Differences in gene expression in the hippocampus of aged rats are associated with better long-term memory performance in a passive avoidance test.

Microarray analysis was used to identify genes differentially expressed in the hippocampus of aged rats showing diverse long-term (3 and 6 h) spatial-associative memory performance in a single-trial inhibitory avoidance task. The transcription of 43 genes (including genes functionally linked to signal transduction, cell growth and differentiation, translation, energy metabolism, and nucleic acid processing) was significantly upregulated in good- versus bad-performing animals, whereas that of 18 genes (including genes functionally linked to transcription, cell growth and differentiation, apoptosis, and protein transport) was significantly downregulated in good- versus bad-performing animals. The differential expression of 14 of these genes was confirmed by real-time polymerase chain reaction.

A ketogenic diet increases succinic dehydrogenase (SDH) activity and recovers age-related decrease in numeric density of SDH-positive mitochondria in cerebellar Purkinje cells of late-adult rats.

Ketogenic diets (KDs) have been applied in the therapy of paediatric epilepsy for nearly a century. Recently, beneficial results have also been reported on metabolic disorders and neurodegeneration, designating aged individuals as possible recipients. However, KDs efficacy decrease after the suckling period, and very little is known about their impact on the aging brain. In the present study, the effect on the neuronal energetic supply of a KD containing 20% of medium chain triglycerides (MCT) was investigated in Purkinje cells of the cerebellar vermis of late-adult (19-month-old) rats. The animals were fed with the KD for 8 weeks, and succinic dehydrogenase (SDH) activity was cytochemically determined. The following parameters of SDH-positive mitochondria were evaluated by the use of a computer-assisted image analysis system connected to a transmission electron microscope: numeric density (Nv), average volume (V), volume density (Vv), and cytochemical precipitate area/mitochondrial area (R). Young, age-matched, and old animals fed with a standard chow were used as controls. We found significantly higher Nv in MCT-KD-fed rats vs. all the control groups, in young vs. late-adult and old controls, and in late-adult vs. old controls. V and Vv showed no significant differences among the groups. R was significantly higher in MCT-KD-fed rats vs. all the control animals, and in old vs. young and late-adult controls. Present data indicate that the ketogenic treatment counteracted age-related decrease in numeric density of SDH-positive mitochondria, and enhanced their metabolic efficiency. Given the central role of mitochondrial impairment in age-related physio-pathological changes of the brain, these findings may represent a starting point to examine novel potentialities for KDs.

Perichromatin fibrils accumulation in hepatocyte nuclei reveals alterations of pre-mRNA processing during aging.

We previously described an unusual accumulation of perichromatin fibrils (PF)-the in situ form of pre-mRNA transcription and early splicing-in hepatocyte nuclei of old rats. Here we have investigated, by immunoelectron microscopy, the nature of such PF, analyzing the presence of transcription, splicing and cleavage factors, polyadenylated RNA, and the incorporation of bromouridine in adult and old rats. Our observations revealed alterations in amount and/or distribution of pre-mRNA transcription, splicing and cleavage factors, as well as of polyadenylated RNA, together with lower bromouridine incorporation in newly transcribed RNA in the hepatocyte nucleoplasm of old rats. Therefore, our data indicate both a decrease in pre-mRNA transcription and a slow down of PF processing and transport during aging.

Neuronal apoptosis in Alzheimer's disease: the role of age-related mitochondrial metabolic competence.

To assess the role of the mitochondrial metabolic competence (MMC) in the development of age-related changes, we measured the levels of immunohistochemically stained (IH) mitochondrial- and nuclear-encoded subunits of cytochrome oxidase (COX II and COX IV, respectively) and compared these data with mRNA in situ hybridization (ISH) of the same subunits and with cytochemically evidenced COX activity in the temporal (TC) and frontal (FC) cortex of adult and late-adult monkeys. Quantitative cytochemistry of COX activity was performed by calculating the ratio (R) of the area of the cytochemical precipitate to the area of the respective organelle. Although ISH studies showed reduced gene expression of both subunits in FC of late-adult monkeys, no significant age-related difference was found either in TC or FC when considering the IH data. R was significantly increased in FC of late-adult animals, and a quartile distribution of the mitochondrial area showed that R is higher in the FC of older animals independent of the organelle size. The assessment of COX genetic and phenotypic parameters reliably reports on MMC because this enzyme is the terminal complex of the electron transport chain. Taken together, the present IH, ISH, and R findings suggest that, with advancing age, compensating mechanisms are activated to preserve the mitochondrial functional metabolic capacities. Although significant mitochondrial defects are currently reported to occur in Alzheimer's disease pathogenesis, our data document that MMC is actively involved in the physiological rearrangement of the age-related neuronal network and may provide substantial metabolic support for the energy demand of neuronal apoptosis.

