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Overfeeding (OF) and obesity increase the risk for brain aging and neurodegenerative diseases due to increased oxidative stress and neuroinflammation, which likely contribute to cellular dysfunction. In contrast, caloric restriction (CR) is an intervention known for its effects on extending both life- and health-span. In the current study, the effects on the aging brain of two short-term feeding regimens, OF and CR, were investigated. We applied these diets for 12 weeks to both young and aged zebrafish. We performed protein and mRNA level analysis to examine diet-mediated effects on any potential age-related alterations in the brain. Markers implicated in the regulation of brain aging, cell cycle, proliferation, inflammation, and cytoskeleton were analyzed. The most prominent result observed was a downregulation in the expression levels of the stem cell marker, Sox2, in CR-fed animals as compared to OF-fed fish. Furthermore, our data highlighted significant age-related downregulations in Tp53, Myca, and L-plastin levels. The multivariate analyses of all datasets suggested that as opposed to OF, the adaptive mechanisms increasing lifespan via CR are likely exerting their effects by reinforcing the stem cell pool and downregulating inflammation. The data reveal important therapeutic targets with respect to the state of nutrient uptake for the slowing down of the detrimental effects of aging, resulting in a healthy and extended lifespan, as well as lowering the risk for neurodegenerative disease.

Increased caloric intake (OF) impairs quality of life causing comorbidities with other diseases and cog-nitive deficits, whereas dietary restriction (DR) increases healthspan by preventing age-related deteriora-tions. To understand the effects of these opposing dietary regimens on the cellular and synaptic dynamics during brain aging, the zebrafish model, which shows gradual aging like mammals, was utilized. Global changes in cellular and synaptic markers with respect to age and a 12 week dietary regimen of OF and DR demonstrated that aging reduces the levels of the glutamate receptor subunits, GLUR2/3, inhibitory synaptic clustering protein, GEP, synaptic vesicle protein, SYP, and early-differentiated neuronal marker, HuC. DR significantly elevates levels of glutamate receptor subunits, GLUR2/3, and NMDA clustering pro-tein, PSD95, levels, while OF subtly increases the level of the neuronal protein, DCAMKL1. These data suggest that decreased caloric intake within the context of aging has more robust effects on synapses than cellular proteins, whereas OF alters cellular dynamics. Thus, patterns like these should be taken into account for possible translation to human subjects. (c) 2021 Elsevier Inc. All rights reserved.

The zebrafish is a popular model organism that has been widely used in developmental biology. Due to its integrated nervous system with homologous structures to those of mammals and displays of similar age-related behavioral and neurobiological changes, it is invaluable for understanding the neuroscience of aging. Besides the advantages that stem from the nature of the zebrafish, several genetic and nongenetic gerontological and disease models are available for understanding the progression of age-related changes. These genetic models provide grounds to study accelerated/decelerated aging phenotypes and neurodegenerative diseases throughout the life span or within a specific time frame of development. Moreover, nongenetic interventions such as dietary restriction, drug applications, and environmental enrichment have been used with both wild-type or mutant strains as possible means of reversing age-related changes. This chapter focuses on the age-related alterations in cognitive, behavioral, and molecular domains in zebrafish, as well as existing models and antiaging interventions.

Understanding the principles underlying age-related changes in motion perception is paramount for improving the quality of life and health of older adults. However, the mechanisms underlying age-related alterations in this aspect of vision, which is essential for survival in a dynamic world, still remain unclear. Using optomotor responses to drifting gratings, we investigated age-related changes in motion detection of adult zebrafish (wild-type/AB-strain and achesb55/ thorn mutants with decreased levels of acetylcholinesterase). Our results pointed out negative optomotor responses that significantly depend on the spatial frequency and contrast level of stimulation, providing supporting evidence for the visual motion-driven aspect of this behavior mainly exhibited by adult zebrafish. Although there were no significant main effects of age and genotype, we found a significant three-way interaction between contrast level, age, and genotype. In the contrast domain, the changes in optomotor responses and thus in the detection of motion direction were ageand genotype-specific. Accordingly, these behavioral findings suggest a strong but complicated relationship between visual motion characteristics and the cholinergic system during neural aging. (c) 2020 Elsevier Inc. All rights reserved.

Intermittent fasting (IF) and its mimetic, rapamycin extend lifespan and healthspan through mechanisms that are not fully understood. We investigated different short-term durations of IF and rapamycin on cellular and molecular changes in the brains of young (6-10 months) and old (26-31 months) zebrafish. Interestingly, our results showed that IF significantly lowered glucose levels while increasing DCAMKL1 in both young and old animals. This proliferative effect of IF was supported by the upregulation of foxm1 transcript in old animals. Rapamycin did not change glucose levels in young and old animals but had differential effects depending on age. In young zebrafish, proliferating cell nuclear antigen and the LC3-II/LC3-I ratio was decreased, whereas glial fibrillary acidic protein and gephyrin were decreased in old animals. The changes in proliferative markers and a marker of autophagic flux suggest an age-dependent interplay between autophagy and cell proliferation. Additionally, changes in glia and inhibitory tone suggest a suppressive effect on neuroinflammation but may push the brain toward a more excitable state. Mammalian target of rapamycin (mTOR) activity in the brain following the IF and rapamycin treatment was differentially regulated by age. Interestingly, rapamycin inhibited mTOR more potently in young animals than IF. Principal component analysis supported our conclusion that the regulatory effects of IF and rapamycin were age-specific, since we observed different patterns in the expression levels and clustering of young and old animals. Taken together, our results suggest that even a short-term duration of IF and rapamycin have significant effects in the brain at young and old ages, and that these are age and treatment dependent.

