Gwendolen E. Haley

Influence of Age and 17β-Estradiol on Kisspeptin, Neurokinin B, and Prodynorphin Gene Expression in the Arcuate-Median Eminence of Female Rhesus Macaques

The neuropeptides kisspeptin, neurokinin B, and dynorphin A (collectively abbreviated as KNDy) are, respectively, encoded by KiSS-1, NKB, and PDYN and are coexpressed by neurons of the hypothalamic arcuate nucleus (ARC). Here, using quantitative real-time PCR, we examined age-related changes in the expression of genes encoding KNDy and associated receptors G protein-coupled receptor 54 (encoded by GPR54), neurokinin 3 receptor (encoded by NK3), and kappa-opioid receptor (encoded by KOR), in the female rhesus macaque ARC-median eminence (ARC-ME). Expression of KiSS-1 and NKB was highly elevated in old perimenopausal compared with young or middle-aged premenopausal animals. To test whether these age-related changes could be attributed to perimenopausal loss of sex steroids, we then examined KNDy, GPR54, NK3, and KOR expression changes in response to ovariectomy (OVX) and exposure to 17beta-estradiol (E(2)). Short-term (7 months) OVX (with or without 1 month of estrogen replacement) failed to modulate the expression of any of the KNDy-related genes. In contrast, long-term ( approximately 4 yr) OVX significantly increased KiSS-1 and NKB expression, and this was reversed by E(2) administration. Finally, we examined the expression of KNDy-related genes in young adult females during the early follicular, late follicular, or midluteal phases of their menstrual cycle but found no difference. Together, the results suggest that short-term alterations in circulating E(2) levels, such as those occurring during the menstrual cycle, may have little effect on the ARC-ME expression of KNDy and associated receptors. Nevertheless, they clearly demonstrate that loss of ovarian steroid negative feedback that occurs during perimenopause plays a major role in modulating the activity of KNDy circuits of the aging primate ARC-ME.

Apolipoprotein E isoform-dependent effects on anxiety and cognition in female TR mice

Compared with apoE3, apoE4 is associated with increased risk to develop age-related cognitive decline, particularly in women. In this study, young, middle-aged, and old female mice expressing human apoE under control of the mouse apoE promoter were behaviorally analyzed. Cognitive performance in the water maze decreased with age in all mice. Compared with apoE2 and apoE3 mice, apoE4 mice showed better cognitive performance and higher measures of anxiety than apoE2 and apoE3 mice. Measures of anxiety correlated with cognitive performance in the water maze and passive avoidance tests and might have contributed to the enhanced cognitive performance of the apoE4 mice. ApoE4 mice showed better water maze learning and higher cortical apoE levels than mice expressing apoE4 in astrocytes under control of the GFAP promoter. This was not seen in apoE3 mice. There were no line differences in either genotype in spatial memory retention in the probe trial following the last day of hidden platform training. Thus, the promoter used to express apoE4 critically modulates its effects on brain function.

Early Effects of Whole-Body 56Fe Irradiation on Hippocampal Function in C57BL/6J Mice

Relatively little is known about early irradiation effects on hippocampal function in wild-type mice. In this study, the effects of 56Fe irradiation on hippocampal function were assessed starting 2 weeks after whole-body irradiation. Compared to sham irradiation, radiation impaired novel object recognition in female and male C57BL/6J wild-type mice. There were no effects of irradiation on contextual fear conditioning or spatial memory retention in the water maze. It is possible that oxidative damage might contribute to radiation-induced cognitive changes. Therefore, hippocampal and cortical levels of 3-nitrotyrosine (3NT) and lipid peroxidation, measures of oxidative damage were assessed. There were no effects of irradiation on these measures of oxidative damage. As 56Fe irradiation can increase reactive oxygen species (ROS) levels, which may contribute to the impairments in novel object recognition, the effects of the antioxidant alpha-lipoic acid (ALA) on cognition following sham irradiation and irradiation were also assessed. ALA did not prevent radiation-induced impairments in novel object recognition and impaired spatial memory retention of sham-irradiated and irradiated mice in the probe trial after the first day of hidden platform training in the water maze. Thus, the novel object recognition test is particularly sensitive to detect early cognitive effects of 56Fe irradiation through a mechanism unlikely involving ROS or oxidative damage.

