Reduced serine racemase expression contributes to age-related deficits in hippocampal cognitive function.

To gain insight into the contribution of d-serine to impaired cognitive aging, we compared the metabolic pathway and content of the amino acid as well as d-serine-dependent synaptic transmission and plasticity in the hippocampus of young and old rats of the Wistar and Lou/C/Jall strains. Wistar rats display cognitive impairments with aging that are not found in the latter strain, which is therefore considered a model of healthy aging. Both mRNA and protein levels of serine racemase, the d-serine synthesizing enzyme, were decreased in the hippocampus but not in the cerebral cortex or cerebellum of aged Wistar rats, whereas the expression of d-amino acid oxidase, which degrades the amino acid, was not affected. Consequently, hippocampal levels of endogenous d-serine were significantly lower. In contrast, serine racemase expression and d-serine levels were not altered in the hippocampus of aged Lou/C/Jall rats. Ex vivo electrophysiological recordings in hippocampal slices showed a marked reduction in N-methyl-d-aspartate-receptor (NMDA-R)-mediated synaptic potentials and theta-burst-induced long-term potentiation (LTP) in the CA1 area of aged Wistar rats, which were restored by exogenous d-serine. In contrast, NMDA-R activation, LTP induction and responses to d-serine were not altered in aged Lou/C/Jall rats. These results further strengthen the notion that the serine racemase-dependent pathway is a prime target of hippocampus-dependent cognitive deficits with aging. Understanding the processes that specifically affect serine racemase during aging could thus provide key insights into the treatment of memory deficits in the elderly.

Preserved memory capacities in aged Lou/C/Jall rats.

Although memory impairments are a hallmark of aging, the degree of deficit varies across animal models, and is likely to reflect different states of deterioration in metabolic and endocrinological properties. This study investigated memory-related processes in young (3-4 months) and old (24 months) Sprague-Dawley rats (SD), which develop age-linked pathologies such as obesity or insulin-resistance and Lou/C/Jall rats, which do not develop such impairments. In short- and long-term memory recognition tasks, old Lou/C/Jall rats were never impaired whereas old SD rats were deficient at 1 and 24h latencies. The expression of N-methyl-d-aspartate receptors (NMDAR)-mediated synaptic plasticity in CA1 hippocampal networks shifted towards lower activity values in old Lou/C/Jall rats whereas long-term potentiation was impaired in age-matched SD rats. Age-related decrease in NR2A subunits occurred in both strains, extended to NR2B, NR1 and GluR1 subunits in older animals (28 months) but only in SD rats. Therefore, the Lou/C/Jall rats can be considered as a model of healthy aging, not only in terms of its preserved metabolism, but also in terms of cognition and synaptic plasticity.

Effect of aging on brain-derived neurotrophic factor, proBDNF, and their receptors in the hippocampus of Lou/C rats.

The interest in understanding healthy aging has prompted scientist to look for animal models presenting this feature. Lou/C rats, an inbred strain of Wistar origin, is an animal model of successful aging with a longer lifespan and preserved memory capacities than most laboratory rat strains. In an attempt to shed light on this remarkable aging feature, we investigated the hippocampal patterns (mRNA and proteins) of some protective and plasticity-related molecules, i.e., brain-derived neurotrophic factor (BDNF), its precursor proBDNF, and its receptors (i.e., TrkB.FL, TrkB.T1, TrkB.T2, and p75). Using different experimental approaches, we compared these characteristics in young and aged Lou/C versus matched Wistar rats (the most appropriate controls). Data showed that young and aged Lou/C rats had higher amounts of BDNF and proBDNF content than Wistar rats. In contrast, proBDNF content was reduced in aged Lou/C rats and increased in aged Wistar rats. With aging, Lou/C rats showed a weaker decrease in TrkB.FL receptors than Wistar rats and no changes in TrkB.T1 receptors, which, contrarily, were increased in aged Wistar rats. Overall, these observations could account for the preserved cognitive performances and memory-dependent mechanisms, such as the unaltered long-term potentiation (LTP), throughout the lifespan recently reported in the Lou/C strain. Data suggest that boosting the expression or activity of these endogenous protective systems may be a promising alternative for combating some age-related cognitive declines. Therefore, Lou/C rats represent an interesting model of healthy aging for studying plasticity-related processes that evolve from youthfulness to aging.

Regulation of the pituitary growth hormone-releasing hormone receptor in ageing male and female LOU rats: new insights into healthy ageing.

