Normal cognitive aging is associated with deficits in memory processes dependent on the hippocampus, along with large-scale changes in the hippocampal expression of many genes. Histone acetylation can broadly influence gene expression and has been recently linked to learning and memory. We hypothesized that cAMP response element binding (CREB)-binding protein (CBP), a key histone acetyltransferase, may contribute to memory decline in normal aging. Here, we quantified CBP protein levels in the hippocampus of young, aged unimpaired and aged impaired rats, classified on the basis of spatial memory capacity documented in the Morris water maze. First, CBP-immunofluorescence was quantified across the principal cell layers of the hippocampus using both low and high resolution laser scanning imaging approaches. Second, digital images of CBP immunostaining were analyzed by a multi-purpose classifier algorithm (WND-CHARM) with validated sensitivity across many types of input materials. Finally, CBP protein levels in the principal subfields of the hippocampus were quantified by quantitative western blotting. CBP levels were equivalent as a function of age and cognitive status in all analyses. The sensitivity of the techniques used was substantial, sufficient to reveal differences across the principal cell fields of the hippocampus, and to correctly classify images from young and aged animals independent of CBP-immunoreactivity. The results are discussed in the context of recent evidence suggesting that CBP decreases may be most relevant in conditions of aging that, unlike normal cognitive aging, involve significant neuron loss.
epigenetics; memory; image analysis; stereology; quantitative microscopy
Preclinical studies in aged, surgically-menopausal rhesus monkeys have revealed powerful benefits of intermittent estrogen injections on prefrontal cortex-dependent working memory, together with corresponding effects on dendritic spine morphology in the prefrontal cortex. This contrasts with the inconsistent effects of hormone therapy (HT) reported in clinical studies in women. Factors contributing to this discrepancy could include differences in the formulation and sequence of HT regimens, resulting in different neurobiological outcomes. The current study evaluated, in aging surgically menopausal rhesus monkeys, the cognitive effects of four HT regimens modeled directly on human clinical practice, including continuous estrogen treatment opposed by progesterone. None of the regimens tested produced any cognitive effect, despite yielding physiologically relevant serum hormone levels, as intended. These findings have implications for the design of regimens that might optimize the benefits of hormone treatment for healthy aging, and suggest that common HT protocols used by women may fail to result in substantial cognitive benefit, at least via direct effects on the prefrontal cortex.
ovarian hormones; aging; macaque; learning; memory; prefrontal; temporal
Epigenetic regulation of gene expression plays an important role in learning and memory, mediating the influence of experience on critical mechanisms of plasticity. Speakers in the opening session of the Summit reviewed research on epigenetic contributions to age-related cognitive decline and discussed strategies for the development of interventions targeting epigenetic mechanisms. The presentations focused on experience-dependent DNA methylation, the regulatory role of microRNAs, histone deacetylases as potential therapeutic targets, and strategies for exploring epigenetic contributions to the aging of brain and cognition. This session established useful mileposts for gaging progress in this rapidly advancing area of research.
Cognitive changes; Epigenetics
Aged rhesus monkeys exhibit deficits in hippocampus-dependent memory, similar to aging humans. Here we explored the basis of cognitive decline by first testing young adult and aged monkeys on a standard recognition memory test (delayed nonmatching-to-sample test; DNMS). Next we quantified synaptic density and morphology in the hippocampal dentate gyrus (DG) outer (OML) and inner molecular layer (IML). Consistent with previous findings, aged monkeys were slow to learn DNMS initially, and they performed significantly worse than young subjects when challenged with longer retention intervals. Although OML and IML synaptic parameters failed to differ across the young and aged groups, the density of perforated synapses in the OML was coupled with recognition memory accuracy. Independent of chronological age, monkeys classified on the basis of menses data as peri/post-menopausal scored worse on DNMS, and displayed lower OML perforated synapse density, than pre-menopausal monkeys. These results suggest that naturally occurring reproductive senescence potently influences synaptic connectivity in the DG OML, contributing to individual differences in the course of normal cognitive aging.
