Crosstalk between short- and long-term calorie restriction transcriptomic signatures with anxiety-like behavior, aging, and neurodegeneration: implications for drug repurposing.

Calorie restriction (CR) is considered an effective intervention for anxiety, aging, and obesity. We investigated the effects of short- and long-term CR on behavior as well as transcriptome profiles in the hypothalamus, amygdala, prefrontal cortex, pituitary, and adrenal glands of Hooded Wistar and Long Evans male rats. A reduction in anxiety-like behavior, as assessed via the elevated plus maze, was observed in both short- and long-term CR. Despite this, short- and long-term CR regulated different sets of genes, leading to distinct transcriptomic signatures. The employed models were able to simultaneously analyze categorical and numerical variables, evaluating the effect of tissue type along with expression data. In all tissues, transcription factors, zinc finger protein 45-like and zinc finger BTB domain-containing two, were the top selected genes by the models in short and long-term CR treatments, respectively. Text mining identified associations between genes of the short-term CR signature and neurodegeneration, stress, and obesity and between genes of the long-term signature and the nervous system. Literature mining-based drug repurposing showed that alongside known CR mimetics such as resveratrol and rapamycin, candidates not typically associated with CR mimetics may be repurposed based on their interaction with transcriptomic signatures of CR. This study goes some way to unravelling the global effects of CR and opens new avenues for treatment for emotional disorders, neurodegeneration, and obesity.

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.

Social support moderates the effect of stress on the cortisol awakening response in dementia family caregivers.

Dementia caregiving has been associated with a range of adverse effects on the physical health of the caregiver. However, the specific mechanisms underlying the relationship between dementia caregiver stress and ill health remain unclear. The aim of this study was to investigate, using available prospective data, the relationship between perceived stress (burden) and pre-clinical indices of ill-health (cortisol awakening response and secretory immunoglobulin A) amongst dementia caregivers. The potential moderating effect of social support on the perceived stress-physiological stress/health relationship was also explored. Participants (N = 31) were caregivers of community-dwelling older adults living with dementia who were enroled in a psychoeducation support program and provided data (study questionnaire and saliva samples) at two timepoints (T1 and T2), 10 weeks apart. Hierarchical regressions were used to determine if changes in stress and social support predicted change in each of the physiological outcomes. Findings indicate that caregivers with more hours of care at T1, or with greater satisfaction with social support, were more likely to exhibit an adaptive cortisol awakening response at T2. Moreover, social support was found to buffer the effect of caregiver stress and hours of caregiving on the cortisol awakening response. Implications for future interventions targeting caregiver health are discussed.

Assessing a hyperarousal hypothesis of insomnia in adults with autism spectrum disorder.

This study aimed to investigate the relationship between sleep, psychopathology (anxiety, depression and presleep arousal) symptoms, and cortisol in adults with autism spectrum disorder (ASD). The sample composed of 29 adults with ASD (51.7% males) and 29 control adults (51.7% males) aged 21-44 years. Thirteen adults with ASD were medicated for a comorbid diagnosis of anxiety and/or depression (ASD-Med), while the remaining 16 adults with ASD were not medicated for such diagnoses (ASD-Only). Participants completed a questionnaire battery, 14-day sleep/wake diary and 14-day actigraphy assessment. On one day during the data collection period, participants collected five saliva samples, hourly, prior to sleep and two morning samples; immediately upon waking and 30 min thereafter for the analysis of cortisol. Cortisol 1 hr prior to habitual sleep onset time was associated with poorer sleep efficiency in both ASD groups and increased wake after sleep onset duration (ASD-Only). Higher subjective somatic arousal was also associated with increased sleep onset latency, regardless of group, and poorer sleep efficiency in the ASD-Only group. ASD-Only participants had significantly greater reductions in evening cortisol concentrations compared to both ASD-Med and control participants. No significant group differences were found for the cortisol awakening response. Findings suggest a hyperarousal hypothesis of insomnia in adults with ASD. Moreover, the low cortisol levels observed in ASD-Only adults suggest dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis. Longitudinal studies exploring the interplay between insomnia, anxiety and HPA axis regulation across the lifespan in those with ASD are warranted. Autism Res 2019, 12: 897-910. © 2019 International Society for Autism Research, Wiley Periodicals, Inc. LAY SUMMARY: Both objective (cortisol) and subjective (somatic) physiological arousal were associated with poor sleep quality in adults on the autism spectrum. Adults with autism spectrum disorder (ASD) who were not medicated for a comorbid diagnosis of anxiety and/or depression also had dampened cortisol secretion, suggesting a dysregulation of the hypothalamic pituitary axis. Longitudinal studies investigating the relationship between sleep, psychopathology symptoms and physiological arousal in autistic individuals are warranted. Autism Res 2019. © 2019 International Society for Autism Research, Wiley Periodicals, Inc.