Ageing is characterised by a decrease of somatotroph functionality, involving growth hormone-releasing hormone receptor (GHRH-R). The present study was conducted in LOU/C/jall (LOU) rats, a strain described as a model of healthy ageing, which is characterised by a low adiposity and long life expectancy without developing severe pathologies. Effects of age and diet (chow versus self-selection), on levels of anterior pituitary GHRH-R mRNA transcripts, were assessed in male and female LOU rats. The effect of age on pituitary GHRH-R functionality was examined in the anterior pituitary of both males and females fed chow diet. Moreover, serum insulin-like growth factor-I (IGF-I), T4 and leptin were measured because changes in their concentration could affect GHRH-R expression. In the pituitary of 18-month-old male and female LOU/C/jall rats fed standard chow, the level of 2.5-kb GHRH-R mRNA transcript, coding for functional GHRH-R, was significantly decreased. In 24- to 34-month-old males and females, it progressively returned to the level of younger animals, suggesting an enrichment of the group with survivors maintaining functional GHRH-R. In males and females repeatedly submitted to self-selection, this phenomenon was not observed. Studies with the GHRH-R agonist, Fluo-GHRH, revealed that 73% of 16-18-month-old male and female rats studied did not show an increase of fluorescence density characteristic of receptor-mediated internalisation upon incubation at 37 degrees C. In the other 27%, the increase of fluorescence was identical to that observed in pituitaries of young rats, suggesting the presence of an optimal level of functional GHRH-R. Serum levels of leptin, free T4 and total IGF-I decreased more drastically in ageing males and in rats fed a self-selection diet. A positive correlation was demonstrated between leptin and IGF-I levels in ageing males and females fed standard chow and ageing females submitted to a self-selection regimen. In conclusion, healthy ageing in LOU rats fed chow diet appears to be associated with a maintenance of functional pituitary GHRH-R levels found in younger rats but not necessarily with those of serum leptin, T4 and IGF-I.

Effect of treatment with dexamethasone on protein intake in adult and old Lou/c/jall rats.

A deleterious decrease of protein intake had been evidenced in Lou/c/jall rats during ageing. This result could be induced by an impaired regulation of feeding behaviour. Glucocorticoids inducing specific amino-acid needs for gluconeogenesis and for the synthesis of inflammatory proteins by the liver, we investigated the age-related effect of a 4-days treatment with dexamethasone (DEX) on caloric and protein intake. Males and females aged 7, 19, 25 and 31 months received 573.6 +/- 65.6 microg/(kg day) of dexamethasone via the drinking water. Body weight (BW), caloric and macronutrients intakes were monitored during treatment and during 10 days after the treatment. A strong hypophagia was seen during treatment in all groups, which was mainly due to a decrease in fat intake. In the same time, rats maintained their protein intake so that protein became the main macronutrient of the diet in most of the groups. However, older males showed a lesser efficiency in adjusting their diet. These results are in agreement with previous data obtained in a protein deprivation study. They lead to the conclusion that the loss of appetite for protein in old age probably does not reflect a loss of ability to choose the needed amount of protein. We can hypothesise that the decrease of protein intake in old rats could be due to some inadequacy of casein to the metabolic requirement of aged animals.

Old as mature LOU/c/jall rats enhance protein selection in response to a protein deprivation.

Previous experiments have shown a strong decrease in protein consumption as rats grow old. This result could be induced by an impaired regulation of feeding behaviour. Present study investigated the ability of ageing rats to adapt their protein intake to protein requirements. Four groups of self-selecting LOU/c/jall male and female rats (4, 16, 22, 28 months at the beginning of the experiments) were submitted to 4-days of protein deprivation periods while their macronutrient intakes were monitored. Moreover, they were submitted to a 4-day fasting period. After reintroduction of proteins, old and senescent rats were able to increase their protein intake in response to the specific protein need created by the protein deprivation. They were also able to increase caloric intake after the fasting period. These results led to the conclusion that the loss of appetite for protein observed with ageing reflects rather an adaptation than an impairment of the regulation of macronutrient choice. Another important observation was that older animals did not recover initial body weight after a body weight loss even if they ate as many calories as younger animals. This data supports a decrease in the efficacious utilization of body fuels in old and senescent rats.

Effect of morphine on caloric intake and macronutrient selection in male and female Lou/c/jall rats during ageing.