Aging occurs due to a combination of several factors, such as telomere attrition, cellular senescence, and stem cell exhaustion. The telomere attrition-dependent cellular senescence is regulated by increased levels of SMAD specific E3 ubiquitin protein ligase 2 (smurf2). With age smurf2 expression increases and Smurf2 protein interacts with several regulatory proteins including, Smad7, Ep300, Yy1, Sirt1, Mdm2, and Tp53, likely affecting its function related to cellular aging. The current study aimed at analyzing smurf2 expression in the aged brain because of its potential regulatory roles in the cellular aging process. Zebrafish were used because like humans they age gradually and their genome has 70% similarity. In the current study, we demonstrated that smurf2 gene and protein expression levels altered in a region-specific manner during the aging process. Also, in both young and old brains, Smurf2 protein was enriched in the cytosol. These results imply that during aging Smurf2 is regulated by several mechanisms including post-translational modifications (PTMs) and complex formation. Also, the expression levels of its interacting partners defined by the STRING database, tp53, mdm2, ep300a, yy1a, smad7, and sirt1, were analyzed. Multivariate analysis indicated that smurf2, ep300a, and sirt1, whose proteins regulate ubiquitination, acetylation, and deacetylation of target proteins including Smad7 and Tp53, showed age- and brain region-dependent patterns. Our data suggest a likely balance between Smurf2- and Mdm2-mediated ubiquitination, and Ep300a-mediated acetylation/Sirt1-mediated deacetylation, which most possibly affects the functionality of other interacting partners in regulating cellular and synaptic aging and ultimately cognitive dysfunction. (C) 2020 IBRO. Published by Elsevier Ltd. All rights reserved.

In this study, we designed and developed a novel low-cost system for anesthetizing and injecting adult zebrafish. The system utilizes a gradual cooling method for the anesthesia and maintains the fish in a stable anesthetic plane, as well as stabilizes the animal so that intraperitoneal injections can be consistently performed. It is a system that any laboratory with access to a workshop can build for their group. Moreover, it is a safe system for researchers, as well as a reliable one for repeated experiments since multiple fish can be injected quickly and there is little physical contact necessary between the investigator and the animal. This will likely reduce any unnecessary stress in the fish, as compared with manual methods of injection. Finally, the system is adaptable so that as the investigators' procedural needs change due to different research questions, that is, gradual rewarming or something of that nature, it could be modified. This work was supported by a European Molecular Biology Organization (EMBO) Installation Grant to Michelle M. Adams.

Progression of cognitive decline with or without neurodegeneration varies among elderly subjects. The main aim of the current study was to illuminate the molecular mechanisms that promote and retain successful aging in the context of factors such as environment and gender, both of which alter the resilience of the aging brain. Environmental enrichment (EE) is one intervention that may lead to the maintenance of cognitive processing at older ages in both humans and animal subjects. EE is easily applied to different model organisms, including zebrafish, which show similar age-related molecular and behavioral changes as humans. Global changes in cellular and synaptic markers with respect to age, gender and 4-weeks of EE applied with sensory stimulation were investigated using the zebrafish model organism. Results indicated that EE increases brain weight in an age-dependent manner without affecting general body parameters like body mass index (BMI). Age-related declines in the presynaptic protein synaptophysin, AMPA-type glutamate receptor subunits and a post-mitotic neuronal marker were observed and short-term EE prevents these changes in aged animals, as well as elevates levels of the inhibitory scaffolding protein, gephyrin. Gender-driven alterations were observed in the levels of the glutamate receptor subunits. Oxidative stress markers were significantly increased in the old animals, while exposure to EE did not alter this pattern. These data suggest that EE with sensory stimulation exerts its effects mainly on age-related changes in synaptic dynamics, which likely increase brain resilience through specific cellular mechanisms.

Brain aging is a complex process, which involves multiple pathways including various components from cellular to molecular. This study aimed to investigate the gene expression changes in zebrafish brains through young-adult to adult, and adult to old age. RNA sequencing was performed on isolated neuronal cells from zebrafish brains. The cells were enriched in progenitor cell markers, which are known to diminish throughout the aging process. We found 176 statistically significant, differentially expressed genes among the groups, and identified a group of genes based on gene ontology descriptions, which were classified as cell adhesion molecules. The relevance of these genes was further tested in another set of zebrafish brains, human healthy, and Alzheimer's disease brain samples, as well as in Allen Brain Atlas data. We observed that the expression change of 2 genes, GJC2 and ALCAM, during the aging process was consistent in all experimental sets. Our findings provide a new set of markers for healthy brain aging and suggest new targets for therapeutic approaches to neurodegenerative diseases. (C) 2020 Elsevier Inc. All rights reserved.

Brain aging is marked by a decline in cognitive abilities and associated with neurodegenerative disorders. Recent studies have shown, neurogenesis continues into adulthood but is known to be decreasing during advancing age and these changes may contribute to cognitive alterations. Advances, which aim to promote better aging are of paramount importance. Dietary restriction (DR) is the only non-genetic intervention that reliably extends life and health-span. Mechanism's of how and why DR and age affect neurogenesis are not well-understood, and have not been utilized much in the zebrafish, which has become a popular model to study brain aging and neurodegenerative disease due to widely available genetic tools. In this study we used young (8-8.5 months) and old (26-32.5 months) zebrafish as the model to investigate the effects of a short-term DR on actively proliferating cells. We successfully applied a 10-week DR to young and old fish, which resulted in a significant loss of body weight in both groups with no effect on normal age-related changes in body growth. We found that age decreased cell proliferation and increased senescence associated beta-galactosidase, as well as shortened telomere lengths. In contrast, DR shortened telomere lengths only in young animals. Neither age nor DR changed the differentiation patterns of glial cells. Our results suggest that the potential effects of DR could be mediated by telomere regulation and whether these are beneficial or negative remains to be determined. (C) 2016 IBRO. Published by Elsevier Ltd. All rights reserved.

