Neurobiology of the aging brain: morphological alterations at synaptic regions

Computer-assisted morphometric studies have been carried out on nerve cell terminal regions in rats of different ages. The numerical density (Nv), the average area (S) and the surface density (Sv) of ethanol phosphotungstic acid stained (E-PTA) synaptic junctions were evaluated in cerebellar glomeruli and dentate gyrus supragranular layers. The volume density (Vv), the average volume (V) and the numerical density (Nvm) of synaptic mitochondria were measured in the cerebellar glomeruli of young (3 months), adult (11 months) and old (28 months) animals. We found that during aging Nv and Sv undergo a significant decrease, whereas S is significantly increased. The mitochondrial Vv is unchanged in all the age groups analysed, whereas in old rats V is increased and Nvm decreased, respectively. We interpret our results as supporting that the old CNS retains part of its remodelling activity and is capable of adaptive morphological response at synaptic terminal regions.     

Age-related morphological rearrangements of synaptic junctions in the rat cerebellum and hippocampus

A quantitative investigation has been carried out on the age-related morphological changes of the synaptic junctions in the cerebellar glomeruli and in the supragranular layer of the hippocampal dentate gyrus of young (3 months), adult (12 months) and old (28-30 months) rats. The numerical (Nv) and surface (Sv) density as well as the average length (L) of E-PTA stained synapses was calculated by means of morphometric methods. The results we obtained showed a similar trend in both these areas of the CNS. Nv significantly increased between young and adult rats and significantly decreased in the old group, when compared both to young and adult animals. Sv appeared to be unchanged comparing young and adult animals, whereas it was significantly reduced in the old group. L showed a decrease between 3 and 12 months of age and appeared to be markedly increased in the old animals when compared to adult values. From the present findings it can be inferred that number (Nv) and size (L) of the synapses are in a close inverse relationship which, through the organism's life span, aims to maintain the constancy of the surface contact area (Sv) among the dendritic network. By considering Nv, L and Sv altogether per age group, we were able to obtain a reliable measurement of the morphological aspect of synaptic plasticity through different periods of the life. With regard to aging we found that, despite the 'compensative synaptogenesis' brought about by the increased size (L) of the synaptic appositions, the reactive capacity of old nerve cells is seriously impaired.     

Dietary restriction modulates synaptic structural dynamics in the aging hippocampus

A computer-assisted morphometric study has been carried out on the synaptic ultrastructural features in the hippocampus of 14-month old (DR14) and 27-month old (DR27) dietary restricted (−50% lipids and −35% carbohydrates) rats. Age-matched controls were maintained on an ad libitum (AL) feeding schedule. Synaptic numeric density (Nv), surface density (Sv) and average area (S) were the parameters measured. In old AL vs. adult AL animals, Nv decreased to a not significant extent, while S increased and Sv decreased significantly. In DR14 rats vs. AL littermates Nv increased significantly, but S and Sv were unchanged. DR27 rats vs. age-matched AL controls showed a significant increase of Nv and Sv while S was significantly decreased. Comparing DR14 vs. DR27, no significant difference due to age was documented. Both in DR14 and in DR27 groups the percent distribution of S showed a marked increase of smaller contact zones. Despite reporting on discrete aspects of synaptic ultrastructure, Nv and S are supported to be in an inverse relationship which aims at maintaining Sv constant. Thus, these three ultrastructural parameters when taken together per experimental group, appear to provide information on synaptic morphological rearrangements. In this context, the percent increase of smaller synapses in DR animals is consistent with the idea of a marked remodelling process. Considering previous data from the same groups of rats reporting significant changes in neuronal membrane lipid composition and fluidity, we interpret our findings to account for a positive modulation of dietary restriction on the synaptic structural dynamics.     