Employment status is related to sleep problems in adults with autism spectrum disorder and no comorbid intellectual impairment.

Both sleep problems and unemployment are common in adults with autism spectrum disorder; however, little research has explored this relationship in this population. This study aimed to explore factors that may be associated with the presence of an International Classification of Sleep Disorders-Third Edition defined sleep disorder in adults with autism spectrum disorder (IQ > 80). A total of 36 adults with autism spectrum disorder and 36 controls were included in the study. Participants completed a 14-day actigraphy assessment and questionnaire battery. Overall, 20 adults with autism spectrum disorder met the International Classification of Sleep Disorders-Third Edition criteria for insomnia and/or a circadian rhythm sleep-wake disorder, while only 4 controls met criteria for these disorders. Adults with autism spectrum disorder and an International Classification of Sleep Disorders-Third Edition sleep disorder had higher scores on the Pittsburgh Sleep Quality Index and were more likely to be unemployed compared to adults with autism spectrum disorder and no sleep disorder. The findings demonstrate, for the first time, that sleep problems are associated with unemployment in adults with autism spectrum disorder. Further research exploring the direction of this effect is required; sleep problems that have developed during adolescence make attainment of employment for those with autism spectrum disorder difficult, or unemployment results in less restrictions required for optimal and appropriate sleep timing.

Assessing the Dim Light Melatonin Onset in Adults with Autism Spectrum Disorder and No Comorbid Intellectual Disability.

This study assessed melatonin levels and the dim light melatonin onset (DLMO) in adults with Autism Spectrum Disorder (ASD) and also investigated the relationships between melatonin and objectively measured sleep parameters. Sixteen adults with ASD (ASD-Only), 12 adults with ASD medicated for comorbid diagnoses of anxiety and/or depression (ASD-Med) and 32 controls participated in the study. Although, the timing of the DLMO did not differ between the two groups, advances and delays of the melatonin rhythm were observed in individual profiles. Overall mean melatonin levels were lower in the ASD-Med group compared to the two other groups. Lastly, greater increases in melatonin in the hour prior to sleep were associated with greater sleep efficiency in the ASD groups.

An experimental examination of the effort-reward imbalance model of occupational stress: Increased financial reward is related to reduced stress physiology.

Effort-reward imbalance in the workplace is linked to a variety of negative health and organisational outcomes, but it has rarely been assessed experimentally. We manipulated reward (while keeping effort constant) in a within-subjects design with female participants (N=60) who were randomly assigned to high and standard reward conditions within a simulated office environment. Self-report, behavioural (task performance), and physiological (heart rate variability, salivary alpha amylase) measures assessed the impact of increased financial reward. Participants reported increased perceptions of reward, performed moderately better on the task, and were less physiologically reactive in the high reward versus the standard condition. These findings highlight the importance of assessing both subjective self-reports of stress together with objective physiological measures of stress, and suggest that increasing monetary rewards has the potential to decrease stress physiological reactivity, and in turn, reduce the risk of ill-health in employees, and may also positively influence task efficacy.

An experimental study of the job demand-control model with measures of heart rate variability and salivary alpha-amylase: Evidence of increased stress responses to increased break autonomy.

