Amyloidosis increase is not attenuated by long-term calorie restriction or related to neuron density in the prefrontal cortex of extremely aged rhesus macaques.

As human lifespan increases and the population ages, diseases of aging such as Alzheimer's disease (AD) are a major cause for concern. Although calorie restriction (CR) as an intervention has been shown to increase healthspan in many species, few studies have examined the effects of CR on brain aging in primates. Using postmortem tissue from a cohort of extremely aged rhesus monkeys (22-44 years old, average age 31.8 years) from a longitudinal CR study, we measured immunohistochemically labeled amyloid beta plaques in Brodmann areas 32 and 46 of the prefrontal cortex, areas that play key roles in cognitive processing, are sensitive to aging and, in humans, are also susceptible to AD pathogenesis. We also evaluated these areas for cortical neuron loss, which has not been observed in younger cohorts of aged monkeys. We found a significant increase in plaque density with age, but this was unaffected by diet. Moreover, there was no change in neuron density with age or treatment. These data suggest that even in the oldest-old rhesus macaques, amyloid beta plaques do not lead to overt neuron loss. Hence, the rhesus macaque serves as a pragmatic animal model for normative human aging but is not a complete model of the neurodegeneration of AD. This model of aging may instead prove most useful for determining how even the oldest monkeys are protected from AD, and this information may therefore yield valuable information for clinical AD treatments.

Cognition in aged rhesus monkeys: effect of DHEA and correlation with steroidogenic gene expression.

Estradiol supplementation has been shown to enhance cognitive performance in old ovariectomized rhesus macaques (Macaca mulatta). To determine if similar benefits could be achieved in perimenopausal animals using alternative hormonal supplements, we administered dehydroepiandrosterone (DHEA) to old ovary-intact female rhesus macaques for ∼2.5 months. Using computerized touch screen memory tasks, including delayed response (DR) and delayed matching-to-sample (DMS), we observed improved performance with time in all of the animals but failed to detect a significant effect of DHEA. On the other hand, gene expression profiling disclosed a significant correlation between cognitive performance and the expression of several steroidogenic and steroid-responsive genes. The DR performance was positively correlated with hippocampal expression of AKR1C3 and STAR and negatively correlated with the expression of SDRD5A1. A positive correlation was also found between DMS performance and prefrontal cortical expression of AKR1C3 and a negative correlation with STAR, as well as a negative correlation with the hippocampal expression of HSD11B1 and NR3C1. Taken together, the results suggest that steroidogenic gene regulation within the brain may help to maintain cognitive function during the perimenopausal transition period, despite a decline in sex-steroid levels in the circulation.

Hormone supplementation during aging: how much and when?

Circulating levels of dehydroepiandrosterone, a major adrenal steroid, show a marked age-related decrease in both humans and nonhuman primates. Because this decrease has been implicated in age-related cognitive decline, we administered supplementary dehydroepiandrosterone to perimenopausal rhesus macaques (Macaca mulatta) to test for cognitive benefits. Although recognition memory improved, there was no benefit to spatial working memory. To address the limitations of this study we developed a hormone supplementation regimen in aged male macaques that more accurately replicates the 24-hr androgen profiles of young animals. We hypothesize that this more comprehensive physiological hormone replacement paradigm will enhance cognitive function in the elderly.

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.

Sex-differences in age-related cognitive decline in C57BL/6J mice associated with increased brain microtubule-associated protein 2 and synaptophysin immunoreactivity.

