BRIEF RESEARCH REPORT published: 05 October 2021 doi: 10.3389/fnins.2021.733984 Olfactory Signatures in the Food Finding Test in Mice With Normal and Alzheimer’s Disease-Pathological Aging With Special Concerns on the Effects of Social Isolation Daniela Marín-Pardo 1,2 and Lydia Giménez-Llort 1,2* 1 Institut de Neurociències, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Department of Psychiatry and Forensic Medicine, School of Medicine, Universitat Autònoma de Barcelona, Barcelona, Spain The temporal course and the severity of the involution of sensory systems through aging can be critical since they ensure the ability to perceive and recognize the world. In older people, sensory impairments significantly increase their risk of biological, psychological, and social impoverishment. Besides this, olfactory loss is considered an early biomarker in Alzheimer’s disease (AD) neurodegenerative process. Here we studied olfactory Edited by: ethograms in middle-aged male and female gold-standard C57BL/6 mice and 3xTg- Sara Invitto, AD mice, a genetic model of AD that presents cognitive dysfunction and a conspicuous University of Salento, Italy neuropsychiatric-like phenotype. A paradigm involving 1-day food deprivation was used Reviewed by: Tommaso Cassano, to investigate the ethological patterns shown in the olfactory inspection of a new cage University of Foggia, Italy and the sniffing, finding, and eating of hidden food pellets. The sniffing–find–eat temporal Daniel W. Wesson, patterns were independent of the loss of weight and unveiled (fast) olfactory signatures University of Florida, United States in Alzheimer’s disease, differing from those (slow progressive) in normal aging. Male *Correspondence: Lydia Giménez-Llort 3xTg-AD mice exhibited an early signature than female mice, opposite to animals with lidia.gimenez@uab.cat normal aging. The sequence of actions was correlated in male and female 3xTg-AD mice in contrast to control mice. Social isolation, naturally occurring in male 3xTg-AD Specialty section: This article was submitted to due to the death of cage mates, emphasized their olfactory patterns and disrupted the Perception Science, behavioral correlates. The paradigm provided distinct contextual, sex, and genotype a section of the journal Frontiers in Neuroscience olfactory ethogram signatures useful to investigate olfactory function in normal and AD- Received: 30 June 2021 pathological aging. Isolation had an impact on enhancing the changes in the olfactory Accepted: 31 August 2021 signature here described, for the first time, in the 3xTg-AD model of Alzheimer’s disease. Published: 05 October 2021 Keywords: neuroethology, 3xTg-AD mice, behavioral neuroscience, methods, smell loss, aging, Alzheimer’s Citation: disease, ethogram Marín-Pardo D and Giménez-Llort L (2021) Olfactory Signatures in the Food Finding Test in Mice With Normal and Alzheimer’s INTRODUCTION Disease-Pathological Aging With Special Concerns on the Effects Throughout life, sensory systems must ensure our ability to perceive and recognize the world. of Social Isolation. Thus, the time course and severity of its involution through the aging process are decisive for Front. Neurosci. 15:733984. individuals since these limit the ability to sustain the quality of sensory stimuli, affecting not only doi: 10.3389/fnins.2021.733984 cognitive processes but also their self-esteem, habits, and styles of a lifetime. In older people, sensory Frontiers in Neuroscience | www.frontiersin.org 1 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD deficiencies significantly increase their risk of biological, counterparts of the same age, a phenomenon that has been related psychological, and social impoverishment (Dziechciaz and Filip, to their neuro-immune-endocrine impairment (Giménez-Llort 2014). Sensory deficits have been proposed as predictors of et al., 2008). Furthermore, in male mice, the rates increase cognitive decline in older adults and as early indicators of exponentially at 12 to 13 months (Giménez-Llort et al., 2008), an the prodromal stages of Alzheimer’s disease. Among them, age mimicking the advanced stages of the disease (Oddo et al., the association between olfactory dysfunction and cognitive 2003; Belfiore et al., 2019), thus providing a naturalistic scenario impairment is of growing interest. Olfactory dysfunction has to study the impact of the loss of life mates. 