archives@tulane.edu / Background: Individuals with human immunodeficiency virus (HIV) and undergoing antiretroviral therapy (ART) exhibit high levels of circulating inflammatory cytokines and proteins, which are strongly correlated with shortened time to death and disease. To target damaging inflammation at the source, the drivers of inflammation must be identified. Adipose tissue is a massive organ that contains adipocytes and immune cells capable of producing pro-inflammatory mediators. Dysregulated adipose tissue is implicated in the pathogenesis of obesity and related diseases, such as type 2 diabetes, that are likewise reported in persons with chronic HIV infection. Adipose tissue was therefore explored as a contributor to circulating inflammation in patients with HIV using the rhesus macaque model. Simian immunodeficiency virus (SIV) closely models HIV regarding pathogenesis, including CD4+ T cell depletion, induction of a viral reservoir, and development of opportunistic infections before succumbing to Acquired Immunodeficiency Syndrome (AIDS) and death. Methods: Subcutaneous adipose tissue (SQAT) from SIV-infected rhesus macaques was characterized using confocal microscopy to describe the major immune cell subsets. Adipose tissue homogenates and plasma were analyzed for expression of genes and proteins related to inflammatory processes using antibody and RNA-based fluorescent multiplex bead technology for protein and gene quantitation, respectively. The functions of adipose tissue immune cells during SIV infection were measured with stimulation and phagocytosis assays. / 1 / Marissa Fahlberg

This thesis presents the development and application of methods to assess cognitive markers of emotion and psychological wellbeing in a species of nonhuman primate, the rhesus macaque (Macaca mulatta). In humans, vulnerability to emotional disorders such as anxiety and depression is characterized by particular cognitive profiles, known as cognitive biases. For example, anxious people automatically attend to threat-relevant information, interpret ambiguous information negatively, and have negative expectations of future events. In this thesis, I first describe two treatments that were used prior to cognitive testing to induce positive and negative shifts in inferred affective state in the monkeys (enrichment and a health-check, respectively) and discuss the impact of these treatments on the monkeys’ behaviour and physiology (Chapters 2 and 3). In the first cognitive study (Chapter 4), I present a method that uses eye-gaze to assess the extent to which threatening (versus non-threatening) stimuli capture visual spatial attention when two stimuli are presented at different locations. In the second study (Chapter 5), I present a simple operant touch-screen task to assess the extent to which a threatening distractor stimulus captures attention and impairs performance on an ongoing task when presented at the same location as the taskrelevant stimulus. In the third study (Chapter 6), I present a Go/NoGo touchscreen task to assess judgements about the reward value of ambiguous stimuli. In all of these studies, the two treatments led to different cognitive profiles in the monkeys. Monkeys showed a) automatic capture of attention by threatening stimuli, which was followed by avoidance following the health-check, but not Post-enrichment; b) impaired task performance when a threatening distractor stimulus was presented Post-health-check, and improved performance on these trials Post-enrichment; and c) a more negative judgement about the reward value of ambiguous stimuli Post-health-check versus Post-enrichment. I discuss these cognitive biases in light of available data from humans, and recent work with nonhuman animals. These data indicate that furthering our understanding of primate and other animal psychological wellbeing, may be achieved through the development of measures of cognitive bias, such as those presented here.

Typically, rhesus mothers begin rejecting their infants' attempts to nurse when the infants are approximately three months of age in order to begin the process of weaning. A small subgroup of mothers begin rejecting their infants earlier, at one or two months of age, typically before infants seek and maintain independence from their mother. The effects of this early maternal rejection on the development of infants and some potential factors that contribute to premature maternal rejection were explored in this study. Infants who were rejected early were hypothesized to subsequently spend less time in positive contact with their mother, have lower activity levels, were groomed less by their mother and, as a consequence of the maternal rejections, display a higher frequency of aggression toward other group members when compared to infants experiencing maternal rejection after the age-typical, three months of age. Mothers who were primiparous and/or had a poor early-rearing experience were hypothesized to be more likely to reject their infants prematurely. Consistent with these hypotheses, infants who were rejected early spent less time on their mother's ventrum and were groomed less by their mother, suggesting that early maternal rejection may lead to less positive mother-infant interactions and a more distant mother-infant relationship. Infants rejected early were also more likely engage in aggression. Given the punitive nature of the maternal rejection, the results suggest that aggression is transmitted from mother to infant through their interactions. Prematurely rejected infants were found to spend significantly more time in a passive, withdrawn behavioral state. When assessing the causes of premature rejections, primiparous mothers were not more likely to prematurely reject their infants, indicating that premature rejection was not simply a lack of experience with an infant. There was evidence that the mothers engaging in early rejection had poor early-rearing experiences, with surrogate-peer-reared mothers showing more early rejections than those who were reared by an adult female, and with mothers who were peer-reared having higher rates of rejection overall. The present results suggest that early rejection is associated with more difficult mother-infant relationships and may lead to increased likelihood of aggression in infants.

