiting FAAH-mediated hydrolysis to promote anandamide signaling may offer a viable therapeutic approach for autism-related social impairment. These findings provide important insights for three broader questions in social neuroscience.
The first question is whether endocannabinoid signaling contributes to social behavior or rather only coincides with general cognitive or emotional systems. In other words, does endocannabinoid signaling simply modulate stress reactivity to allow for social interactions to occur independently, or does it facilitate the social interaction itself? This question subserves the overarching idea that neural systems for social behavior may have evolved from existing systems, such as pain or anxiety, so that social systems are both overlapping and distinct (Panksepp,
99
1997). The existence of an opioid system that affects responses to both social and other rewards indicates social evolution may have co-opted existing reward systems. On the other hand, the characteristics of oxytocin signaling indicates the existence of a distinctly social system. Mice lacking the oxytocin gene show social amnesia, but are normal in other forms of non-social memory (Ferguson et al., 2000). Accordingly, the present discovery that oxytocin neurons in the hypothalamus drives anandamide-mediated endocannabinoid signaling in the nucleus accumbens argues in favor of a distinct, circuit-based role for endocannabinoid signaling in social behavior.
This emphasizes a recruitment mechanism whereby a distinctly social signal in oxytocin exploits existing endocannabinoid signaling as a component of its overall function. In this way, oxytocin- driven anandamide signaling may be patently social. Still, endocannabinoids recruited by other circuits may have overlapping features that are more generalizable. For example, we found 2-AG signaling to not be driven by oxytocin, and generalizable to another natural reward in high-fat diet. 2-AG also has a slower accrual than anandamide does, suggesting that it is more distal to the sensory-stimulating social cues that activate the oxytocin system. Thus, for key themes in addiction research, it may be worthwhile to target 2-AG during the co-facilitating acquisition of social and other rewards, whereas oxytocin-anandamide may be involved in social buffering against addiction. Overall, our findings contribute to the notion that the identity of the endocannabinoids involved, their timing, and the social circuits activated are all determining factors in whether endocannabinoid signaling is involved as a bona fide social brain system. Our findings provide an example of cooperativity between signaling systems that can lead to a distinctly social signal.
This recruitment-cooperativity also raises a second question. If oxytocin drives endocannabinoid signaling, endocannabinoid signaling may carry out functions of oxytocin. These functional
100 hand-offs could vary in as-yet unimagined contexts. Nevertheless, we can speculate upon two possible scenarios, which may or may not be overlapping. The first scenario would be that endocannabinoid acts as a mediator to carry out core oxytocin functions. Research may then be directed at testing the facilitatory role of endocannabinoid signaling in social processes such as maternal separation or social cognition. Indeed, support for such a hypothesis comes from studies suggesting a role for endocannabinoid signaling in maternal separation (Schechter et al., 2012) and cognition (reviewed in Mechoulam and Parker, 2013). These roles have not been linked to oxytocin, however, and experiments attempting to do so may offer new insights. The second scenario would be that oxytocin takes advantage of endocannabinoid-mediated modulatory control to carry out forms of social modulation that result in core cannabinoid effects. This type of modulation may be best understood in therapeutic contexts – perhaps socially induced oxytocin recruits endocannabinoid signaling to protect against addiction, pain, or anxiety. In line with this concept are known endocannabinoid effects in buffering against mental illness
(reviewed in Mechoulam and Parker), such as depression, and the placebo effect (Benedetti et al.,
2011; Peciña et al., 2014). Furthermore, our preliminary results support this concept – socially mobilized endocannabinoid signaling in the periaqueductal grey may buffer against physical pain
(Chapter 5). In sum, the prosocial situations that call for anti-stress and anti-pain – a set of core cannabinoid effects – may use oxytocin-driven endocannabinoid modulation. A first step toward collecting evidence for these two scenarios is determining the anatomical sites where oxytocin recruitment of endocannabinoids occurs. Our results suggest that, even though only anandamide in the nucleus accumbens may be tightly controlled by oxytocin in a social-reward context, more widespread oxytocin-driven endocannabinoid signaling is possible. Investigations grounded in other contexts may prove useful to follow up the widespread actions of oxytocin-induced
101 endocannabinoid changes. A different approach would be to uncover the identities and interactions of molecular players involved in the catalysis of anandamide formation from oxytocin receptor activation. The signaling cascade of the oxytocin receptor, in its only known Gq- coupled form, would putatively be assumed to activate a phospholipase C. In contrast to the canonical phospholipase D-mediated catalysis of N-arachidonoyl-phosphatidylethanolamine
(NAPE) precursors, one might hypothesize an alternative route of anandamide synthesis. There is evidence that this might involve a phosphatase, such as PTPN22 (Liu et al., 2006; 2008). If new synthetic machinery is identified, this approach could help inform the anatomy of where oxytocin drives anandamide.