delayed nonmatching-to-sample; disector method; estrogen; hippocampus; menopause; outer molecular layer; perforated synapse; post-synaptic density; recognition memory
Spatiotemporal and recognition memory are affected by aging in humans and macaque monkeys. To investigate whether these deficits are coupled with atrophy of memory-related brain regions, T1-weighted magnetic resonance images were acquired and volumes of the cerebrum, ventricles, prefrontal cortex (PFC), calcarine cortex, hippocampus, and striatum were quantified in young and aged rhesus monkeys. Subjects were tested on a spatiotemporal memory procedure (delayed response [DR]) that requires the integrity of the PFC and a medial temporal lobe-dependent recognition memory task (delayed nonmatching to sample [DNMS]). Region of interest analyses revealed that age inversely correlated with striatal, dorsolateral prefrontal cortex (dlPFC), and anterior cingulate cortex volumes. Hippocampal volume predicted acquisition of the DR task. Striatal volume correlated with DNMS acquisition, whereas total prefrontal gray matter, prefrontal white matter, and dlPFC volumes each predicted DNMS accuracy. A regional covariance analysis revealed that age-related volumetric changes could be captured in a distributed network that was coupled with declining performance across delays on the DNMS task. This volumetric analysis adds to growing evidence that cognitive aging in primates arises from region-specific morphometric alterations distributed across multiple memory-related brain systems, including subdivisions of the PFC.
age-related memory impairment; medial temporal lobe; MRI; prefrontal cortex; rhesus monkey
Rhesus monkeys provide a valuable model for studying the basis of cognitive aging because they are vulnerable to age-related decline in executive function and memory in a manner similar to humans. Some of the behavioral tasks sensitive to the effects of aging are the delayed response working memory test, recognition memory tests including the delayed nonmatching-to-sample and the delayed recognition span task, and tests of executive function including reversal learning and conceptual set-shifting task. Much effort has been directed toward discovering the neurobiological parameters that are coupled to individual differences in age-related cognitive decline. Area 46 of the dorsolateral prefrontal cortex (dlPFC) has been extensively studied for its critical role in executive function while the hippocampus and related cortical regions have been a major target of research for memory function. Some of the key age-related changes in area 46 include decreases in volume, microcolumn strength, synapse density, and α1- and α2-adrenergic receptor binding densities. All of these measures significantly correlate with cognitive scores. Interestingly, the critical synaptic subtypes associated with cognitive function appear to be different between the dlPFC and the hippocampus. For example, the dendritic spine subtype most critical to task acquisition and vulnerable to aging in area 46 is the thin spine, whereas in the dentate gyrus, the density of large mushroom spines with perforated synapses correlates with memory performance. This review summarizes age-related changes in anatomical, neuronal, and synaptic parameters within brain areas implicated in cognition and whether these changes are associated with cognitive decline.
Aging; Area 46; Dentate gyrus; Executive function; Recognition memory; Perforated synapse
Preclinical animal models have provided strong evidence that estrogen therapy (ET) enhances cognition and induces spinogenesis in neuronal circuits. However, clinical studies have been inconsistent, with some studies revealing adverse effects of ET, including an increased risk of dementia. In an effort to bridge this disconnect between the preclinical and clinical data, we have developed a non-human primate (NHP) model of ET combined with high-resolution dendritic spine analysis of dorsolateral prefrontal cortical (dlPFC) neurons. Previously, we reported cyclic ET in aged, ovariectomized NHPs increased spine density on dlPFC neurons. Here, we report that monkeys treated with cyclic E treatment paired with cyclic progesterone (P), continuous E combined with P (either cyclic or continuous), or unopposed continuous E failed to increase spines on dlPFC neurons. Given that the most prevalent form of ET prescribed to women is a combined and continuous E and P, these data bring into convergence the human neuropsychological findings and preclinical neurobiological evidence that standard hormone therapy in women is unlikely to yield the synaptic benefit presumed to underlie the cognitive enhancement reported in animal models.
Preclinical animal models have provided strong evidence that estrogen (E) therapy (ET) enhances cognition and induces spinogenesis in neuronal circuits. However, clinical studies have been inconsistent, with some studies revealing adverse effects of ET, including an increased risk of dementia. In an effort to bridge this disconnect between the preclinical and clinical data, we have developed a nonhuman primate (NHP) model of ET combined with high-resolution dendritic spine analysis of dorsolateral prefrontal cortical (dlPFC) neurons. Previously, we reported cyclic ET in aged, ovariectomized NHPs increased spine density on dlPFC neurons. Here, we report that monkeys treated with cyclic E treatment paired with cyclic progesterone (P), continuous E combined with P (either cyclic or continuous), or unopposed continuous E failed to increase spines on dlPFC neurons. Given that the most prevalent form of ET prescribed to women is a combined and continuous E and P, these data bring into convergence the human neuropsychological findings and preclinical neurobiological evidence that standard hormone therapy in women is unlikely to yield the synaptic benefit presumed to underlie the cognitive enhancement reported in animal models.
