Nonhuman primate models of Early Life Stress Relevance

Nonhuman primate models of Early Life Stress: Relevance for Drug Addiction Mar M Sanchez, Ph. D Professor Dept. of Psychiatry & Behavioral Sciences School of Medicine and Core Scientist Yerkes National Primate Research Center Emory University

Early Life Stress is a major risk factor for the development of psychopathology, including anxiety and mood disorders, drug addiction and social deficits. Early ADVERSE experiences: - Parental loss - Trauma (e. g. earthquake) - Poverty (low SES) - Maltreatment: Abuse/neglect BIOLOGICAL MECHANISMS? ? ? Does Early Life Stress lead to repeated/chronic stress activations, which leads to elevated levels of stress hormones (e. g. cortisol), which leads to alterations in brain development ? ? ? , Which leads to… Gx. E Health RISKS: - Depression - Anxiety disorders - Drug addiction - Cardiovascular disease - Diabetes - Obesity

Early Life Stress is a major risk factor for the development of psychopathology, including anxiety and mood disorders, drug addiction and social deficits. Early ADVERSE experiences: - Parental loss - Trauma (e. g. earthquake) - Poverty (low SES) - Maltreatment: Abuse/neglect Health RISKS: - Depression - Anxiety disorders - Drug addiction - Cardiovascular disease - Diabetes - Obesity Biological mechanisms ? ? ? But…we don’t fully understand how these adverse experiences get “under the skin” and unfold during development in humans (e. g. difficult to perform prospective, longitudinal studies). Translational Animal Models (with ethological/construct/predictive validity) are important to understand developmental and biological mechanisms, using prospective/longitudinal designs with high experimental control.

Early Life Stress is a major risk factor for the development of psychopathology, including anxiety and mood disorders, drug addiction and social deficits. Early ADVERSE experiences: - Parental loss - Trauma (e. g. earthquake) - Poverty (low SES) - Maltreatment: Abuse/neglect Health RISKS: - Depression - Anxiety disorders - Drug addiction - Cardiovascular disease - Diabetes - Obesity Biological mechanisms ? ? ? s if d o m C on i t a ic ES N Iadverse experiences get But…we don’t fully understand how these O T E RM Oduring N ENdevelopment in humans “under the skin” and O unfold I IG P S H ATprospective, (e. g. difficult to Sperform longitudinal studies). /E E M R R ST FLAM ULA - IN EC L - MO Translational Animal Models (with ethological/construct/predictive validity) are important to understand developmental and biological mechanisms, using prospective/longitudinal designs with high experimental control.

Childhood Maltreatment • It is a devastating experience. Major public health problem that afflicts an estimated 3 million children annually in the US. • In addition to the obvious risk of physical injury or death, child maltreatment is associated with high risk for psychopathology, including depression and anxiety disorders, substance abuse, social and cognitive deficits (e. g. Heim et al, 2000/2001; Bremmer & Vermetten, 2001; Glaser, 2001; Gunnar & Vazquez, 2005; Sinha, 2008; Hyman et al, 2007 & 2008; Douglas et al, 2010, Enoch, 2011 –review-). • The earlier the age at onset of maltreatment, the more severe the adverse outcomes (e. g. , maltreatment starting in infancy or preschool years is associated with increased risk for anxiety and mood disorders -Kaplow and Widom, 2007 -). Critical periods.

Childhood Maltreatment • It is a devastating experience in humans. Major public health problem that afflicts an estimated 1 million children annually in the US. • In addition to the obvious risk of physical injury or death, child maltreatment is associated with increased risk for psychopathology, including depression and anxiety disorders, substance abuse, social and cognitive deficits (e. g. Heim et al, 2000/2001; Bremmer & Vermetten, 2001; Glaser, 2001; Gunnar & Vazquez, 2005; Sinha, 2008; Hyman et al, 2007 & 2008; Douglas et al, 2010, Enoch, 2011 –review-). . • The earlier the age at onset of maltreatment, the more severe the adverse outcomes (e. g. , maltreatment starting in infancy or preschool years is associated with increased risk for anxiety and mood disorders -Kaplow and Widom, 2007 -). • Human studies face many limitations (limited prospective studies, co-morbidities, heterogeneity) • Childhood maltreatment has also been reported in nonhuman primates, both in captivity and in the wild (Brent et al, 2002; Johnson et al. , 1996; Maestripieri, 1998 & 1999; Troisi et al. , 1982), with a 2 -5% prevalence. This provides an incredible research opportunity to use a NATURALISTIC animal model of infant maltreatment to perform prospective, longitudinal studies since birth with strong experimental control, examine how the brain and behavioral alterations unfold during development, and the mechanisms involved, which is very difficult in human studies.

