Throughout life, sensory systems must ensure our ability to perceive and recognize the world. Thus, the time course and severity of its involution through the aging process are decisive for individuals since these limit the ability to sustain the quality of sensory stimuli, affecting not only cognitive processes but also their self-esteem, habits, and styles of a lifetime. In older people, sensory deficiencies significantly increase their risk of biological, psychological, and social impoverishment (Dziechciaz and Filip, 2014). Sensory deficits have been proposed as predictors of cognitive decline in older adults and as early indicators of the prodromal stages of Alzheimer’s disease. Among them, the association between olfactory dysfunction and cognitive impairment is of growing interest. Olfactory dysfunction has attracted much attention among researchers in the field of brain damage and is a well-known marker for many neurological diseases (Wilson et al., 2009). By causing such vulnerability and/or disability, sensory deficiencies significantly increase the risk of the impoverishment of older people on a biological, psychological, and social level (Volkers and Scherder, 2011). More importantly, emergent literature unveiled anosmia as being more predictive of 5-year mortality risk than cardiovascular disease (Van Regemorter et al., 2020).

The complete and adequate evaluation of patients plays a role in any diagnostic work to demonstrate the etiology, pathogenesis, diagnosis, and possible prevention/therapy in neurodegenerative disorders (Kondo et al., 2020). Olfactory loss, which is known to occur in bacterial and viral infections and is considered an early biomarker in the neurodegenerative processes of Alzheimer’s and Parkinson’s diseases (Kovács, 2004), has also been reported as an early indicator of current SARS-CoV-2 infection (Mahalaxmi et al., 2020). Sniffing impairment or deficiency is present in almost 90% of the population with Alzheimer’s disease (AD) (Conti et al., 2013). Furthermore, in certain cases with mild cognitive impairment, this loss has also been observed during the transition phase between normal aging and dementia. Previous studies have found that olfactory impairment or deficiency may be an indicator that people affected by mild cognitive impairment are more likely to develop dementia (Invitto et al., 2018). A first diagnostic screening based on olfactory tests would allow the rapid implementation of preventive approaches and thus improve cognition and brain and mental health (Brai et al., 2020).

Male and female sexes can be regarded as two exceptional natural scenarios to study the role of biological, psychological, and social factors, their functional interplay, and their impact on the crosstalk of homeostatic networks in health and disease through life cycle (Giménez-Llort et al., 2014). At the translational level, in the present brief report, we have studied the olfactory signatures in male and female mice with normal and neurodegenerative aging associated with advanced stages of AD. For this purpose, we used 3xTg-AD mice (Oddo et al., 2003) and age-matched non-transgenic mice, both with a C57BL/6J genetic background (Baeta-Corral and Giménez-Llort, 2014). The 3xTg-AD mouse is a genetic model of Alzheimer’s disease that presents not only AD cognitive dysfunction but also a striking phenotype modeling some “behavioral and psychological symptoms of dementia” (BPSD), including neuropsychiatric symptoms such as anxiety, apathy, and depression-like behaviors (Giménez-Llort et al., 2006, 2007; Torres-Lista and Giménez-Llort, 2013; Baeta-Corral et al., 2018a).

In accordance with the increased mortality rates in dementia compared to the older population (van Dijk et al., 1991), the mortality of 3xTg-AD mice is higher than that of their wild-type counterparts of the same age, a phenomenon that has been related to their neuro-immune-endocrine impairment (Giménez-Llort et al., 2008). Furthermore, in male mice, the rates increase exponentially at 12 to 13 months (Giménez-Llort et al., 2008), an age mimicking the advanced stages of the disease (Oddo et al., 2003; Belfiore et al., 2019), thus providing a naturalistic scenario to study the impact of the loss of life mates.

The present work characterizes AD genotype and sex effects on olfactory ethograms in the food finding test that define olfactory signatures in male and female non-transgenic and 3xTg-AD mice.

The food finding test (Deacon et al., 2009) is an easy and ready-to-do test that can be used in any animal department setting without the need to buy anything since it is based on the ethological response of animals to fasting. Therefore, using this behavioral paradigm also allows doing a neuroethological assessment of sniffing function. Approaches that really take into account the specific characteristics of the behavior of the species and use ethological methods will be the most useful to interpret these behavioral findings and to better understand the biological mechanisms in brain function. The use of ethologically relevant stimuli is of particular importance to assess cognitive performance and dissect the effects of preventive/therapeutical interventions or the impact of risk factors and hazards (Torres-Lista et al., 2015, 2019; Giménez-Llort and Torres-Lista, 2021).

Throughout the experimental process in the food finding test, weight is monitored to assess the impact of food finding (Deacon et al., 2009). In the context of aging, body weight also serves as a good indicator of the health status of the animal (Barron et al., 2013). In the case of 3xTg-AD mice, we have shown that weight loss depends on intrinsic factors such as increased nocturnal activity (Baeta-Corral et al., 2018b) and hypermetabolic state (Escrig et al., 2020) compared to age-matched NTg mice. We have also shown that weight loss is useful to monitor the aggravation of the AD-pathogenic processes (Torres-Lista et al., 2019).