Age-related decreases in SYN levels associated with increases in MAP-2, apoE, and GFAP levels in the rhesus macaque prefrontal cortex and hippocampus

Loss of synaptic integrity in the hippocampus and prefrontal cortex (PFC) may play an integral role in age-related cognitive decline. Previously, we showed age-related increases in the dendritic marker microtubule associated protein 2 (MAP-2) and the synaptic marker synaptophysin (SYN) in mice. Similarly, apolipoprotein E (apoE), involved in lipid transport and metabolism, and glial fibrillary acidic protein (GFAP), a glia specific marker, increase with age in rodents. In this study, we assessed whether these four proteins show similar age-related changes in a nonhuman primate, the rhesus macaque. Free-floating sections from the PFC and hippocampus from adult, middle-aged, and aged rhesus macaques were immunohistochemically labeled for MAP-2, SYN, apoE, and GFAP. Protein levels were measured as area occupied by fluorescence using confocal microscopy as well as by Western blot. In the PFC and hippocampus of adult and middle-aged animals, the levels of SYN, apoE, and GFAP immunoreactivity were comparable but there was a trend towards higher MAP-2 levels in middle-aged than adult animals. There was significantly less SYN and more MAP-2, apoE, and GFAP immunoreactivity in the PFC and hippocampus of aged animals compared to adult or middle-aged animals. Thus, the age-related changes in MAP-2, apoE, and GFAP levels were similar to those previously observed in rodents. On the other hand, the age-related changes in SYN levels were not, but were similar to those previously observed in the aging human brain. Taken together, these data emphasize the value of the rhesus macaque as a pragmatic translational model for human brain aging.

Circadian Activity Associated With Spatial Learning and Memory in Aging Rhesus Monkeys

In rodents, spatial learning and memory tests require navigation, whereas in nonhuman primates these tests generally do not involve a navigational component, thus assessing nonhomologous neural systems. To allow closer parallels between rodent and primate studies, we developed a navigational spatial learning and memory task for nonhuman primates and assessed the performance of elderly (19-25 years) female rhesus monkeys (Macaca mulatta). The animals were allowed to navigate in a room containing a series of food ports. After they learned to retrieve food from the ports, a single port was repeatedly baited and the animals were tested until they learned the correct location. The location of the baited port was then changed (shift position). We also determined whether test performance was associated with circadian activity measured with accelerometers. Performance measures included trials to criterion, search strategies, and several indices of circadian activity. Animals learned the task as reflected in their search strategies. Correlations were found between the number of initial or shift trials and circadian activity parameters including day activity, dark:light activity ratio, sleep latency, and wake bouts. Thus, disruptions in circadian rhythms in nonhuman primates are associated with poorer performance on this novel test. These data support the usefulness of this spatial navigational test to assess spatial learning and memory in rhesus monkeys and the importance of circadian activity in performance.

Novel image–novel location object recognition task sensitive to age-related cognitive decline in nondemented elderly

Traditional tests used in the clinic to identify dementia, such as the mini-mental state examination (MMSE), are useful to identify severe cognitive impairments but might be less sensitive to detect more subtle age-related cognitive changes. Previously, the novel image–novel location (NINL) object recognition test was shown to be sensitive to detect effects of apolipoprotein E4, a risk factor for developing age-related cognitive decline and Alzheimer’s disease, in nondemented elderly. In the present longitudinal study, performance on the MMSE and the NINL tests were compared over a 4-year period. Individual NINL scores over this period were highly correlated. In addition, while MMSE scores did not change over the 4-year period, NINL scores did. In a final testing session of a subset of the participants, NINL scores correlated with logical memory and word recall lists, cognitive tasks used to detect dementia in the clinic, as well as clinical dementia rating scales. These results support that the NINL might be a valuable tool to assess age-related cognitive decline.