<p>Making decisions is fundamental to everything we do, yet it can be impaired in various disorders and conditions. While research into the neural basis of decision-making has flourished in recent years, many questions remain about how decisions are instantiated in the brain. Here we explored how primates make abstract decisions and decisions in social contexts, as well as one way to non-invasively modulate the brain circuits underlying decision-making. We used rhesus macaques as our model organism. First we probed numerical decision-making, a form of abstract decision-making. We demonstrated that monkeys are able to compare discrete ratios, choosing an array with a greater ratio of positive to negative stimuli, even when this array does not have a greater absolute number of positive stimuli. Monkeys’ performance in this task adhered to Weber’s law, indicating that monkeys—like humans—treat proportions as analog magnitudes. Next we showed that monkeys’ ordinal decisions are influenced by spatial associations; when trained to select the fourth stimulus from the bottom in a vertical array, they subsequently selected the fourth stimulus from the left—and not from the right—in a horizontal array. In other words, they begin enumerating from one side of space and not the other, mirroring the human tendency to associate numbers with space. These and other studies confirmed that monkeys’ numerical decision-making follows similar patterns to that of humans, making them a good model for investigations of the neurobiological basis of numerical decision-making. </p><p>We sought to develop a system for exploring the neuronal basis of the cognitive and behavioral effects observed following transcranial magnetic stimulation, a relatively new, non-invasive method of brain stimulation that may be used to treat clinical disorders. We completed a set of pilot studies applying offline low-frequency repetitive transcranial magnetic stimulation to the macaque posterior parietal cortex, which has been implicated in numerical processing, while subjects performed a numerical comparison and control color comparison task, and while electrophysiological activity was recorded from the stimulated region of cortex. We found tentative evidence in one paradigm that stimulation did selectively impair performance in the number task, causally implicating the posterior parietal cortex in numerical decisions. In another paradigm, however, we manipulated the subject’s reaching behavior but not her number or color comparison performance. We also found that stimulation produced variable changes in neuronal firing and local field potentials. Together these findings lay the groundwork for detailed investigations into how different parameters of transcranial magnetic stimulation can interact with cortical architecture to produce various cognitive and behavioral changes.</p><p>Finally, we explored how monkeys decide how to behave in competitive social interactions. In a zero-sum computer game in which two monkeys played as a shooter or a goalie during a hockey-like “penalty shot” scenario, we found that shooters developed complex movement trajectories so as to conceal their intentions from the goalies. Additionally, we found that neurons in the dorsolateral and dorsomedial prefrontal cortex played a role in generating this “deceptive” behavior. We conclude that these regions of prefrontal cortex form part of a circuit that guides decisions to make an individual less predictable to an opponent.</p> / Dissertation