Chapter 1 Across the world, the aging population is expanding due to an increasing average life expectancy. The percentage of elderly over the age of 65 is expected to be more than 15% of the total world population by 2025. As the lifespan increases, there will be a need for maintaining a healthy state for these individuals. Our current knowledge on types and durations of potential anti-aging therapies is quite limited. Recently the zebrafish has emerged as a promising model for understanding the cognitive and neurobiological changes during aging, as well as its use with potential anti-aging interventions. Like humans this model organism ages gradually, displays similar behavioral properties and social characteristics, and in addition, there is a wealth of molecular and genetic tools to uncover the cellular mechanism that contribute to age-related cognitive declines. Drug effect and toxicity can be easily tested in the zebrafish. Therefore, this animal model can provide information about potential therapies that could be translated directly into human populations or provide a more focused treatment direction for testing in other mammalian animal models. The zebrafish will be a powerful tool for uncovering the mysteries of the aging brain.

The zebrafish has become a popular model for studying normal brain aging due to its large fecundity, conserved genome, and available genetic tools; but little data exists about neurobiological age-related alterations. The current study tested the hypothesis of an association between brain aging and synaptic protein loss across males and females. Western blot analysis of synaptophysin (SYP), a presynaptic vesicle protein, and postsynaptic density-95 (PSD-95) and gephyrin (GEP), excitatory and inhibitory postsynaptic receptor-clustering proteins, respectively, was performed in young, middle-aged, and old male and female zebrafish (Danio rerio) brains. Univariate and multivariate analyses demonstrated that PSD-95 significantly increased in aged females and SYP significantly decreased in males, but GEP was stable. Thus, these key synaptic proteins vary across age in a sexually dimorphic manner, which has been observed in other species, and these consequences may represent selective vulnerabilities for aged males and females. These data expand our knowledge of normal aging in zebrafish, as well as further establish this model as an appropriate one for examining human brain aging.

WOS: 000415310800007 PubMed ID: 29144972 Science is ideally suited to connect people from different cultures and thereby foster mutual understanding. To promote international life science collaboration, we have launched "The Science Bridge'' initiative. Our current project focuses on partnership between Western and Middle Eastern neuroscience communities. Medical Research Council [MC_UP_1202/5]

Age-related memory impairments show a progressive decline across lifespan. Studies have demonstrated equivocal results in biological and behavioral outcomes of aging. Thus, in the present study we examined the novel object recognition task at a delay period that has been shown to be impaired in aged rats of two different strains. Moreover, we used a strain of rats, Fisher 344XBrown Norway, which have published age-related biological changes in the brain. Young (10 month old) and aged (28 month old) rats were tested on a standard novel object recognition task with a 50-minute delay period. The data showed that young and aged rats in the strain we used performed equally well on the novel object recognition task and that both young and old rats demonstrated a righthanded side preference for the novel object. Our data suggested that novel object recognition is not impaired in aged rats although both young and old rats have a demonstrated side preference. Thus, it may be that genetic differences across strains contribute to the equivocal results in behavior, and genetic variance likely influences the course of cognitive aging.

Chapter 11 Normal aging is accompanied by cognitive impairment with subtle cellular and molecular changes in the brain, whereas, pathological brain aging manifests as severe behavioral impairments with cellular pathology. Understanding the factors that contribute to both states is undoubtedly important for determining appropriate interventions that alter their progression. Mammalian target of rapamycin (mTOR) signaling has been implicated in affecting lifespan and age-related diseases such as cancer. The relationship of mTOR signaling with pathological brain aging has been more extensively studied, whereas the association with normal brain aging is not well understood. In this chapter we present information about normal and pathological brain aging, the relationship with mTOR signaling and use information from other age-related diseases to suggest that mTOR may have a role in promoting the cellular and molecular changes that underlie age-related cognitive changes. Future work should be directed towards understanding the precise role of mTOR signaling in brain aging. © 2016 Elsevier Inc. All rights reserved.

Using fluorescent indicators and several serum albumins, a sensing system for fatty acids was developed. Fatty acids were able to be differentiated based on their carbon chain length and degree of unsaturation. A sensing ensemble was then applied to study complex mixtures of triglycerides, namely, edible oils. The oils, with different fatty acid compositions, were successfully differentiated using principal component analysis.

The saponin fraction QS-21 from Quillaja saponaria has been demonstrated to be a potent immunological adjuvant when mixed with keyhole limpet hemocyanin conjugate vaccines, as well as with other classes of subunit antigen vaccines. QS-21 adjuvant is composed of two isomers that include the apiose and xylose forms in a ratio of 65:35, respectively. The chemical syntheses of these two isomers in pure form have recently been disclosed. Herein we describe detailed in vivo immunological evaluations of these synthetic QS-21 isomeric constituents, employing the GD3-KLH melanoma antigen. With this vaccine construct, high antibody titers against GD3 ganglioside and KLH were elicited when GD3-KLH was co-administered with adjuvant, either as the individual separate synthetic QS-21 isomers (SQS-21-Api or SQS-21-Xyl), or as its reconstituted 65:35 isomeric mixture (SQS-21). These antibody titer levels were comparable to that elicited by vaccinations employing naturally derived QS-21 (PQS-21). Moreover, toxicities of the synthetic saponin adjuvants were also found to be comparable to that of naturally derived PQS-21. These findings demonstrate unequivocally that the adjuvant activity of QS-21 resides in these two principal isomeric forms, and not in trace contaminants within the natural extracts. This lays the foundation for future exploration of structure-function correlations to enable the discovery of novel saponins with increased potency, enhanced stability, and attenuated toxicity.

The success of antitumor and antiviral vaccines often requires the use of an adjuvant, a substance that significantly enhances the immune response to a co-administered antigen. Only a handful of adjuvants have both sufficient potency and acceptable toxicity for clinical investigation. One promising adjuvant is QS-21, a saponin natural product that is the immunopotentiator of choice in many cancer and infectious disease vaccine clinical trials. However, the therapeutic promise of QS-21 adjuvant is curtailed by several factors, including its scarcity, difficulty in purification to homogeneity, dose-limiting toxicity, and chemical instability. Here we report the design, synthesis, and evaluation of chemically stable synthetic saponins. These novel, amide-modified, non-natural substances exhibit immunopotentiating effects in vivo that rival or exceed that of QS-21 in evaluations with the GD3-KLH melanoma conjugate vaccine. The highly convergent synthetic preparation of these novel saponins establishes new avenues for discovering improved molecular adjuvants for specifically tailored vaccine therapies.