Previous studies have showed a shift of preferences from carbohydrate to fat in the Lou/c/jall rat with advancing age when they are submitted to a self-selection procedure. Protein intake also decreased according to the age, earlier for males (after 16 months) than for females (29 months). The present study aimed at investigating the mechanism underlying these modifications. We analysed the effect of the reference mu agonist, morphine (5 mg/kg subcutaneous), on the caloric intake, body weight and macronutrient intake of 30 male and 30 female rats divided in four age groups: young adults (10), mature (17), old (24) and senescent rats (29 months). During the experiment, animals had the choice between separate sources of the three pure macronutrients. Morphine injection reduced total daily caloric intake and induced a decrease in body weight. The weight loss was age- and sex-related (males and old rats were more affected by the drugs). The injection of morphine evoked a triphasic influence on the chronology of the intake. A brief (1 h) hypophagia was followed by an hyperphagia (3 h) and a persistent hypophagia (8 h). No modification in the diet composition was observed. These results did not support a clear involvement of the opioid system concerning the modifications in macronutrient rates in diet previously observed across ageing.

Effects of peripheral and central administration of GHRH on feeding in aging LOU rats.

In aging LOU rats, a decreased protein intake is restored by GH administration. To study the contribution of GHRH to macronutrient selection, hGHRH NH(2) was administered sc. (1 mg/kg B.W./day/14 days) or icv. (4 and 40 pmol/rat) to 11-, 19-, 24- and 28-month-old rats. Sc. administration induced a decreased food and lipid intakes from 24 months of age and a transient stimulation of protein intake in 19-month-old and older low protein eaters (<10% protein/total intake). Icv. administration induced decreased food and lipid intakes in all age groups. These results suggest that GHRH may regulate feeding through pituitary and/or hypothalamic GHRH receptor mechanisms.

A moderate swimming exercise regularly performed throughout the life induces age and sex-related modifications in adaptive macronutrients choice.

The ability of laboratory rats to adapt food intake to needs is well-known. The present study investigates changes in this adaptive behavior when animals grow old. A cohort of male and female Lou/c/jall rats was regularly submitted to an exercise throughout their life (6 consecutive days of moderate intensity training (3x15 min/day)). Caloric intake and macronutrients selection during exercise and post-exercise periods were compared to the pre-training period. During swimming, a decrease in both caloric intake and fat selection was observed and an increase in protein intake was specifically seen in female groups. However, males were unable to modify the diet composition (macronutrient rate) from 16 months of age, whereas females were able to do it until 24 months of age. The present results suggest a sex-dependent loss of capacity of adjusting feeding behavior to metabolic needs when animals grow old, may be due to a deterioration of the central control of food intake.

Age-related changes in nociception and effect of morphine in the Lou rat.

Little is known about how the ageing process affects pain sensitivity and a relevant animal model is therefore required. The effect of age on pain reactivity in animals has been investigated by several experimenters but the results are conflicting. Four groups of male and female Lou/C/Jall rats (4-29 months old) were used for our study. Four pain tests based on evaluation of reflex or more integrated behaviours after a thermal (tail immersion test) or mechanical (paw pressure test and von Frey test) stimulation were used. With mechanical stimulus, a significant decrease in the pain threshold across age was observed, females were more sensitive than males. This increase in nociceptive sensitivity to mechanical stimulation was more pronounced on integrated behaviours (struggle reaction) than on withdrawal reflex. An age-related increase in sensitivity was found on von Frey test. No effect on the latency of reflex induced by thermal stimulation was observed. In addition, a decrease in the spontaneous motor activity during exploration was observed across ageing; this effect was more marked for the females. The effect of morphine at doses of 1, 3 and 9 mg/kg (s.c.) decreased in intensity across ageing. These data demonstrate the need to use (1) various noxious stimuli because differences were observed in the modification of pain reactivity according to the nature of the stimulus; (2) various pain parameters and particularly integrated behaviours; (3) several age groups. In addition, Lou/C/Jall rat could be a useful model for studying of effect of age on pain.

Age-related changes in adaptive macronutrient intake in swimming male and female Lou rats.