We assessed in an experimental design whether the stress response towards a work task was moderated by the autonomy to choose a break during the assigned time to complete the task. This setting is defined in accordance with the theoretical framework of the job-demand-control (JDC) model of work related stress. The findings from naturalistic investigations of a stress-buffering effect of autonomy (or 'buffer hypothesis') are equivocal and the experimental evidence is limited, especially with relation to physiological indices of stress. Our objective was to investigate if increased autonomy in a particular domain (break time control) was related with adaptive physiology using objective physiological markers of stress; heart rate variability (HRV) and salivary alpha amylase (sAA). We used a within-subject design and the 60 female participants were randomly assigned to an autonomy (free timing of break) and standard conditions (fixed timing of break) of a word processing task in a simulated office environment in a random order. Participants reported increased perceptions of autonomy, no difference in demand and performed worse in the task in the break-time autonomy versus the standard condition. The results revealed support for the manipulation of increased autonomy, but in the opposing direction. Increased autonomy was related with dysregulated physiological reactivity, synonymous with typical increased stress responses. Potentially, our findings may indicate that autonomy is not necessary a resource but could become an additional stressor when it adds additional complexity while the amount of work (demands) remains unchanged. Further, our findings underscore the need to collect objective physiological evidence of stress to supplement self-reported information. Self-report biases may partially explain the inconsistent findings with the buffer hypothesis.

Increased systolic blood pressure reactivity to acute stress is related with better self-reported health.

The stress reactivity hypothesis posits that the magnitude of cardiovascular reactions to acute stress tasks is related with future blood pressure status, heart hypertrophy, and atherosclerosis. We assessed the stress reactivity hypothesis and aimed to identify which physiological indices (blood pressure, heart-rate, cortisol, salivary immunoglobulin A (sIgA)) related to self-reported mental and physical health. We also assessed if physiological reactions elicited by an acute stressor were more related than basal assessments. Participants provided physiological samples, self-reported stress and health-data before and after an assessed 5-7 minute academic oral presentation. In hierarchical regression models, increased systolic and reduced sIgA reactivity was associated with better perceptions of mental health. Reactivity data were more related to self-reported data than basal data. In line with the only 2 studies to assess the reactivity hypothesis with self-perceived health, increased systolic reactivity was best associated with better perceived physical and mental health. The findings suggest that increased SBP reactivity may also be associated with positive health outcomes. Further research is required to determine if increased or decreased sIgA reactivity is most predictive of future morbidity.

Calorie restriction dose-dependently abates lipopolysaccharide-induced fever, sickness behavior, and circulating interleukin-6 while increasing corticosterone.

In mice a 50% calorie restriction (CR) for 28days attenuates sickness behavior after lipopolysaccharide (LPS) and these mice demonstrate a central anti-inflammatory bias. This study examined the dose-dependent effect of CR on sickness behavior (fever, anorexia, cachexia) and peripheral immune markers post-LPS. Male Sprague-Dawley rats fed ad libitum or CR by 50% for 14, 21, or 28days were injected on day 15, 22, or 29 with 50µg/kg of LPS or saline (1mL/500g). Changes in body temperature (Tb), locomotor activity, body weight, and food intake were determined. A separate cohort of rats was fed ad libitum or CR by 50% for 28days and serum levels of corticosterone (CORT), interleukin 6 (IL-6), and IL-10 were determined at 0, 2, and 4h post-LPS. The rats CR for 28days demonstrated the largest attenuation of sickness behavior: no fever, limited reduction in locomotor activity, no anorexia, and reduced cachexia following LPS. Rats CR for 14 and 21days demonstrated a partial attenuation of sickness behavior. Rats CR for 14days demonstrated a larger increase in Tb, larger reduction in locomotor activity, and larger weight loss compared to rats CR for 21days. Serum CORT was increased at 2h post-LPS in ad libitum and CR groups; however it was two times larger in the CR animals. Levels of IL-6 were significantly attenuated at 2h post-LPS in the CR animals. IL-10 levels were similar post-LPS. CR results in an enhanced anti-inflammatory response in the form of increased CORT and diminished pro-inflammatory signals.

Effort-reward imbalance at work and pre-clinical biological indices of ill-health: the case for salivary immunoglobulin A.

Physiological indices of stress and ill-health (cortisol and salivary immunoglobulin A) were assessed to determine if they were predicted by Siegrist's effort-reward imbalance model (ERI) with an aim of identifying employees at risk of illness. Male Australian dairy farmers (N=66) completed the Perceived Stress Scale, Work related Questions II & III, Eysenck Personality Questionnaire Revised--Short and demographic questions and provided morning saliva samples (at awakening and 30 min post awakening) on a working day, which were subsequently analysed for cortisol and salivary immunoglobulin A (sIgA) concentration levels. A high percentage (45.5%) of the sample reported an imbalance between efforts and rewards in the workplace that may place them 'at risk' for ill-health. After controlling for disposition, sIgA scores were more successfully predicted by the ERI than the cortisol assessments. Although both efforts and rewards were significantly associated with sIgA, efforts were most strongly associated. The dispositional trait overcommitment, did not moderate the experience of stress on the physiologic indices. The current investigation supports the continued use of sIgA in studies that use biomarkers to assess occupational stress. ERI ratio scores >1 aligned with previous findings that suggest elevated risk of illness for these employees.