BACKGROUND: Natural killer (NK) cells are traditionally considered part of the innate immune system but have recently been shown to possess adaptive qualities similar to T cells in response to an infection with a pathogen. In addition to possessing adaptive features, NK cells have also been found to reside in different organs such as the liver, spleen, and lymph nodes and differ based on phenotypic markers and their responses to different cytokines. Utilizing these findings, several groups have isolated and identified CD49a as a marker for tissue-resident NK cells. In the liver, CD49a has also been shown to be a positive indicator for NK cell memory-like responses in murine models. Building off work that demonstrated antigen-specific responses in rhesus macaques, this project focuses on characterizing the phenotypic markers and functional profile of CD49a+ NK cells in non-human primates. To better understand the role of CD49a in memory-like NK cells outside of the liver, this project utilized spleen samples from rhesus macaques infected with SIV/SHIV. This work aims to help us better understand the dysfunction NK cells experience as a result of HIV-1 infection in humans and also to demonstrate the changes NK cells experience as the disease progresses. A thorough understanding of the adaptive capabilities of NK cells can pave the way for targeted therapies to increase NK cell antiviral activity in HIV and other infections. METHODS: To characterize the functional and phenotypic profiles of CD49a+ NK cells by multiparameter flow cytometry, thirteen samples of spleen from rhesus macaques were thawed and then stained with two different protocols. A phenotyping protocol involved staining with antibodies against surface markers as well as intracellular markers T-Bet and Eomes. For the functional characterization protocol, the same thirteen samples were stained intracellularly after being stimulated with a cocktail of PMA and ionomyocin. The antibodies used in this were for functional markers. Of the thirteen samples used, six were infected with SHIVSF162P3, two were infected with SIVmac239X, and the remaining five were uninfected. After staining, these samples were analyzed on an BD LSRII from BD Biosciences. The data obtained were then analyzed using FlowJo software to study NK cells, which were characterized as CD45+CD14-CD20-CD3-CD159+. RESULTS: The analysis compared NK cells with T cells, B cells, and NKB cells. Some increases were seen among CD49a+ NK cells in the frequency of CD336+ (NCR2/NKp44), CD337+ (NKp30), and CD366+ (Tim-3) after infection. Although there were some mild increases in CD107a and TNF- in infected samples compared to uninfected, a significant increase was observed in the frequency of IFN-ɣ among infected CD49a+ NK cells compared to uninfected. CONCLUSION: When comparing samples that were infected vs uninfected, it appears there were some mild decreases after infection in the ratio of NK cells to other lymphocytes. In addition, there did not appear to be a significant increase in the frequency of CD49a+ among these NK cells as a result of the infection. However, among the CD49a+ subpopulation, there were some observed non-significant decreases in CD56-CD16+ cells. Furthermore, there was found to be an almost significant increase in TNF- (p = 0.06) among CD49a+ cells after infection. These findings demonstrate an increase in cytotoxic activity in splenic NK cells associated with an adaptation to the virus. Although there does not appear to be significant changes in the ratio of NK cell populations in the spleen, the changes observed in phenotypic and functional markers associated with CD49a+ demonstrate an increase in the cytotoxic activity of NK cells as a result of infection with SIV/SHIV. However, it remains to be seen if CD49a is a direct indicator of this type of infection. Future work geared toward memory-like NK cells in non-human primate splenic tissue could look at the contrast in CD49a+ NK cells from different states of infection with HIV-1 and/or SIV (acute vs chronic) to better understand the integrin’s role in adaptation.

Studies suggest that adoptees are at risk for a number of psychopathological behaviors. To understand the etiology of this risk, 150 socially housed rhesus macaques were studied, including 107 infants reared with their biological mothers and 43 infants reared with unrelated adoptive mothers. Mother-infant behaviors were recorded across the first 6 months of life. Analyses were performed using a hierarchical linear mixed model. All reported results were tested at p<0.05. Adopted infants were observed on average to approach and leave their mothers more frequently, explore the environment and locomote longer, exhibit more anxiety-like behavior, spend less time being held to their mother's breast, and were rejected by their mothers more when compared to nonadopted infants, indicating they are more likely responsible for maintaining the relationship. They also direct and receive more noncontact aggression on average to other social group members, and showed evidence of higher anxiety exhibiting high levels of anxiety-like self-directed behavior when compared to nonadopted infants. Also, results indicate that adopted infants have significantly lower levels of the CSF serotonin metabolites 5-hydroxyindoleacetic acid when compared to nonadopted infants.

Natal dispersal may have considerable social, ecological and evolutionary consequences. While speciesspecific dispersal strategies have received much attention, individual variation in dispersal decisions and its fitness consequences remain poorly understood. We investigated causes and consequences of natal dispersal age in rhesus macaques (Macaca mulatta), a species with male dispersal. Using long-term demographic and genetic data from a semi-free ranging population on Cayo Santiago, Puerto Rico, we analysed how the social environment such as maternal family, group and population characteristics affected the age at which males leave their natal group. While natal dispersal age was unrelated to most measures of group or population structure, our study confirmed earlier findings that sons of high-ranking mothers dispersed later than sons of low-ranking ones. Natal dispersal age did not affect males\\\' subsequent survival, but males dispersing later were more likely to reproduce. Late dispersers were likely to start reproducing while still residing in their natal group, frequently produced extra-group offspring before natal dispersal and subsequently dispersed to the group in which they had fathered offspring more likely than expected. Hence, the timing of natal dispersal was affected by maternal rank and influenced male reproduction, which, in turn affected which group males dispersed to.