A ketogenic diet increases succinic dehydrogenase activity in aging cardiomyocytes.

Impairment of energy metabolism and an increase of reactive oxygen species (ROS) production seem to play a major role in age-related apoptotic loss of cardiomyocytes. Succinic dehydrogenase (SDH) is an important marker of the mitochondrial capability to provide an adequate amount of ATP. Moreover, because of its unique redox properties, SDH activity contributes to maintain the reduced state of the ubiquinone pool. Recent reports have shown that ketone body intake improves cardiac metabolic efficiency and exerts a cardioprotective antioxidant action, we therefore performed a cytochemical investigation of SDH activity in cardiomyocytes of late-adult (19-month-old) rats fed for 8 weeks with a medium-chain triglycerides ketogenic diet (MCT-KD). Young, age-matched and old animals fed with a standard chow were used as controls. The overall area of the precipitates (PA) from SDH activity and the area of the SDH-positive mitochondria (MA) were measured. The percent ratios PA/MA and MA/total myocardial tissue area (MA/TA) were the parameters taken into account. We found that PA/MA was significantly higher in young control rats and in MCT-KD-fed rats versus late-adult and old control rats and in young control versus MCT-KD-fed rats. MA/TA of MCT-KD-fed rats was significantly higher versus age-matched and old control rats and tended to be higher versus young control rats; this parameter was significantly higher in young versus old control rats. Thus, MCT-KD intake partially recovers age-related decrease of SDH activity and increases the myocardial area occupied by metabolically active mitochondria. These effects might counteract metabolic alterations leading to apoptosis-induced myocardial atrophy and failure during aging.

Effect of two medium chain triglycerides-supplemented diets on synaptic morphology in the cerebellar cortex of late-adult rats.

Ketogenic diets (KDs) have shown beneficial effects in experimental models of neurodegeneration, designating aged individuals as possible recipients. However, few studies have investigated their consequences on aging brain. Here, late-adult rats (19 months of age) were fed for 8 weeks with two medium chain triglycerides-supplemented diets (MCT-SDs) and the average area (S), numeric density (Nv(s)), and surface density (S(v)) of synapses, as well as the average volume (V), numeric density (Nv(m)), and volume density (V(v)) of synaptic mitochondria were evaluated in granule cell layer of the cerebellar cortex (GCL-CCx) by computer-assisted morphometric methods. MCT content was 10 or 20%. About 10%MCT-SD induced the early appearance of senescent patterns (decreased Nv(s) and Nv(m); increased V), whereas 20%MCT-SD caused no changes. Recently, we have shown that both MCT-SDs accelerate aging in the stratum moleculare of CA1 (SM CA1), but are "antiaging" in the outer molecular layer of dentate gyrus (OML DG). Since GCL-CCx is more vulnerable to age than OML DG but less than SM CA1, present and previous results suggest that the effects of MCT-SDs in the aging brain critically depend on neuronal vulnerability to age, besides MCT percentage.

Melatonin prevents age-related mitochondrial dysfunction in rat brain via cardiolipin protection.

Reactive oxygen species (ROS) are considered a key factor in brain aging process. Complex I of the mitochondrial respiration chain is an important site of ROS production and hence a potential contributor to brain functional changes with aging. Appropriate antioxidant strategies could be particularly useful to limit this ROS production and associated mitochondrial dysfunction. Melatonin has been shown to possess antioxidant properties and to reduce oxidant events in brain aging. The mechanism underlying this protective effect of melatonin is not well established. In the present study, we examined the effects of long-term treatment of aged rats with melatonin on various parameters related to mitochondrial bioenergetics in brain tissue. After isolation of mitochondria from control, aged, and melatonin-treated young and aged rats, various bioenergetic parameters were evaluated such as complex I activity, rates of state 3 respiration, mitochondrial hydrogen peroxide (H2O2) production, and membrane potential. The mitochondrial content of normal and oxidized cardiolipin was also evaluated. We found that all these mitochondrial parameters were significantly altered with aging, and that melatonin treatment completely prevented these age-related alterations. These effects appear to be due, at least in part, to melatonin's ability to preserve the content and structural integrity of cardiolipin molecules, which play a pivotal role in mitochondrial bioenergetics. The melatonin's ability to prevent complex I dysfunction and cardiolipin peroxidation was also demonstrated by in vitro experiments on brain mitochondria treated with tert-butyl hydroperoxide. In summary, this study documents a decline of mitochondrial bioenergetic functions in brain with aging and the beneficial effect of melatonin.