To evaluate the age-related changes in capacity to adjust the nutrient intake to needs, self-selecting male and female Lou/C/jall rats of 4, 6, 12, 16 and 23 months of age were submitted to a swimming exercise. They were given 6 consecutive days of moderate intensity training (3 x 15 minutes per day). Exercise and postexercise periods were compared with results from the pretraining period. During swimming, a body weight loss and a decrease in both caloric intake and fat selection were observed. This effect was more marked in older groups compared to 4 month-old groups. An increase in protein intake was observed in females, specially in older groups, whereas no effect was seen in males. The ability to increase caloric ingestion and regain weight during the postexercise period decreased with advancing age and was better in females than in males. We also showed an age-related effect on the recovery of initial nutrient intake rate that was more pronounced and more precocious for males. Moreover, males tended to decrease their protein intake, whereas females significantly increased it. The present findings suggest a decrease of capacity of adjusting feeding behavior to metabolic needs in aged rats, may be due to a deterioration of the central control of food intake.

Peripheral injection of growth hormone stimulates protein intake in aged male and female Lou rats.

It is well established that growth hormone (GH) induces growth rate and food efficiency and stimulates protein accretion in young mammals. Senescence is characterized by metabolic and hormonal disorders, particularly a decrease in protein turnover, which could be correlated to a decrease in GH and insulin-like growth factor I (IGF-I) secretion. We have shown that body weight, protein intake, and IGF-I plasma levels are greatly decreased with aging in Lou/C rats, particularly in males. In order to specify the GH effect on protein intake during aging, males and females (6, 19, and 24 mo) placed on a self-selection regimen were injected daily with a physiological dose of human GH (0.023 mg/rat sc). No GH effect on caloric intake was observed. Nevertheless, GH treatment stimulated body weight in older rats. It also increased protein intake in females and older males (19-24 mo). This stimulating effect was positively correlated with the degree of weight loss in senescent rats, suggesting that the decrease in protein intake observed with aging could be a marker of senescence.

Age-related changes in adaptive mechanisms of macronutrient self-selection: evidence for a sexual dimorphism.

The effect of aging on patterns of food intake and nutrient selection was investigated using a longitudinal study. Male (n = 10) and female (n = 10) Wistar-Lou rats from 4 to 28 months of age were repeatedly submitted to a macronutrient self-selection (S-S) regimen while controls were maintained under a standard chow diet (Std). An age-related shift of preferences from CHO to fat diets, and a decrease in protein intake for both males and females were evident. Nevertheless, all these modifications were more pronounced and precocious for males. Physical exercise (45 min/day of swimming, on 6 days) induced a body weight loss and an hypophagia more pronounced for males than for females. S-S regimen results revealed that hypophagia concerned exclusively fat intake and that females significantly increased protein intake during and after the exercise period. Study of longevity curves showed a decrease of the mortality in S-S submitted male rats compared to control rats. This study shows that aging induces a sex-difference in feeding patterns which undoubtedly reveals a sex-difference metabolic requirements. Moreover, these results suggest that allowing rats to select macronutrient intakes could delay the process of senescence.

Dietary self-selection can compensate an age-related decrease of rat liver 20 S proteasome activity observed with standard diet.

Aged Lou female rats (33 months) submitted to a self-selection regimen showed a decrease in protein intake (down to 11% of the total intake), whereas mature rats (18 months) selected a high percentage of protein (20% of the total intake) similar to the protein content of the standard diet. To find out if this decrease in protein intake would prevent an observed age-related decrease in proteasome activity, four peptidase activities and oxidized protein degradation were tested with proteasome purified from the liver of 18- and 33-month-old rats. The peptidylglutamyl-peptide hydrolase activity, which is decreased with age for rats fed the standard diet, was restored in the self-selecting old rats to the level observed for the mature rats. Degradation of oxidized glutamine synthetase, which is also decreased with age for rats fed the standard diet, was partly restored. Proteasome from self-selecting old rats showed a slight increase in trypsin-like and chymotrypsin-like activities as compared to proteasome from old rats fed the standard diet. Two-dimensional gel electrophoresis followed by quantitative analysis of the pattern of proteasome subunits revealed an increase in the intensity of two protein spots for proteasome from old rats fed the standard diet as compared with proteasome from either mature rats or self-selecting old rats. These findings may have important implications in aging for proteasome-mediated proteolysis and subsequent accumulation of oxidatively damaged protein.

Changes in pattern of macronutrient intake during aging in male and female rats.