Understanding cognitive aging is becoming more important as the elderly population grows. Here, the effects of age and sex on learning and memory performance were compared in female and male young (3-4 months old) middle-aged (10-12 months old) and old (18-20 months old) wild-type C57BL/6J mice. Old males and females performed worse than young or middle-aged mice in novel location, but not novel object recognition tasks. Old mice, of both sexes, also showed impaired spatial water maze performance during training compared with young or middle-aged mice, however only old females failed to show robust spatial bias during probe trials. While there was no age-difference in passive avoidance performance for males, females showed an age-related decline. There was no difference in cognitive performance between young and middle-age mice of either sex on any task. Cognitive performance was associated with alterations in immunoreactivity of microtubule-associated protein 2-positive dendrites and synaptophysin-positive pre-synaptic terminals in hippocampal CA1, CA3, and dentate, entorhinal cortex, and central nucleus of amygdala. Overall, microtubule-associated protein 2 immunoreactivity was increased in old females compared with both young and middle-age females with no significant difference in males. In contrast, synaptophysin immunoreactivity increased from young to middle-age in females, and from middle-age to old in males; females had higher levels of synaptophysin immunoreactivity than males in middle-age only. Elevated levels of microtubule-associated protein 2 and synaptophysin may constitute a compensatory response to age-related functional decline in mice.

Changes in blood chemistry and hematology variables during aging in captive rhesus macaques (Macaca mulatta). J Med Primatol 30:161-173, 2001.

The Primate Aging Database (PAD) is being developed to assist research using nonhuman primate models for various gerontological applications. We provide now an update of an earlier report providing data on hematological and blood chemistry values for rhesus monkeys across the adult lifespan. These data were collected from several research colonies and have been submitted to rigorous statistical analyses to identify relationships with chronological age.

Changes in blood chemistry and hematology variables during aging in captive rhesus macaques (Macaca mulatta).

Identifying changes with age in physiological variables of captive nonhuman primates will aid in the proper treatment and clinical diagnosis of these animals, as well as enhance our understanding of nonhuman primate models for human aging. Information for 33 physiological variables was obtained from the Primate Aging Database, a multi-centered database being developed for clinical and research use. Using multiple regression analyses, we investigated the relationship of age to hematological variables, blood chemistry and body weight in 345 captive rhesus monkeys (age range 7-30 years) from three different primate research facilities. The analyses revealed that 15 of these variables show a significant relationship with chronological age and are altered in older as compared with adult animals. Here we present the first phase of a project that will: a) identify changes with age in physiological variables among adult captive rhesus macaques; and b) characterize normative values for the aging rhesus population.

A developmental increase in the expression of messenger ribonucleic acid encoding a second form of gonadotropin-releasing hormone in the rhesus macaque hypothalamus.

GnRH-I is thought to represent the primary neuroendocrine link between the brain and the reproductive axis. Recently, however, a second molecular form of this decapeptide (GnRH-II) was found to be highly expressed in the brains of humans and nonhuman primates. In this study, in situ hybridization was used to examine the regional expression of GnRH-II messenger ribonucleic acid in the hypothalamus of immature (0.6 yr) and adult (10-15 yr) male and female rhesus macaques (Macaca mulatta). Overall, no sex-related differences were observed. In all of the animals (n = 3 animals/group), intense hybridization of a monkey GnRH-II riboprobe was evident in the paraventricular nucleus and supraoptic nucleus and to a lesser extent in the suprachiasmatic nucleus, but no age- or sex-related differences were apparent. Intense hybridization of the riboprobe also occurred in the mediobasal hypothalamus, and this was markedly greater in the adults than in the immature animals. These data show that the expression of GnRH-II messenger ribonucleic acid increases developmentally in a key neuroendocrine center of the brain. Moreover, because GnRH-II can stimulate LH release in vivo, it is plausible that changes in its gene expression represent an important component of the mechanism by which the hypothalamus controls reproductive function.

Oestrogen upregulates noradrenaline release in the mediobasal hypothalamus and tyrosine hydroxylase gene expression in the brainstem of ovariectomized rhesus macaques.