Ketogenic diets cause opposing changes in synaptic morphology in CA1 hippocampus and dentate gyrus of late-adult rats.

Ketogenic diets (KDs) have beneficial effects on several diseases, such as epilepsy, mitochondriopathies, cancer, and neurodegeneration. However, little is known about their effects on aging individuals. In the present study, late-adult (19-month-old) rats were fed for 8 weeks with two medium chain triglycerides (MCT)-KDs, and the following morphologic parameters reflecting synaptic plasticity were evaluated in stratum moleculare of hippocampal CA1 region (SM CA1) and outer molecular layer of hippocampal dentate gyrus (OML DG): average area (S), numeric density (Nv(s)), and surface density (Sv) of synapses, and average volume (V), numeric density (Nv(m)), and volume density (Vv) of synaptic mitochondria. In SM CA1, MCT-KDs induced the early appearance of the morphologic patterns typical of old animals (higher S and V, and lower Nv(s) and Nv(m)). On the contrary, in OML DG, Sv and Vv of MCT-KDs-fed rats were higher (as a result of higher Nv(s) and Nv(m)) versus controls; these modifications are known to improve synaptic function and metabolic supply. The opposite effects of MCT-KDs might reflect the different susceptibility to aging processes: OML DG is less vulnerable than SM CA1, and the reactivation of ketone bodies uptake and catabolism might occur more efficiently in this region, allowing the exploitation of their peculiar metabolic properties. Present findings provide the first evidence that MCT-KDs may cause opposite morphologic modifications, being potentially harmful for SM CA1 and potentially advantageous for OML DG. This implies risks but also promising potentialities for their therapeutic use during aging.

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.

Long-term visual object recognition memory in aged rats.

Aging is associated with memory impairments, but the neural bases of this process need to be clarified. To this end, behavioral protocols for memory testing may be applied to aged animals to compare memory performances with functional and structural characteristics of specific brain regions. Visual object recognition memory can be investigated in the rat using a behavioral task based on its spontaneous preference for exploring novel rather than familiar objects. We found that a behavioral task able to elicit long-term visual object recognition memory in adult Long-Evans rats failed in aged (25-27 months old) Wistar rats. Since no tasks effective in aged rats are reported in the literature, we changed the experimental conditions to improve consolidation processes to assess whether this form of memory can still be maintained for long term at this age: the learning trials were performed in a smaller box, identical to the home cage, and the inter-trial delays were shortened. We observed a reduction in anxiety in this box (as indicated by the lower number of fecal boli produced during habituation), and we developed a learning protocol able to elicit a visual object recognition memory that was maintained after 24 h in these aged rats. When we applied the same protocol to adult rats, we obtained similar results. This experimental approach can be useful to study functional and structural changes associated with age-related memory impairments, and may help to identify new behavioral strategies and molecular targets that can be addressed to ameliorate memory performances during aging.

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.

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.

Platelet as a physiological model to investigate apoptotic mechanisms in Alzheimer beta-amyloid peptide production.

Although neuronal apoptosis in Alzheimer's disease is generally interpreted as the consequence of the toxicity of extracellular beta-amyloid (Abeta) peptide aggregates, some experimental results provide evidence that the Abeta overproduction can be the result of a primary neuronal degeneration. As platelets are considered a good model where to study proteolytic processing of the amyloid precursor protein (APP), we exposed platelets to the proapoptotic agent ionomycin and analyzed Abeta40 and Abeta42 levels in the intracellular and extracellular compartments. The activation of apoptotic pathways in platelets has been verified by mitochondrial membrane depolarization, exposure of phosphatidylserine, protease activation and morphological changes. A significative increase in intraplatelet Abeta40, but not Abeta42, was observed after 10 min treatment with ionomycin. Thus, the activation of apoptotic pathways in platelets determines an altered processing of APP leading to elevated levels of intracellular Abeta40. The specific intracellular production of Abeta40 represents a potential threat to the cells since very high local Abeta40 concentration increases the risk of its aggregation and toxicity. As a result, Abeta40 might be dangerous even before it becomes secreted rendering neurons highly vulnerable.

Brain aging: The zinc connection.