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 MATERIALS AND METHODS and/or disability, sensory deficiencies significantly increase the risk of the impoverishment of older people on a biological, Animals psychological, and social level (Volkers and Scherder, 2011). A total number of 97 13-month-old male and female homozygous More importantly, emergent literature unveiled anosmia as being 3xTg-AD (n = 58, 22 male and 36 female) and non-transgenic more predictive of 5-year mortality risk than cardiovascular (NTg, n = 39, 24 male and 15 female) mice on a C57BL/6J disease (Van Regemorter et al., 2020). background (after embryonic transfer and backcrossing at least The complete and adequate evaluation of patients plays a role 10 generations) established in the Universitat Autònoma de in any diagnostic work to demonstrate the etiology, pathogenesis, Barcelona (Baeta-Corral and Giménez-Llort, 2014) were used diagnosis, and possible prevention/therapy in neurodegenerative in this study. The 3xTg-AD mice harboring transgenes were disorders (Kondo et al., 2020). Olfactory loss, which is known to genetically engineered at the University of California Irvine, occur in bacterial and viral infections and is considered an early as previously described (Oddo et al., 2003). The animals were biomarker in the neurodegenerative processes of Alzheimer’s and maintained under standard laboratory conditions of food and Parkinson’s diseases (Kovács, 2004), has also been reported as water ad lib, 22 ± 2◦ C, 12-h light/dark cycle with lights on at 8:00 an early indicator of current SARS-CoV-2 infection (Mahalaxmi am, and relative humidity of 50–60%. et al., 2020). Sniffing impairment or deficiency is present in almost 90% of the population with Alzheimer’s disease (AD) Social Conditions (Conti et al., 2013). Furthermore, in certain cases with mild Animals of the same genotype and sex were maintained cognitive impairment, this loss has also been observed during the in groups of three to four mice per cage (Macrolon, transition phase between normal aging and dementia. Previous 35 cm × 35 cm × 25 cm), which was filled with 5 cm of clean studies have found that olfactory impairment or deficiency wood cuttings (Ecopure, Chips6, DateSand, United Kingdom; may be an indicator that people affected by mild cognitive Uniform cross-cut wood granules with 2.8–1.0 mm chip size) impairment are more likely to develop dementia (Invitto et al., and nesting materials (Kleenex, Art: 08834060, 21 cm × 20 cm, 2018). A first diagnostic screening based on olfactory tests White). In the current work, a natural social isolation scenario would allow the rapid implementation of preventive approaches was found among male 3xTg-AD mice due to increased and thus improve cognition and brain and mental health mortality rates in male mice of this genotype occurring at this age (Brai et al., 2020). (Giménez-Llort et al., 2008). Thus, short isolation (2–3 months) Male and female sexes can be regarded as two exceptional was observed in seven of the 22 male 3xTg-AD that recently natural scenarios to study the role of biological, psychological, lost their cage mates after 10 months of living in a standard and social factors, their functional interplay, and their impact social environment. In all the cases, the standard home cages on the crosstalk of homeostatic networks in health and covered with a metallic grid allow the perception of olfactory and disease through life cycle (Giménez-Llort et al., 2014). At the auditory stimuli from the rest of the colony. translational level, in the present brief report, we have studied the olfactory signatures in male and female mice with normal and Behavioral Assessment neurodegenerative aging associated with advanced stages of AD. Physical Status For this purpose, we used 3xTg-AD mice (Oddo et al., 2003) and To monitor the effects of fasting used in the food finding age-matched non-transgenic mice, both with a C57BL/6J genetic paradigm, body weight was measured before (Wpre) and after background (Baeta-Corral and Giménez-Llort, 2014). The 3xTg- (Wpost) food deprivation. The recovery of weight was monitored AD mouse is a genetic model of Alzheimer’s disease that presents at 24 h later (Wfinal). not only AD cognitive dysfunction but also a striking phenotype modeling some “behavioral and psychological symptoms of The Food Finding Test of Olfactory Ability dementia” (BPSD), including neuropsychiatric symptoms such To investigate olfactory function, the food finding test (FFT) as anxiety, apathy, and depression-like behaviors (Giménez-Llort olfactory paradigm (Deacon et al., 2009) was used. The mice were et al., 2006, 2007; Torres-Lista and Giménez-Llort, 2013; Baeta- deprived of food for 14 h before testing (starting with lights off at Corral et al., 2018a). 