Rhesus monkey (a. k. a. Macaca mulatta) • Highly social species with strong mother-infant bonds • Primate species used to study the developmental effects of early social environment, due to: - social and biological similarities with humans - well-developed brain (e. g. prefrontal cortex) - resilient to disease - adaptability to captivity • Important nonhuman primate model for substance abuse: drug self-administration paradigms. Translational value (Attachment Theory –Bowlby-, Harlow studies) Rule of thumb: multiply Rhesus age x 4 to compare with Human age

Yerkes National Primate Research Center Field Station (Lawrenceville, GA) * Complex social environment (outdoor enclosures with indoor access) * Social groups composed of 2 -3 adult males, and 20 -50 females with their sub-adult and juvenile offspring (50 -175 animals/group) Experimental setting: complex social groups to provide species-typical social experiences

Infant maltreatment in rhesus monkeys • Physical Abuse: violent behaviors exhibited by mom (infant dragging, throwing, crushing, sitting or stepping on) that cause pain and distress in infants. • These are atypical behaviors (2 -5% prevalence), never exhibited by control mothers • Exhibited during the first 2 -3 months of life (Early Critical period; first year in humans). • Abuse is repeated with successive infants (maternal trait). • Intergenerational transmission along the maternal line (abused females often perpetuate abuse with their own infants). • Severe cases of physical abuse are not studied. Abuse results in infant distress and superficial scratches/bruises. • We don’t study primiparous (first-time) mothers due to high incidence of neglect/abuse • Physical Abuse is co-morbid with high Maternal Rejection Translational value

Rates of Physical Abuse & Rejection received by infants Abuse Rejection Kai Mc. Cormack (Ph. D. work) control Rejections/hour maltreatment month 1 month 2 Infant’s age Mc. Cormack, Sanchez et al (2006), Developmental Psychobiology [Replicated in several studies (including ongoing studies using crossfostering & random assignment design, and bigger sample size)]: Maternal trait. month 3

Infant maltreatment is a stressful experience… blood Maltreated infants had higher BASAL cortisol: • at month 1 (when physical abuse rates are the highest) • from birth to 6 months of age (hair cortisol accumulation) Month 1 Stalder & Kirschbaum 2012 Cortisol in hair (signature of CHRONIC STRESS)

Behavioral outcomes (INFANT period): Increased Emotional Reactivity higher rates of SCREAMS more TANTRUMS Mc. Cormack, Sanchez et al, (2006), Dev Psychobiol Elevated ANXIETY Summary: - behavioral signs of Distress - elevated Anxiety - delayed Independence from mom - less Social Play - increased Behavioral Inhibition (during Free Play –Lab. TAB) Mc. Cormack et al & Sanchez (2009), Hormones & Behavior

Behavioral outcomes (JUVENILE period & ADOLESCENCE) • Increased emotional reactivity (persistent) - Higher levels of fear (freezing) and anxiety (self-directed behaviors)… - …and more impulsivity (impulsive aggression, impaired threat responses) Translational value: Measures of internalizing & externalizing behaviors Howell et al & Sanchez, Dev. Psychobiology (2014): APPROACH-AVOIDANCE TASK • Altered Social behavior: - Higher social aggression (threat bias measures) - less affiliative and play behaviors:

Behavioral outcomes (JUVENILE period & ADOLESCENCE) • Increased emotional reactivity (persistent) - Higher levels of fear (freezing) and anxiety (self-directed behaviors)… - …and more impulsivity (impulsive aggression, impaired threat responses) Translational value: Measures of internalizing & externalizing behaviors Howell et al & Sanchez, Dev. Psychobiology (2014): APPROACH-AVOIDANCE TASK • Altered Social behavior: - Higher social aggression (threat bias) - less affiliative and play behaviors:

Neurobiological alterations: Brain serotonin (5 HT) & emotional/stress reactivity • The brain 5 -HT system plays an important role in emotional regulation and the control of impulsive and aggressive behaviors. • Due to high density of 5 -HT innervation found in limbic structures: üamygdala, BNST, prefrontal cortex, hippocampus, hypothalamus, striatum Macaques & squirrel monkeys [Friedman & Shi, 2001; Bauman & Amaral, 2006; Sadikot & Parent, 1990]