Effects of ε4 on Object Recognition in the Non-Demented Elderly

Previously we reported that Apolipoprotein E (ApoE) e4 negatively affects performance in the novel-image-novel- location (NINL) object recognition test in healthy non-demented elderly human study participants. In this study, the participants were invited to return for testing sessions 6 and 18 months after the baseline session. Using a longitudinal study design, effects of e4 on NINL test performance were assessed in study “dropouts”, participants that did not return for the second and/or third session(s), and “finishers”, participants that returned for all sessions. There were effects of e4 on dropout rates and NINL total scores as well as sub-scores in both dropouts and finishers. NINL total score was a predictor of e4 participant dropout. Compared to non-e4 dropouts, e4 dropouts had lower NINL scores. In contrast, e4 finishers had higher NINL scores than non-e4 finishers. Thus, the NINL test could be a valuable tool in detecting preclinical signs of age-related cognitive impairments, particularly those associated with e4 risk.

Hippocampal M1 receptor function associated with spatial learning and memory in aged female rhesus macaques

Of the acetylcholine muscarinic receptors, the type 1 (M1) and type 2 (M2) receptors are expressed at the highest levels in the prefrontal cortex (PFC) and hippocampus, brain regions important for cognition. As equivocal findings of age-related changes of M1 and M2 in the nonhuman primate brain have been reported, we first assessed age-related changes in M1 and M2 in the PFC and hippocampus using saturation binding assays. Maximum M1 receptor binding, but not affinity of M1 receptor binding, decreased with age. In contrast, the affinity of M2 receptor binding, but not maximum M2 receptor binding, increased with age. To determine if in the elderly cognitive performance is associated with M1 or M2 function, we assessed muscarinic function in elderly female rhesus macaques in vivo using a scopolamine challenge pharmacological magnetic resonance imaging and in vitro using saturation binding assays. Based on their performance in a spatial maze, the animals were classified as good spatial performers (GSP) or poor spatial performers (PSP). In the hippocampus, but not PFC, the GSP group showed a greater change in T2*-weighted signal intensity after scopolamine challenge than the PSP group. The maximum M1 receptor binding and receptor binding affinity was greater in the GSP than the PSP group, but no group difference was found in M2 receptor binding. Parameters of circadian activity positively correlated with the difference in T2*-weighted signal intensity before and after the challenge, the maximum M1 receptor binding, and the M1 receptor binding affinity. Thus, while in rhesus macaques, there are age-related decreases in M1 and M2 receptor binding, in aged females, hippocampal M1, but not M2, receptor function is associated with spatial learning and memory and circadian activity.

Association of microtubule associated protein-2, synaptophysin, and apolipoprotein E mRNA and protein levels with cognition and anxiety levels in aged female rhesus macaques

The dendritic protein microtubule associated protein 2 (MAP-2), the presynaptic marker synaptophysin (SYN), and apolipoprotein E (APOE), a protein which plays a role in lipid transport and metabolism and affects synaptic activity show changes with age. We analyzed post-mortem tissue from aged female rhesus macaques cognitively tested in a spatial maze and classified as good spatial performers (GSP) or poor spatial performers (PSP) and behaviorally tested in a playroom and classified as bold or reserved animals. MAP2, SYN, and APOE mRNA and protein levels in the prefrontal cortex (PFC), hippocampus, and amygdala, were assessed using qRT-PCR and western blot. In the amygdala, bold monkeys had higher levels of MAP2 and SYN mRNA than reserved monkeys. MAP2 mRNA correlated positively with amygdala size on the right, left, and combined left and right sides, while SYN mRNA levels correlated positively with the size of the right amygdala. In the hippocampus, SYN and APOE protein levels were higher in GSP than PSP animals. Thus, in aged nonhuman primates, classification of measures of anxiety is associated with differences in selected mRNA, but not protein, levels. In contrast, classification of cognitive performance is associated with differences in selected protein, but not mRNA, levels.