Background WDR81 (WD repeat-containing protein 81) is associated with cerebellar ataxia, mental retardation and disequilibrium syndrome (CAMRQ2, [MIM 610185]). Human and mouse studies suggest that it might be a gene of importance during neurodevelopment. This study aimed at fully characterizing the structure of the wdr81 transcript, detecting the possible transcript variants and revealing its expression profile in zebrafish, a powerful model organism for studying development and disease. Results As expected in human and mouse orthologous proteins, zebrafish wdr81 is predicted to possess a BEACH (Beige and Chediak-Higashi) domain, a major facilitator superfamily domain and WD40-repeats, which indicates a conserved function in these species. We observed that zebrafish wdr81 encodes one open reading frame while the transcript has one 5′ untranslated region (UTR) and the prediction of the 3′ UTR was mainly confirmed along with a detected insertion site in the embryo and adult brain. This insertion site was also found in testis, heart, liver, eye, tail and muscle, however, there was no amplicon in kidney, intestine and gills, which might be the result of possible alternative polyadenylation processes among tissues. The 5 and 18 hpf were critical timepoints of development regarding wdr81 expression. Furthermore, the signal of the RNA probe was stronger in the eye and brain at 18 and 48 hpf, then decreased at 72 hpf. Finally, expression of wdr81 was detected in the adult brain and eye tissues, including but not restricted to photoreceptors of the retina, presumptive Purkinje cells and some neurogenic brains regions. Conclusions Taken together these data emphasize the importance of this gene during neurodevelopment and a possible role for neuronal proliferation. Our data provide a basis for further studies to fully understand the function of wdr81. Electronic supplementary material The online version of this article (doi:10.1186/s12868-015-0229-4) contains supplementary material, which is available to authorized users.

Insulin-like growth factor-1 (IGF-1) and growth hormone play a major role in the growth and development of tissues throughout the mammalian body. Plasma IGF-1 concentrations peak during puberty and decline with age. We have determined that chronic treatments to restore plasma IGF-1 concentrations to adult levels attenuate spatial learning deficits in aged rats, but little is known of the acute actions of IGF-1 in the brain. To this end, we utilized hippocampal slices from young Sprague-Dawley rats to characterize the acute effects of des-IGF-1 on excitatory synaptic transmission in the CA1 region. We observed a 40% increase in field excitatory postsynaptic potential (fEPSP) slope with application of des-IGF-1 (40 ng/ml) and used whole cell patch-clamp recordings to determine that this enhancement was due to a postsynaptic mechanism involving α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) but not N-methyl-d-aspartate receptors. Furthermore, the enhancement was completely blocked by the broad-spectrum tyrosine kinase inhibitor, genistein (220 μM), and significantly reduced by the PI3K blockers wortmannin (1 μM) and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (10 μM), suggesting that the effect was predominantly dependent on PI3K activation. This characterization of the acute actions of des-IGF-1 at hippocampal excitatory synapses may provide insight into the mechanism by which long-term increases in plasma IGF-1 impart cognitive benefits in aged rats. Increases in AMPA receptor-mediated synaptic transmission may contribute directly to cognitive improvement or initiate long-term changes in synthesis of proteins such as brain-derived neurotrophic factor that are important to learning and memory.

Increasing data suggest that glutamate might act as a cell-signaling molecule in non-neuronal tissues such as the skin. Here we demonstrate the presence of functional N-methyl-D-aspartate (NMDA)-type glutamate receptors in human keratinocytes. NMDA receptor expression strongly reflects the degree of cell-to-cell contact. Wounding polarizes the expression of NMDA receptors in keratinocytes involved in re-epithelialization, and the process of re-epithelialization is inhibited by NMDA receptor activation. We also demonstrate that squamous cell carcinomas lack NMDA receptors. Our data suggest that Ca2+ entry through NMDA receptors influences the cycle of keratinocyte proliferation, differentiation, and migration during epithelialization. Moreover, NMDA receptor activation might play a role in contact-mediated inhibition of growth, a process that is absent during neoplastic pathology. This receptor may serve as a pharmacological target for modulating keratinocyte behavior and treating cutaneous disorders.

Estrogen interacts with N-methyl-D-aspartate (NMDA) receptors to regulate multiple aspects of morphological and functional plasticity. In hippocampus, estrogen increases both dendritic spine density and synapse number, and NMDA antagonists block these effects. Thus, estrogen-mediated hippocampal plasticity may be of particular importance in the context of age-related changes in endocrine status and cognitive performance. NR1 levels per synapse are increased in CA1 by estrogen in aged rats but not young rats, although no information is available on estrogen-induced synaptic alterations in other NMDA receptor subunits that might impact function. Therefore, the present study was designed to investigate the effect of estrogen on the synaptic and subsynaptic distributions of the NMDA receptor subunits, NR2A and NR2B in CA1 pyramidal cells, within the context of aging. Our results demonstrated that the overall synaptic levels of NR2A and NR2B are similar in young and aged female rats, regardless of estrogen treatment. However, in the aged CA1, estrogen restores NR2B levels back to young levels in the lateral portions of the active synaptic zone. Thus, estrogen may impact the mobility of NMDA receptors across the synapse and, in the process, restore a more youthful synaptic profile. These findings have important implications for the mechanism of estrogen-induced alterations in NMDA receptor-mediated processes, particularly in the context of aging. J. Comp. Neurol. 474:419–426, 2004. © 2004 Wiley-Liss, Inc.

The ramifications of endocrine and neural senescence converge in the hippocampus, particularly with respect to glutamatergic synapses. In this review, we will focus on current literature suggesting that potential synaptic alterations induced by estrogen in the hippocampus are mediated through interactions between ER-alpha and NMDA receptors. In addition, we will examine the data suggesting that these interactions may be uncoupled with aging. These studies demonstrate that while estrogen helps retain a youthful synaptic phenotype by some measures, the aged synapse may differ from the young synapse in several key respects that impact plasticity in general, and endocrine influences on the synapse, in particular.