2000h), the equivalent to 1-day food deprivation. In accordance with the increased mortality rates in dementia The test was performed under dim white light (20 lx) during compared to the older population (van Dijk et al., 1991), the their light phase of the light/dark cycle (from 10:00 to 11:00 a.m.) mortality of 3xTg-AD mice is higher than that of their wild-type using a novel cage (50 cm × 22 cm × 14 cm) with 1 cm of Frontiers in Neuroscience | www.frontiersin.org 2 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD FIGURE 1 | Genotype and sex differences in body weight monitoring and food finding test in 13-month-old NTg and 3xTg-AD mice. The results are expressed as mean ± SEM. (A) Food deprivation: Wpre, initial weight before fasting; Wpost, weight after fasting;%Wloss, percent of weight lost overnight; Final W, final weight at 24 h later. (B) Food finding test—latencies in the food finding test: LatSniffing, latency to sniffing the hidden pellet; LatFF, latency to find the food (hidden pellet); LatEat, latency to eat the pellet. Statistics: **p < 0.01, ***p < 0.001 vs. NTg mice of the same sex; S, p < 0.05; SS, p < 0.01; SSS, p < 0.001 vs. male of the same genotype. beddings. Eight food pellets (45 mg) were placed in the central retrospective analysis. All procedures followed the Spanish zone of the cage and covered by 1 cm of wood chip bedding. The legislation on the “Protection of Animals Used for Experimental mouse was placed in a corner of the cage, facing the walls, and and Other Scientific Purposes” and the EU Directive its behavior was observed. The latencies of three goal-directed (2010/63/UE) on this subject. The study complies with the behaviors toward hidden food were recorded, namely, sniffing— ARRIVE guidelines developed by the NC3Rs and aims to reduce when the nose makes a direct contact with a surface and the the number of animals used (Kilkenny et al., 2010). vibrissae are in maximum extension and are in contact with the surface in the same way (Welker, 1964); finding (digging)— “finding a food pellet” was defined as digging, touching, and Statistical Analysis holding the pellet in the front paws for more than 3 s; and The results are expressed as mean ± SEM. SPSS 20.0 software was eating the hidden food—holding the pellet in the front paws for used. A 2 × 2 factorial design was used to analyze the effects of (G) more than 3 s of continuous eating. The beddings were renewed genotype and (S) sex factors. Differences were studied through between each mouse. multivariate general linear model analysis, followed by post hoc Behavioral assessments were performed in a counterbalanced Duncan’s test (multiple comparisons). In analyzing the effects of manner by direct observation by an observer blind to the social isolation, the differences between two independent groups genotype and the support of video recordings (ViewPoint were measured with Student’s t-test. In all the tests, p < 0.05 was Behavior Technology, Lion, France) for complementary considered statistically significant. Frontiers in Neuroscience | www.frontiersin.org 3 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD RESULTS p < 0.001] reached statistical significance. Sex differences were found between male mice (faster) and female mice (slower) just Effect of Food Deprivation on Body in the actions of sniffing [Lat Sniffing, sex∗ , F(1, 86) = 5.558, p < 0.05], but not in the food finding and eating action [Lat FF, Weight but Not Related to the Olfactory n.s., F(1, 86) = 2.811, p = 0.097; Lat Eating, n.s., F(1, 86) = 0.131, Signature p = 0.718]. The body weight of animals was monitored throughout Male 3xTg-AD mice were faster in all the actions compared to experimental procedures (Figure 1A). At the beginning of NTg mice, with statistically significant differences in the latency the experiments, before the animals were submitted to food of finding the hidden food [male, Lat FF∗∗∗ , F(3, 86) = 8.716, deprivation for 14 h, the weight of 3xTg-AD mice was lower p < 0.001] and eating it once it was found [male, Lat Eat∗∗∗ , F(3, than that of controls [Wpre, genotype∗∗∗ , F(1, 86) = 34.369, 86) = 17.064, p < 0.001], while those in the latency to sniffing the p < 0.001]. After the food finding test, all the groups of food did not reach statistical significance. animals lost weight, but genotype differences persisted [Wpos, Moreover, very short delays between actions in the male 3xTg- genotype∗∗∗ , F(1, 86) = 36.481, p < 0.001]. Sexual dimorphism AD mice indicated an immediate execution of actions, which was found in