A third question involves the idea of reward sensitization during development. It bears repeating that social experience during development is crucial to social behavior in the long term, and affects social impairment broadly, including increasing susceptibility to mental illness, such as addiction and autism spectrum disorder. A commonly referred-to concept is that development is crucial for the ‘calibration’ of reward sensitivity (Volkow et al., 2014). Unclear, however, are the mechanisms of this calibration and the role of endocannabinoid signaling. To address this question, we can synthesize our work with the broader literature on social reward. While the present work focuses on social reward in adolescent peers, other forms of social reward include maternal attachment and pair affiliation. Oxytocin’s role in peer reward is only recently coming to light (Dölen et al., 2013), whereas its role in attachment has been established (Insel, 2010; described in General Introduction). Development of neural systems must aid in the transition between these social relationships. Indeed, a recent study on CNTNAP2 mice (contactin- associated protein-like 2), which model cortical dysplasia and focal epilepsy (high penetrance for autism spectrum disorder), found that these mice have an early postnatal decrease in oxytocin
102 neurons and brain oxytocin levels, and that developmental oxytocinergic activation during a critical window led to long-term social recovery (Peñagarikano et al., 2015). We can consider in parallel the endocannabinoid system based on: (i) our work; (ii) the role of anandamide in social play, a behavior which peaks during adolescence and is considered important for social development (Trezza et al., 2008; 2012; described in General Introduction); (iii) the findings that developmental cannabinoid exposure leads to long-term deficits (Schneider et al., 2005;
Rubino and Parolaro, 2008; 2015; described in General Introduction) and, in a logically consistent manner, that enhanced functional CB1 activity prolongs the adolescent behavioral repertoire into adulthood (Schneider et al., 2015; described in General Introduction). It becomes reasonable to postulate that social development could involve mutual interactions between social- endocannabinoid signaling and configuration of the reward system. If so, inappropriate cannabinoid activation during development, whether due to abnormal social experience (as in autism) or exogenous cannabinoid intake (as in teenager marijuana usage), may have long-term consequences for the endocannabinoid system in reward sensitivity. There is support for these postulates. Epidemiological studies have found that adolescents are more vulnerable to marijuana addiction (Lopez-Quintero et al., 2011), early usage associates with other illicit drug liability
(Agrawal et al., 2004), and persistent usage associates with long-term cognitive declines
(Schweinsburg et al., 2008; Meier et al., 2012) and psychosis (Andreasson et al., 1987; Henquet et al., 2005; Arseneault et al., 2004). In support of a causal role for early cannabinoid exposure in these vulnerabilities, animal studies have found that adolescent exposure to synthetic cannabinoid agonists and Δ9-tetrahydrocannabinol increases later sensitivity to opiate drugs (Ellgren et al.,
2007). This effect is thought to be based on a decrease in the expression of striatal D2 receptors, which is predictive of drug-seeking behavior (Hurd et al., 2014). Even in light of these studies,
103 however, the postulate of mutual social-endocannabinoid interactions in reward-system configuration requires additional investigation. Whether it holds true for social behavior similarly to other rewards, and how social endocannabinoid signaling changes reward sensitivity mechanistically, remain open questions. As a first step, our preliminary experiments suggest that early cannabinoid exposure persistently increases anandamide levels, thereby rendering anandamide mobilization insensitive to social stimulation and disrupting the expression of social reward (Chapter 4).
In conclusion, oxytocin-induced endocannabinoid signaling links the social domain with the cannabinoid domain. It offers new insights on the social recruitment of endocannabinoid function (for anandamide), and non-recruitment in other contexts (2-AG). It may provide new perspectives toward understanding the social modulation and treatment of neuropsychiatric conditions, as well as the social development of reward sensitivity.
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