Noradrenaline plays a key role in the initiation of ovulation in nonprimate species. A similar noradrenaline role in the primate has not been established experimentally. We utilized the ovariectomized-oestrogen-supplemented (OVX + E) rhesus macaque to examine the effects of intravenous (i.v.) infusion of oestradiol-17beta (E2) on the activity of the brain noradrenaline system. Experiment 1 established the induction of a preovulatory surge-like release of luteinizing hormone in OVX + E monkeys by i.v. infusion of E2 (OVX + E + E2). In experiment 2, a marked increase in hypothalamic microdialysate noradrenaline concentrations occurred after identical E2 infusion into the OVX + E monkeys that were used in experiment 1. In experiment 3, tyrosine hydroxylase (TH) mRNA expression in the locus coeruleus of the brainstem increased at various times after E2 infusion as determined by semiquantitative in situ hybridization. The amount of TH mRNA in OVX + E + E2 animals was higher (P < 0.05) than that in either the OVX + E or OVX monkeys; no difference was found in the latter two groups. Moreover, selected locus coeruleus sections from E2-infused monkeys were examined for the localization of oestrogen receptors (ER) by in situ hybridization. Both ER-alpha and ER-beta mRNAs were expressed in the locus coeruleus, although the expression was greater for ER-alpha than for ER-beta. We conclude that i.v. infusion of E2, which induces a preovulatory surge-like release of LH, stimulates brain noradrenaline activity; this enhanced activity likely involves an ER-mediated process and is reflected by hypothalamic noradrenaline release and locus coeruleus TH mRNA expression. The results support the concept that noradrenaline can influence the E2-stimulated ovulation in nonhuman primates and that the brainstem is one of the components in this neuroendocrine process.

Distribution of estrogen receptor beta (ERbeta) mRNA in hypothalamus, midbrain and temporal lobe of spayed macaque: continued expression with hormone replacement.

This study used in situ hybridization (ISH) to examine the distribution of estrogen receptor beta (ERbeta) mRNA in hypothalamic, limbic, and midbrain regions of monkey brain and its regulation by estrogen (E) and progesterone (P). Monkey-specific ERbeta cDNAs were developed with human primers and reverse transcription and polymerase chain reaction (RT-PCR) using mRNA extracted from a rhesus monkey prostate gland. ERbeta 5' (262 bases) and 3' (205 bases) riboprobes were used in combination for ISH. Ovariectomized and hysterectomized (spayed) pigtail macaques (Macaca nemestrina; four per treatment group) were either untreated spayed-controls, treated with E (28 days), or treated with E plus P (14 days E+14 days E and P). Dense ERbeta hybridization signal was seen in the preoptic area, paraventricular nucleus, and ventromedial nucleus of the hypothalamus; the substantia nigra, caudal linear, dorsal raphe, and pontine nuclei of the midbrain; the dentate gyrus, CA1, CA2, CA3, CA4, and the prosubiculum/subiculum areas of the hippocampus. Expression in the suprachiasmatic region, supraoptic nucleus, arcuate nucleus, and amygdala was less intense. Image analysis of the dense areas showed no significant difference in the hybridization signal in individual regions of the hypothalamus, midbrain, or hippocampus between any of the treatment groups. However, P treatment decreased overall ERbeta signal in the hypothalamus and hippocampus when several different subregions were combined. The localization of ERbeta in monkey brain by ISH is in general agreement with that previously described in rodents. The presence of monkey ERbeta mRNA in brain regions that lack ERalpha should help to clarify the molecular mechanisms by which E acts in the central nervous system to influence hormone secretion, mood disorders, cognition, and neuroprotection.

Two molecular forms of gonadotropin-releasing hormone (GnRH-I and GnRH-II) are expressed by two separate populations of cells in the rhesus macaque hypothalamus.

Gonadotropin-releasing hormone represents the primary neuroendocrine link between the brain and the reproductive axis, and at least two distinct molecular forms of this decapeptide (GnRH-I and GnRH-II) are known to be expressed in the forebrain of rhesus macaques (Macaca mulatta). Although the distribution pattern of the two corresponding mRNAs is largely dissimilar, their expression appears to show some overlap in specific regions of the hypothalamus; this raises the possibility that some cells express both molecular forms of GnRH. To resolve this issue, double-label histochemistry was performed on hypothalamic sections from six male rhesus macaques, using a monoclonal antibody to GnRH-I and a riboprobe to monkey GnRH-II mRNA. In total, more than 2000 GnRH neurons were examined but in no instance were GnRH-I peptide and GnRH-II mRNA found to be coexpressed. This finding emphasizes that GnRH-I and GnRH-II are synthesized by two distinct populations of hypothalamic neurons, and suggests that they may be regulated by different neuroendocrine pathways.

