Neuronal vulnerability to brain aging and neurodegeneration in cognitively impaired marmoset monkeys (Callithrix jacchus).

The investigation of neurobiological and neuropathological changes that affect synaptic integrity and function with aging is key to understanding why the aging brain is vulnerable to Alzheimer's disease. We investigated the cellular characteristics in the cerebral cortex of behaviorally characterized marmosets, based on their trajectories of cognitive learning as they transitioned to old age. We found increased astrogliosis, increased phagocytic activity of microglial cells and differences in resting and reactive microglial cell phenotypes in cognitively impaired compared to nonimpaired marmosets. Differences in amyloid beta deposition were not related to cognitive trajectory. However, we found age-related changes in density and morphology of dendritic spines in pyramidal neurons of layer 3 in the dorsolateral prefrontal cortex and the CA1 field of the hippocampus between cohorts. Overall, our data suggest that an accelerated aging process, accompanied by neurodegeneration, that takes place in cognitively impaired aged marmosets and affects the plasticity of dendritic spines in cortical areas involved in cognition and points to mechanisms of neuronal vulnerability to aging.

Letrozole treatment alters hippocampal gene expression in common marmosets (Callithrix jacchus).

Aromatase inhibitors (AIs) are a class of drugs commonly given to patients with estrogen receptor (ER)-dependent breast cancers to reduce estrogenic stimulation. However, AIs like Letrozole are associated with negative side effects such as cognitive deficits, sleep disturbances and hot flashes. We have previously shown that these negative effects can be recapitulated in common marmosets (Callithrix jacchus) treated with Letrozole (20 µg daily) for 4 weeks and that marmosets treated with Letrozole show increased levels of estradiol in the hippocampus (Gervais et al., 2019). In order to better understand the mechanisms through which AIs affect cognitive function and increase steroid levels in the hippocampus, we used bulk, paired-end RNA-sequencing to examine differentially expressed genes among Letrozole-treated (LET; n = 8) and vehicle-treated (VEH; n = 8) male and female animals. Gene ontology results show significant reduction across hundreds of categories, some of the most significant being inflammatory response, stress response, MHC Class II protein complex binding, T-cell activation, carbohydrate binding and signaling receptor binding in LET animals. GSEA results indicate that LET females, but not LET males, show enrichment for hormonal gene sets. Based on the transcriptional changes observed, we conclude that AIs may differentially affect the sexes in part due to processes mediated by the CYP-450 superfamily. Ongoing studies will further investigate the longitudinal effects of AIs on behavior and whether AIs increase the risk of stress-induced neurodegeneration.

Potential trade-off between olfactory and visual discrimination learning in common marmosets (Callithrix jacchus): Implications for the assessment of age-related cognitive decline.

Olfactory dysfunction has been identified as an early biomarker for dementia risk but has rarely been assessed in nonhuman primate models of human aging. To better characterize common marmosets as such models, we assessed olfactory discrimination performance in a sample of 10 animals (5 females), aged 2.5-8.9 years old. The monkeys were proficient in the discrimination and reversal of visual stimuli but naïve to odor stimuli. For olfactory discrimination, the monkeys performed a series of six discriminations of increasing difficulty between two odor stimuli. We found no evidence for an age-related decline as both young and older individuals were able to perform the discriminations in roughly the same number of trials. In addition, the older monkeys had faster responses than the younger animals. However, we noted that when adjusted for age, the speed of acquisition of the first discrimination in the olfactory modality was inversely correlated to the speed of acquisition of their first discrimination of two visual stimuli months earlier. These results suggest that marmosets may compensate for sensory deficits in one modality with higher sensory performance in another. These data have broad implications for the assessment of age-related cognitive decline and the categorization of animals as impaired or nonimpaired.

Sex differences in cognitive aging: a 4-year longitudinal study in marmosets.