Reduced CSF levels of 5 -HT and 5 -HIAA in Maltreated Monkeys (juvenile & adolescent periods) In primates cerebrospinal fluid (CSF) levels of 5 -HT and its metabolite 5 -HIAA are used as indices of serotonergic production and turnover, respectively. Low 5 -HT and 5 -HIAA CSF levels in monkeys exposed to HIGH maternal rejection rates ** 80 LOW rejection HIGH rejection 70 ng/ml CSF 60 50 40 5 * 0 5 HIAA 5 HT Sanchez et al, 2007, Mol Psychiatry * p=0. 026 (5 -HT; t-test) ** p=0. 001 (5 -HIAA; t-test) …and the low levels were associated more strongly with high levels of maternal rejection experienced in their first 6 months of life, than with physical abuse.

Neurodevelopmental alterations: Prefrontal-Amygdala & Prefrontal-Nucleus Accumbens circuits MRI analysis: developed age-specific rhesus infant brain Atlases & longitudinal tools (in collaboration with Martin Styner-UNC) 2 wk Shi et al, 2017. Frontiers in Neuroscience 3 mo 6 mo

Early Life Stress weakens Prefrontal cortex (PFC) Functional Connectivity with ventral striatum (Nucleus Accumbens) & amygdala: Increased risk for drug abuse FC FC Maltreatment also reduces PFC functional connectivity (FC) with N. Accumbens during ADOLESCENCE

. . . leading to NIDA R 01 -Adolescence grant (Sanchez/Howell): Title: “Early life stress and adolescence cocaine abuse: neurobiological vulnerabilities” Goal: To use a translational macaque model of Early Life Stress (infant maltreatment) to examine neurobiological mechanisms underlying increased vulnerability to cocaine abuse and relapse during adolescence. Focus on Prefrontal cortex connectivity and 5 HT systems controlling DA (PET). - Follows the same population of macaques longitudinally through Adolescence based on a Crossfostering experimental design with random asignment of infants at birth to experimental groups (MALT, CONTROL) to disentangle the effect of experience from heritable factors. Study Age (months): 0 Infant Maltreatment Model: Cross-fostering Design abuse + rejection infancy 0. 5 3 Juvenile period 6 R 01 (NIH/NIDA DA 038588 -01). MPIs: Sanchez/Howell 12 18 ADOLESCENCE (NIDA grant)

Experimental Design (Adolescence): Title: Early life stress and adolescence cocaine abuse: neurobiological vulnerabilities. Principal Investigator: M. Sanchez, L. Howell (MPI) Agency/Source: National Institute of Drug Abuse (NIH/NIDA) Type: R 01 (DA 038588 -01) • • Emotional Regulation (fear-conditioning: fear-potentiated startle paradigm (AX+/BX-) Stress physiology (HPA axis function: baseline, stress-reactivity, negative feedback) Neuroimaging: MRI (structural & functional connectivity), PET (DA, 5 HT receptors) Cocaine i. v. Self-Administration; Phases: – Acquisition – Maintenance – Limited and Extended Access – Extinction – Reinstatement

. . . leading to NIDA R 01 -Adolescence grant (Sanchez/Howell): Title: “Early life stress and adolescence cocaine abuse: neurobiological vulnerabilities”. Goal: To use a translational macaque model of Early Life Stress (infant maltreatment) to examine neurobiological mechanisms underlying increased vulnerability to cocaine abuse and relapse during adolescence. Focus on Prefrontal cortex connectivity and 5 HT systems controlling DA (PET). - Follows the same population of macaques longitudinally through Adolescence based on a Crossfostering experimental design with random asignment of infants at birth to experimental groups (MALT, CONTROL) to disentangle the effect of experience from heritable factors. Study Age (months): 0 Infant Maltreatment Model: Cross-fostering Design abuse + rejection infancy 0. 5 3 Juvenile period 6 12 18 AIM 3: Test pharmacological intervention with a selective 5 HT 2 C receptor agonist (WAY 163909) to reduce cocaine intake (by decreasing DA release in NAcc, which decreases reinforcing effects of psychostimulants): Alison Wakeford, Ph. D (postdoctoral work) R 01 (NIH/NIDA DA 038588 -01). MPIs: Sanchez/Howell ADOLESCENCE (NIDA grant)

