Aims

Above I have examined the function, evolution, mechanism and development of aging in general, and introduced the dog as an emerging model species not only for human cognition, physiology and disease, but also for aging research. In order to study aging in dogs I have established the Senior Family Dog Project at the Department of Ethology, Eötvös Loránd University in 2016. The group explores the cognitive aging of family dogs using an interdisciplinary approach, integrating methods of ethology, neuroscience and genetics/molecular biology (Figure 1). Overall, our main aim is (1) to provide empirical evidence that the dog model holds unmatched potentials for aging research which is still debated regarding translational aspects and (2) to promote healthy aging for the benefit of both owners and dogs. More specifically, we aim to characterise the canine aging phenotype with cross-sectional and longitudinal investigations, and identify the underlying processes. We use large scale surveys and develop sensitive and standardized behaviour tests to enable veterinarians, dog trainers, and behaviour counsellors to assess dogs’ cognitive abilities, document changes occurring over time and detect early signs of pathological cognitive decline.

We also aim to identify neural markers of aging with non-invasive neuroimaging techniques and uncover the genetic background of extremely long life by sequencing the genome of methuselah dogs. Our recently established Canine Brain and Tissue Bank provides samples for both gross anatomical evaluation and also for tracing cellular changes (e.g. by RNA-analysis, immunohistochemistry, and proteomics). The database of the Bank includes both molecular

and behavioural data from individuals and offers a unique opportunity for obtaining specific canine brain tissues. The results are expected to aid our knowledge about the rate of successful and pathological aging, help canine welfare initiatives through guidelines for a healthy lifestyle toward successful aging, and the understanding of the biology of human cognitive aging.

In the thesis I present published research on age-related differences in the demographics and health, cognition, emotional and face processing, personality, and intraspecific relationships in companion dogs and our recent initiatives about studying brain activity during sleep and the genetic background of individuals with an exceptionally long lifespan. Ongoing research is presented in the Perspectives chapter.

Figure 1. Visual abstract of the Senior Family Dog Project. ((f)MRI (functional) magnetic resonance imaging, UHR-CT:

ultrahigh resolution computertomography, EEG: electroencepalography, qPCR: quantitative polymerase chain reaction, RNA-seq: ribonucleic acid sequencing).

Specific questions

I. Demography and health

Chapter 7: The prevalence of age-related cognitive decline in companion dogs across the entire adult lifespan

Before conducting any study about dog aging, we have to estimate the prevalence of cognitive decline. However, differentiating between dogs showing signs of typical aging or early signs

of cognitive dysfunction based on direct behavioural measures has proven to be a challenging task. Specific questionnaires have been developed for veterinarians and owners, but these show huge variation in their estimation of the proportion of affected dogs (ranging from 14 to 68%, depending on the measurement tool and the population, Szabó et al., 2016). Whether changes regarding the prevalence of these behaviours are detectable before 8 years of age has not been investigated. Findings regarding other risk factors such as sensory impairment, body size, sex, and neuter status have been also contradictory. Our goal is to investigate the impact of these factors (sensory deficits, sex, neutered status, training) on the occurrence of behavioural signs associated with cognitive decline, taking into account the differences in the expected lifespan of small and large dogs. (The gathered information will be reflective about the relationship between the putative behavioural signs of cognitive decline and relative age, sensory impairments and certain demographic factors across the whole adult lifespan. Diagnosis of cognitive dysfunction needs veterinary examination). We expect that beside relative age (mean lifespan for each breed/ actual age of the dog), impairments in every sensory domain and training history are associated with behavioural problems reflecting cognitive decline.

Chapter 8: Demographics of companion dogs across age groups and identifying the key variables associated with health status

Dogs go through similar stages of development as humans, and their living conditions and nutritional requirements can change considerably as they age. However, many owners do not consider their dog’s life stage when selecting a diet and are unable to recognize if their pets are overweight or obese (Davies, 2012; Holmes, Morris, Abdulla, Hackett, & Rawlings, 2007).

Therefore, e.g. in the UK, up to 60% of dogs are now classified as overweight or obese (Courcier, Thomson, Mellor, & Yam, 2010; Holmes et al., 2007; YouGov, 2017). We utilize an on-line questionnaire to examine the link between the age and health of the dog, and owner and dog demographics in a cross-sectional sample. We expect to identify key variables associated with health status. Since this study is exploratory, we include a total of 27 dog and owner demographic factors in our analysis.

II. Cognition, emotion and face processing

Chapter 9: Developing a behaviour test for assessing discrimination and reversal learning A large body of evidence indicates that functional decline in cognitive domains, such as learning, memory, executive function, and spatial function, occurs similarly in dogs and humans as they age. However, several tests involve laboratory dogs, large apparatus and prolonged training. The aim of this study is to develop a reversal learning task which could detect age-related changes in the learning abilities of companion dogs without overt medical problems in a short time-frame (about 1 hour). We design a simple and reproducible version of a reversal learning task, which does not require large or complex equipment or several weeks of training, in contrast to previous tests. We also aim at developing tests that could be repeated over time, to monitor the progress of the condition. Therefore, we test the dogs with different

stimuli, either with the location or the physical attributes of the rewarded object, in order to explore whether these would affect their performance.