Longitudinal studies are essential to understand healthy and pathological neurocognitive aging such as Alzheimer's Disease, but longitudinal designs are rare in both humans and non-human primate models of aging because of the difficulty of tracking cognitive change in long-lived primates. Common marmosets (Callithrix jacchus) are uniquely suited for aging studies due to their naturally short lifespan (10-12 years), sophisticated cognitive and social abilities and Alzheimer Disease-like neuropathology. We report the first longitudinal study of cognitive aging in marmosets (N = 28) as they transitioned from middle- (∼5 years) to old age (∼9 years). We characterized aging trajectories using reversal learning with different stimuli each year. Marmosets initially improved on cognitive performance due to practice, but worsened in the final year, suggesting the onset of age-related decline. Cognitive impairment emerged earlier in females than males and was more prominent for discrimination than for reversal learning. Sex differences in cognitive aging could not be explained by differences in motivation or motor abilities, which improved or remained stable across aging. Likewise, males and females did not differ in aging trajectories of overall behavior or reactivity to a social stressor, with the exception of a progressive decline in the initiation of social behavior in females. Patterns of cognitive aging were highly variable across marmosets of both sexes, suggesting the potential for pathological aging for some individuals. Future work will link individual cognitive trajectories to neuropathology in order to better understand the relationships between neuropathologic burden and vulnerability to age-related cognitive decline in each sex.

Nonhuman primates at the intersection of aging biology, chronic disease, and health: An introduction to the American Journal of Primatology Special Issue on aging, cognitive decline, and neuropathology in nonhuman primates.

Aging across the Primate Order is poorly understood because ages of individuals are often unknown, there is a dearth of aged animals available for study, and because aging is best characterized by longitudinal studies which are difficult to carry out in long-lived species. The human population is aging rapidly, and advanced age is a primary risk factor for several chronic diseases and conditions that impact healthspan. As lifespan has increased, diseases and disorders of the central nervous system (CNS) have become more prevalent, and Alzheimer's disease and related dementias have become epidemic. Nonhuman primate (NHP) models are key to understanding the aging primate CNS. This Special Issue presents a review of current knowledge about NHP CNS aging across the Primate Order. Similarities and differences to human aging, and their implications for the validity of NHP models of aging are considered. Topics include aging-related brain structure and function, neuropathologies, cognitive performance, social behavior and social network characteristics, and physical, sensory, and motor function. Challenges to primate CNS aging research are discussed. Together, this collection of articles demonstrates the value of studying aging in a breadth of NHP models to advance our understanding of human and nonhuman primate aging and healthspan.

Comparative neuropathology in aging primates: A perspective.

While humans exhibit a significant degree of neuropathological changes associated with deficits in cognitive and memory functions during aging, non-human primates (NHP) present with more variable expressions of pathological alterations among individuals and species. As such, NHP with long life expectancy in captivity offer an opportunity to study brain senescence in the absence of the typical cellular pathology caused by age-related neurodegenerative illnesses commonly seen in humans. Age-related changes at neuronal population, single cell, and synaptic levels have been well documented in macaques and marmosets, while age-related and Alzheimer's disease-like neuropathology has been characterized in additional species including lemurs as well as great apes. We present a comparative overview of existing neuropathologic observations across the primate order, including classic age-related changes such as cell loss, amyloid deposition, amyloid angiopathy, and tau accumulation. We also review existing cellular and ultrastructural data on neuronal changes, such as dendritic attrition and spine alterations, synaptic loss and pathology, and axonal and myelin pathology, and discuss their repercussions on cellular and systems function and cognition.

The marmoset as an important primate model for longitudinal studies of neurocognitive aging.

Age-related cognitive decline has been extensively studied in humans, but the majority of research designs are cross-sectional and compare across younger and older adults. Longitudinal studies are necessary to capture variability in cognitive aging trajectories but are difficult to carry out in humans and long-lived nonhuman primates. Marmosets are an ideal primate model for neurocognitive aging as their naturally short lifespan facilitates longitudinal designs. In a longitudinal study of marmosets tested on reversal learning starting in middle-age, we found that, on average, the group of marmosets declined in cognitive performance around 8 years of age. However, we found highly variable patterns of cognitive aging trajectories across individuals. Preliminary analyses of brain tissues from this cohort also show highly variable degrees of neuropathology. Future work will tie together behavioral trajectories with brain pathology and provide a window into the factors that predict age-related cognitive decline.

