Benjamin J. Ragen

Translational Evidence for a Role of Endocannabinoids in the Etiology and Treatment of Posttraumatic Stress Disorder

INTRODUCTION Posttraumatic stress disorder (PTSD) is a prevalent, chronic, and disabling anxiety disorder that may develop following exposure to a traumatic event. Despite the public health significance of PTSD, relatively little is known about the etiology or pathophysiology of this disorder, and pharmacotherapy development to date has been largely opportunistic instead of mechanism-based. Recently, an accumulating body of evidence has implicated the endocannabinoid system in the etiology of PTSD, and targets within this system are believed to be suitable for treatment development. METHODS Herein, we describe evidence from translational studies arguing for the relevance of the endocannabinoid system in the etiology of PTSD. We also show mechanisms relevant for treatment development. RESULTS There is convincing evidence from multiple studies for reduced endocannabinoid availability in PTSD. Brain imaging studies show molecular adaptations with elevated cannabinoid type 1 (CB1) receptor availability in PTSD which is linked to abnormal threat processing and anxious arousal symptoms. CONCLUSION Of particular relevance is evidence showing reduced levels of the endocannabinoid anandamide and compensatory increase of CB1 receptor availability in PTSD, and an association between increased CB1 receptor availability in the amygdala and abnormal threat processing, as well as increased severity of hyperarousal, but not dysphoric symptomatology, in trauma survivors. Given that hyperarousal symptoms are the key drivers of more disabling aspects of PTSD such as emotional numbing or suicidality, novel, mechanism-based pharmacotherapies that target this particular symptom cluster in patients with PTSD may have utility in mitigating the chronicity and morbidity of the disorder.

Effects of defeat stress on behavioral flexibility in males and females: modulation by the mu-opioid receptor.

Behavioral flexibility is a component of executive functioning that allows individuals to adapt to changing environmental conditions. Independent lines of research indicate that the mu opioid receptor (MOR) is an important mediator of behavioral flexibility and responses to psychosocial stress. The current study bridges these two lines of research and tests the extent to which social defeat and MOR affect behavioral flexibility and whether sex moderates these effects in California mice (Peromyscus californicus). Males and females assigned to social defeat or control conditions were tested in a Barnes maze. In males, defeat impaired behavioral flexibility but not acquisition. Female performance was unaffected by defeat. MOR binding in defeated and control mice in the orbitofrontal cortex (OFC), striatum and hippocampus was examined via autoradiography. Stressed males had reduced MOR binding in the OFC whereas females were unaffected. The MOR antagonist beta‐funaltrexamine (1 mg/kg) impaired performance in males naïve to defeat during the reversal phase but had no effect on females. Finally, we examined the effects of the MOR agonist morphine (2.5 and 5 mg/kg) on stressed mice. As expected, morphine improved behavioral flexibility in stressed males. The stress‐induced deficits in behavioral flexibility in males are consistent with a proactive coping strategy, including previous observations that stressed male California mice exhibit strong social approach and aggression. Our pharmacological data suggest that a down‐regulation of MOR signaling in males may contribute to sex differences in behavioral flexibility following stress. This is discussed in the framework of coping strategies for individuals with mood disorders.

Presence of a pair-mate regulates the behavioral and physiological effects of opioid manipulation in the monogamous titi monkey (Callicebus cupreus)

The role of opioid receptors in infant-mother attachment has been well established. Morphine, a preferential μ opioid receptor (MOR) agonist, attenuates separation distress vocalizations and decreases physical contact between infant and mother. However, there is little research on how opioid receptors are involved in adult attachment. The present study used the monogamous titi monkey (Callicebus cupreus) to explore the role of opioid receptors in the behavioral and physiological components of pair-bonding. In Experiment 1, paired male titi monkeys (N=8) received morphine (0.1, 0.5, or 1.0mg/kg), the opioid antagonist naloxone (1.0mg/kg), vehicle, or a disturbance control and were filmed with their pair-mate for 1h. In Experiment 2, the same eight males received morphine (0.25mg/kg), naloxone (1.0mg/kg), vehicle, or a disturbance control and were filmed for an hour without their pair-mates. All video sessions were scored for social and non-social behaviors. Blood was sampled immediately prior to drug administration and at the end of the hour session. Plasma was assayed for cortisol, oxytocin, and vasopressin. In Experiment 1, opioid manipulation had no effect on affiliative behaviors; however, morphine dose-dependently decreased locomotor behavior and increased scratching. In Experiment 2 in which males were separated from their pair-mates, naloxone increased locomotion. Morphine dose-dependently attenuated the rise in cortisol, while naloxone potentiated the increase of cortisol. The cortisol increase following naloxone administration was greater when a male was alone compared to when the male was with his pair-mate. Naloxone increased vasopressin but only when the male was tested without his pair-mate. The present study found that the absence of a pair-mate magnified naloxones effects on stress-related hormones and behaviors, suggesting that the presence of a pair-mate can act as a social buffer against the stress-inducing effects of naloxone.