Chapter 10: Positivity effect in dogs: do old dogs experience less negative emotions?

Age-related changes in the brain can alter how emotions and the social environment are processed. In humans, attention and memory shift from negative to positive stimuli with increasing age, i.e. older people experience fewer negative emotions, which is known as the

“positivity effect”. We measure how fast young and old dogs react to human vocalisations, with positive, negative, and neutral emotional valence, during a sound playback test. We hypothesise that old dogs are less responsive and react slower to negative vocalisations than young dogs, suggesting that dogs have the same positivity effects as humans.

Chapter 11: Age-related effects in looking at faces of humans and conspecifics

The gaze of others is informative for both humans and dogs. In humans, responsiveness to the eyes and gaze change with age. We show pictures of conspecific and human faces to dogs and expect that older dogs orient less towards them due to decreased perception. It is also possible that they react differently to the pictures (e.g. orient more towards the human face but less towards the dog head) than young dogs, due to altered processing of social stimuli. We also investigate the effect of head shape and breed function on the looking behaviour.

III. Personality and intraspecific relationships

Chapter 12: Age-related changes in human-based personality traits and associations with owner and dog demographics

Numerous cross-sectional studies in humans have reported mean-level changes in personality traits across the lifespan, and that significant life events and educational experiences can influence personality traits. People tend to show increased confidence, warmth, self-control, and emotional stability with age. The dog has been suggested as a possible model for human personality development. We adapt a human personality questionnaire, and analyse the relationship between canine personality traits, age and other variables on a large sample with more than 10,000 adult individuals.

Chapter 13. Interventions to increase play and training motivation may alleviate the negative effects of aging

The literature is contradictory regarding the number and definitions of canine personality traits.

Inconsistencies may be due to the fact that different methods were used to obtain the trait scores, and/or different age groups and age ranges were examined. The Dog Personality Questionnaire has been shown to demonstrate reliability and validity, and has been used in numerous studies to measure personality in dogs via owner report. The aim of this study is to investigate the effects of age on dog personality on a different sample with a different questionnaire than in our previous study. We predict a strong influence of dog age on dog personality and confirm our previous results with an independent survey.

Chapter 14. The relationship between age, personality, dominance and leadership in a group of dogs

Movement interactions and the underlying social structure in groups have relevance across many social-living species. Decisions about which individual leads a group can be based on an

‘egalitarian’ system, but in practice it is often influenced by underlying social network structures and by individual characteristics such as older individuals being dominant and/or leaders of the group. Our goal is to identify how a group of family dogs decides the direction of their movements and whether there is a leader, or is decision-making based on an egalitarian system. We collect GPS trajectory data from an owner and a group of dogs during several walks. We assume that younger individuals will follow the older, more experienced ones. We also examine the role of personality and dominance in connection with leadership.

Chapter 15. Dominance status and age in companion dogs sharing the same household Dominance describes long-term dominant-subordinate social relationships within a dyad or group. An age-related hypothesis has been suggested to explain dominance in dogs. According to this, a simple rule of thumb could explain formal dominance in dogs: young dogs perform affiliative behaviours towards older group members. However, the literature does not always support correlations of dominance rank with age, and it remains unexplored whether the age related hypothesis is a better predictor of formal dominance than dominance displays. We launch a questionnaire study to investigate owner-derived estimates of dominance in dog dyads sharing the same household. We suppose that that dog age does not explain the occurrence of dominance-related behaviours over the owners' estimate of dominance status.

IV. Steps towards understanding the mechanisms of aging

Chapter 16. Age related differences in the spindling activity of the sleeping brain

The dog brain is still relatively unknown compared to the brains of other model animals.

Mechanistic and correlational evidence from humans and rats support sleep spindle involvement in memory consolidation. Spindle amplitude and occurrence are lower in the elderly, while frequency increases. If findings on humans generalize to dog sleep spindles (0.5-5 seconds long, 9-16 Hz oscillations, observed mainly in the non-REM EEG signal), it would strengthen the argument that dogs are good model animals. We investigate a large sample of intact and neutered family dogs of both sexes, varying in breed and age, searching for sleep spindles in segments of non-REM sleep. We predict a similar pattern for spindle occurrence as in humans.

Chapter 17. The genetic background of longevity based on whole-genome sequence data of two methuselah dogs

The genetic research of aging is generally concerned with mutations found to increase longevity. The oldest known dog (†29) lived 130% longer than the average lifespan of dogs (13), in contrast to humans where centenarians live at most 50% longer compared to the average human lifespan. By studying methuselah dogs (individuals with an exceptionally long

lifespan), we aim to extend our understanding on extreme longevity. We use the whole-genome sequence of two extremely old dogs, which lived 22 and 27 years (90-135% longer than the average lifespan of dogs) to investigate the genetic background of longevity and determine why these dogs were successful in aging. We also compare the results to that of human centenarians.

We expect to detect rare genetic variants linked to canine longevity, which could be prime candidates for age-related research in the future.