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Chronobiology International
The Journal of Biological and Medical Rhythm Research
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/icbi20
Aging-related changes on social synchronization
of circadian activity rhythm in a diurnal primate
(Callithrix jacchus)
Fabiana B. Gonçalves , Bruno S. B. Gonçalves , Jeferson S. Cavalcante &
Carolina V. M. Azevedo
To cite this article: Fabiana B. Gonçalves , Bruno S. B. Gonçalves , Jeferson S. Cavalcante & Carolina V. M. Azevedo (2020) Aging-related changes on social synchronization of circadian activity rhythm in a diurnal primate (Callithrix�jacchus), Chronobiology International, 37:7, 980-992, DOI: 10.1080/07420528.2020.1773495
To link to this article: https://doi.org/10.1080/07420528.2020.1773495
Published online: 23 Jun 2020.
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Aging-related changes on social synchronization of circadian activity rhythm in
a diurnal primate (Callithrix jacchus)
Fabiana B. Gonçalvesa, Bruno S. B. Gonçalvesb, Jeferson S. Cavalcantec, and Carolina V. M. Azevedo d
aEscola Multicampi de Ciências Médicas do Rio Grande do Norte, Universidade Federal do Rio Grande do Norte, Caicó, RN, Brazil; bEscola de Artes,
Ciências e Humanidades, Universidade de São Paulo, São Paulo, SP, Brazil; cLaboratório de Estudos Neuroquímicos, Departamento de Fisiologia
e Comportamento, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil; dLaboratório de Cronobiologia, Departamento de Fisiologia e
Comportamento, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil ABSTRACT
The input of environmental time cues and expression of circadian activity rhythms may change with aging. Among nonphotic zeitgebers, social cues from conspecific vocalizations may contribute to the stability and survival of individuals of social species, such as nonhuman primates. We evaluated aging- related changes on social synchronization of the circadian activity rhythm (CAR) in a social diurnal primate, the common marmoset. The activity of 18 male marmosets was recorded by actiwatches in two conditions. (1) Experimental – 4 young adult (5 ± 2 yrs of age) and 4 older (10 ± 2 yrs of age) animals maintained under LD 12/12 h and LL in a room with full insulation for light but only partial insulation for sound from vocalizations of conspecifics maintained outdoors in the colony; and (2) Control – 10 young adult animals maintained outdoors in the colony (5 animals as a control per age group). In LL, the CAR of young adults showed more stable synchronization with controls. Among the aged marmosets, two free-ran with τ > 24 h, whereas the other two showed relative coordination during the first 30 days in LL, but free-ran thereafter. These differences were reflected in the “social” phase angles (ψon and ψoff) between rhythms of experimental and control animal groups. Moreover, the activity patterns of aged animals showed lower social synchrony with controls compared to young adults, with the time lags of the time series between each experimental group and control group being negative in aged and positive in young adult animals (t-test, p < 0.05). The index of stability of the CAR showed no differences according to age, while the intradaily variability of the CAR was higher in the aged animals during LD-resynchronization, who took additional days to resynchronize. Thus, the social modulation on CAR may vary with age in marmosets. In the aged group, there was a lower effect of social synchronization, which may be associated with aging-related changes in the synchro-nization and generation of the CAR as well as in system outputs.
ARTICLE HISTORY
Received 2 March 2020 Revised 18 May 2020 Accepted 19 May 2020
KEYWORDS
Circadian activity rhythm; aging; social entrainment; nonphotic cues; behavioral rhythms; marmosets
Introduction
Synchronization is a fundamental property that allocates biological functions in a propitious temporal manner that favors the adaptability and survival of organisms through phase and period (τ) adjustments of rhythms by time cues (Roenneberg et al. 2003). The light–dark cycle (LD) by its recurrent and regular oscillation is the main
zeitgeber of circadian rhythms. However, environmental
scenarios have a broad temporal representation, in which social relations mediated by auditory and olfac-tory cues can also act as significant nonphotic zeitgebers (Hastings et al. 1998; Mistlberger and Skene 2004).