At variance with other organs, where the functional and structural units are repeated, the brain is a composite assembly of groups of cells with different metabolic features and functional units. Deterioration of brain function occurs when the number of neurons or their connections decrease below a critical reserve level and coping with environmental stimulation is seriously hampered. Physiopathological alterations of the synaptic junctional areas are reported to play a central role in the process of brain aging. Current research is documenting an age-related numeric loss of synapses which is paired by a significant enlargement of the persisting contact zones: the final outcome of these balanced changes is a significant reduction of the overall synaptic junctional area per unit volume of neuropil. The progressive decline of the mitochondrial metabolic competence, i.e. the capacity of select pools of organelles to provide adequate amounts of adenosine triphosphate is supposed to represent a key determinant in synaptic aging. Cytochemical estimations of the activity of cytochrome oxidase confirm that mitochondrial dysfunctions play an early role in synaptic deterioration. Zinc ions act as physiological neuromodulators at glutamatergic synapses, however, in order to avoid neurotoxic damage, the intracellular free Zn(++) concentration ([Zn(++)](i)) must be tightly controlled by: (i) extrusion (Zn(++) transporters); (ii) buffering (metallothioneins) and (iii) sequestration (mitochondria) systems. In physiological aging, if any of these systems is impaired and/or not adequately coordinated with the other two, the resulting significant rise of ([Zn(++)](i)) may inhibit the cellular energy providing systems and affect mitochondria as primary target.

Immunoproteasome in Macaca fascicularis: no age-dependent modification of abundance and activity in the brain and insight into an in silico structural model.

In this study, we investigated proteasome composition and activity in the brain of Macaca fascicularis, in order to test whether this nonhuman primate species might be a suitable animal model for anti-aging therapies in the central nervous system, addressed to the ubiquitin-proteasome system. We detected the catalytic beta subunits of constitutive proteasome, as well as the PA28 regulator and a subunit of immunoproteasome (i.e., beta1i [LMP2]), in seven adult, six old, and one young nonhuman primate brains. Subunit expression and proteasome activity were not influenced by the age of the animal in any of the brain regions (temporal and frontal cortex and cerebellum) we studied. However, an area-specific susceptibility to aged-related oxidative stress emerged. On the whole, the results suggest that, compared to humans, Macaca fascicularis primates may have a different age-dependent regulation of the ubiquitin-proteasome system and, possibly, of neuroinflammation in the brain. An in silico model of the 20S immunoproteasome containing the Macaca fascicularis alpha and beta subunits, present in database or identified by our group (i.e., LMP2), has been developed. Additional information was obtained by de novo sequencing of the beta1 (delta) subunit of Macaca fascicularis. A comparison with humans suggests that in multiprotein complexes some functional subunits, such as alpha subunits, appear to be preferentially conserved during evolution.

Alterations of synaptic turnover rate in aging may trigger senile plaque formation and neurodegeneration.

The changes of synaptic ultrastructure were investigated by morphometry in the frontal (FC) and temporal (TC) cortex of adult and aged monkeys, to assess the potential role of age-related synaptic deterioration in neurodegeneration. The average synaptic size (S), the synaptic numeric density (Nv: number of synapses/microm(3) of tissue), the synaptic surface density (Sv: overall area of synaptic junctional zones/microm(3) of tissue), and the number of synapses/neuron (Syn/Neur) were calculated. In FC, significant differences of Nv and Sv due to age were not revealed, while the S value was significantly increased in the aged animals. In TC, Sv did not change in relation to age, whereas Nv was significantly decreased and S significantly increased in aged monkeys. A percent distribution of S showed that the fraction of enlarged synapses (>0.20 microm(2)) was higher in TC than in FC, regardless of the age of the animals (21.3% versus 16.9% in adult and 33.9% versus 26.0% in aged monkeys, respectively). In aged animals, Syn/Neur was not significantly decreased in TC and not significantly increased in FC (4.4%). The above morphometric parameters account for the ongoing rearrangements of synaptic ultrastructure, reacting to the environmental stimuli. Our findings provide evidence of an age-related decline of synaptic plasticity in the brain of aged monkeys that is statistically significant in TC. According to current literature data on synaptic structural dynamics, this decay may represent an early and subtle alteration able to trigger the development of senile plaques and neurodegenerative events.

Preservation of mitochondrial volume homeostasis at the early stages of age-related synaptic deterioration.