Synaptic remodeling in hippocampal CA1 region of aged rats correlates with better memory performance in passive avoidance test

Aging is associated with deficits in long-term declarative memory formation, and wide differences in performance can be observed among aged individuals. The cellular substrates of these deficits and the reasons for such marked individual differences are not yet fully understood. In the present study, morphologic parameters of synapses and synaptic mitochondria in stratum molecolare of CA1 hippocampal region were investigated in aged (26- to 27-month-old) female rats after a single trial inhibitory avoidance task. In this memory protocol animals learn to avoid a dark compartment in which they received a mild, inescapable foot shock. Rats were tested 3 and 6 or 9 hours after the training, divided into good and bad responders according to their performance (retention times above or below 100 seconds, respectively) and immediately sacrificed. The number of synapses and synaptic mitochondria per cubic micrometer of tissue (numeric density), the average area of synapses and volume of synaptic mitochondria, the total area of synapses per cubic micrometer of tissue, the percentage of perforated synapses and the overall volume of mitochondria per cubic micrometer of tissue were evaluated. In the good responder group, the numeric density of synapses and mitochondria was significantly higher and the average mitochondrial volume was significantly smaller 9 hours versus 6 hours after the training. No significant differences were observed among bad responders. Thus, better performances in passive avoidance memory task are correlated with more efficient plastic remodeling of synaptic contacts and mitochondria in hippocampal CA1. Present findings indicate that maintenance of synaptic plastic reactivity during aging is a critical requirement for preserving long-term memory consolidation.     

Decreased presence of perforated synapses in a triple-transgenic mouse model of Alzheimer's disease

Transgenic mouse models of Alzheimer's disease (AD) are useful tools to further our understanding of AD genotype-phenotype interaction. The triple transgenic mice harboring mutant forms of APP/PS1/Tau (3xTg-AD) exhibit beta-amyloid (Abeta) plaques (by 6 months of age) as well as neurofibrillary tangles (by 10-12 months of age). In this study, we characterized morphological alterations of hippocampal synapses obtained from 13-month-old 3xTg-AD and age-matched control (PS1-KI) mice. Numeric density of synapses (Nv, number of junctions/microm(3) of tissue), average synaptic contact area (S), and synaptic surface density (Sv, total synaptic contact area/microm(3) of tissue) were investigated by morphometric methods in the AD vulnerable CA1 pyramidal cell layer. Comparisons between 3xTg-AD and control mice showed no statistically significant differences in any of the three parameters; however, a significant decrease (by 28.5%) in the fraction of perforated junctional areas (PS) was observed in the 3xTg-AD mice. As PS is a reliably indirect index of synaptic plasticity, a decreased PS number might represent a subtle and early sign of synaptic dysfunction occurring in the 3xTg-AD mice, and lend support to the hypothesis that altered synaptic function is a critical feature of AD.     

Synaptic pathology in the brain cortex of old monkeys as an early alteration in senile plaque formation

Synaptic numeric density (Nv), average size (area: S), surface density (Sv) and number of synapses/neurone (Syn/Neur) were morphometrically measured in frontal (FC) and temporal (TC) cortex of adult and old monkeys. Sv was constant, a clear age-related trend to decrease by Nv and increase by S were observed in both areas investigated. Syn/Neur significantly decreased in TC of aged animals (-21.1%), whereas FC showed a not significant reduction (-2.6%). The present data support the hypothesis of an increased sensitivity to deterioration of TC synapses in aged monkeys, which might constitute a predisposing condition to the development of senile plaques.     

Synaptic mitochondria and ageing: computer-assisted morphometry in rat cerebellar glomeruli

The ultrastructural features of synaptic mitochondria have been investigated by means of computer-assisted morphometry in the cerebellar glomeruli of young, adult and old rats. The volume fraction occupied by mitochondria (volume density: Vv), the number of organelles/ microm(3) (numerical density: Nv), the average volume (V) and the average length (Sk) of the mitochondria were the parameters measured in electron microscopic photos taken at an enlargement of x 8000. Vv showed a lifespan constancy. Nv was increased in the adult group vs. the young, but it was decreased in old animals as compared with the other 2 groups of age studied. V and Sk showed the same age-dependent changes: a significant decrease in the adults and a significant increase in the old rats vs. the other groups analysed, respectively. We interpret these findings to represent age-dependent morphofunctional adaptations occurring at synaptic mitochondria as a response to actual energy demands from the synaptic connectivity they subserve. Although impaired, the dynamic morphology of synaptic mitochondria appears to be capable of consistent compensative ultrastructural rearrangements in the central nervous system of old individuals.     

