Long-Term Calorie Restriction Alters Anxiety-like Behaviour and the Brain and Adrenal Gland Transcriptomes of the Ageing Male Rat.

Further examination of the molecular regulators of long-term calorie restriction (CR), reported to have an anxiolytic effect, may highlight novel therapeutic targets for anxiety disorders. Here, adult male Hooded Wistar rats were exposed to a 25% CR whilst anxiety-like behaviour was assessed at 6-, 12-, and 18-months of age via the elevated plus maze, open field, and acoustic startle tests. Next-generation sequencing was then used to measure transcriptome-wide gene expression in the hypothalamus, amygdala, pituitary, and adrenal glands. Results showed an anxiolytic behavioural profile across early, middle, and late adulthood by CR, with the strongest effects noted at 6-months. Transcriptomic analysis by seven attribute weighting algorithms, including Info Gain Ratio, Rule, Chi Squared, Gini Index, Uncertainty, Relief, and Info Gain, led to the development of a signature of long-term CR, independent of region. Complement C1q A chain (C1qa), an extracellular protein, expression was significantly decreased by CR in most regions examined. Furthermore, text mining highlighted the positive involvement of C1qa in anxiety, depression, neurodegeneration, stress, and ageing, collectively identifying a suitable biomarker candidate for CR. Overall, the current study identified anxiety-related phenotypic changes and a novel transcriptome signature of long-term CR, indicating potential therapeutic targets for anxiety, depression, and neurodegeneration.

Paternal calorie restriction prior to conception alters anxiety-like behavior of the adult rat progeny.

The maternal environment influences a broad range of phenotypic outcomes for offspring, with anxiety-like behavior being particularly susceptible to maternal environmental perturbations. Much less is known regarding paternal environmental influences. To investigate this, adult male rats were exposed to 25% calorie restriction (CR) or glucocorticoid elevation (CORT; 200 µg/ml of corticosterone in drinking water) for ∼ 6 weeks prior to breeding. Elevated plus maze (EPM), open field (OF), predator odor (cat urine), and acoustic startle/pre-pulse inhibition (AS/PPI) were characterised in the adult male offspring. Plasma concentrations of corticotrophin-releasing hormone (CRF), adrenocorticotropin hormone (ACTH), and serum leptin were characterised in both sires and offspring. Maternal care received by litters was additionally observed. Expectedly, CR and CORT treatment attenuated weight gain, whilst only CR induced anxiolytic behavior in the EPM. The adult offspring sired by CR males also demonstrated a reduction in weight gain, food intake and serum leptin levels when compared to controls. Moreover, CR offspring demonstrated an anxiolytic-like profile in the EPM and OF, enhanced habituation to the AS pulse, reduced PPI, but no alteration to predator odor induced defensiveness compared to control. CORT offspring failed to demonstrate any behavioral differences from controls, however, exhibited a trend towards reduced ACTH and leptin concentration. Collectively, the results indicate that a reduction in calories in males prior to conception can affect the behavior of adult offspring. The phenotypic transmission of CR experiences from fathers to the progeny could potentially be mediated epigenetically. The role of glucocorticoid elevation and maternal care are also discussed.

Calorie restriction and corticosterone elevation during lactation can each modulate adult male fear and anxiety-like behaviour.

Early life events, such as calorie restriction (CR) and elevated glucocorticoids, can calibrate the lifelong behavioural and physiological profile of an individual. Stress reactivity in adulthood is particularly sensitive to early life events; however, the consequence to fear and anxiety-like behaviour is less clear. Consequently, the current study sought to examine the effects of post-natal CR and glucocorticoid elevation, long considered powerful programming stimuli, on the subsequent fear and anxiety behaviour of the adult offspring. Rat dams received either corticosterone (200 µg/ml) supplementation in drinking water (CORT) or a 25% CR from post-natal day (PND) 1 to 11. Responses to the elevated plus maze (EPM), open field and a predator odour (TMT; 2,5-dihydro-2,4,5-trimethylthiazoline) were characterised in the adult male offspring. Both treatment conditions resulted in enhanced fear responses to TMT, characterised by heightened risk assessment and increased avoidance of TMT. CORT nursed offspring further demonstrated an anxiogenic profile in the open field. Basal hypothalamic-pituitary-adrenal function was unchanged in CORT adult offspring, whilst corticosterone concentration was elevated by post-natal CR. CR and CORT treated dams both exhibited greater anxiety-like behaviour in the EPM. A modest and temporary enhancement of maternal care was observed in CR and CORT treated dams, with CR dams further exhibiting rapid pup retrieval latencies. The results indicate enhanced emotionality in the adult male progeny of dams exposed to CR and corticosterone supplementation during the post-natal period. The modest enhancement of maternal care observed by both treatments is unlikely to have influenced the behavioural profile of the offspring.