Social synchronization contributes to the coordinate action of individuals living in groups, favoring survival and the perpetuation of the life of species – as observed in bees (Fuchikawa et al. 2016) to vertebrates (Favreau et al. 2009) – through subsistence, defense of predators
and mating (Davidson and Menaker 2003). In mam-mals, the effect of social cues on circadian rhythms undergoes ontogenetic modification. In the early stages of development, the maternal nursing rhythm, observed in altricial species, acts as zeitgeber in rabbits (Jilge 1993,
1995), rats (Viswanathan and Davis 1995), nonhuman primates (e.g. capuchin monkeys – Serón-Ferré et al.
2013) and humans (Weinert 2005; Weinert et al. 1994). In marmosets (Callithrix jacchus), social synchroni-zation has been studied only in juvenile and adult ani-mals (Bessa et al. 2018; Erkert and Schardt 1991; Melo et al. 2013). These social synchronization studies indi-cate (1) positive masking and relative coordination (Erkert et al. 1986; Gonçalves et al. 2009; Silva et al.
2014), (2) acceleration of photic resynchronization (Mendes et al. 2008) and (3) steady entrainment (Bessa et al. 2018). Social synchronization has been studied in
CONTACT Carolina V. M. Azevedo carolina@cb.ufrn.br Departamento de Fisiologia – Centro de Biociências, Universidade Federal do Rio Grande do Norte, Natal, RN, Brazil
2020, VOL. 37, NO. 7, 980–992
https://doi.org/10.1080/07420528.2020.1773495
elderly humans, since aging is a long and slow process and can be easily monitored by subjective and objective data. For the aged, social cues promote physical and mental health, since it helps maintain regularity of bio-logical functions (Monk et al. 1992). Irregular rhythms are associated with impairment in the performance of motor activities (Mistlberger and Skene 2004) that may result from improper exposure to light and impaired synchronization (Ancoli-Israel et al. 2003; Mishima et al. 2001).
Erkert and Schardt (1991) suggested social cues act as a weak zeitgeber in marmosets, acting in specific condi-tions, such as similar endogenous periods or a great degree of acquaintance between animals, whereas Gerber et al. (2002) emphasize social bonds are essential for the physiological stability in this species. Conspecific vocalizations are possible time cues for these small arboreal diurnal primates who live in social groups of up to 15 individuals (Stevenson and Rylands 1988). In habitats with dense vegetation, where visual contact is difficult, vocalizations may transmit information about food availability, presence of predators and important social functions (Bezerra and Souto 2008; Epple 1968), contributing to the wellbeing of the group. In a previous study, we showed a shortening of circadian period (τ) in response to conspecific vocalization playbacks repro-duced daily for 1 h for a duration of 30 days in animals maintained under total darkness (Silva et al. 2014).
The few studies addressing the ontogenesis of activity rhythm in marmosets indicate the strength of the circa-dian component increases progressively from birth, becoming stable after the 16th week of life (Menezes et al. 1996). In adulthood, activity is strictly diurnal and the circadian τ < 24 h (23.2 ± 0.3 h) under constant light (Erkert 1989). In aged individuals, we observed prolonga-tion of circadian τ of the activity rhythm, reducprolonga-tion of total daily activity, with signs of lower stability and greater fragmentation of the rhythm, and photic desynchroniza-tion in a case study (Gonçalves et al. 2016). Structural and neurochemical changes were observed in marmosets aged 9 to 12 yrs, such as reduction of the number and diameter of neurons in the SCN, and in serotonin and neuropep-tide Y (Engelberth et al. 2014).
In mammals, aging is frequently associated with the reduction of amplitude and increase of fragmentation of the 24 h activity rhythm, as described in mice (Gutman et al. 2011; Weinert and Weinert 1998), nonhuman pri-mates (Aujard et al. 2007; Gomez et al. 2012; Zhdanova et al. 2011) and humans (Van Someren et al. 1997). Another important change associated with age occurs in endogenous τ, with different responses, such as short-ening (Rosenberg et al. 1991; Van Gool et al. 1987) and prolongation (Possidente et al. 1995; Valentinuzzi et al.
1997) in rodents, as well as shortening (Aujard et al.
2006), prolongation (Gonçalves et al. 2016) and also absence of alteration in primates (Zhdanova et al. 2011).