Morphometric investigations of the mitochondrial damage in ceroid lipopigment accumulation due to vitamin E deficiency

Numeric (Nv) and volume (Vv) densities, as well as the average size (skeleton: Sk) of synaptic mitochondria from adult, normally fed and adult, vitamin E deficient animals (11 months of age) were semiautomatically measured by computer-assisted morphometry in the cerebellar granular layer. Nv, Vv and the average mitochondrial volume (V) were measured on perikaryal Purkinje cell organelles preferentially stained for succinic dehydrogenase (SDH) activity. Adult vitamin E deficient animals showed a significant decrease of Nv, a significant increase of Sk and an unchanged value of Vv. While in adult normally fed animals the mitochondria of increased size (Sk>5 microm) were 5.3%, in the adult vitamin E deficient rats this fraction accounted for 25.5%. In Purkinje cell perikarya, vitamin E deficiency resulted in a significant decrease of Vv, Nv and V, as well as a steeper reduction of the percentage of SDH-positive mitochondria of larger size. Taken together, these findings document that vitamin E deficiency is responsible of mitochondrial morphometric alterations in adult rats. Structurally deteriorated mitochondria are reported to play a role in producing increased amounts of free radicals, which can facilitate the accumulation of ceroid pigment.     

Synaptic disinhibition during maintenance of long-term potentiation in the CA1 hippocampal subfield.

Long-term potentiation (LTP) in the CA1 region of the hippocampus is widely believed to occur through a strengthening of efficacy of excitatory synapses between afferent fibers and pyramidal cells. An alternative mechanism of LTP, reduction of efficacy of synaptic inhibition, was examined in the present report. The present study demonstrates that the maintenance of LTP in the CA1 hippocampal subfield of guinea pigs is accompanied by impairment of type A gamma-aminobutyric acid (GABA) receptor function, particularly at apical dendritic sites of CA1 pyramidal cells. Enhanced excitability of GABAergic interneurons during LTP represents a strengthening of inhibitory efficacy. The net effect of opposite modifications of synaptic inhibition during LTP of CA1 pyramidal cells is an overall impairment of the strength of GABAergic inhibition, and disinhibition could contribute importantly to CA1 pyramidal cell LTP.     

The effect of chronic hydergine treatment on the plasticity of synaptic junctions in the dentate gyrus of aged rats

The number of synapses (Nv), the surface density of contact zones (Sv) as well as the average size (S) of E-PTA stained synapses in the supragranular layer of the dentate gyrus from adult (12 months), old (30 month), and Hydergine-treated old (30 months) rats were measured by using quantitative morphometric techniques. In old animals, Nv and Sv were significantly reduced, whereas S was significantly increased as compared with the values in adult rats. Hydergine (Codergocrine mesylate) treatment of old animals (3 mg/Kg/day for 4 weeks) influenced these three parameters, differentially. The Sv in aged animals receiving Hydergine, relative to that in untreated old rats, was significantly increased; the number and size of synapses in the treated old rats were significantly higher and smaller, respectively, than that in old controls. We interpret the present findings to indicate a modulating effect of Hydergine on the morphological plasticity of synaptic junctions in the dentate gyrus of aged rats.     

Aging-like alterations of SDH activity in Purkinje cell mitochondria of adult vitamin-E deficient rats

The ultrastructural features of perikaryal mitochondria positive to the copper ferrocyanide cytochemical reaction due to SDH activity were investigated in Purkinje cells of adult rats fed a vitamin E (α-tocopherol) deficient diet (AVED) for 11 months. The mitochondrial volume fraction (volume density: Vv), the number of organelles/μm³ of tissue (numeric density: Nv) and their average volume (V) were estimated by computer-assisted morphometry. The data obtained were compared with our previous results on 3, 12 and 24 month-old normally fed rats. In a comparison with age-matched controls, AVED animals showed significant decreases of the three morphometric parameters taken into account. These reductions were also observed in old, normally fed rats vs. the young and adult groups, but in AVED rats Vv and V decreased to a higher extent. In adult control animals, the percent of larger organelles (0.32 μm³ >) decreases to less than 1%. Vitamin E deficiency resulted in a steeper reduction of this fraction of organelles, i.e. only 0.5% in the 0.24–0.32 μm³ size range accounted for the largest mitochondria in the AVED group. Taken together, these data document a significant impairment of mitochondrial efficiency in old and AVED rats. We interpret these findings to support that the underlying processes of aging and vitamin E deficiency may share common mechanisms. Considering the antioxidant action of α-tocopherol and the SDH role in cellular bioenergetics, inadequate protection from free radical attacks appears to represent an important determinant in the age-related decline of the mitochondrial metabolic competence.     