Calorie restriction inhibits relapse behaviour and preference for alcohol within a two-bottle free choice paradigm in the alcohol preferring (iP) rat.

Among its many beneficial effects, calorie restriction (CR) has also been found to reduce anxiety related behavior in the rodent. With heightened levels of stress and anxiety implicated as a key precipitating factor of relapse and alcohol addiction, it was found that a 25% CR in addition to inducing anxiolytic effects also had the capacity to reduce intake of alcohol and inhibit relapse within a model of operant self-administration. The aim of this study was to investigate if a 25% CR would also display similar effects in a two-bottle free choice paradigm, whereby 24 h ad libitum access to both 10% ethanol and water is provided. All animals were initially tested on the elevated plus maze (EPM) and open field test prior to commencing the two-bottle free choice paradigm. Differences between control and CR25% animals demonstrated the anxiolytic effects of CR, with the CR25% group displaying greater percentage of open arm/total arm duration and open arm/total arm entries in the EPM. During the acquisition phase of the two-bottle free choice paradigm, CR25% animals showed a reduced intake of 10% ethanol in ml/kg, in comparison to the control group. Whilst control animals displayed a strong preference for 10% ethanol, the CR25% group consumed both 10% ethanol and water equally with no differences found in total fluid intake between groups. Similarly this was also the case following forced deprivation. In addition to reduced intake and lack of preference for 10% ethanol, CR 25% animals unlike controls failed to display a typical alcohol deprivation effect following abstinence. Taken collectively the results of this study suggest that CR may act as a protective factor against addiction and relapse in the alcohol preferring (iP) rat. In addition, given CR25% animals did not display a preference for 10% ethanol, results also suggest that CR may be altering the hedonic impact of ethanol within this group.

The effects of calorie restriction on operant-responding for alcohol in the alcohol preferring (iP) rat.

Calorie restriction (CR) is well established in the research literature to have several beneficial effects on health and has also been found to induce anxiolytic effects in the rat. Heightened levels of stress and anxiety are often regarded as key precipitating factors of relapse to substance abuse and alcohol addiction. In this study, the potential implication of a 25% CR diet in altering drug-seeking and relapse like behaviour through its capacity to influence anxiolytic-like behavioural changes was investigated.Anxiety was assessed in all rats with the elevated plus maze (EPM) and open field test prior to being trained to operantly self-administer either 10% ethanol, or water. Differences were found between the groups in the percentage of open arm/total arm duration and open arm/total arm entries in the EPM,demonstrating the anxiolytic effects of CR25%. Both control and CR25% groups showed preference for alcohol vs. water, however, controls responded more for alcohol during the conditioning phase than the CR25% group. Controls exhibited an alcohol deprivation-effect (ADE) post abstinence, and a cue-induced reinstatement of alcohol-seeking post extinction however the CR25% did not. These results demonstrate that the anxiolytic effects of CR25% reduces operant responding for ethanol and inhibits relapse behaviour.Taken collectively, the results of this study suggest that in line with past research a CR25% dietary regime can induce anxiolytic effects in the alcohol preferring (iP) rat. Furthermore, it also reduces the intake of ethanol and inhibits the ADE and cue-induced relapse that is characteristic of addiction in this strain.

Calorie restriction at increasing levels leads to augmented concentrations of corticosterone and decreasing concentrations of testosterone in rats.