Challenges to the pair bond: Neural and hormonal effects of separation and reunion in a monogamous primate

Social monogamy at its most basic is a group structure in which two adults form a unit and share a territory. However, many socially monogamous pairs display attachment relationships known as pair bonds, in which there is a mutual preference for the partner and distress upon separation. The neural and hormonal basis of this response to separation from the adult pair mate is under-studied. In this project, we examined this response in male titi monkeys (Callicebus cupreus), a socially monogamous New World primate. Males underwent a baseline scan, a short separation (48 h), a long separation (approximately 2 weeks), a reunion with the female pair mate and an encounter with a female stranger (with nine males completing all five conditions). Regional cerebral glucose metabolism was measured via positron emission tomography (PET) imaging using [18F]-fluorodeoxyglucose (FDG) co-registered with structural magnetic resonance imaging (MRI), and region of interest (ROI) analysis was carried out. In addition, plasma was collected and assayed for cortisol, oxytocin (OT), vasopressin (AVP), glucose and insulin concentrations. Cerebrospinal fluid (CSF) was collected and assayed for OT and AVP. We used generalized estimating equations (GEE) to examine significant changes from baseline. Short separations were characterized by decreases in FDG uptake, in comparison to baseline, in the lateral septum (LS), ventral pallidum (VP), paraventricular nucleus of the hypothalamus (PVN), periaqueductal gray (PAG), and cerebellum, as well as increases in CSF OT, and plasma cortisol and insulin. Long separations differed from baseline in reduced FDG uptake in the central amygdala (CeA), reduced whole brain FDG uptake, increased CSF OT and increased plasma insulin. The response on encounter with a stranger female depended on whether or not the male had previously reproduced with his pair mate, suggesting that transitions to fatherhood contribute to the neurobiology underlying response to a novel female. Reunion with the partner appeared to stimulate coordinated release of central and peripheral OT. The observed changes suggest the involvement of OT and AVP systems, as well as limbic and striatal areas, during separation and reunion from the pair mate.

The effects of morphine, naloxone, and κ opioid manipulation on endocrine functioning and social behavior in monogamous titi monkeys (Callicebus cupreus).

The μ opioid receptor (MOR) and κ opioid receptor (KOR) have been implicated in pair-bond formation and maintenance in socially monogamous species. Utilizing monogamous titi monkeys (Callicebus cupreus), the present study examined the potential role opioids play in modulating the response to separation, a potent challenge to the pair-bond. In Experiment 1, paired male titi monkeys were separated from their pair-mate for 30-min and then received saline, naloxone (1.0mg/kg), morphine (0.25mg/kg), or the KOR agonist, U50,488 (0.01, 0.03, or 0.1mg/kg) in a counter-balanced fashion, immediately prior to a 30-min reunion with their mate. Blood samples were collected immediately prior to and after the reunion. Males receiving morphine approached females less, initiated contact less, and females broke contact with the males less. The increase in cortisol in response to naloxone was greater compared to vehicle, and the increase in cortisol in response to the high dose of U50,488 compared to vehicle approached significance. In Experiment 2, paired males were treated with the KOR antagonist, GNTI (0.1, 0.3, or 1.0mg/kg), or saline 24h prior to a 60-min separation from their mate. Blood samples were collected at the time of injection and immediately before and after separation. Administration of the low dose of GNTI decreased the locomotor component of the separation response compared to vehicle. The present study found that the opioid system is involved in both the affiliative and separation distress components of a pair-bond, and these components are regulated by different opioid receptors.