the initial body weight [Wpre, sex∗∗∗ , F(1, differed from the progressive time increase from one action to 86) = 152.694, p < 0.001]. Thus, while the weight of male the other exhibited by male NTg mice. Besides this, the delays 3xTg-AD mice was lower than C57BL/6J, the body weight of between actions also indicated a 10-fold faster time pattern in female mice was independent of the genotype. As expected, food male 3xTg-AD mice than NTg mice (Table 1). deprivation did not modify sexual dimorphism [Wpos, sex∗∗∗ , In female mice, both groups exhibited longer latencies F(1, 86) = 210.954, p < 0.001]. Regarding the percentage of weight compared to male mice, and the time pattern of their ethogram loss [%Wloss, sex∗ , F(1, 86) = 6.585, p < 0.05], it was in the for sniffing–finding–eating food was slower [Figure 2, female, range of 5–10% of their weight, independently of the genotype LatSniffing, S, p < 0.05; LatFF, SSS, p < 0.001; LatEat, SS, but higher in female mice than in male mice in the 3xTg-AD p < 0.01]. However, the time delay between actions in the female genotype. At 24 h after fasting, the animals of both genotypes and 3xTg-AD mice was very short, as described for male 3xTg-AD sexes almost regained their initial weight and showed genotype mice, but with two- to threefold increase compared to them. In [Final W, genotype∗∗∗ , F(1, 86) = 52.252, p < 0.001] and sex female NTg, the delays were shorter than those of male NTg, with [Final W, sex∗∗∗ , F(1, 86) = 223.375, p < 0.001] differences. values that were half of those recorded for them (see Table 1). The effects of food deprivation and the food finding test in the Therefore, the male–female ratios in NTg and 3xTg-AD mice male sex indicated that the loss of weight, expressed either in units were in an opposite sense. of weight or as a percentage, was not related to the statistically significantly shorter latency of 3xTg-AD mice to find the food and to eat it. Food Deprivation and Food Finding Test in Naturalistic Social Isolation of Male Sex- and Genotype-Dependent 3xTg-AD Mice Signatures in the Food Finding Test As detailed in Table 2, the naturalistic social isolation of 3xTg-AD As indicated in Figure 1B, the FFT paradigm to investigate the mice did not result in changes in the temporal patterns compared olfactory function in normal and AD-pathological aging elicited to group-housed mice of the same genotype. On the other hand, different sex- and genotype-dependent olfactory ethogram the genotype-dependent statistical differences in the ethogram signatures. There was a significant genotype difference between were enhanced in the isolated group, thus the latency of finding the 3xTg-AD and NTg animals regarding the three actions, albeit the hidden food [Table 2, Lat FF, genotype∗∗∗ , F(2, 43) = 20.275, only finding [Lat FF, genotype∗∗∗ , F(1, 86) = 12.686, p < 0.001] p < 0.001] and eating the food [LatEating, genotype∗∗∗ , F(2, and eating the food [Lat Eating, genotype∗∗∗ , F(1, 86) = 41.984, 43) = 10.930, p < 0.001]. TABLE 1 | AD-genotype and sex effects in the time delay between actions. NTg, male NTg, female 3xTg-AD, male 3xTg-AD, female Time delay (s) (n = 24) (n = 15) (n = 15) (n = 36) Mean ± SEM Mean ± SEM Ratio S Mean ± SEM Ratio G Mean ± SEM Ratio S Sniffing—finding 173.27 ± 45.5 87.93 ± 27.1 1/2 × 16.75 ± 4.9*** 10× 47.06 ± 19.5 2× Finding–eating 488.13 ± 134.8 265.13 ± 28.4 1/2× 40.58 ± 13.8*** 10× 71.67 ± 43.2 3× Sniffing—eating 661.40 ± 137.6 353.07 ± 38.4S 1/2× 57.33 ± 13.7*** 10× 118.72 ± 47.9* 2× The results are expressed as mean ± SEM. Time delay (s) between actions: sniffing and finding food, finding food and eating it, and sniffing and eating it. Ratio S, fold increase vs. male mice of the same genotype; Ratio G, fold increase vs. male NTg mice. *p < 0.05, ***p < 0.001 vs. NTg mice of the same sex; S, p < 0.05 vs. mice of the same genotype. Frontiers in Neuroscience | www.frontiersin.org 4 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD FIGURE 2 | Olfactory signatures in mice with normal and AD-pathological aging in the food finding test. (A) Experimental design and ethogram tablature for “sniffing, finding, and eating the hidden food pellet” by 13-month-old male and female 3xTg-AD and NTg mice and natural isolation male 3xTg-AD mice. (B) Meaningful correlation analysis in food finding test. Graphical representation of the significant Pearson r correlations between the three olfactory actions. All of them were positive. The figure was created with BioRender.com. Correlation Analysis Between the DISCUSSION Actions of Olfactory Signatures in the The