Serotonergic Modulation via 5 -HT 2 C activation Dopamine in NAc Reinforcing effects of psychostimulants Mesolimbic DA pathway *Diagram from Howell & Cunningham, 2015: Pharmacol Rev. 67(1): 176 -197

Conclusions • Naturalistic rhesus monkey model of Early Life Stress (infant maltreatment): - physical abuse, high infant rejection (& low maternal sensitivity/responsiveness) - poor maternal care early in life is stressful for the infants - intense during the first 3 -6 months of life: critical period for infant development. • Developmental outcomes: - Increased emotional reactivity (fear/anxiety) and aggression; reduced affiliation - Elevated cortisol during infancy & juvenile period - Neurobiological alterations: + Reduced brain serotonin/DA function (associated with increased emotional reactivity, impulsivity, perpetuation of abuse and inflammation). + Alterations in development of prefrontal-amygdala-ventral striatal circuits: [weaker PFC-Amygdala & PFC-nucleus accumbens functional connectivity] - Increased cocaine intake and sensitivity to reinforcing properties of cocaine. - Cognitive deficits (Prefrontal-Hippocampal dependent): “quitting” & poor executive function SUMMARY: - Maternal care early in life is critical for proper brain and behavioral development. - Poor maternal care is a stressful experience that results in increased emotional reactivity and brain alterations contributing to risk for behavioral disorders, including addiction.

ACKNOWLEDGEMENTS: SANCHEZ LAB (Dept. Psychiatry & Yerkes Primate Ctr. , Emory Univ. ): Kai Mc. Cormack (Ph. D & post-doc work; now faculty, Spelman College) Brittany Howell (Ph. D & post-doc work; now post-doc Univ Minnesota) Elyse Morin (Ph. D work) Alison Wakeford (post-doc) Sara Bramlett (Ph. D work) Desiree Sharpe, Tori Vratanina, Alison Grand (Ph. D work) Kathy Reding, Jodi Godfrey, Melanie Pincus, Desiree De. Leon (Ph. D work) Zsofia Kovacs-Balint (postdoc) Kaela Kuitchuoa, Jacquie Steele, Maggie Kyle, Caroline Fu, Divya Padmanabhan, Felicia Lombardi (undergrads) Erin Siebert, Anne Glenn, Christine Marsteller, Jennifer Miles, Matt Boudreau (Research Specialists, Lead & Senior) COLLABORATORS: Mark Wilson (Yerkes National Primate Research Center): Social subordination model Dario Maestripieri (Univ Chicago) & Kai Mc. Cormack (Spelman College, Atlanta): Maltreatment model Jocelyne Bachevalier, Lisa Parr (Yerkes National Primate Center) Leonard Howell, John Nye, Andy Kazama, Rob Hampton (YNPRC) Andrew Miller (Dept. Psychiatry, Emory Univ): inflammation studies Xiaoping Hu, Longchuan Li, Xiaodong Zhang (Emory Univ. ): MRI/DTI Martin Styner (UNC), Damien Fair (OHSU), Clare Kelly/Xavier Castellanos (NYU): infant rhesus atlases & rs-f. MRI Stacy Drury (Tulane. Univ): Telomere length Early Experience, Stress and Neurobehavioral Development Center (NIMH P 50 MH 078105) Gunnar (PI), Fisher, Fox, Suomi, Pollak, Dozier, Sullivan, Tottenham, Plotsky, Dallman, (Levine). Emory Autism Center of Excellence (P 50 MH 100029; PI: Ami Klin) Klin (PI), Bachevalier, Parr, Li, Hu, Payne, Feczko, Jones, Ramsay. Styner (UNC), Fair (OHSU). FUNDING: Maltreatment model: NIMH (P 50 MH 078105 & P 50 MH 078105 -01 A 2 S 1; MH 650046), NICHD (R 21 HD 055255); NIDA (R 01 DA 038588) Social Subordination model: NIMH (MH 079100) & NICHD (“Stress & Obesity” R 01: HD 077623) Emory Autism Center of Excellence (P 50 MH 100029; PI: Ami Klin) Conte Center for Oxytocin and Social Cognition (P 50 MH 100023; PI: Larry Young) NARSAD, NSF (CBN venture grant) & KTGF Yerkes Primate Center Base Grant (RR-00165)