Papio ursinus are dietary generalists that exploit a diverse repertoire of food resources. This study explored the foraging strategies of a group of Papio ursinus and the foraging differences between subgroups classified by age and sex. Food resource preference, nutritional properties of food resources, and nutritional demands were examined to test the hypotheses that the foraging strategies exhibited by the subgroups would differ and that food selection is driven by nutritional demands. Adult females and juveniles were found to seek out food resources higher in proteins, while adult males were found to prefer food resources higher in carbohydrates. The findings support the alternative hypotheses and suggest that nutritional pressures are the best predictor of foraging optimization. A pilot study, involving the observation of a provisioned group of Macaca mulatta, that proved elemental to the formulation of the research design employed in the study of Papio ursinus, is also described.

Self-injurious behavior (SIB) is a complex phenotype that occurs with an increasing prevalence of about 7-34% in humans and 10-12% in non-human primates (NHPs). This study evaluated the efficacy of probiotic Lactobacillus reuteri as a treatment for self-injurious behavior (SIB) and sleep disruption in rhesus macaques. The treatment was proposed to alleviate mild self-biting, sleep disruption, and reduce chronically elevated hypothalamic-pituitary-adrenocortical (HPA) axis activity, all hallmark features of monkeys with this condition. The probiotic preparation included two strains of L. reuteri (L. reuteri ATCC PTA 6475 & L. reuteri DSM 17938) containing on average 200 million colony forming units per chewable tablet. The study was conducted on 14 rhesus macaque monkeys (9 males) housed at the University of Massachusetts Amherst. To our knowledge this is the first time that a Lactobacillus strain has been used as a treatment for SIB in rhesus macaques. This study utilizes motion-activated infrared camera technology, modified enzyme-immunosorbent-assays (EIAs) techniques to measure hair cortisol concentrations, and daily behavioral observations to provide an overall assessment of the behavioral, physiological, and sleep associated implications of probiotic treatment on SIB and control non-human primates (NHPs). Administration of L reuteri modestly decreased biting behavior in monkeys with SIB (F(2,12) = 5.64, p= 0.02) and showed overall decrease in nighttime activity across all subjects but did not normalize SIB to nonSIB values. Hair cortisol values are pending. These findings and the findings of previous work further strengthen the argument for probiotics as an efficacious treatment for SIB behavior.

Operant conditioning using positive reinforcement techniques has been used extensively in the management of nonhuman primates in both zoological and laboratory settings. Based on a large body of previous research that demonstrates the utility of such techniques in reducing stress, abnormal behavior, and aggression, this research project was intended to develop and test the usefulness of habituation and counter-conditioning techniques in reducing the fear-responses of singly-housed male rhesus macaques living in the laboratory environment. Additionally, we investigated the variable of temperament as it relates to the reduction of fear-responsivity and overall training success. Based on a Wilcoxon Matched-Pairs Sign Test, we found that animals exposed to desensitization training were significantly likely to show a reduction in the rate at which they engaged in cringing toward humans (exact significance = .016, one-tailed, N ties = 6), cringing in general (exact significance = .016, one-tailed, N ties = 6), and in stress-related behaviors (exact significance = .016, one-tailed, N ties = 6). Animals exposed to basic husbandry training or exposed to no training at all were not significantly likely to show a reduction in the rates of these behaviors. When these same behaviors were analyzed in terms of duration of behavior, desensitization-exposed animals were significantly likely to show reduction in the amount of time spent cringing toward humans (exact significance = .016, one-tailed, N ties = 6), but not in cringing behaviors in general or in stress-related behaviors. Neither the husbandry-exposed group nor the group exposed to no training showed a significant number of subjects exhibiting a reduction in duration of any of these behaviors. Additionally, initial temperament assessments were found to significantly predict the relative ability of subjects exposed to training to acquire trained behaviors such that animals generally ranked as more inhibited in terms of temperament also ranked as slower learners based on a Wilcoxon Matched-Pairs Signed-Ranks test, z = -.316, p = .752 (two-tailed). Results of this study could enhance both laboratory animal welfare and laboratory animal research, and could be a first step in developing techniques for reducing fearful behavior in rhesus monkeys in the laboratory environment.