Stereologic study of the sinoatrial node of rats -- age related changes

Changes in the sinoatrial node represent the major mechanism of sudden death in humans, and because of the sparse knowledge about the effects of aging on this structure, light microscopic and quantitative studies of the sinoatrial node were undertaken. Twenty-one hearts were studied, seven rat hearts from each of the following age groups: three months of age, twelve months of age and eighteen months of age. In the stereo logic study, the following parameters were studied: Vv_[nc] and Vv_{[interstitium]} % (the volume densities of the nodal cell and interstitium, determined by the point-couting method), and Nv_[nc](1/mm³) (the numerical density of the nodal cell, determined by the disector method). The mean volume of the nodal cell (V_[nc]) (μm³) was also determined. The comparisons showed that in the oldest animals, the volume density of the nodal cells decreased, while the volume density of the interstitium increased. Although numerical density of the nodal cell per volume of sinus node decreased, the nodal cells displayed increased mean volume with age. In conclusion, the aging process implies changes in the cell and fiber content of the sinoatrial node. This revised version was published online in July 2006 with corrections to the Cover Date.     

Human pancreatic growth hormone-releasing factor (1-44) stimulates GH cells in an anuran amphibian (Rana perezi).

Subcellular responses of amphibian growth hormone (GH)-producing cells to in vivo administration of human pancreatic growth hormone-releasing factor (1-44) (hpGRF) were investigated. The volume density (Vv) of secretory granules (SG), rough endoplasmic reticulum (RER), and Golgi complex (GC) and the numerical density (Nv) of the granules were estimated by ultrastructural morphometry. Immunogold staining was applied to ultrathin sections using an antiserum to ovine GH to identify GH-producing cells. In vivo treatment with hpGRF significantly decreased the Vv of the SG and the Nv of medium SG after 6 hr. The peptide also stimulated development of the cellular biosynthetic machinery and increased the number of small SG 24 hr after stimulation. Four-day-stimulated GH cells did not recover control morphological appearances. These morphological results suggest that: (1) In vivo administered hpGRF stimulates GH cells in Rana perezi by inducing hormone release and enhances biosynthetic activity; (2) after four injections, the cellular response is more intense, indicating that GH cells remain hyperactive, probably because the exogenous hpGRF overcomes the endogenous inhibitory control of GH secretion.     

不同月龄大鼠大脑中低密度脂蛋白受体相关蛋白的表达

目的　探讨不同月龄大鼠大脑额叶、海马、纹状体低密度脂蛋白受体相关蛋白 (LRP 1)表达的变化。方法　雄性SD大鼠 15只 ,流式细胞技术检测并计算 1,3,10月龄大鼠大脑额叶、海马、纹状体匀浆单细胞悬液LRP 1表达的平均荧光指数 ;免疫组织化学技术测定大脑海马CA1区神经细胞、微血管LRP 1表达 ,并应用MIAS图像分析系统进行平均吸光度测定。结果　流式细胞研究显示 ,大鼠海马 1、3月龄LRP 1表达强度均显著高于 10月龄大鼠 ;而额叶及纹状体各月龄组间差异无显著性意义。 1、3月龄大鼠海马区LRP 1表达强度均显著高于额叶及纹状体。免疫组织化学结果显示 ,LRP 1在海马CA1区微血管、神经细胞均有表达。随着月龄的增加 ,LRP 1在微血管的表达显著下降 ,而在海马CA1区神经细胞的表达各月龄组差异无显著性意义。结论　随着月龄的增加 ,海马区LRP 1的表达有下降趋势 ,尤其是海马CA1区微血管LRP 1的表达显著下降。     

RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.     