The influence of calorie restriction (CR) on increasing life span, enhancing immunocompetence, and reducing the incidence of age-related diseases is well established. Evidence points to the involvement of neuroendocrine alterations in these beneficial effects. Accordingly, we hypothesized that CR will result in significant alterations to the hormones investigated. Little attention has been directed toward ascertaining the doses of CR required to obtain such alterations and, indeed, whether a dose-response exists. Adult rats were subjected to 1 of 5 dietary regimens: control, CR12.5%, CR25%, CR37.5%, or CR50%. Rats were decapitated 3 weeks following the onset of restriction; and trunk blood was collected and assayed for concentrations of serum adrenocorticotropic hormone, corticosterone, and testosterone, as well as plasma concentrations of noradrenalin and adrenalin. No effect was found as a result of dietary manipulation for serum concentrations of adrenocorticotropic hormone. However, all doses of CR resulted in increased serum corticosterone in a dose-response trend. A dose-response was also observed for serum testosterone, with higher doses of CR associated with lower testosterone. Concentrations of noradrenalin were not found to be altered by any CR dose, although a trend toward a down-regulation at CR50% was observed. Plasma adrenalin displayed a biphasic distribution with reductions observed at CR25% and CR50%, although the down-regulations only attained statistical significance relative to the CR37.5% and not the control group. As well as reporting the effect of CR on multiple hormones within individual animals, these results go some way in determining the optimal levels of CR needed to induce neuroendocrinologic alterations.

HPA and sympathoadrenal activity of adult rats perinatally exposed to maternal mild calorie restriction.

Developmental programming of neuroendocrine systems is profoundly influenced by environmental cues such as caloric availability. The focus of investigations in this area has been on the effects of under- and malnutrition while there is a paucity of research examining the effects of more mild levels of calorie restriction (CR). Rat dams and their offspring were subjected to one of five dietary regimens: control, CR50% for 3 days preconception, CR25% during gestation, CR25% during lactation, and CR25% during gestation, lactation, and post-weaning (lifelong). Adult male offspring were decapitated and trunk blood collected to assay for basal concentrations of serum adrenocorticotropic hormone (ACTH) and corticosterone (CORT), as well as plasma concentrations of adrenalin (A) and noradrenalin (NA). Basal serum ACTH was reduced by 35-43% in all dietary regimens except the lifelong group. Although a similar trend was observed in the concentrations of serum CORT, only the decrease in the lactation group attained statistical significance. A was reduced by 33-49% as a result of all dietary regimens and NA was reduced in the gestation and lifelong groups by 51% and 39%, respectively. The potential mechanisms underlying these neuroendocrine alterations are discussed.

The effects of calorie restriction olfactory cues on conspecific anxiety-like behaviour.

Olfactory stimuli and calorie restriction (CR) have both been found to reduce anxiety-like behaviour and alter anxiety-related neurochemical mechanisms in rats. The aim of this study was to determine if exposure to olfactory cues from 25% CR male rats leads to anxiolytic-like behaviour in male rats fed ad libitum. Animals were divided into four groups: control (fed ad libitum and given new bedding every 5 days), control olfactory group (fed ad libitum and given the bedding from the control group every 5 days), CR (fed a 25% CR regime and given new bedding every 5 days), and CR olfaction (fed ad libitum and given the bedding from the CR group every 5 days). All animals were assessed on two measures of anxiety-like behaviour: the open field and the elevated plus maze. The CR group demonstrated anxiolytic-like behavioural responses in the open field test, characterised by more time spent in the aversive central zone and a higher frequency of central and middle zone entries compared to all other groups. Intriguingly, the CR olfaction group demonstrated anxiolytic-like behaviour in the elevated plus maze test, characterised by more time spent on the open arms, and a higher ratio of open compared to total arm entries relative to the control and control olfaction groups. After the completion of behavioural testing, serum corticosterone assays were conducted on trunk blood. However, only the CR group demonstrated an increase in corticosterone. Olfactory cues from conspecifics on a CR regime significantly reduced anxiety-like behaviour in rats fed ad libitum, similar to the reduction in anxiety-like behaviour following CR. This may have implications for the development of more efficacious novel treatments for anxiety disorders.

Testosterone, social and sexual behavior of perinatally and lifelong calorie restricted offspring.

Calorie restriction (CR) during sensitive perinatal periods has consistently been demonstrated to alter the development of a variety of physiological systems, which consequently affect behavior. This study compared the social behavior and sexual behavior of the adult male offspring of mothers administered a 25% CR at one of four times in the perinatal period: a brief period preconception, during gestation, during lactation, or a lifelong restriction (beginning at conception and continuing throughout life). Levels of serum testosterone were also determined in these animals. Social interaction increased in the gestation and lifelong CR groups. The lifelong group also exhibited more dominant type behaviors. CR during preconception and lactation resulted in offspring that displayed an enhanced and more efficient copulatory pattern compared to all other conditions. This was demonstrated by a reduced frequency of intromissions, shorter latency to ejaculation, and a greater frequency of ejaculations by the preconception and lactation group compared to some, if not all of the other CR groups and controls. Serum testosterone was significantly higher in the preconception group compared to controls. These findings indicate that CR during specific periods of development can differentially alter the social behavioral phenotype and hormone levels in adulthood.