Investigational drugs under development for the treatment of PTSD

Introduction: Posttraumatic stress disorder (PTSD) is a prevalent, chronic and disabling anxiety disorder that may develop following exposure to a traumatic event. There is currently no effective pharmacotherapy for PTSD and therefore the discovery of novel, evidence-based treatments is particularly important. This review of potential novel treatments could act as a catalyst for further drug investigation. Areas covered: In this review, the authors discuss the heterogeneity of PTSD and why this provides a challenge for discovering effective treatments for this disorder. By searching for the neurobiological systems that are disrupted in individuals with PTSD and their correlation with different symptoms, the authors propose potential pharmacological treatments that could target these symptoms. They discuss drugs such as nabilone, d-cycloserine, nor-BNI, 7,8-dihydroxyflavone and oxytocin (OT) to target systems such as cannabinoids, glutamate, opioids, brain-derived neurotrophic factor and the OT receptor, respectively. While not conclusive, the authors believe that these brain systems include promising targets for drug discovery. Finally, the authors review animal studies, proof-of-concept studies and case studies that support our proposed treatments. Expert opinion: A mechanism-based approach utilizing techniques such as in vivo neuroimaging will allow for the determination of treatments. Due to the heterogeneity of the PTSD phenotype, focusing on symptomology rather than a categorical diagnosis will allow for more personalized treatment. Furthermore, there appears to be a promise in drugs as cognitive enhancers, the use of drug cocktails and novel compounds that target specific pathways linked to the etiology of PTSD.

Differences in Titi Monkey (Callicebus cupreus) Social Bonds Affect Arousal, Affiliation, and Response to Reward

Titi monkeys (Callicebus cupreus) are a monogamous, New World primate. Adult pair‐mates form a bidirectional social bond and offspring form a selective unidirectional bond to their father. Some of the neurobiology involved in social bonds and maternal behavior is similar to the neural circuitry involved in nonsocial reward. Due to these overlapping mechanisms, social states may affect responses to external rewarding stimuli. We sought to determine whether having a social attachment, and/or being in the presence of that attachment figure, can affect an individuals response to a rewarding stimulus. In addition, we compared affiliative bonds between pair‐mates to those between offspring and fathers. Eighteen adult male titi monkeys were either living alone (Lone), with a female pair‐mate (Paired), or with the natal group (Natal; N = 6/condition). Each individual went through eight 30‐min preference tests for a sweet substance, Tang. For Paired and Natal males, half of the test sessions were with their attachment figure and half were alone. Lone males were always tested alone. Preference scores for Tang, time spent drinking, affiliative, and arousal behaviors were measured. Paired and Natal males emitted significantly more isolation peeps and locomoted more when tested alone compared to when tested with their attachment figure, and paired males engaged in more affiliative behavior than Natal males. Lone males engaged in significantly more behaviors indicative of behavioral arousal such as locomotion and piloerection compared to Paired and Natal males. Finally, Paired males drank significantly more Tang and had a significantly greater preference for Tang compared to Lone and Natal males. These results indicate that offspring undergo a behavioral separation response upon separation from their father that persists into adulthood, Lone males are more behaviorally reactive, and that living with an attachment figure and the type of attachment relationship result in different responses to a rewarding sweet stimulus. Am. J. Primatol. 74:758‐769, 2012.

Oxytocin, Vasopressin and Related Peptides in the Regulation of Behavior: Oxytocin and vasopressin in non-human primates