Selective decline of the metabolic competence of oversized synaptic mitochondria in the old monkey cerebellum

The morphofunctional features of synaptic mitochondria, positive to the activity of cytochrome oxidase (COX), were investigated in the cerebellar cortex of adult and old monkeys (Macaca fascicularis) to assess the potential age-related changes in the energy metabolism occurring at the neuronal synaptic compartment. The following mitochondrial ultrastructural parameters-numeric density (Nv), volume density (Vv), average volume (V), and average length (Fmax)-were measured by computer-assisted morphometric methods. The ratio (R) area of the COX cytochemical precipitate/area of the mitochondrion was semi-automatically calculated and considered as an estimation of the mitochondrial metabolic competence (MMC), that is, the capacity of single organelles to provide adequate amounts of adenosinetriphosphate. No age-related significant differences were found in any of the ultrastructural parameters taken into account, whereas a significant decrease of R was observed in old animals. In these animals, the quartile distribution of the COX-positive organelles, according to their respective cross-sectional area, showed no significant difference of R when comparing small (I quartile), medium-sized (II quartile), and large (III quartile) mitochondria, while a significant decrease of R was evident in oversized mitochondria (IV quartile). Although our data document an age-related preservation of the morphological features of COX-positive mitochondria in the monkey cerebellum, the significant decrease of R in old animals needs to be considered from the functional standpoint. Since COX is the terminal enzyme of the mitochondrial respiratory chain, the estimation of its activity is regarded as a reliable MMC index; thus our findings, by matching preferential cytochemistry and morphometry, support the hypothesis that the specific functional impairment of enlarged synaptic mitochondria may seriously affect information processing and cell-to-cell communication at synaptic junctional areas with aging.     

大鼠海马神经元及其线粒体增龄性改变的形态计量分析

目的探讨大鼠海马神经元及其线粒体形态结构增龄性改变的程度及性质。方法应用组织化学及电镜技术，通过计算机图像分析系统对海马神经元及其线粒体结构进行形态计量分析。结果海马ＣＡ１和ＣＡ３区神经元随增龄出现细胞皱缩，ＣＡ３区神经元数密度在老年组较青年组显著减少（Ｐ＜０．０５），ＣＡ１区神经元数密度各月龄组之间差异无显著性（Ｐ＞０．０５）。ＣＡ３区神经元胞体内线粒体体密度、数密度、比表面及嵴膜密度随增龄而减少，线粒体平均体积及平均截面积随增龄而增大。结论海马神经元及其线粒体形态结构随增龄发生显著性改变，这些形态结构的改变可能是大鼠海马老化的指征。     

Estrogen increases synaptic connectivity between single presynaptic inputs and multiple postsynaptic CA1 pyramidal cells: a serial electron-microscopic study

Dendritic spines are sites of the vast majority of excitatory synaptic input to hippocampal CA1 pyramidal cells. Estrogen has been shown to increase the density of dendritic spines on CA1 pyramidal cell dendrites in adult female rats. In parallel with increased spine density, estrogen has been shown also to increase the number of spine synapses formed with multiple synapse boutons (MSBs). These findings suggest that estrogen-induced dendritic spines form synaptic contacts with preexisting presynaptic boutons, transforming some previously single synapse boutons (SSBs) into MSBs. The goal of the current study was to determine whether estrogen-induced MSBs form multiple synapses with the same or different postsynaptic cells. To quantify same-cell vs. different-cell MSBs, we filled individual CA1 pyramidal cells with biocytin and serially reconstructed dendrites and dendritic spines of the labeled cells, as well as presynaptic boutons in synaptic contact with labeled and unlabeled (i.e., different-cell) spines. We found that the overwhelming majority of MSBs in estrogen-treated animals form synapses with more than one postsynaptic cell. Thus, in addition to increasing the density of excitatory synaptic input to individual CA1 pyramidal cells, estrogen also increases the divergence of input from individual presynaptic boutons to multiple postsynaptic CA1 pyramidal cells. These findings suggest the formation of new synaptic connections between previously unconnected hippocampal neurons.