Transversal and longitudinal studies of the natural patterns of food intake were made in male (n = 65) and female (n = 52) Lou rats, from 4 to 34 months of age. The efficiency of body weight control, especially for females, seems to be a main feature of this strain of rat: these rats are small eaters, have lighter body weight, show no development of obesity with age, and have a longer life expectancy than other common strains of rat. A better adaptation to feeding schedules was shown by female groups throughout the experiments. Moreover, an increased consumption for the oldest animals (from 28 months) enables them to keep their body weight constant up to age 34 months, whereas the male rats gradually lost weight with old age (from 28 months). When male and female rats of different age groups were allowed to choose their diet from pure macronutrient sources (protein, fat, and carbohydrate), an age-related shift of preferences from carbohydrate to fat diets was evidenced during daily energy intake. Protein intake also decreased according to age, earlier for males (around 21 months) than for females (34 months).

Administration of LHRH analog can improve working memory in aged female rats.

We had previously found that an age-related deficit in working memory appeared in middle-aged females, possibly related to their ageing reproductive system. This experiment investigates the effect of administering an analog of luteinizing hormone-releasing hormone (LHRH), D-Trp6-LHRH, to 22-mo-old female rats submitted to successive trainings in a water-reinforced matching to sample task since the age of three months. Animals were injected SC with a long-lasting microcapsule formulation (800 micrograms/kg continuously releasing 5 micrograms D-Trp6-LHRH per day for at least 28 days). The action of the analog on ovary weight and estradiol and progesterone levels was also studied. Administration of D-Trp6-LHRH counteracted the deleterious effect of age on performance and inhibited ovary function. However, hormone plasma levels were not modified. These results suggest that the hypothalamo-ovarian axis might be implicated in memory deficits of middle-aged females.

Effect of administration of an analog of LHRH on appetitive learning in young and middle-aged female rats.

Hypothalamic luteinizing hormone-releasing hormone (LH-RH) had been reported to induce changes in defensive learning. In middle age, females exhibit a decline in their reproductive axis. Several studies in rodents suggested that hypothalamic LHRH function deteriorated in middle-aged females. Our experiments compare T-maze learning in young and middle-aged female rats and study the effect of administration of an analog of LHRH, D-Trp6-LHRH. The ovarian action of the analog was studied and a gonadectomized control group was added. No differences were observed between young and middle-aged females in acquisition, retention, and reversal of a simple discrimination in the T-maze. However, after removal of motor and spatial cues acquisition of the discrimination on visual cues was impaired in middle-aged females compared to young mature ones. Administration of D-Trp6-LHRH enhanced performance during the visual discrimination in younger females and had no action in middle-aged ones, whereas it inhibited ovary function in both groups. Ovariectomy had no effect. These results suggest a direct effect of the analog of LHRH on the CNS and show that this peptide fails to counteract the deleterious effect of age on performance.

Food-restriction interacts with vasopressin modulation of activity.

The effects of peripheral injection of various doses of lysine-vasopressin (LVP), administered 30 min before a 5-min session in a hole-board apparatus, were compared as a function of food restriction. Comparison of performance for various indices of general activity clearly showed that the food-restricted rats were more active and exhibited less photophobia than normally fed ones. The differences between the two groups were maintained in a second session 24 hours later. There was no sign of behavioral habituation to the apparatus among restricted animals. Different doses (0.2, 1, 2 micrograms of LVP) affected food-restricted animals differently from the rats fed ad lib. Only the highest dose reduced activity in both groups. A posttest injection of the smallest dose (0.2 micrograms) had an opposite effect on the activity in the hole-board, measured 24 hours after the injection. A second experiment showed that plasma and adrenal corticosterone were higher in deprived rats. The administration of 0.2 micrograms of LVP was followed by an increase in corticosterone. In food-restricted rats this increase was bigger and was still observed 24 hours after the injection. There is an interaction between feeding conditions and LVP injections which affects both the internal hormonal state and spontaneous reactivity to environment. These findings are of relevance to the effect of vasopressin on behavior.

Acquisition of a matching-to-sample task in young and middle-aged male and female rats.

Male and female rats were trained in a water-reinforced matching-to-sample task. The rule was acquired more rapidly by the females. After initial training, the retention of the rule was measured when the rats were twelve and 14 months old. The performance of neither group was modified at twelve months, but at 14 months, while no change appeared in the performance of males, females showed a drop in correct responses. Training in a modified device which made the task more difficult induced no changes in performance. Final training (at 18 months) showed the drop in performance in females had persisted. The decline of performance in middle-aged females might be related to the ageing female reproductive system.