Piracetam, an AMPAkine drug, facilitates memory consolidation in the day-old chick.

Piracetam is an AMPAkine drug that may have a range of different mechanisms at the cellular level, and which has been shown to facilitate memory, amongst its other effects. This series of experiments demonstrated that a 10mg/kg dose of piracetam facilitated memory consolidation in the day-old chick when injected from immediately until 120min after weak training (i.e. using a 20% v/v concentration of methyl anthranilate) with the passive avoidance learning task. Administration of piracetam immediately after training led to memory facilitation which lasted for up to 24h following training. This dose of the AMPAkine was not shown to facilitate memory reconsolidation. These findings support the contention that application of the AMPAkine piracetam facilitates memory using a weak training task, and extend the range of actions previously noted with NMDA-related agents to those which also facilitate the AMPA receptor.

Brain-derived neurotrophic factor facilitates memory consolidation and reconsolidation of a weak training stimulus in the day-old chick.

Recent research has pointed to a role for brain-derived neurotrophic factor (BDNF) in long-term potentiation and memory. The present series of experiments examined the effects of the application of exogenous BDNF on memory consolidation and reconsolidation of a weak training stimulus with the day-old chick, using the passive avoidance learning paradigm. Chicks injected intracranially with 12.5 µg/mL recombinant BDNF immediately after a single-trial training event displayed enhanced retention relative to saline up to 24h post-training. Furthermore, this dose was also shown to enhance retention when administered following initial weak training. Thus, exogenous BDNF was shown to enhance both consolidation and reconsolidation of memory when administered acutely to the day-old chick.

Memantine facilitates memory consolidation and reconsolidation in the day-old chick.

Memantine is a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist that has been approved for the treatment of the cognitive deficits noted in Alzheimer's disease. While there is a body of research that supports memantine's facilitative action upon memory compromise, this series of studies aimed to investigate the effects of this drug in healthy animals with intact memory functioning. A 0.1 mM dose of memantine injected immediately after a weakly aversive training event (i.e. 20% v/v methyl anthranilate) was found to enhance passive avoidance learning for this event in day-old chicks up to 24 h following training. The same dose of memantine was also observed to enhance memory for the training event when it was administered in conjunction with a reminder trial. These results suggest that memantine is capable of facilitating both memory consolidation as well as memory reconsolidation. It was concluded that memantine's mechanism may involve the short-term or intermediate memory phases of the Gibbs and Ng model of memory, and that the current findings represent enhancement of intact memory, rather than amelioration of memory compromise.

Coping in an intermittent swim stress paradigm compromises natural killer cell activity in rats.

The effects of intermittent swim stress and stressor controllability on natural killer cell activity (NKCA) was examined. Significant decreases in splenic NKCA were observed immediately post-stress, but only when the stress was controllable. Although decreased NKCA was also observed in yoked rats subjected to the same stressor, it failed to attain statistical significance. Previous results suggest these effects are not due to corticosterone. The results suggest a cost of coping on the acute, in vitro immune measure of NKCA.

Stress resilience and vulnerability: the association with rearing conditions, endocrine function, immunology, and anxious behavior.

BACKGROUND: The current study explored the underlying behavioral, endocrine, and immune markers of vulnerability to stress-induced depression, and the impact of rearing environments on adult functioning. METHOD: Adult Sprague-Dawley rats (n=195) were reared in either Maternal Separation (MS), Early Weaning and Isolation (EWI), or Non-Handled (NH) conditions. Anxiety behavior was assessed using the emergence test at mean postnatal day (PND) 60. Stress-induced depressive behavior was measured at mean PND 86 using an intermittent cold water swim stress and swim escape test (SET) paradigm. Immediately following the SET, and in a sample of naïve controls (N=31), trunk blood was collected to assay for serum corticosterone (CORT) and spleens were removed for determination of Concanavalin A (Con-A) stimulated T-cell proliferation. RESULTS: Stress vulnerable rats (top tertile of SET swim time) were characterised by increased anxiety-like behavior, greater post-stress CORT concentrations, and a significantly higher Con-A induced T-cell proliferative response compared to stress resilient rats (bottom tertile of SET swim time). The EWI rearing condition was a contributing factor in predicting total swim escape time, however MS was not. MS offspring did have double the basal level of CORT than NH offspring, suggestive of a hyperfunctioning HPA axis. CONCLUSION: The swim stress animal model enabled observation of stress vulnerability and resilience; results point towards the existence of distinct behavioral, endocrine, and immunological profiles of the vulnerable and resilient animal, which may have important implications for mental health and stress research.