present work characterizes AD genotype and sex effects on Food Finding Test olfactory ethograms in the food finding test that define olfactory As shown in Figure 2B, the meaningful correlation analysis signatures in male and female non-transgenic and 3xTg- evidenced that, in the 3xTg-AD mice, the actions were closely AD mice. related and predictable, mostly in female mice where statistical The food finding test (Deacon et al., 2009) is an easy and correlation increased in magnitude. In male mice living in ready-to-do test that can be used in any animal department naturalistic isolation conditions, the latency of sniffing was setting without the need to buy anything since it is based on predictive of eating, but not of finding the food, and some of these the ethological response of animals to fasting. Therefore, using correlations were lost. In male NTg mice, correlations were found this behavioral paradigm also allows doing a neuroethological between the finding and eating for male mice, and correlations assessment of sniffing function. Approaches that really take were enhanced in order of magnitude in female mice. into account the specific characteristics of the behavior of Frontiers in Neuroscience | www.frontiersin.org 5 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD TABLE 2 | Effects of naturalistic isolation of male 3xTg-AD mice in food deprivation, food finding test, and the time delay between actions. NTg, male 3xTg-AD, male Ratio G 3xTg AD-ISO, male Ratio G (n = 24) (n = 15) (n = 7) Mean ± SEM Mean ± SEM Mean ± SEM Food deprivation Wpre (g) 42.63 ± 1.00 32.23 ± 1.06*** 30.2 ± 0.82*** Wpos 28.49 ± 1.11 29.53 ± 0.48 28.19 ± 0.75 %Wloss (g) 6.92 ± 2.50 7.40 ± 1.51 6.66 ± 0.28 Final W (g) 31.74 ± 0.91 32.77 ± 0.65*** 31.33 ± 0.75*** Food finding test Latency of sniffing (s) 251.00 ± 63.88 135.71 ± 16.28 85.14 ± 14.58 Latency of finding food (s) 424.27 ± 57.92 152.46 ± 15.79 111.86 ± 17.16*** Latency of eating (s) 912.40121.02 193.04 ± 17.80 138.14 ± 23.62*** Time (s) delay Sniffing—finding 173.27 45.50 16.75 ± 4.90*** 10× 26.71 ± 10.77** 6× Finding—eating 488.13 134.81 40.58 ± 13.85*** 10× 26.29 ± 14.97*** 19× Sniffing—eating 661.40 137.57 57.33 ± 13.75*** 10× 53.00 ± 12.92*** 12× The results are expressed as mean ± SEM. Time (s) between actions: sniffing and finding food, finding food and eating it, and sniffing and eating food. Wpre, initial weight before fasting; Wpost, weight after fasting;%Wloss, percent of weight lost overnight; Final W, final weight at 24 h later; LatSniffing, latency to smell the hidden pellet; LatFF, latency to find the hidden pellet; LatEat, latency to eat the pellet; Ratio G, fold increase vs. male NTg mice. **p < 0.01, ***p < 0.001. the species and use ethological methods will be the most that olfactory deterioration during normal aging implies the useful to interpret these behavioral findings and to better loss of different olfaction skills, such as recognizing hidden understand the biological mechanisms in brain function. The food odor (Murphy et al., 1997; Schiffman, 1997). As referred use of ethologically relevant stimuli is of particular importance to in the “Introduction”, recent literature on olfaction as a to assess cognitive performance and dissect the effects of reflection of general health status also unveil a strong link preventive/therapeutical interventions or the impact of risk between olfactory impairment and mortality, with underlying factors and hazards (Torres-Lista et al., 2015, 2019; Giménez- mechanisms being suspected to be associated with the impact Llort and Torres-Lista, 2021). of olfactory loss on intrinsic (nutrition, accelerated brain Throughout the experimental process in the food finding aging, and neurodegenerative diseases) and extrinsic (life- test, weight is monitored to assess the impact of food finding threatening situations and social interactions) mechanisms (Deacon et al., 2009). In the context of aging, body weight also (Van Regemorter et al., 2020). serves as a good indicator of the health status of the animal In the present work, male mice with normal and AD- (Barron et al., 2013). In the case of 3xTg-AD mice, we have pathological aging were equally delayed in their first contact with shown that weight loss depends on intrinsic factors such as food pellets, while in the female sex this latency was increased increased nocturnal activity (Baeta-Corral et al., 2018b) and but also depended on the genotype. In contrast, female 3xTg- hypermetabolic state (Escrig et al., 2020) compared to age- AD performed the three-action (sniffing–find–eat) ethogram