The exploratory behavior implies an internal conflict between the attraction for the novelty of space and the risk it contains since they could be exposing themselves to potentially dangerous environments (Giménez-Llort et al., 1995). In the case of rodents, most of this behavior depends on olfaction, a sense that allows not only the detection of odors in the surrounding environment necessary to identify food, predators, and sexual partners but also to get the spatial orientation (Invitto et al., 2018; Coronas-Sámano et al., 2014) being indispensable for survival. Once the animals had inspected the arena, the latencies to sniffing, finding, and eating the food pellets were found to have progressively increased in male mice with normal aging but consecutively developed in 3xTg-AD mice. It is known that olfactory deterioration during normal aging implies the loss of different olfaction skills, such as recognizing hidden food odor (Murphy et al., 1997; Schiffman, 1997). As referred to in the “Introduction”, recent literature on olfaction as a reflection of general health status also unveil a strong link between olfactory impairment and mortality, with underlying mechanisms being suspected to be associated with the impact of olfactory loss on intrinsic (nutrition, accelerated brain aging, and neurodegenerative diseases) and extrinsic (life-threatening situations and social interactions) mechanisms (Van Regemorter et al., 2020).

In the present work, male mice with normal and AD-pathological aging were equally delayed in their first contact with food pellets, while in the female sex this latency was increased but also depended on the genotype. In contrast, female 3xTg-AD performed the three-action (sniffing–find–eat) ethogram compared to their males cage mates. This could be because the sniffing in female mice is prone to be more affected during this stage of the disease, developing a situation in which the sensitivity to detect odors is more deficient (Mitrano et al., 2021).

Sex differences were found in the latency of the actions, with female mice being delayed compared to male mice, and also in the delay to develop the sequence, in this latter case being in a genotype-dependent manner. Thus, both groups of female mice exhibited longer delays in starting the ethogram than male mice, but the temporal pattern of their ethogram (delays between actions) to sniffing–finding–eating the food was two- to threefold times faster in the 3xTg-AD mice, but 1/2 in the case of NTg mice. This paradoxical genotype and sex modulation of the ethogram could be described as an accordion.

In the present work, the effects of naturalistic isolation (2–3 months) in a subgroup of 3xTg-AD that recently lost their cage mates after 10 months of living in a standard social environment were also assessed. This short isolation was enough to enhance the AD-genotype olfactory patterns shown by group-housed mice of the same genotype. When bearing different explanatory hypotheses, it is important to note that the body weight and the loss of weight after overnight fasting of isolated male 3xTg-AD were similar to those in the group-housed male 3xTg-AD, suggesting similar food motivation. The current report also shows that the ethogram was independent of body weight. Another explanation for their ability to find the hidden food faster could be related to a more intact olfactory function to detect food and recognize it over the other cage smells. In this case, the results would also suggest these mice as being somehow resilient to insults upon their nervous system, and according to the strong link between olfaction loss and mortality (Van Regemorter et al., 2020), this could explain why they lived longer than their cage mates. However, 100% of the group-housed 3xTg-AD mice could also be considered survivors with respect to the mean life span of the colony, and the female 3xTg-AD mice—that have better survival than male mice—were delayed compared to male mice. The most plausible interpretation could be that the shorter “inspection” of the new environment performed by 3xTg-AD mice living in social conditions is related to their anxiety-like profile and reduced to a minimum inspection in the isolated animals. In fact, we have recently reported increased anxiety in naturalistic isolated 3xTg-AD mice (Muntsant and Giménez-Llort, 2020) and distinct digging signatures, an ethological behavior of animals whose enhancement in the 3xTg-AD models neuropsychiatric-like behaviors associated with AD (Giménez-Llort and Alveal-Mellado, 2020) and where the olfactory function is also involved. However, since female 3xTg-AD are more anxious than male 3xTg-AD, other neuronal mechanisms may also be underlying these behavioral observations.

The time interval between actions to inspect the beddings was 10 times faster in the 3xTg-AD animals than in the NTg mice with normal aging. The control animals confirm that the sense of sniffing deteriorates during normal aging, and the different signatures that characterize sniffing, such as the detection of hidden pellet odor, are lost.

Meaningful correlations between the three actions in the food finding test indicate that the actions were correlated in the AD-genotype as could be inferred because the temporal pattern consisted of actions immediately consecutively performed. In the case of 3xTg-AD mice, the accordion effect resulted in correlations with a stronger statistical significance. Interestingly, the lower number of correlations found in the NTg mice, despite the bigger sample size in this group, would suggest that behavioral patterns driven by olfactory function coexisted with behaviors driven by other cognitive functions. It is important to note that the variables were independent of the loss of body weight. In isolated 3xTg-AD mice, the correlations were mainly lost, but we cannot discard this due to the small sample size limitation.

In summary, the present results on the olfactory signatures in male and female 3xTg-AD mice compared to C57BL/6 non-transgenic mice show the following: (1) the sniffing–find–eat temporal pattern was independent of the loss of weight, (2) olfactory signatures in male and female 3xTg-AD mice were characterized by a fast ethogram and strong behavioral correlates, (3) olfactory signatures in male and female controls, the gold-standard C57BL/6 mice, were slow and progressively developed as part of a more complex ethogram, as also supported by disrupted behavioral correlates between the three actions assessed in the food finding test, (4) social isolation, naturally occurring only in 3xTg-AD mice, enhanced the genotype differences and disrupted the behavioral correlates.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The animal study was reviewed and approved by CEEAH/Universitat Autònoma de Barcelona and DARP/Generalitat de Catalunya.

LG-L: conceptualization. DM-P: performance and analysis of behavior, illustrations and draft manuscript. Both authors equally contributed to the scientific discussions, the edit writing, and approval of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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