Studies in experimental animals and humans have stressed the role of the cerebellum in motor skill learning. Yet, the relative importance of the cerebellar cortex and deep nuclei, as well as the nature of the dynamic functional changes occurring between these and other motor-related structures during learning, remains in dispute. Using functional magnetic resonance imaging and a motor sequence learning paradigm in humans, we found evidence of an experience-dependent shift of activation from the cerebellar cortex to the dentate nucleus during early learning, and from a cerebellar–cortical to a striatal–cortical network with extended practice. The results indicate that intrinsic modulation within the cerebellum, in concert with activation of motor-related cortical regions, serves to set up a procedurally acquired sequence of movements that is then maintained elsewhere in the brain.

Estrogens and N-methyl-D-aspartate (NMDA) receptors regulate multiple aspects of morphological and functional plasticity in young animals. For example, estrogens increase spine density in the hippocampus, and NMDA antagonists block these effects. Few studies have examined the effects of age, postovariectomy interval, and duration of estrogen replacement in the hippocampus and more specifically on NMDA receptor subunits. Therefore, the present study was designed to investigate the effects of short- and long-term estrogen replacement or deprivation on mRNA levels of three NMDA receptor subunits, NR1, NR2A, and NR2B, in the hippocampus of aging female Sprague-Dawley rats. Young (3- to 4-month-old) and middle-aged (12- to 13-month-old) rats were ovariectomized for 1 month and then treated with estrogen or vehicle for either 2 days or 2 weeks. Another set of middle-aged and aged (24-to 25-month-old) animals were ovariectomized for 6 months and treated with estrogen or vehicle for 2 days or 2 weeks. RNase protection assay was used to assess changes in the NMDA receptor subunit mRNA levels. Our results demonstrated significant effects of age and length of ovariectomy on NMDA receptor mRNA levels, with little effect of the estrogen status of the animals on these parameters. The largest effect was seen for the length of the postovariectomy interval, with the results demonstrating that rats with a short-term ovariectomy have substantially higher NMDA receptor subunit mRNA levels than animals with long-term ovariectomy. The most dramatic effects of aging were seen for NR1 and NR2B mRNAs in ventral hippocampus, with large age-related increases. These data suggest that age and duration of ovariectomy impact NMDA receptor mRNA levels in the hippocampus, potentially affecting the stoichiometry and/or function of these receptors. These findings have important implications for postmenopausal or hysterectomy/oophorectomy estrogen depletion and replacement in humans.

Hippocampal N-methyl-D-Aspartate (NMDA) receptors mediate mechanisms of cellular plasticity critical for spatial learning in rats. The present study examined the relationship between spatial learning and NMDA receptor expression in discrete neuronal populations, as well as the degree to which putative age-related changes in NMDA receptors are coupled to the effects of normal aging on spatial learning. Young and aged Long-Evans rats were tested in a Morris water maze task that depends on the integrity of the hippocampus. Levels of NR1, the obligatory subunit for a functional NMDA receptor, were subsequently quantified both biochemically by Western blot in whole homogenized hippocampus, and immunocytochemically by using a high-resolution confocal laser scanning microscopy method. The latter approach allowed comprehensive, regional analysis of discrete elements of excitatory hippocampal circuitry. Neither method revealed global changes, nor were there region-specific differences in hippocampal NR1 levels between young and aged animals. However, across all subjects, individual differences in spatial learning ability correlated with NR1 immunofluorescence levels selectively in CA3 neurons of the hippocampus. Parallel confocal microscopic analysis of the GluR2 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA) receptor failed to reveal reliable differences as a function of age or spatial learning ability. This analysis linking age, performance, and NR1 levels demonstrates that although dendritic NR1 is generally preserved in the aged rat hippocampus, levels of this receptor subunit in selective elements of hippocampal circuitry are linked to spatial learning. These findings suggest that NMDA receptor abundance in CA3 bears a critical relationship to learning mediated by the hippocampus throughout the life span. J. Comp. Neurol. 432:230‐243, 2001. © 2001 Wiley-Liss, Inc.

The present study examined the long-standing concept that changes in hippocampal circuitry contribute to age-related learning impairment. Individual differences in spatial learning were documented in young and aged Long-Evans rats by using a hippocampal-dependent version of the Morris water maze. Postmortem analysis used a confocal laser-scanning microscopy method to quantify changes in immunofluorescence staining for the presynaptic vesicle glycoprotein, synaptophysin (SYN), in the principal relays of hippocampal circuitry. Comparisons based on chronological age alone failed to reveal a reliable difference in the intensity of SYN staining in any region that was examined. In contrast, aged subjects with spatial learning deficits displayed significant reductions in SYN immunoreactivity in CA3 lacunosum-moleculare (LM) relative to either young controls or age-matched rats with preserved learning. SYN intensity values for the latter groups were indistinguishable. In addition, individual differences in spatial learning capacity among the aged rats correlated with levels of SYN staining selectively in three regions: outer and middle portions of the dentate gyrus molecular layer and CA3-LM. The cross-sectional area of SYN labeling, by comparison, was not reliably affected in relation cognitive status. These findings are the first to demonstrate that a circuit-specific pattern of variability in the connectional organization of the hippocampus is coupled to individual differences in the cognitive outcome of normal aging. The regional specificity of these effects suggests that a decline in the fidelity of input to the hippocampus from the entorhinal cortex may play a critical role.