The marmoset is a good model for the study of aging (Fischer and Austad 2011; Lacreuse and Herndon 2009), since they are diurnal primates that reach sexual matur-ity ~1.5 yr of age (Abbott et al. 2003; Tardif et al. 2008) and are considered elderly at 6 (Engelberth et al. 2013; Geula et al. 2002) or 8 yrs of age (Abbott et al. 2003). Another advantage derives from their average lifetime of 4 to 6 yrs in captivity (Tardif et al. 2008), with a variable life expectancy that can extend to 17 (Abbott et al. 2003) to 22 yrs (Nishijima et al. 2012) of age. Thus, marmosets enable a detailed study of aging because they do not have a short life expectancy, like rodents, but neither a long life expectancy, like Rhesus monkeys, which minimize the difficulties and costs of conducting long-term chron-obiological studies (Roth et al. 2004).
Social cues can promote synchronization in mammals. However, studies on the importance of social synchroniza-tion in several species have focused on the early life span of the mother–infant relationship. Except for a few studies on humans, little is known about the importance of social synchronization in the later stages of life. The aim of this study was to evaluate changes associated with aging on the social synchronization of the circadian activity rhythm (CAR) in the marmoset, the first study in the field of social synchronization in aging entailing a nonhuman diurnal primate species, which presents social complexity. Based on the assumption conspecific vocalizations act as
zeitge-bers of the CAR in this primate, we aimed to test the
hypothesis that aging is accompanied by changes in social synchronization, so that the rhythm of aged marmosets would show lower stability of phase relationships with the rhythm of conspecifics, specifically the onset and offset of activity, and additionally lower social synchrony between their CAR profiles and those of their conspecifics.
Materials and methods Animals
The study was carried out with eight male common mar-mosets (Callithrix jacchus) divided into two experimental groups: four young adults (5 ± 2 yrs of age) and four older animals (10 ± 2 yrs age). For the evaluation of social synchronization, 10 adult male animals (4.6 ± 1.07 yrs) compose two control groups (5 each), one for each experi-mental group. The motor activity of animals of the control group served as a reference to the activity patterns of animals maintained outdoors in the colony that showed regular daily behavioral rhythms (Menezes et al. 1993). Difficulty in the availability of aged animals to use in
experiments precluded the organization of an aged-specific control group. All animals were descendants of breeding pairs from the primate colony of the Universidade Federal
do Rio Grande do Norte (UFRN). Age classification of
animals was done according to Abbott et al. (2003), which considered animals to be aged when ≥8 yrs old.
All the experiments were conducted according to the Brazilian guidelines for animal research (Comissão de Ética no Uso de Animais – CEUA/UFRN, protocol nº 022/2011) and international ethical standards for research of animal biological rhythm (Portaluppi et al.
2010). All efforts were made to minimize the number and stress of the animals used.
Experimental conditions and procedures
All animals of the experimental group were housed under natural light (L):dark (D) condition (5°50´S, 35° 12´W – photophase: ~05:15 to ~17:15 h) in individual outdoor cages (H2.0 x L1.0 x W2.0 m) before transfer to the experimental room, where they were kept in indivi-dual iron cages (H90 x L60 x W60 cm), the nest box (animal resting place) being made of wood or opaque plastic. In the light phase (LD 12:12/03:00 to 15:00 h) and in constant light (LL), room lighting was provided by ten 32-watt lamps (~350 lx – digital photometer MLM-1011 Minipa). In the dark phase of the LD cycle, room lighting was provided by a small white incandes-cent lamp of 7 watts (~2 lx). Environmental conditions of ambient temperature (adult: 25.4 ± 0.7 °C/aged: 25.9 ± 0.8 °C) and relative air humidity (adult: 69.6 ± 10.2%/aged: 71.6 ± 13.0%) were maintained stable through air conditioning and exhaust fan.
The experimental room was fully insulated for light but only partially insulated for sound to allow penetra-tion of vocalizapenetra-tions emitted by conspecifics kept in outdoor cages of the colony (about 170 marmosets at the time of the experiment) that acted as nonphotic cues.
Before the experiment, their sounds were recorded in the empty room using a high sensitivity microphone (MKH416-P48 – Sennheiser, USA) connected to a recording system (TASCAM HD-P2 – TEAC Corp., Tokyo, Japan) for two consecutive days throughout the active phase of the outdoor animals, from 05:30 to 17:30 h, which varied during the day (Figure 1).