Robustness of sex-differences in functional connectivity over time in middle-aged marmosets.

Nonhuman primates (NHPs) are an essential research model for gaining a comprehensive understanding of the neural mechanisms of neurocognitive aging in our own species. In the present study, we used resting state functional connectivity (rsFC) to investigate the relationship between prefrontal cortical and striatal neural interactions, and cognitive flexibility, in unanaesthetized common marmosets (Callithrix jacchus) at two time points during late middle age (8 months apart, similar to a span of 5-6 years in humans). Based on our previous findings, we also determine the reproducibility of connectivity measures over the course of 8 months, particularly previously observed sex differences in rsFC. Male marmosets exhibited remarkably similar patterns of stronger functional connectivity relative to females and greater cognitive flexibility between the two imaging time points. Network analysis revealed that the consistent sex differences in connectivity and related cognitive associations were characterized by greater node strength and/or degree values in several prefrontal, premotor and temporal regions, as well as stronger intra PFC connectivity, in males compared to females. The current study supports the existence of robust sex differences in prefrontal and striatal resting state networks that may contribute to differences in cognitive function and offers insight on the neural systems that may be compromised in cognitive aging and age-related conditions such as mild cognitive impairment and Alzheimer's disease.

Age-related decline in executive function as a hallmark of cognitive ageing in primates: an overview of cognitive and neurobiological studies.

Executive function (EF) is a complex construct that reflects multiple higher-order cognitive processes such as planning, updating, inhibiting and set-shifting. Decline in these functions is a hallmark of cognitive ageing in humans, and age differences and changes in EF correlate with age-related differences and changes in association cortices, particularly the prefrontal areas. Here, we review evidence for age-related decline in EF and associated neurobiological changes in prosimians, New World and Old World monkeys, apes and humans. While EF declines with age in all primate species studied, the relationship of this decline with age-related alterations in the prefrontal cortex remains unclear, owing to the scarcity of neurobiological studies focusing on the ageing brain in most primate species. In addition, the influence of sex, vascular and metabolic risk, and hormonal status has rarely been considered. We outline several methodological limitations and challenges with the goal of producing a comprehensive integration of cognitive and neurobiological data across species and elucidating how ageing shapes neurocognitive trajectories in primates with different life histories, lifespans and brain architectures. Such comparative investigations are critical for fostering translational research and understanding healthy and pathological ageing in our own species. This article is part of the theme issue 'Evolution of the primate ageing process'.

Baseline cortisol levels and social behavior differ as a function of handedness in marmosets (Callithrix jacchus).

Population hand preferences are rare in nonhuman primates, but individual hand preferences are consistent over a lifetime and considered to reflect an individual's preference to use a particular hemisphere when engaged in a specific task. Previous findings in marmosets have indicated that left-handed individuals tend to be more fearful than their right-handed counterparts. Based on these findings, we tested the hypotheses that left-handed marmosets are (a) more reactive to a social stressor and (b) are slower than right-handed marmosets in acquiring a reversal learning task. We examined the hand preference of 27 male and female marmosets (ages of 4-7 years old) previously tested in a social separation task and a reversal learning task. Hand preference was determined via a simple reaching task. In the social separation task, monkeys were separated from their partner and the colony for a single 7-hr session. Urinary cortisol levels and behavior were assessed at baseline, during the separation and 24 hr postseparation. Hand preferences were equally distributed between left (n = 10), right-handed (n = 10), and ambidextrous (n = 7) individuals. The separation phase was associated with an increase in cortisol levels and behavioral changes that were similar across handedness groups. However, cortisol levels at baseline were positively correlated with right-handedness, and this relationship was stronger in females than in males. In addition, the occurrence of social behaviors (pre- and postseparation) was positively correlated with right-handedness in both sexes. Baseline cortisol levels did not correlate significantly with social behavior. Acquisition of the reversals was poorer in females than males but did not differ as a function of handedness. We conclude that (a) both stress reactivity and cognitive flexibility are similar across handedness groups and (b) left-handers exhibit less social behavior and have lower basal cortisol levels than ambidextrous and right-handed subjects. The underlying causes for these differences remain to be established.