We investigated the patterns of projections from the pulvinar to visual areas V1, V2, V4, and MT, and their relationships to pulvinar subdivisions based on patterns of calbindin (CB) immunostaining and estimates of visual field maps (P(1), P(2) and P(3)). Multiple retrograde tracers were placed into V1, V2, V4, and/or MT in 11 adult macaque monkeys. The inferior pulvinar (PI) was subdivided into medial (PI(M)), posterior (PI(P)), central medial (PI(CM)), and central lateral (PI(CL)) regions, confirming earlier CB studies. The P(1) map includes PI(CL) and the ventromedial portion of the lateral pulvinar (PL), P(2) is found in ventrolateral PL, and P(3) includes PI(P), PI(M), and PI(CM). Projections to areas V1 and V2 were found to be overlapping in P(1) and P(2), but those from P(2) to V2 were denser than those to V1. V2 also received light projections from PI(CM) and, less reliably, from PI(M). Neurons projecting to V4 and MT were more abundant than those projecting to V1 and V2. Those projecting to V4 were observed in P(1), densely in P(2), and also in PI(CM) and PI(P) of P(3). Those projecting to MT were found in P(1)- P(3), with the heaviest projection from P(3). Projections from P(3) to MT and V4 were mainly interdigitated, with the densest to MT arising from PI(M) and the densest to V4 arising from PI(P) and PI(CM). Because the calbindin-rich and -poor regions of P(3) corresponded to differential patterns of cortical connectivity, the results suggest that CB may further delineate functional subdivisions in the pulvinar.

Behavioral and neurophysiological studies suggest that skill learning can be mediated by discrete, experience-driven changes within specific neural representations subserving the performance of the trained task. We have shown that a few minutes of daily practice on a sequential finger opposition task induced large, incremental performance gains over a few weeks of training. These gains did not generalize to the contralateral hand nor to a matched sequence of identical component movements, suggesting that a lateralized representation of the learned sequence of movements evolved through practice. This interpretation was supported by functional MRI data showing that a more extensive representation of the trained sequence emerged in primary motor cortex after 3 weeks of training. The imaging data, however, also indicated important changes occurring in primary motor cortex during the initial scanning sessions, which we proposed may reflect the setting up of a task-specific motor processing routine. Here we provide behavioral and functional MRI data on experience-dependent changes induced by a limited amount of repetitions within the first imaging session. We show that this limited training experience can be sufficient to trigger performance gains that require time to become evident. We propose that skilled motor performance is acquired in several stages: “fast” learning, an initial, within-session improvement phase, followed by a period of consolidation of several hours duration, and then “slow” learning, consisting of delayed, incremental gains in performance emerging after continued practice. This time course may reflect basic mechanisms of neuronal plasticity in the adult brain that subserve the acquisition and retention of many different skills.

Previous immunohistochemical studies combined with retrograde tracing in macaque monkeys have demonstrated that corticocortical projections can be differentiated by their content of neurofilament protein. The present study analyzed the distribution of nonphosphorylated neurofilament protein in callosally projecting neurons located at the V1/V2 border. All of the retrogradely labeled neurons were located in layer III at the V1/V2 border and at an immediately adjacent zone of area V2. A quantitative analysis showed that the vast majority (almost 95%) of these interhemispheric projection neurons contain neurofilament protein immunoreactivity. This observation differs from data obtained in other sets of callosal connections, including homotypical interhemispheric projections in the prefrontal, temporal, and parietal association cortices, that were found to contain uniformly low proportions of neurofilament protein-immunoreactive neurons. Comparably, highly variable proportions of neurofilament protein-containing neurons have been reported in intrahemispheric corticocortical pathways, including feedforward and feedback visual connections. These results indicate that neurofilament protein is a prominent neurochemical feature that identifies a particular population of interhemispheric projection neurons at the V1/V2 border, and suggest that this biochemical attribute may be critical for the function of this subset of callosal neurons.

Cataloged from PDF version of article. In rats, as in humans, normal aging is characterized by a decline in hippocampal-dependent learning and memory, as well as in glutamatergic function. Both growth hormone (GH) and insulin-like growth factor-I (IGF-I) levels have been reported to decrease with age, and treatment with either GH or IGF-I can ameliorate age-related cognitive decline. Interestingly, acute GH and IGF-I treatments enhance glutamatergic synaptic transmission in the rat hippocampus of juvenile animals. However, whether this enhancement also occurs in old rats, when cognitive impairment is ameliorated by GH and IGF-I (des-IGF-I), remains to be determined. To address this issue, we used an in vitro CA1 hippocampal slice preparation and extracellular recording techniques to study the effects of acute application of GH and IGF-I on compound field excitatory postsynaptic potentials (fEPSPs), as well as AMPA- and NMDA-dependent fEPSPs, in young adult (10 months) and old (28 months) rats. The results indicated that both GH and IGF-I increased compound-, AMPA-and NMDA-dependent fEPSPs to a similar extent in slices from both age groups and that this augmentation was likely mediated via a postsynaptic mechanism. Initial characterization of the signaling cascades underlying these effects revealed that the GH-induced enhancement was not mediated by the JAK2 signaling element in either young adult or old rats but that the IGF-Iinduced enhancement involved a PI3K-mediated mechanism in old, but not young adults. The present findings are consistent with a role for a GH-or IGF-I-induced enhancement of glutamatergic transmission in mitigating age-related cognitive impairment in old rats. © 2012 American Aging Association.

Previous studies of the primate cerebral cortex have shown that neurofilament protein is present in pyramidal neuron subpopulations displaying specific regional and laminar distribution patterns. In order to characterize further the neurochemical phenotype of the neurons furnishing feedforward and feedback pathways in the visual cortex of the macaque monkey, we performed an analysis of the distribution of neurofilament protein in corticocortical projection neurons in areas V1, V2, V3, V3A, V4, and MT. Injections of the retrogradely transported dyes Fast Blue and Diamidino Yellow were placed within areas V4 and MT, or in areas V1 and V2, in 14 adult rhesus monkeys, and the brains of these animals were processed for immunohistochemistry with an antibody to nonphosphorylated epitopes of the medium and heavy molecular weight subunits of the neurofilament protein. Overall, there was a higher proportion of neurons projecting from areas V1, V2, V3, and V3A to area MT that were neurofilament protein-immunoreactive (57-100%), than to area V4 (25-36%). In contrast, feedback projections from areas MT, V4, and V3 exhibited a more consistent proportion of neurofilament protein-containing neurons (70-80%), regardless of their target areas (V1 or V2). In addition, the vast majority of feedback neurons projecting to areas V1 and V2 were located in layers V and VI in areas V4 and MT, while they were observed in both supragranular and infragranular layers in area V3. The laminar distribution of feedforward projecting neurons was heterogeneous. In area V1, Meynert and layer IVB cells were found to project to area MT, while neurons projecting to area V4 were particularly dense in layer III within the foveal representation. In area V2, almost all neurons projecting to areas MT or V4 were located in layer III, whereas they were found in both layers II-III and V-VI in areas V3 and V3A. These results suggest that neurofilament protein identifies particular subpopulations of corticocortically projecting neurons with distinct regional and laminar distribution in the monkey visual system. It is possible that the preferential distribution of neurofilament protein within feedforward connections to area MT and all feedback projections is related to other distinctive properties of these corticocortical projection neurons.