The feeding scheme consisted of primate rations, rai-sins and water ad libitum. In addition, animals received two meals at random times during the activity phase. The first meal was a portion of banana-based nutritional mix-ture (~60 mL) enriched with food supplementation (Aminomix mini pet-VETNIL® and Hemolitan). This meal was followed by a portion of tropical fruits and vegetables. Two or three times a week jelly beans, insect larvae (Tenebrio molitor), chicken and boiled eggs were offered as food supplementation. The health of the ani-mals was monitored by the veterinarian of the colony using records of motor activity, observations of behavior and body weight measurements.
In the experimental room, animals were submitted to LD 12/12 h for 120 days (aged) and 75 days (adults). Difference in the duration of this stage was the conse-quence of technical failures, making it necessary to extend the number of days for aged animals. The first 10 days in LD were considered the animals’ adaptation to experimental conditions. The difference between the LD schedules in the experimental room (photophase 03:00 to 15:00 h) versus outdoor (photophase ~05:15 to ~17:15 h) promoted the difference between the activ-ity phase in the CAR of controls and experimental animals, making it possible to visualize the phase adjust-ment between the CAR of the animals in the room and those of co-specifics kept outdoors during the process of social synchronization.
After LD 12/12 h, experimental animals were sub-mitted to constant light (LL) for 50 days to evaluate the effect of vocalizations as social zeitgebers. Maintenance
Figure 1. Daily frequency vocalizations from conspecifics located outdoors in the colony registered inside the experimental room during two consecutive days before the experiment.
of animals in LL allowed us to evaluate the effect of social cues of conspecifics as temporal cue on the CAR of experimental animals, since in this condition, the other time cues are maintained constant (the light/ dark, temperature and feeding cycles). Due to technical problems in data collection, this phase for the aged group was extended for 5 days. After LL, experimental animals were returned to LD 12/12 h, being evaluated for the first 30 days to observe resynchronization of CAR with a stable τ of 24 h. The experimental protocol is described in Figure 2. In parallel to LL of the experi-mental groups, control animals were housed outdoor in individual cages exposed to natural conditions of tem-perature (~25 °C), humidity (~80%), rainfall (~2.0 mm3) and illumination from the sun (sunrise: ~05:15 h and sundown: ~17:15 h).
Data collection and analysis
Motor activity of animals was continuously recorded by actiwatches (Actiwatch AW 16 model, Mini Mitter Company, Oregon, USA, 16 KB of onboard memory) at 5 min intervals. Transfer of data to the computer was done approximately every 30 days through the software Actiware-sleep version 3.4 (Mini Mitter). Each marmoset carried an actiwatch housed in a plastic container that mimicked a backpack. The weight of the set formed by the
backpack and actiwatch was ~33 g, amounting to ~8.7% of the average weight of the adult animals (378.2 g ± 23.0 g) and 9.9% of the aged ones (332 g ± 22.5 g). Thus, it did not exceed the limit of 10% of the weight of the animals (Melo et al. 2013). All animals were provided 15 days of adaptation to the equipment before the experiment.
We analyzed data of the 20 days after the first interval of 10 days (decade) of adaptation (that is, the second and third decades) in LD 12/12 h as the baseline stage; the 50 days in LL as the LL stage; and the 20 days after the first decade of resynchronization to LD 12/12 h as the resyn-chronization to LD stage. For the analysis of control animals, we considered the 50 days that coincided with the LL stage of the experimental groups.
Several tools of the El temps program (A. Díez-Noguera, Universitat de Barcelona, http://www.el-temps.com) were used to evaluate the CAR: (1) actogram for visual analysis of the time series; (2) Sokolove-Bushell Periodogram to obtain period (τ), which was analyzed for the age factor through Spearman’s Correlation; (3) waveform to calculate indexes of variability (IV) and stability (IS) and (4) two flanks to obtain the beginning and end time of the activity of the animals.
IV indicates the amount of fragmentation of the rhythm, through the frequency and extension of transi-tions between inactive and activity phases, calculated by
Figure 2. Timeline of experimental stages: (A) LD cycle (12:12/03:00 to 15:00 h/~350:~2 lx), (B) constant light (LL ~350 lx) and (C) resynchronization LD 12/12 h.
the ratio of the average squares of differences between successive hours; IS indicates the degree of coupling between the biological rhythm and the zeitgeber, nor-malized to the τ of the activity rhythm and number of data (Van Someren et al. 1997). These indexes were compared between ages and stages by ANOVA test (post hoc Tukey).