matched NTg mice. We have also shown that weight loss is compared to their males cage mates. This could be because the useful to monitor the aggravation of the AD-pathogenic processes sniffing in female mice is prone to be more affected during this (Torres-Lista et al., 2019). stage of the disease, developing a situation in which the sensitivity The exploratory behavior implies an internal conflict between to detect odors is more deficient (Mitrano et al., 2021). the attraction for the novelty of space and the risk it contains Sex differences were found in the latency of the actions, with since they could be exposing themselves to potentially dangerous female mice being delayed compared to male mice, and also environments (Giménez-Llort et al., 1995). In the case of in the delay to develop the sequence, in this latter case being rodents, most of this behavior depends on olfaction, a sense in a genotype-dependent manner. Thus, both groups of female that allows not only the detection of odors in the surrounding mice exhibited longer delays in starting the ethogram than male environment necessary to identify food, predators, and sexual mice, but the temporal pattern of their ethogram (delays between partners but also to get the spatial orientation (Invitto et al., actions) to sniffing–finding–eating the food was two- to threefold 2018; Coronas-Sámano et al., 2014) being indispensable for times faster in the 3xTg-AD mice, but 1/2 in the case of NTg mice. survival. Once the animals had inspected the arena, the latencies This paradoxical genotype and sex modulation of the ethogram to sniffing, finding, and eating the food pellets were found to could be described as an accordion. have progressively increased in male mice with normal aging In the present work, the effects of naturalistic isolation but consecutively developed in 3xTg-AD mice. It is known (2–3 months) in a subgroup of 3xTg-AD that recently lost Frontiers in Neuroscience | www.frontiersin.org 6 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD their cage mates after 10 months of living in a standard social temporal pattern was independent of the loss of weight, (2) environment were also assessed. This short isolation was enough olfactory signatures in male and female 3xTg-AD mice were to enhance the AD-genotype olfactory patterns shown by group- characterized by a fast ethogram and strong behavioral correlates, housed mice of the same genotype. When bearing different (3) olfactory signatures in male and female controls, the gold- explanatory hypotheses, it is important to note that the body standard C57BL/6 mice, were slow and progressively developed weight and the loss of weight after overnight fasting of isolated as part of a more complex ethogram, as also supported by male 3xTg-AD were similar to those in the group-housed male disrupted behavioral correlates between the three actions assessed 3xTg-AD, suggesting similar food motivation. The current report in the food finding test, (4) social isolation, naturally occurring also shows that the ethogram was independent of body weight. only in 3xTg-AD mice, enhanced the genotype differences and Another explanation for their ability to find the hidden food disrupted the behavioral correlates. 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 DATA AVAILABILITY STATEMENT resilient to insults upon their nervous system, and according to the strong link between olfaction loss and mortality (Van The raw data supporting the conclusions of this article will be Regemorter et al., 2020), this could explain why they lived longer made available by the authors, without undue reservation. 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 ETHICS STATEMENT have better survival than male mice—were delayed compared to male mice. The most plausible interpretation could be that the The animal study was reviewed and approved by shorter “inspection” of the new environment performed by 3xTg- CEEAH/Universitat Autònoma de Barcelona and DARP/ AD mice living in social conditions is related to their anxiety-like Generalitat de Catalunya. 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- AUTHOR CONTRIBUTIONS Llort, 2020) and distinct digging signatures, an ethological LG-L: conceptualization. DM-P: performance and analysis of behavior of animals whose enhancement in the 3xTg-AD models behavior, illustrations and draft manuscript. Both authors equally neuropsychiatric-like behaviors associated with AD (Giménez- contributed to the scientific discussions, the edit writing, and Llort and Alveal-Mellado, 2020) and where the olfactory function approval of the manuscript. 