Rhesus monkeys provide a valuable model for studying the neurobiological basis of cognitive aging, because they are vulnerable to age-related memory decline in a manner similar to humans. In this study, young and aged monkeys were first tested on a well-characterized recognition memory test (delayed nonmatching-to-sample; DNMS). Then, electron microscopic immunocytochemistry was performed to determine the subcellular localization of two proteins in the hippocampal dentate gyrus (DG): the GluA2 subunit of the glutamate alpha-amino-3-hydroxyl-5-methyl-4-isoxazole propionate (AMPA) receptor and the atypical protein kinase C ζ isoform (PKMζ). PKMζ promotes memory storage by regulating GluA2-containing AMPA receptor trafficking. Thus, we examined whether the distribution of GluA2 and PKMζ is altered with aging in DG axospinous synapses and whether it is coupled with memory deficits. Monkeys with faster DNMS task acquisition and more accurate recognition memory exhibited higher proportions of dendritic spines coexpressing GluA2 and PKMζ. These double-labeled spines had larger synapses, as measured by postsynaptic density area, than single- and unlabeled spines. Within this population of double-labeled spines, aged monkeys compared to young expressed a lower density of synaptic GluA2 immunogold labeling, which correlated with lower recognition accuracy. Additionally, higher density of synaptic PKMζ labeling in double-labeled spines correlated with both faster task acquisition and better retention. Together, these findings suggest that age-related impairment in maintenance of GluA2 at the synapse in the primate hippocampus is coupled with memory deficits.
AMPA receptor; delayed nonmatching-to-sample test; GluR2; immunogold; PKMζ; recognition memory
Some patients experience enduring cognitive impairment after cancer treatment, a condition termed “chemofog”. Animal models allow assessment of chemotherapy effects on learning and memory per se, independent of changes due to cancer itself or associated health consequences such as depression. The present study examined the long-term learning and memory effects of a chemotherapy cocktail used widely in the treatment of breast cancer, consisting of 5-fluorouracil (5FU) and cyclophosphamide (CYP). Eighty 5-month old male F344 rats received contextual and cued fear conditioning before treatment with saline, or a low or high dose drug cocktail (50 mg/kg CYP and 75 mg/kg 5FU, or 75 mg/kg CYP and 120 mg/kg 5FU, i.p., respectively) every 30 days for 2 months. After a 2-month, no-drug recovery, both long-term retention and new task acquisition in the water maze and 14-unit T-maze were assessed. Neither dose of the CYP/5FU cocktail impaired retrograde fear memory despite marked toxicity documented by enduring weight loss and 50% mortality at the higher dose. Acquisition in the water maze and Stone maze was also normal relative to controls in rats treated with CYP/5FU. The results contribute to a growing literature suggesting that learning and memory mediated by the hippocampus can be relatively resistant to chemotherapy. Future investigation may need to focus on assessments of processing speed, executive function and attention, and the possible interactive contribution of cancer itself and aging to the post-treatment development of cognitive impairment.
Chemofog; Chemobrain; spatial memory; fear conditioning; cognition
Age-related memory impairment occurs in many mammalian species including humans. Moreover, women undergoing the menopausal transition often complain of problems with memory. We recently reported that rhesus monkeys display age- and menopause-related recognition memory impairment on a hippocampus-reliant test (delayed nonmatching-to-sample; DNMS). In the same monkeys, perforated synapse densities in the dentate gyrus outer molecular layer (OML) correlated with DNMS recognition accuracy, while total axospinous synapse density was similar across age and menses groups. The current study examined whether synaptic characteristics of OML axonal boutons are coupled with age- or menopause-related memory deficits. Using serial section electron microscopy, we measured the frequencies of single-synapse boutons (SSBs), multiple-synapse boutons (MSBs), and boutons with no apparent synaptic contacts (non-synaptic boutons, NSBs) in the OML. Aged females had double the percentage of NSBs as compared to young females and this measure correlated positively and inversely with DNMS acquisition (number of trials to criterion) and delay performance (average accuracy), respectively. Aged compared to young females also had a lower frequency of MSBs and a lower number of synaptic contacts per MSB, and the latter variable inversely correlated with DNMS acquisition. Although proportions of NSBs, SSBs and MSBs were similar across menses groups, compared to pre-menopausal monkeys, peri/post-menopausal monkeys had fewer MSBs contacting one or more segmented perforated synapse and the abundance of this bouton subtype positively correlated with DNMS performance. These results suggest that age- and menopause-related shifts in OML synaptic subtypes may be coupled with deficits in task acquisition and recognition memory.