A morphometric study on synaptic mitochondria was performed in the frontal (FC) and temporal (TC) cortex of adult and aged monkeys to seek ultrastructural alterations due to age. The overall volume covered by mitochondria (volume density: Vv), the number of mitochondria/microm(3) of tissue (numeric density: Nv), the average mitochondrial size (average volume: V), and the average mitochondrial shape (average length: Fmax) were calculated. Either in FC and TC, no significant age-related differences were revealed for any of the above-mentioned morphometric parameters. Namely, in FC of aged monkeys, a decrease of Vv (2%) and Nv (6%) was observed, whereas V and Fmax were increased by 5% and 2%, respectively. In TC of aged animals, both Vv and Nv increased by 7%, V decreased by 2%, and Fmax increased by 1%. The above morphometric parameters account for changes in single aspects of mitochondrial ultrastructure; nonetheless, when considered together per experimental group, they provide information regarding the structural rearrangements occurring on discrete populations of organelles. Considering these assumptions, the present findings document a preservation of the mitochondrial volume homeostasis in the brain of aged monkeys. Because our data from a previous investigation on the same animals showed early signs of synaptic deterioration in FC and TC during aging, this seems to be in contrast with the results of the present study. However, the clear age-related preservation of the mitochondrial potential for structural dynamics may be interpreted as a reactive response to early signs of synaptic deterioration.

Release of beta-amyloid from high-density platelets: implications for Alzheimer's disease pathology.

The main component of Alzheimer's disease (AD) senile plaques in the brain is amyloid-beta peptide (Abeta), a proteolytic fragment of the amyloid precursor protein (APP). Platelets contain both APP and Abeta and much evidence suggests that these cells may represent a useful tool to study both amyloidogenic and nonamyloidogenic pathways of APP processing. It has been demonstrated that platelets activated by physiological agonists, such as thrombin and collagen, specifically secrete Abeta ending at residue 40. To verify whether APP beta-processing could be observed also in an in vitro system of highly concentrated platelets, we measured the Abeta released in the incubation media of 5 x 10(9) platelets/mL by enzyme-linked immunosorbent assay (ELISA). The activation status of platelets was investigated by ultrastructural analysis. We found that Abeta(40) levels were significantly higher in incubation media of 5 x 10(9)/mL platelets in comparison with 10(8)/mL platelets (normalized values), while Abeta(42) levels were not affected by cell density. The ultrastructural analysis showed platelets at different phases of activation: some platelets were at earlier stage, characterized by granule swelling and dilution, others had granules concentrated in a compact mass in the cell centers within constricted rings of circumferential microtubules (later stage). Normally concentrated cells had the characteristic morphology of resting platelets. Our data suggest that high-density platelets undergo activation likely by increased frequency of platelet-platelet collisions. This, in turn, determines the activation of APP beta-processing with consequent release of Abeta(40). Investigating the biochemical pathways triggering Abeta secretion in platelets might provide important information for developing tools to modulate this phenomenon in AD brains.

Synaptic and mitochondrial morphometry provides structural correlates of successful brain aging.

Average synaptic size (S), synaptic numeric density (Nv) and surface density (Sv), average mitochondrial volume, mitochondrial numeric density, and mitochondrial volume density were measured by morphometry in the frontal (FC) and temporal (TC) cortex from adult and old monkeys (Macaca fascicularis). In relation to aging, Sv did not change, while Nv was significantly decreased in TC, but not in FC. S was significantly increased in FC and TC. No significant difference due to age was found with regard to mitochondrial ultrastructure. Considering the functional significance of the above parameters, their substantial age-related constancy suggests that they may reasonably represent structural correlates of successful brain aging.

Effects of ageing on the fine distribution of the circadian CLOCK protein in reticular formation neurons.

Many biochemical, physiological and behavioural processes, from bacteria to human, exhibit roughly 24 h cyclic oscillations defined as circadian rhythms. However, during ageing, numerous aspects of the circadian biology undergo alterations; in particular, the sleep pattern changes, with more frequent awakenings and shorter sleep time. The basic mechanism of the circadian clock relies on intracellular molecular pathways involving interlocking transcriptional/translational feedback loops, and CLOCK protein, a transcription factor, is essential for normal circadian rhythms. In this study, the fine distribution of CLOCK protein has been analysed, in adult and old rats, at different phases of the daily cycle in the neurons of the medullary reticular formation, involved in the control of the sleep-wake cycle. The results demonstrate quali-quantitative modifications of CLOCK protein in the neurons of old animals, suggesting that such a deregulation of the intracellular clock mechanism may play some role in the degeneration of the sleep-wake circadian cycle.