Ovarian hormones differentially influence immunoreactivity for dopamine beta- hydroxylase, choline acetyltransferase, and serotonin in the dorsolateral prefrontal cortex of adult rhesus monkeys.

Recent studies have shown that ovariectomy reduces, and subsequent hormone replacement restores the density of axons immunoreactive for tyrosine hydroxylase in the dorsolateral prefrontal cortex of adult female rhesus monkeys. The present study indicates that three additional extrathalamic frontal lobe afferents are also sensitive to changes in the ovarian hormone environment. Specifically, the combination of hormone manipulation with qualitative and quantitative analysis of immunocytochemistry for dopamine beta-hydroxylase, choline acetyltransferase, and serotonin in the primate prefrontal cortex revealed quantitative responses in both cholinergic and monoaminergic axons to changing ovarian hormone levels. However, whereas ovariectomy produced a modest net decrease in the density of fibers immunoreactive for choline acetyltransferase, this same treatment markedly increased the density of axons immunoreactive for dopamine beta-hydroxylase and for serotonin. Further, the effects of ovariectomy on these afferent systems were differentially attenuated by estrogen verses estrogen plus progesterone hormone replacement. Estrogen was as effective as estrogen plus progesterone in stimulating normal prefrontal immunoreactivity for choline acetyltransferase and dopamine beta-hydroxylase. The dual replacement of estrogen plus progesterone, however, was a much more potent influence than estrogen alone for serotonin immunoreactivity. Thus, ovarian hormones appear to provide stimulation that differentially affects each of four chemically identified extrathalamic prefrontal afferent systems examined to date, and may have roles in maintaining the normal balance and functional interactions between these neurotransmitter systems.

Regional expression of mRNA encoding a second form of gonadotropin-releasing hormone in the macaque brain.

In mammals, reproduction is thought to be controlled by a single neuropeptide, gonadotropin-releasing hormone (GnRH-I), which regulates the synthesis and secretion of gonadotropins from the pituitary gland. However, another form of this decapeptide (GnRH-II), of unknown function, also exists in the brain of many vertebrate species, including humans; it is encoded by a different gene and its amino acid sequence is 70% identical to that of GnRH-I. Here we report the cloning of a GnRH-II cDNA from the rhesus macaque (Macaca mulatta), and show for the first time by in situ hybridization that GnRH-II mRNA is expressed in the primate midbrain, hippocampus and discrete nuclei of the hypothalamus, including the supraoptic, paraventricular, suprachiasmatic and arcuate. Because the regional distribution pattern of cells containing GnRH-II mRNA is largely dissimilar to that of cells containing GnRH-I mRNA, it is likely that these two cell populations receive distinct neuroendocrine inputs and thus regulate GnRH synthesis and release differently.

Regional distribution of glutamic acid decarboxylase (GAD65 and GAD67) mRNA in the hypothalamus of male rhesus macaques before and after puberty.

Glutamic acid decarboxylase (GAD) is the rate-limiting enzyme in the gamma-aminobutyric acid (GABA) biosynthetic pathway, and is coded for by two mRNAs, GAD65 and GAD67. Using in situ hybridization, we examine the distribution pattern of both GAD mRNAs in the hypothalamus and thalamus of prepubertal and adult male rhesus macaques. Qualitatively, GAD65 and GAD67 mRNAs showed a similar wide, but highly specific distribution pattern, supporting the view that GABAergic neurons play an important role in modulating neuroendocrine function. However, no quantitative difference in the intensity of hybridization signal was detected between prepubertal and adult animals in any of the hypothalamic or thalamic nuclei. Therefore, although GABAergic neurons are anatomically well-placed to control the secretion of gonadotropin-releasing hormone (GnRH) in primates, it is unlikely that the onset of puberty and the associated increase in GnRH secretion is triggered by a change in GAD gene transcription.