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 FUNDING 10 times faster in the 3xTg-AD animals than in the NTg mice This work has been supported by 2017-SGR-1468 and ArrestAD with normal aging. The control animals confirm that the sense H2020 Fet-OPEN-1-2016-2017-737390 to LG-L. of sniffing deteriorates during normal aging, and the different signatures that characterize sniffing, such as the detection of hidden pellet odor, are lost. ACKNOWLEDGMENTS Meaningful correlations between the three actions in the food finding test indicate that the actions were correlated in the AD- We thank Prof. Frank M. LaFerla, Institute for Memory genotype as could be inferred because the temporal pattern Impairments and Neurological Disorders, Department consisted of actions immediately consecutively performed. In of Neurobiology and Behavior, University of California, the case of 3xTg-AD mice, the accordion effect resulted in Irvine, United States, for kindly providing the progenitors correlations with a stronger statistical significance. Interestingly, of the Spanish colonies of 3xTg-AD and NTg mice. the lower number of correlations found in the NTg mice, The illustrations were created with BioRender.com. despite the bigger sample size in this group, would suggest that Frontiers APCs thank Envellir bé-Saber envejecer— behavioral patterns driven by olfactory function coexisted with HealthyAging.org Memorial Mercedes Llort Sender behaviors driven by other cognitive functions. It is important 2021/80/24091941/03. 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 SUPPLEMENTARY MATERIAL size limitation. In summary, the present results on the olfactory signatures The Supplementary Material for this article can be found online in male and female 3xTg-AD mice compared to C57BL/6 non- at: https://www.frontiersin.org/articles/10.3389/fnins.2021. transgenic mice show the following: (1) the sniffing–find–eat 733984/full#supplementary-material Frontiers in Neuroscience | www.frontiersin.org 7 October 2021 | Volume 15 | Article 733984 Marín-Pardo and Giménez-Llort Olfactory Signatures in Aging and AD REFERENCES Kondo, K., Kikuta, S., Ueha, R., Suzukawa, K., and Yamasoba, T. (2020). Age- related olfactory dysfunction: epidemiology, pathophysiology, and clinical Baeta-Corral, R., Castro-Fuentes, R., and Giménez-Llort, L. (2018a). Sexual management. Front. Aging Neurosci. 12:208. doi: 10.3389/fnagi.2020.00208 dimorphism in the behavioral responses and the immunoendocrine status in Kovács, T. (2004). Mechanisms of olfactory dysfunction in aging and d-galactose-induced aging. J. Gerontol. A Biol. 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Effects of the systemic absence of any commercial or financial relationships that could be construed as a administration of kainic acid and NMDA on exploratory activity in rats. potential conflict of interest. Pharmacol. Biochem. Behav. 51, 205–210. doi: 10.1016/0091-3057(94)00420-n Giménez-Llort, L., and Torres-Lista, V. (2021). Social nesting, animal welfare, and Publisher’s Note: All claims expressed in this article are solely those of the authors disease monitoring. Animals (Basel) 11:1079. doi: 10.3390/ani11041079 and do not necessarily represent those of their affiliated organizations, or those of Giménez-Llort, L., Torres-Lista, V., and De la Fuente, M. (2014). Crosstalk the publisher, the editors and the reviewers. Any product that may be evaluated in between behavior and immune system during the prodromal stages of this article, or claim that may be made by its manufacturer, is not guaranteed or Alzheimer’s disease. Curr. Pharm. Des. 20, 4723–4732. doi: 10.2174/ endorsed by the publisher. 1381612820666140130205500 Invitto, S., Piraino, G., Ciccarese, V., Carmillo, L., Caggiula, M., Trianni, G., et al. Copyright © 2021 Marín-Pardo and Giménez-Llort. This is an open-access article (2018). Potential Role of OERP as Early Marker of Mild Cognitive Impairment. distributed under the terms of the Creative Commons Attribution License (CC BY). Front. Aging Neurosci. 10:272. doi: 10.3389/fnagi.2018.00272 The use, distribution or reproduction in other forums is permitted, provided the Kilkenny, C., Browne, W. J., Cuthill, I. C., Emerson, M., and Altman, D. G. (2010). original author(s) and the copyright owner(s) are credited and that the original Improving bioscience research reporting: the ARRIVE guidelines for reporting publication in this journal is cited, in accordance with accepted academic practice. No animal research. 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