© Cambridge University Press 2013. Neuroanatomy of OT and AVP in non-human primates OT and AVP immunoreactive cells and fibers in the brain, spinal cord, and pineal gland Atunes and Zimmerman (1978) performed one of the first studies using antisera to AVP, OT, and neurophysins to visualize the OT and AVP system in the hypothalamus of non-human primates. They found that there were OT-immunoreactive cells (-ir) and AVP-ir neurons in the paraventricular nucleus (PVN) and the supraoptic nucleus of the hypothalamus (SON). Additionally, cells containing OT and AVP also contained the neurophysin estrogen-stimulated neurophysin (ESN) and nicotine-stimulated neurophysin (NSN), respectively. These two neurophysins are transporter molecules that are important for the trafficking of OT and AVP molecules after translation (de Bree, 2000). OT-ESN cells and AVP-NSN cells were evenly distributed in the PVN while they were localized within the SON. Subsequent studies examining other non-human primate species found similar results. Currently, OT and AVP neurons have been visualized in three species of macaque (rhesus macaques: Macaca mulatta, crab-eating macaques: Macaca fasciculata, and Japanese macaques: Macaca fuscata)(Atunes and Zimmerman, 1978; Caffe et al., 1989; Ginsberg et al., 1994; Ichimiya et al., 1988; Kawata and Sano, 1982; Sladek and Zimmerman, 1982; Sofroniew et al., 1981), the New World squirrel monkey (Saimiri sciureus) (Sofroniew et al., 1981) and the common marmoset (Callithrix jacchus) (Wang et al., 1997a; Wang et al., 1997b). While there is great similarity between species in the location of OT and AVP cell bodies and fibers, species differences do appear to exist (Table 16.1 details abbreviations; Tables 16.2 and 16.3). It is sometimes difficult to determine whether these species differences are genuine due to the fact that different studies focus on specific brain areas, particular parts of cells, and use different methodologies.

Laboratory simulations of mate‐guarding as a component of the pair‐bond in male titi monkeys, Callicebus cupreus

Mate‐guarding and territorial aggression (both intra‐ and inter‐sexual) are behavioral components of social monogamy seen in male coppery titi monkeys (Callicebus cupreus) both in the field and in the laboratory. Methodology for studying these behaviors in captivity facilitates the translation of questions between field and laboratory. In this study, we tested whether exposure to a mirror would stimulate mate‐guarding behavior in male titi monkeys, and whether this exposure was accompanied by hormonal changes. Eight males were exposed to a mirror condition (treatment) or the back of the mirror (control) for five sessions, and behavioral responses were filmed. Blood samples were taken to measure levels of cortisol, oxytocin, and vasopressin. Lipsmacks (P < 0.0001), arching (P < 0.0001), tail‐lashing (P = 0.009), restraining (P = 0.015), and approaches to the female (P = 0.0002) were all higher during the mirror condition, while tail‐twining tended to decline during the mirror condition (P = 0.076). Hormones did not vary by experimental treatment, but were correlated with certain behaviors during the presentation of the mirror. While social behaviors changed with mirror exposure, self‐directed and mirror‐guided behaviors did not, indicating a lack of self‐recognition. Use of a mirror was a safe and effective means of investigating mate‐guarding behavior in response to a simulated intrusion, with the added benefit of not needing another animal to serve as an intruder; and thus may be of use in providing a laboratory model for natural behavior. Especially, as it eliminates the need for a stimulus animal, it would also be of possible use in investigating responses to a simulated intruder in wild populations of titis and other pithecines. Am. J. Primatol. 78:573–582, 2016.

Serotonin 1A agonism decreases affiliative behavior in pair-bonded titi monkeys

Relatively little is known about serotonergic involvement in pair-bonding despite its putative role in regulating social behavior. Here we sought to determine if pharmacological elevation of serotonin 1A (5-HT1A) receptor activity would lead to changes in social behavior in pair-bonded male titi monkeys (Callicebus cupreus). Adult males in established heterosexual pairs were injected daily with the selective 5-HT1A agonist 8-OH-DPAT or saline for 15days using a within-subjects design. Social behavior with the female pair-mate was quantified, and plasma concentrations of oxytocin, vasopressin, and cortisol were measured. When treated with saline, subjects showed reduced plasma oxytocin concentrations, while 8-OH-DPAT treatment buffered this decrease. Treatment with 8-OH-DPAT also led to decreased plasma cortisol 15minutes post-injection and decreased social behavior directed toward the pair-mate including approaching, initiating contact, lipsmacking, and grooming. The reduction in affiliative behavior seen with increased activity at 5-HT1A receptors indicates a substantial role of serotonin activity in the expression of social behavior. In addition, results indicate that the effects of 5-HT1A agonism on social behavior in adulthood differ between rodents and primates.