Spatial and temporal dissociation of AQP4 and Kir4.1 expression during induction of refractive errors.

PURPOSE: Spatial co-localization of aquaporin water channels (AQP4) and inwardly rectifying potassium ion channels (Kir4.1) on the endfeet regions of glial cells has been suggested as the basis of functionally interrelated mechanisms of osmoregulation in brain edema. The aim of this study was to investigate the spatial and temporal changes in the expression of AQP4 and Kir4.1 channels in an avascular retina during the first week of the optical induction of refractive errors. METHODS: Three-day-old hatchling chicks were randomly assigned to three groups and either did not wear lenses or were monocularly goggled with +/-10D lenses for varying times up to 7 days before biometric assessment. Retinal tissue was prepared either for western blot analysis to show the presence of the AQP4 and Kir4.1 protein in the chick retina or for immunolocalization using AQP4 and Kir4.1 antibodies to determine the regional distribution and intensity of labeling during the induction of refractive errors. RESULTS: As expected, ultrasonography demonstrated that all eyes showed rapid elongation post hatching. Negative lens-wearing eyes elongated faster than fellow eyes or normal non goggled eyes and became progressively more myopic with time post lensing. Positive lens-wearing eyes showed reduced ocular growth compared to normal controls and developed a hyperopic refraction. Quantitative immunohistochemistry revealed the upregulation of AQP4 channel expression on Müller cells in the retinal nerve fiber layer during the first 2 days of negative lens wear. Kir4.1 channel upregulation in the inner plexiform layer was only found on day 4 of positive lens wear during the development of refractive hyperopia. CONCLUSIONS: These results indicate that the expression of AQP4 and Kir4.1 channels on Müller cells is associated with the changes in ocular volume seen during the induction of refractive errors. However, the sites of greatest expression and the temporal pattern of the upregulation of AQP4 and Kir4.1 were dissimilar, indicating a dissociation of AQP4 and Kir4.1 function during refractive error development. Increased AQP4 expression in the nerve fiber layer is suggested to contribute to the rapid axial elongation and movement of fluid into the vitreous cavity in the presence of minus lenses; whereas, upregulation of Kir4.1 channels appears to play a role in limiting axial elongation in the presence of plus lenses.

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.

Gamma-butyrolactone (GBL) disruption of passive avoidance learning in the day-old chick appears to be due to its effect on GABAB not gamma-hydroxybutyric [corrected] acid (GHB) receptors.

Gamma-butyrolactone (GBL) is a prodrug to gamma-hydroxybutyric acid (GHB) and metabolises to GHB when ingested. Discrimination stimulus studies report generalisation of effects of GHB to GBL. While amnesia is one of the most commonly reported symptoms of GHB's ingestion in human users, as yet few studies have examined this effect. Although an endogenous GHB specific receptor is present in the brain, several studies have indicated that the clinical effects of exogenous doses of GBL/GHB are due to its action on GABA(B) receptors rather than on the GHB receptor. In this series of studies, New Hampshire x White leghorn cockerels were trained using a modified version of the passive avoidance learning task. Subcutaneous injections of GBL induced a memory deficit by 10 min post-training, which persisted for at least 24 h. No effect on memory was seen with administration of the specific GHB agonist NCS-356 (gamma-p-chlorophenyl-trans-4-hydroxycrotonate). The GBL-induced memory deficit appeared similar to the deficit produced by baclofen, where the antagonist facilitated learning. Additionally, GBL-induced memory deficit was ameliorated by application of a GABA(B) antagonist. The results support the hypothesis that GBL exerts its influence on memory via the GABA(B) receptor rather than by the specific GHB receptor.

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.