Ovarian hormones influence the morphology, distribution, and density of tyrosine hydroxylase immunoreactive axons in the dorsolateral prefrontal cortex of adult rhesus monkeys.

The maturation, adult functioning and dysfunction of the prefrontal cortex in disorders such as schizophrenia show gender biases in human and non-human primates. Although the basis for the hormone influence suggested in these observations is unknown, one possibility is that circulating hormones stimulate catecholamine innervation in the frontal lobe. This innervation is essential for prefrontal cortical function, and gonadal, especially ovarian hormones, profoundly influence catecholamine function and physiology in subcortical structures. This study was undertaken to determine whether influence is also exerted upon the catecholamine innervation of the association cortex by combining ovarian hormone manipulation with immunocytochemistry for tyrosine hydroxylase in the dorsolateral prefrontal cortex of adult female macaque monkeys. Qualitative and quantitative analyses of immunoreactive fibers were carried out and compared in cortices of ovariectomized animals, ovariectomized animals treated with estrogen, ovariectomized animals treated with estrogen followed by progesterone, and in intact, age- and sex-matched controls. These analyses revealed striking, layer-specific anomalies in fiber morphology and profound reductions in fiber density in ovariectomized animals. While hormone replacement with estrogen alone had limited influence, estrogen followed by progesterone was particularly effective in restoring tyrosine hydroxylase innervation in ovariectomized animals. Thus, ovarian hormones appear to be potent regulators of the catecholamine innervation of the primate prefrontal cortex. Such regulation is anticipated in the gender differences observed in prefrontal cortical development and function, and may also be relevant for the prefrontal dysfunction in disorders such as schizophrenia.

Regional distribution of glutamate receptor mRNA in the monkey hippocampus and temporal cortex: influence of estradiol.

The distribution of N-methyl-D-aspartate (NMDA) and alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) glutamate receptor subunits was examined in the hippocampus and temporal cortex of adult ovariectomized female rhesus macaques, some of which received estradiol replacement. In situ hybridization revealed a generalized overlap of NR1, GluR1, and GluR2 subunit mRNAs, but no effect due to estradiol treatment. However, regional differences in expression were noted for each subunit.

Distribution of glutamate receptor subunits in the primate temporal cortex and hippocampus.

The distribution of subunits for the N-methyl-D-aspartate (NR1, NR2A/B), alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (GluR1, GluR2/3, GluR4) and low affinity kainate (GluR5/6/7) ionotropic glutamate receptors was examined by immunocytochemistry in the temporal cortex and hippocampus of the rhesus macaque (Macaca mulatta). Neurons expressing NR1, NR2A/B, GluR2/3, and GluR4 subunits were widely distributed in all of the cortical layers but the overall density of the GluR4-immunopositive neurons was very low. Neurons expressing the GluR1 subunit were found predominantly in cortical layers V and VI while those expressing the GluR5/6/7 subunits were concentrated in layer V and were readily distinguishable by the thick elongate shape of their primary apical dendrites. Subcellular differences in the immunostaining pattern were also noted between the different glutamate receptor subunits. NR1 and NR2A/B immunoreactivity was most pronounced in somatic and primary dendritic compartments and to a lesser extent in cortical and hippocampal molecular layers. GluR1 immunoreactivity was more intense than GluR2/3 in the hippocampal molecular layers whereas GluR4 was undetectable. GluR5/6/7 immunoreactivity was very intense in the dentate molecular layer, and the CA1 pyramidal cells had a subcellular distribution of GluR5/6/7 that was similar to the cortical neurons. Overall, the distribution patterns of the different glutamate receptor subunits was identical in animals that had been ovariectomized and in ovariectomized animals that had subsequently undergone estradiol or estradiol/progesterone hormone replacement. Taken together, these findings demonstrate a differential spatial arrangement of glutamate receptor subunits in the primate temporal cortex and hippocampus, which may have functional significance for the integration of excitatory inputs to these areas. Furthermore, they show that in adult macaques, sex steroids do not play a major role in determining the distribution patterns of these receptor subunits.

