The Effect of Chronic Caloric Restriction on Anxiety-like Behaviours of Adult Male Rats

Anxiety-like behaviours (ALB), such as cautious exploration and avoidance of an area, occur to minimise the risk of an encounter with a threat. Such behaviours can become dysfunctional when they inappropriately mitigate opportunities due to a perceived increase in threat imminence. Recent studies have shown that calorie restriction (CR) of at least 18 days in duration can reduce ALB, particularly in adult male rats, although results have been inconsistent. Several factors have contributed to the inconsistency of findings, including variability in feeding and testing schedules, both of which impact locomotion and exploration. Critically, conventional tests of ALB in rodent research, such as the elevated plus maze (EPM), do not directly measure exploration but instead infer this behaviour from aggregated avoidance-focused ALB indices. The first aim of this thesis was to propose an innovative measure of exploration of rodent anxiogenic testing environments by evaluating the breadth and timing of movement. The current thesis proposed measuring the number of first visits to each unique area of the EPM to capture avoidance and exploration of them simultaneously. Findings from the current thesis showed that changes in the number of first visits over time, or Novel Exploration Growth (NEG), correspond to three behavioural phenotypes: exploratory, delayed and avoidant, which operationalises the conflict between safety and exploratory motivations. The conditional differences between the trends in NEG of these phenotypes were used to infer the exploratory and avoidance preferences over time and contrast them with conventional measures to provide a novel measure of ALB. Because dietary manipulation, feeding, and testing schedules impact the locomotion of rats during ALB testing, an ALB-independent measure of anxiety is used in the current thesis.Heart rate variability (HRV) is considered a valid indicator of defensive emotional states, such as anxiety, by assessing the parasympathetic and sympathetic branches of the autonomic nervous system. HRV is observed through the changes in the interval between peaks of the R wave of the QRS complex produced by cardiac ventricular depolarisation. Parasympathetic branch activity typically increases this interval and has been conventionally assessed through several measures in the time and frequency domains. Chief amongst these is the spectral power in the high frequency band (between 0.75 Hz and 2.5 Hz in rats), which is expressed in absolute units, the natural log, or normalised over total band power. Previous attempts to measure sympathetic influence on the length of R wave intervals have been controversial, and the ClassA framework was recently developed to address this gap. This framework resolves cardiac acceleration trends over different time windows to quantify the amount of parasympathetic or sympathetic dominance present. The current thesis measured sympathetic dominance via the ClassA framework alongside conventional measures demonstrating parasympathetic activity to assess their impact during ALB testing. The second aim of this thesis was to examine the effect of CR on HRV and ALB on adult male rats during exposure to the EPM. 14 rats were implanted with biopotential telemeters, 7 of which had their caloric intake restricted by 25% for 21 days prior to testing. It was hypothesised that CR would increase HRV, which was partially supported by an increase in normalised high frequency power. The current thesis also found that CR reduced sympathetic dominance during testing in EPM by an average of 5%, with the reduction becoming more pronounced as testing progressed. It was also hypothesised that CR would reduce ALB in the EPM, which was supported as CR increased latency to enter and the absolute and proportional number of entries and amount of time spent in the open arms of the EPM. Additionally, CR rats showed equivalent trends in their NEG of the open and closed arms, demonstrating a single, arm-agnostic phase of exploration. To assess whether the effects of CR demonstrated during testing in the EPM would persist despite an increase in threat imminence, the current thesis introduced a predator’s scent (i.e cat urine) into an open field arena. The final aim of this thesis was to examine the effect of CR on HRV and ALB on adult male rats during exposure to odour of a predator located inside an open field arena. It was hypothesised that CR would increase HRV during exposure to cat urine, which was supported by higher natural log and normalised high frequency powers compared to ad libitum-fed counterparts. Additionally, CR reduced sympathetic dominance by an average of 7% during exposure to the predator odour, with the reduction becoming more pronounced as testing progressed. It was also hypothesised that CR would reduce ALBs during exposure to the odour of a predator. However, this hypothesis was only supported by an increase in the duration of time spent in the scent zone. Conversely, when the absolute distance to the scent was analysed, CR and ad libitum-fed rats were equidistant from the cat urine throughout testing. Additionally, NEG was equivalent between the two groups for the total arena and scent zones during exposure to the odour. This study showed that exploration by rats in tests of ALB could be directly measured by tracking first visits into zones of interest. NEG was able to uniquely demonstrate that CR produced parity in the exploration of the arm types of the EPM. Importantly, NEG provides a singular measure of the conflict between exploration and safety in rats. Additionally, results from the current thesis showed that CR increased HRV independently of ALB during exposure to the odour but not during the less threatening EPM. By uniquely combining these cardiovascular and behavioural measures, the current thesis has demonstrated for the first time that the anxiolytic effects of CR are not consistent between behaviour and cardiac responses, and relies on a predator not being detected via their odour. These findings are relevant to future studies assessing CR due to the impact of exploration and the level of perceived threat imminence when assessing the presence and intensity of its anxiolytic effects on behaviour.