Cataloged from PDF version of article. Alterations in the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPA-R) and N-methyl-D-aspartate receptor (NMDA-R) have been documented in aged animals and may contribute to changes in hippocampal-dependent memory. Growth hormone (GH) regulates AMPA-R and NMDA-R-dependent excitatory transmission and decreases with age. Chronic GH treatment mitigates age-related cognitive decline. An in vitro CA1 hippocampal slice preparation was used to compare hippocampal excitatory transmission and plasticity in old animals treated for 6–8 months with either saline or GH. Our findings indicate that GH treatment restores NMDA-Rdependent basal synaptic transmission in old rats to young adult levels and enhances both AMPA-R-dependent basal synaptic transmission and long-term potentiation. These alterations in synaptic function occurred in the absence of changes in presynaptic function, as measured by paired-pulse ratios, the total protein levels of AMPA-R and NMDA-R subunits or in plasma or hippocampal levels of insulin-like growth factor-I. These data suggest a direct role for GH in altering age-related changes in excitatory transmission and provide a possible cellular mechanism through which GH changes the course of cognitive decline. © 2012 Elsevier Inc. All rights reserved.

QS-21 is a potent immunostimulatory saponin that is currently under clinical investigation as an adjuvant in various vaccines to treat infectious diseases, cancers, and cognitive disorders. Herein, we report the design, synthesis, and preclinical evaluation of simplified QS-21 congeners to define key structural features that are critical for adjuvant activity. Truncation of the linear tetrasaccharide domain revealed that a trisaccharide variant is equipotent to QS-21, while the corresponding disaccharide and monosaccharide congeners are more toxic and less potent, respectively. Modification of the acyl chain domain in the trisaccharide series revealed that a terminal carboxylic acid is well-tolerated while a terminal amine results in reduced adjuvant activity. Acylation of the terminal amine can, in some cases, restore adjuvant activity and enables the synthesis of fluorescently labeled QS-21 variants. Cellular studies with these probes revealed that, contrary to conventional wisdom, the most highly adjuvant active of these fluorescently labeled saponins does not simply associate with the plasma membrane, but rather is internalized by dendritic cells.

The aim of this chapter is to introduce the reader to differential sensing, to detail the history of the electronic nose and tongue that led to the development of differential sensing, and to provide a spectrum of examples where differential sensing has been employed to classify and/or detect single analytes and mixtures of analytes. Using the information presented in this chapter, we hope to provide a solid background for the reader so that the differential sensing approach can be extrapolated to any desired chemical sensing purpose. Keywords: differential sensing; array sensing; pattern recognition; sensing; electronic nose and tongue; amino acids; nucleotides; peptides; proteins; ion mixtures; beverages; biomolecules

Caloric restriction (CR) is a reduction of total caloric intake without a decrease in micronutrients or a disproportionate reduction of any one dietary component. While CR attenuates age-related cognitive deficits in tasks of hippocampal-dependent memory, the cellular mechanisms by which CR improves this cognitive decline are poorly understood. Previously, we have reported age-related decreases in key synaptic proteins in the CA3 region of the hippocampus that are stabilized by lifelong CR. In the present study, we examined possible age-related changes in the functional microcircuitry of the synapses in the stratum lacunosum-moleculare (SL-M) of the CA3 region of the hippocampus, and whether lifelong CR might prevent these age-related alterations. We used serial electron microscopy to reconstruct and classify SL-M synapses and their postsynaptic spines. We analyzed synapse number and size as well as spine surface area and volume in young (10 months) and old (29 months) ad libitum fed rats and in old rats that were calorically restricted from 4 months of age. We limited our analysis to SL-M because previous work demonstrated age-related decreases in synaptophysin confined to this specific layer and region of the hippocampus. The results revealed an age-related decrease in macular axo-spinous synapses that was not reversed by CR that occurred in the absence of changes in the size of synapses or spines. Thus, the benefits of CR for CA3 function and synaptic plasticity may involve other biological effects including the stabilization of synaptic proteins levels in the face of age-related synapse loss. © 2010 IBRO.

We introduce a novel sensing mechanism for nitric oxide (NO) detection with a particular easily synthesized embodiment (NO(550)), which displays a rapid and linear response to NO with a red-shifted 1500-fold turn-on signal from a dark background. Excellent selectivity was observed against other reactive oxygen/nitrogen species, pH, and various substances that interfere with existing probes. NO(550) crosses cell membranes but not nuclear membranes and is suitable for both intra- and extracellular NO quantifications. Good cytocompatibility was found during in vitro studies with two different cell lines. The high specificity, dark background, facile synthesis, and low pH dependence make NO(550) a superior probe for NO detection when used as an imaging agent.