Alpha-adrenergic receptor antagonism and N-methyl-D-aspartate (NMDA) induced luteinizing hormone release in female rhesus macaques.

The stimulatory influence of N-methyl-D-aspartate (NMDA), a glutamate receptor agonist, on LH secretion is well established in several mammalian species including the rhesus macaque. Although the mechanism of excitation appears to involve enhanced GnRH secretion, it is unclear whether the GnRH neurons respond directly to this excitation or whether stimulatory inter-neurons are involved. This study investigated the possibility that noradrenergic afferents play a major role in mediating the response of the primate hypothalamo-pituitary reproductive axis to NMDA. In situ hybridization histochemistry, using a cRNA probe coding for the NMDAR1 receptor subunit, revealed abundant mRNA in the locus coeruleus, a brain area rich in noradrenergic neurons. Furthermore, using double-label fluorescence immunocytochemistry, the tyrosine hydroxylase immunopositive neurons of the locus coeruleus showed immunoreactivity for the NMDAR1 receptor subunit protein. A second experiment examined whether prazosin, an alpha 1-adrenergic receptor antagonist, could attenuate NMDA-induced stimulation of LH release. Prazosin (either 1 or 5 mg/kg b.wt., i.v.) was administered to female rhesus macaques during the luteal phase of the menstrual cycle, 40 min before administration of NMDA (10 mg/kg b.wt., i.v.). Regardless of the prazosin pre-treatment, plasma LH concentrations showed a significant increase (P < 0.01) within 10 min of the administration of NMDA. Therefore, in spite of the evidence that at least some of the noradrenergic neurons of the primate hindbrain express the NMDAR1 receptor subunit, it is unlikely that noradrenergic inter-neuronal pathways alone play a major role in mediating the stimulatory action of NMDA on GnRH/LH secretion in primates. Indeed, because the GnRH neurons of the rhesus macaque are located diffusely in various regions of the hypothalamus and medial-septal/preoptic area, their net response to excitatory amino acids is likely to be more complicated, involving a combination of both stimulatory and inhibitory inter-neurons, and possibly also a direct interaction.

Ovarian steroid regulation of tryptophan hydroxylase mRNA expression in rhesus macaques.

Progesterone (P) stimulates prolactin secretion through an unknown neural mechanism in estrogen (E)-primed female monkeys. Serotonin is a stimulatory neurotransmitter in prolactin regulation, and this laboratory has shown previously that E induces progestin receptors (PR) in serotonin neurons. Therefore, we questioned whether E and/or E+P increased serotonin neural function. The expression of mRNA for tryptophan hydroxylase (TPH) was examined in ovariectomized (spayed) control, E-treated (28 d), and E+P-treated monkeys (14 d E and 14 d E+P) using in situ hybridization and a 249 bp TPH cRNA probe generated with RT-PCR (n = 5 animals/group). Densitometric analysis of film autoradiographs revealed a ninefold increase in TPH mRNA in E-treated macaques compared to spayed animals (p < 0.05). With supplemental P treatment, TPH mRNA signal was increased fivefold over spayed animals (p < 0.05), but was not significantly different compared to E-treated animals. These results were verified by grain counts from photographic emulsion-coated slides. There were significantly higher single-cell levels of TPH mRNA in serotonergic neurons of the dorsal raphe in E- and E+P-treated groups (p < 0.05). These data indicate that E induces TPH gene expression in nonhuman primates and that the addition of P has little additive effect on TPH gene expression. Thus, the action of P on prolactin secretion is probably not mediated at the level of TPH gene transcription. However, because P increases raphe serotonin content in E-primed rodents, the possibility remains that P may have other actions on post-translational processing or enzyme activity.