delayed nonmatching-to-sample test; menopause; multiple-synapse bouton; serial sections; recognition memory
Mounting evidence linking epigenetic regulation to memory-related synaptic plasticity raises the possibility that altered chromatin modification dynamics might contribute to age-dependent cognitive decline. Here we show that the coordinated orchestration of both baseline and experience-dependent epigenetic regulation seen in the young adult hippocampus is lost in association with cognitive aging. Using a well-characterized rat model that reliably distinguishes aged individuals with significant memory impairment from others with normal memory, no single epigenetic mark or experience-dependent modification in the hippocampus uniquely predicted differences in the cognitive outcome of aging. The results instead point to a multivariate pattern in which modification-specific, bidirectional chromatin regulation is dependent on recent behavioral experience, chronological age, cognitive status, and hippocampal region. Whereas many epigenetic signatures were coupled with memory capacity among young adults and aged rats with preserved cognitive function, such associations were absent among aged rats with deficits in hippocampal memory. By comparison with the emphasis in current preclinical translational research on promoting chromatin modifications permissive for gene expression, our findings suggest that optimally successful hippocampal aging may hinge instead on enabling coordinated control across the epigenetic landscape.
In rat hippocampus, estrogen receptor-alpha (ER-α) can initiate non-genomic signaling mechanisms that modulate synaptic plasticity in response to either circulating or locally synthesized estradiol (E). Here we report quantitative electron microscopic data demonstrating that ER-α is present within excitatory synapses in dorsolateral prefrontal cortex (dlPFC) of young and aged ovariectomized female rhesus monkeys with and without E treatment. There were no treatment or age effects on the percentage of excitatory synapses containing ER-α, nor were there any group differences in distribution of ER-α within the synapse. However, the mean size of synapses containing ER-α was larger than unlabeled excitatory synapses. All monkeys were tested on delayed response (DR), a cognitive test of working memory that requires dlPFC. In young ovariectomized monkeys without E treatment, presynaptic ER-α correlated with DR accuracy across memory delays. In aged monkeys that received E treatment, ER-α within the postsynaptic density (30–60 nm from the synaptic membrane) positively correlated with DR performance. Thus, while the lack of group effects suggests that ER-α is primarily in synapses that are stable across age and treatment, synaptic abundance of ER-α is correlated with individual performance in two key age/treatment groups. These data have important implications for individual differences in the cognitive outcome among menopausal women and promote a focus on cortical estrogen receptors for therapeutic efficacy with respect to cognition.
estradiol; menopause; aging; cognition; dendritic spines; electron microscopy
Age-related impairments in hippocampus-dependent learning and memory tasks are not associated with a loss of hippocampal neurons, but may be related to alterations in synaptic integrity. Here we used stereological techniques to estimate spine number in hippocampal subfields using immunostaining for the spine-associated protein, spinophilin, as a marker. Quantification of the immunoreactive profiles was performed using the optical disector/fractionator technique. Aging was associated with a modest increase in spine number in the molecular layer of the dentate gyrus and CA1 stratum lacunosum-moleculare. By comparison, spinophilin protein levels in the hippocampus, measured by Western blot analysis, failed to differ as a function of age. Neither the morphological nor the protein level data were correlated with spatial learning ability across individual aged rats. The results extend current evidence on synaptic integrity in the aged brain, indicating that a substantial loss of dendritic spines and spinophilin protein in the hippocampus are unlikely to contribute to age-related impairment in spatial learning.
Hippocampus; Spinophilin; Rat; Aging; Morris water-maze; Learning; Memory; Stereology
A wide spectrum of outcomes in the cognitive effects of aging is routinely observed in studies of the elderly. Individual differences in neurocognitive aging are also a characteristic of other species, such as rodents and non-human primates. In particular, investigations at behavioral, brain systems, cellular and molecular levels of analysis have provided much information on the basis for individual differences in neurocognitive aging among healthy outbred rats. These findings are likely to be relevant to an understanding of the effects of aging on the brain, apart from neurodegenerative conditions, such as Alzheimer’s disease, which do not naturally occur in rodents. Here we review and integrate those findings in a model supporting the concept that certain features of cognitive decline are caused by distributed alterations in the medial temporal lobe, which alter the information processing functions of the hippocampal formation. An additional emerging concept from this research is that preserved abilities at older ages may depend on adaptive changes in the hippocampal system that distinguish successful aging.
spatial memory; hippocampal formation; successful aging; rats; cognitive impairment