One-year change in cognitive flexibility and fine motor function in middle-aged male and female marmosets (Callithrix jacchus).

The common marmoset (Callithrix jacchus) is uniquely suited for longitudinal studies of cognitive aging, due to a relatively short lifespan, sophisticated cognitive abilities, and patterns of brain aging that resemble those of humans. We examined cognitive function and fine motor skills in male and female marmosets (mean age ∼5 at study entry) followed longitudinally for 2 years. Each year, monkeys were tested on a reversal learning task with three pairs of stimuli (n = 18, 9 females) and a fine motor task requiring them to grasp small rewards from two staircases (Hill and Valley test, n = 12, 6 females). There was little evidence for a decline in cognitive flexibility between the two time points, in part because of practice effects. However, independent of year of testing, females took longer than males to reach criterion in the reversals, indicating impaired cognitive flexibility. Motivation was unlikely to contribute to this effect, as males refused a greater percentage of trials than females in the reversals. With regards to motor function, females were significantly faster than males in the Hill and Valley task. From Year 1 to Year 2, a slight slowing of motor function was observed in both sexes, but accuracy decreased significantly in males only. This study (1) demonstrates that marmosets exhibit sex differences in cognitive flexibility and fine motor function that resemble those described in humans; (2) that changes in fine motor function can already be detected at middle-age; and (3) that males may experience greater age-related changes in fine motor skills than females. Additional data points will determine whether these sex and age differences persist over time.

Adverse Effects of Aromatase Inhibition on the Brain and Behavior in a Nonhuman Primate.

Breast cancer patients using aromatase inhibitors (AIs) as an adjuvant therapy often report side effects, including hot flashes, mood changes, and cognitive impairment. Despite long-term use in humans, little is known about the effects of continuous AI administration on the brain and cognition. We used a primate model of human cognitive aging, the common marmoset, to examine the effects of a 4-week daily administration of the AI letrozole (20 µg, p.o.) on cognition, anxiety, thermoregulation, brain estrogen content, and hippocampal pyramidal cell physiology. Letrozole treatment was administered to both male and female marmosets and reduced peripheral levels of estradiol (E2), but unexpectedly increased E2 levels in the hippocampus. Spatial working memory and intrinsic excitability of hippocampal neurons were negatively affected by the treatment possibly due to increased hippocampal E2. While no changes in hypothalamic E2 were observed, thermoregulation was disrupted by letrozole in females only, indicating some impact on hypothalamic activity. These findings suggest adverse effects of AIs on the primate brain and call for new therapies that effectively prevent breast cancer recurrence while minimizing side effects that further compromise quality of life.SIGNIFICANCE STATEMENT Aromatase inhibitors (AIs) are used as an adjuvant therapy for estrogen-receptor-positive breast cancer and are associated with side effects, including hot flashes, depression/anxiety, and memory deficits severe enough for many women to discontinue this life-saving treatment. AIs are also used by men, yet sex differences in the reported side effects have not been systematically studied. We show that AI-treated male and female marmosets exhibit behavioral changes consistent with these CNS symptoms, as well as elevated hippocampal estradiol and compromised hippocampal physiology. These findings illustrate the need for (1) a greater understanding of the precise mechanisms by which AIs impact brain function and (2) the development of new treatment approaches for breast cancer patients that minimize adverse effects on the brain.

Age-related decline in cognitive flexibility in female chimpanzees.

Data on cognitive aging in chimpanzees are extremely sparse, yet can provide an invaluable phylogenetic perspective, especially because Alzheimer disease (AD)-like neuropathology has recently been described in the oldest chimpanzee brains. This finding underscores the importance of data on cognitive aging in this fellow hominin, our closest biological relative. We tested 30 female chimpanzees, 12-56 years old, on a computerized analog of the Wisconsin Card Sort test. This test assesses cognitive flexibility, which is severely impaired in normal aging and AD. Subjects selected stimuli according to color or shape; the rewarded dimension (i.e., color or shape) switched without warning and the chimpanzee had to adapt her responses accordingly. We found that increasing age was associated with an increased number of perseverative errors and an increased number of trials to reach criterion in each switching dimension. The number of aborted trials was similar across age groups. These data show that similar to humans, chimpanzees show a clear age-related decline in cognitive flexibility that is already observed at middle age.