17 beta-Estradiol (E) increases axospinous synapse density in the hippocampal CA1 region of young female rats, but not in aged rats. This may be linked to age-related alterations in signaling pathways activated by synaptic estrogen receptor a (ER-a) that potentially regulate spine formation, such as LIM-kinase (LIMK), an actin depolymerizing factor/cofilin kinase. We hypothesized that, as with ER-alpha, phospho-LIM-kinase (pLIMK) may be less abundant or responsive to E in CA1 synapses of aged female rats. To address this, cellular and subcellular distribution of pLIMK-immunoreactivity (IR) in CA1 was analyzed by light and electron microscopy in young and aged female rats that were ovariectomized and treated with either vehicle or E. pLIMK-IR was found primarily in perikarya within the pyramidal cell layer and dendritic shafts and spines in stratum radiatum (SR). While pLIMK-IR was occasionally present in terminals, post-embedding quantitative analysis of SR showed that pLIMK had a predominant post-synaptic localization and was preferentially localized within the postsynaptic density (PSD). The percentage of pLIMK-labeled synapses increased (30%) with E treatment (P

Caloric restriction (CR) is a daily reduction of total caloric intake without a decrease in micronutrients or disproportionate reduction of any one dietary component. CR can increase lifespan reliably in a wide range of species and appears to counteract some aspects of the aging process throughout the body. The effects on the brain are less clear, but moderate CR seems to attenuate age-related cognitive decline. Thus, we determined the effects of age and CR on key synaptic proteins in the CA3 region of the hippocampus and whether these changes were correlated with differences in behavior on a hippocampal-dependent learning and memory task. We observed an overall, age-related decline in the NR1, N2A and N2B subunits of the N-methyl-d-aspartate (NMDA)-type and the GluR1 and GluR2 subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA)-type ionotropic glutamate receptors. Interestingly, we found that CR initially lowers the glutamate receptor subunit levels as compared to young AL animals, and then stabilizes the levels across lifespan. Synaptophysin, a presynaptic vesicle protein, showed a similar pattern. We also found that both CR and ad libitum (AL) fed animals exhibited age-related cognitive decline on the Morris water maze task. However, AL animals declined between young and middle age, and between middle age and old, whereas CR rats only declined between young and middle age. Thus, the decrease in key synaptic proteins in CA3 and cognitive decline occurring across lifespan are stabilized by CR. This age-related decrease and CR-induced stabilization are likely to affect CA3 synaptic plasticity and, as a result, hippocampal function.

Caloric restriction (CR) extends life span and ameliorates the aging-related decline in hippocampal-dependent cognitive function. In the present study, we compared subunit levels of NMDA and AMPA types of the glutamate receptor and quantified total synapses and multiple spine bouton (MSB) synapses in hippocampal CA1 from young (10 months), middle-aged (18 months), and old (29 months) Fischer 344xBrown Norway rats that were ad libitum (AL) fed or caloric restricted (CR) from 4 months of age. Each of these parameters has been reported to be a potential contributor to hippocampal function. Western blot analysis revealed that NMDA and AMPA receptor subunits in AL animals decrease between young and middle age to levels that are present at old age. Interestingly, young CR animals have significantly lower levels of glutamate receptor subunits than young AL animals and those lower levels are maintained across life span. In contrast, stereological quantification indicated that total synapses and MSB synapses are stable across life span in both AL and CR rats. These results indicate significant aging-related losses of hippocampal glutamate receptor subunits in AL rats that are consistent with altered synaptic function. CR eliminates that aging-related decline by inducing stable NMDA and AMPA receptor subunit levels.

Whole-brain irradiation is used for the treatment of brain tumors, but can it also induce neural changes, with progressive dementia occurring in 20-50% of long-term survivors. The present study investigated whether 45 Gy of whole-brain irradiation delivered to 12-month-old Fischer 344 x Brown Norway rats as nine fractions over 4.5 weeks leads to impaired Morris water maze (MWM) performance 12 months later. Compared to sham-irradiated rats, the irradiated rats demonstrated impaired MWM performance. The relative levels of the NR1 and NR2A but not the NR2B subunits of the NMDA receptor were significantly higher in hippocampal CA1 of irradiated rats compared to control rats. No significant differences were detected for these NMDA subunits in CA3 or dentate gyrus. Further analysis of CA1 revealed that the relative levels of the GluR1 and GluR2 subunits of the AMPA receptor and synaptophysin were not altered by whole-brain irradiation. In summary, a clinically relevant regimen of fractionated whole-brain irradiation led to significant impairments in spatial learning and reference memory and alterations in the relative levels of subunits of the NMDA, but not the AMPA, receptors in hippocampal CA1. These findings suggest for the first time that radiation-induced cognitive impairments may be associated with alterations in glutamate receptor composition.

Estrogen replacement increases both the number of dendritic spines and the density of axospinous synapses in the hippocampal CA1 region in young rats, yet this is attenuated in aged rats. The estrogen receptor-alpha (ER-alpha) is localized within select spines of CA1 pyramidal cells in young animals and thus may be involved locally in this process. The present study investigated the effects of estrogen on the ultrastructural distribution of ER-alpha in the CA1 of young (3-4 months) and aged (22-23 months) Sprague Dawley rats using postembedding immunogold electron microscopy. Within dendritic spines, most ER-alpha immunoreactivity (IR) was seen in plasmalemmal and cytoplasmic regions of spine heads, with a smaller proportion within 60 nm of the postsynaptic density. In presynaptic terminals, ER-alpha-IR was clustered and often associated with synaptic vesicles. Significant effects of both aging and estrogen were observed. Quantitative analysis revealed that nonsynaptic pools of ER-alpha-IR within the presynaptic and postsynaptic compartments were decreased (35 and 27%, respectively) in the young estrogen-replaced animals compared with those that received vehicle. Such localized regulation of ER-alpha in response to circulating estrogen levels might directly affect synaptic signaling in CA1 pyramidal cells. No estrogen treatment-related differences were observed in the aged animals. However, 50% fewer spines contained ER-alpha in the aged compared with young hippocampus. These data suggest that the decreased responsiveness of hippocampal synapses to estrogen in aged animals may result from age-related decrements in ER-alpha levels and its subcellular localization vis-à-vis the synapse. Such a role for spinous ER-alpha has important implications for age-related attenuation of estrogen-induced hippocampal plasticity.