Anxiety-like behaviour in adult rats perinatally exposed to maternal calorie restriction.

Environmental stimuli such as caloric availability during the perinatal period exert a profound influence on the development of an organism. Studies in this domain have focused on the effects of under- and malnutrition while the effects of more mild levels of restriction have not been delineated. Rat dams and their offspring were subjected to one of five dietary regimens: control, CR50% for 3 days preconception, CR25% during gestation, CR25% during lactation, and CR25% during gestation, lactation, and post-weaning (lifelong). The pup retrieval test and maternal observations were conducted during lactation to quantify maternal care. In the pup retrieval test, dams that were concurrently experiencing CR (i.e., from the lactation and lifelong groups) displayed shorter latencies to retrieve all pups than the control and preconception groups and the lactation group constructed better nests than all groups. Adult offspring were tested in three tests of anxiety: the elevated plus maze, open field, and emergence test. No differences were observed in the elevated plus maze; however, in the open field preconception animals made fewer entries and spent more time in the central zone than controls. In addition, preconception offspring exhibited longer latencies to full body emergence, spent less time fully emerged, and spent more time engaged in risk assessment behaviours than all other groups. Offspring from the preconception group were also on average 11% heavier than control rats throughout life and displayed 37% higher serum leptin concentrations than controls. A potential role for leptin in the anxiogenic effect of preconception CR is discussed.

Alterations in male sexual behaviour, attractiveness and testosterone levels induced by an adult-onset calorie restriction regimen.

Despite an abundance of research on calorie restriction (CR) altering gonadal and appetite regulating hormones, the sexual behavioural consequences of CR remain to be examined systematically. This study compared the sexual behaviour, partner preference, serum testosterone and leptin levels of male adult Hooded Wistar rats administered a CR (continuous 25%, 50% CR or a temporary restriction) with ad libitum fed controls. The temporary restriction (Previous CR) failed to alter sexual behaviour, partner preference and levels of testosterone and leptin. The moderately 25% CR males did not demonstrate an impairment in sexual behaviour but did demonstrate a reduced level of attractiveness to females in one measure of partner preference. Sexual performance was affected by a substantial CR, as the CR 50% group exhibited a longer latency to the first intromission, indicating alteration in sexual arousal. Females also consistently demonstrated a clear preference for the control group compared to the CR 50% group. These findings indicate a possible reduction in the overall reproductive potential of the substantially CR animals. Testosterone levels were equally suppressed by both the 25% and 50% CR, while leptin levels were only reduced in the CR 50% group. Leptin, rather than testosterone, may have influenced the impairment in sexual behaviour only demonstrated by the substantially CR animals. Testosterone, may, however, play a role in modulating the preference of control over CR males, as attractiveness was totally reduced by a substantial CR, and partially reduced by a moderate restricted regimen.

Effects of adult-onset calorie restriction on anxiety-like behavior in rats.

Calorie restriction (CR) has consistently been shown to increase lifespan and ameliorate disease outcomes. Its effects on behavior are less clear, although anxiolytic-like effects have been observed. Rats were subjected to 1 of 4 dietary regimens: control, CR25%, CR50% and, an acute episode of CR and tested in 3 tests of anxiety: the open field test, the elevated plus maze, and the modified open field test. In the open field test, the CR25% and CR50% groups made more central zone entries than the control and Acute groups, which was primarily due to differences in the initial 5 min of the test. Moreover, both CR groups engaged in greater exploration of the central zone than the control group in the initial 5 min of the test. The Acute group also exhibited significantly longer latencies to leave the central zone at test onset than the control and CR50% group. In the elevated plus maze, the Acute group also displayed longer latencies to open arm entry as compared to the control and CR50% group and showed a lower ratio of open to total arm entries compared to all other groups. There were no effects of CR on any variable of the modified open field test. Possible neurochemical mechanisms underlying the anxiolytic-like effect of CR are discussed.