Glutamine/glutamate (Glx) concentration in prefrontal cortex predicts reversal learning performance in the marmoset.

This study used Magnetic Resonance Spectroscopy (MRS) to identify potential neurometabolitic markers of cognitive performance in male (n = 7) and female (n = 8) middle-aged (∼5 years old) common marmosets (Callithrix jacchus). Anesthetized marmosets were scanned with a 4.7 T/40 cm horizontal magnet equipped with 450 mT/m magnetic field gradients and a 20 G/cm magnetic field gradient insert, within 3 months of completing the CANTAB serial Reversal Learning task. Neurometabolite concentrations of N-Acetyl Asparate, Myo-Inositol, Choline, Phosphocreatine + creatine, Glutamate and Glutamine were acquired from a 3 mm3 voxel positioned in the Prefrontal Cortex (PFC). Males acquired the reversals (but not simple discriminations) faster than the females. Higher PFC Glx (glutamate + glutamine) concentration was associated with faster acquisition of the reversals. Interestingly, the correlation between cognitive performance and Glx was significant in males, but not in females. These results suggest that MRS is a useful tool to identify biochemical markers of cognitive performance in the healthy nonhuman primate brain and that biological sex modulates the relationship between neurochemical composition and cognition.

Ovarian hormones, sleep and cognition across the adult female lifespan: An integrated perspective.

Loss of ovarian function in women is associated with sleep disturbances and cognitive decline, which suggest a key role for estrogens and/or progestins in modulating these symptoms. The effects of ovarian hormones on sleep and cognitive processes have been studied in separate research fields that seldom intersect. However, sleep has a considerable impact on cognitive function. Given the tight connections between sleep and cognition, ovarian hormones may influence selective aspects of cognition indirectly, via the modulation of sleep. In support of this hypothesis, a growing body of evidence indicates that the development of sleep disorders following menopause contributes to accelerated cognitive decline and dementia in older women. This paper draws from both the animal and human literature to present an integrated view of the effects of ovarian hormones on sleep and cognition across the adult female lifespan.

The middle-aged ovariectomized marmoset (Callithrix jacchus) as a model of menopausal symptoms: Preliminary evidence.

Menopausal women often suffer from hot flashes and sleep disturbances that significantly impact their quality of life. Both human and animal studies suggest that loss of estrogens during menopause contribute to these symptoms. In the female rat, both core body temperature (CBT) and sleep are sensitive to 17ß-estradiol (E2) levels, but important differences between the rat and the human patterns limit the interpretation of the results. The sleep and thermoregulation of the common marmoset (Callithrix jacchus) more closely resemble human patterns. However, no study to date has examined whether E2 influences sleep and thermoregulation in this species. The main goal of the present study was to investigate the suitability of the ovariectomized (OVX) marmoset for studying two major menopausal symptoms experienced by women, sleep disturbance and thermodysregulation. Two middle-aged OVX marmosets (6years old) were implanted with a telemeter that records electroencephalograms (EEG), electromyograms (EMG), and CBT. Sleep patterns and CBT were recorded under baseline, two E2 replacement (6 and 12µg/kg/day, p.o.) conditions and two E2 withdrawal conditions. Relative to both baseline and withdrawal, high E2 replacement was associated with lower nighttime CBT. In addition, fewer nighttime arousals were observed under low E2 replacement compared to baseline. Higher delta power was observed under both E2 replacement conditions suggesting enhanced sleep quality. These preliminary results suggest that E2 modulates sleep and thermoregulation in the OVX marmoset, making it a promising model for studying menopausal symptoms.

Sex differences in inhibitory control in socially-housed baboons (Papio papio).

Inhibitory control is an important component of executive function. An emerging literature in humans suggests that inhibitory control is sexually dimorphic and modulated by sex steroids, but evidence for such a link in nonhuman animals is scarce. In this study, we examined the effects of menstrual cycle and biological sex on response inhibition, as measured by a Stop-Signal task, in the baboon (Papio papio). The monkeys (n=13) were socially-housed, with voluntary access to multiple touchscreen computerized stations. The task required monkeys to inhibit prepotent responses (touching a target, "Go" trials) following the appearance of a visual stop signal on 25% of the trials ("Stop" trials). The cognitive data, consisting of computerized records of the monkeys' performance on the Stop-Signal task over a year of testing, were matched to records of female sexual swellings. Same-day menstrual and cognitive data were available for 5 females, aged 5-18 years. These data were compared to those of 8 males (5-14 years old) performing the Stop-Signal task over the same time period. Contrary to our hypothesis, performance on the task was not significantly affected by the phase (ovulatory vs. luteal) of the cycle in females. However, males were slower than females on Go trials and were less efficient in inhibiting responses on Stop trials. Slower responses in males were indicative of a speed-accuracy trade-off, as overall accuracy was also better in males than in females. Analyses of trial history indicated that males did not speed as much as females following a successful Go trial, but did not differ from females in post-error slowing or post-inhibiting responses. Overall, the data show that biological sex modulates Stop-Signal performance in the baboon, with males exhibiting slower response execution overall, less efficient inhibition, but greater accuracy than females. This pattern of sex differences may reflect motivational sex differences in which males emphasize accuracy rather than speed. Interestingly, these sex differences do not seem to vary as a function of ovarian hormones in females. Males' greater focus on accuracy is possibly due to enhanced sensitivity to reward mediated by testosterone levels.

Reversal learning in gonadectomized marmosets with and without hormone replacement: are males more sensitive to punishment?

This study examined sex differences in executive function in middle-aged gonadectomized marmosets (Callithrix jacchus) with or without hormonal replacement. We tested ten castrated male (mean age 5.5 years) marmosets treated with testosterone cypionate (T, n = 5) or vehicle (n = 5) on Reversal Learning, which contributes to cognitive flexibility, and the Delayed Response task, measuring working memory. Their performance was compared to that of 11 ovariectomized females (mean age = 3.7 years) treated with Silastic capsules filled with 17-ß estradiol (E2, n = 6) or empty capsules (n = 5), previously tested on the same tasks (Lacreuse et al. in J Neuroendocrinol 26:296-309, 2014. doi: 10.1111/jne.12147). Behavioral observations were conducted daily. Females exhibited more locomotor behaviors than males. Males and females did not differ in the number of trials taken to reach criterion on the reversals, but males had significantly longer response latencies, regardless of hormone replacement. They also had a greater number of refusals than females. Additionally, both control and T-treated males, but not females, had slower responses on incorrect trials, suggesting that males were making errors due to distraction, lack of motivation or uncertainty. Furthermore, although both males and females had slower responding following an incorrect compared to a correct trial, the sex difference in response latencies was disproportionally large following an incorrect trial. No sex difference was found in the Delayed Response task. Overall, slower response latencies in males than females during Reversal Learning, especially during and following an incorrect trial, may reflect greater sensitivity to punishment (omission of reward) and greater performance monitoring in males, compared to females. Because these differences occurred in gonadectomized animals and regardless of hormone replacement, they may be organized early in life.

Neurocognitive effects of estrogens across the adult lifespan in nonhuman primates: State of knowledge and new perspectives.

This article is part of a Special Issue "Estradiol and cognition". This review discusses the unique contribution of nonhuman primate research to our understanding of the neurocognitive effects of estrogens throughout the adult lifespan in females. Mounting evidence indicates that estrogens affect many aspects of hippocampal, prefrontal and cholinergic function in the primate brain and the underlying mechanisms are beginning to be elucidated. In addition, estrogens may also influence cognitive function indirectly, via the modulation of other systems that impact cognition. We will focus on the effects of estrogens on sleep and emphasize the need for primate models to better understand these complex interactions. Continued research with nonhuman primates is essential for the development of therapies that are optimal for the maintenance of women's cognitive health throughout the lifespan.