Social synchronization was analyzed in two ways: (1) phase-angle differences between activity onset (ψon) and
offset (ψoff) of each experimental animal (kept in LL) in
relation to control ones (kept in natural LD – represented by the mean value of the activity onset of control animals by day); and (2) social synchrony through calculation of the cross-correlation between the activity profiles of experimental and control animals. This correlation indi-cates the degree of correspondence between the activity profiles of circadian as well as ultradian rhythms, since it considers the complete record of the activity phase (Melo et al. 2013). In the general correlation, the temporal series of each animal was correlated with that of the outdoor animals (built by the mean of activity profiles off all animals each day), whose contact occurred through voca-lization cues. To obtain the maximum correlation, this procedure involved a correlation between two time series displaced at different time intervals (5, 10, 15 . . . 720 min), termed LAG. The highest value obtained in this proce-dure corresponds to the maximum correlation character-ized by a specific LAG, which represents the phase relation between two oscillations. All indexes obtained were compared between adult and aged animals using the Student’s t-test for independent samples.
The resynchronization rate of the CAR to the baseline LD 12/12 h was measured by the number of transient days until a stable phase difference was obtained between the beginning of the activity and the beginning of the light phase (~30 min – Mendes et al. 2008). This parameter was compared between adult and aged animals by Student’s
t-test. One animal (“Boris”) was excluded from this
ana-lysis, since he remained in free-running state throughout this stage. For all tests used in the study, the level of statistical significance was 5%.
Results
Visual analysis of the baseline condition (LD 12/12) actograms revealed most animals exhibited a CAR char-acterized by strictly diurnal activity, and that the adult marmosets adjusted more rapidly to the new LD cycle in the laboratory (12:12/03:00 to 15:00 h), while the aged animals exhibited more episodes of activity during dark phase. Both groups showed stable synchronization to the new LD schedule after the first 10 days (adaptation) in this condition (Figure 3).
In LL, vocalizations of conspecifics acted as temporal cues for the majority of the animals, in spite of the great interindividual variability of the activity pattern. All young adults expressed free-running τ < 24 h during the first 20 days. After ~30 days, there was maintenance of phase relationship between the activity onset and offset of the experimental animals with those residing outdoors, an indication of social synchronization. This occurred when the auditory cues of conspecifics coincided with the final half of the active phase of animals kept in the experimen-tal room. In contrast, aged animals displayed distinct responses on τ (= 24 h or > 24 h). Two animals (“Boris” and “Chaves”) showed free-running with τ > 24 h, whereas the other two other animals showed relative coordination during the first 30 days in LL, with τ ˜
24 h, but thereafter they free-ran (Figure 4, Table 1). The IV was similar between groups (F(1,5) = 1.84; p > 0.05), but differed among the experimental stages.
The LL and LD resynchronization were associated with higher values than the first LD (F(2,10) = 10.27; p < 0.05;
Tukey, p < 0.05), with interaction between age and
experimental condition, so that adult animals showed
lower values than aged animals in LD resynchronization (F(2,10) = 0.31; p > 0.05; Tukey, p < 0.05 – Figure 5A). IS
did not differ by age (F(1,5) = 4.19; p = 0.09), but was
reduced in LL (F(2,10) = 22.5; p < 0.05; Tukey, p < 0.05),
without interaction between age and experimental
con-dition (F(2,17) = 1.80; p > 0.05 – Figure 5B).
In LL under the influence of the social cues, the onset and offset of activity of five experimental animals (62.5%) showed gradual adjustment to such of the outdoor ani-mals. For most adults, ψon and ψoff were positive,
repre-sentative of the activity phase anticipated in relation to outdoor animals; the only exception was one animal that presented activity onset in phase with outdoor animals during most of the LL condition. In the aged group, two animals (“Boris” and “Chaves”) exhibited phase delays with negative ψon and ψoff, while the two others showed
phase synchronization up to the 40th day, one of them with positive ψon and ψoff. The “social” phase relationship
showed differences between adult and aged groups (ψon:
test t = 18.01; p < 0.01/ψoff: test t = 17.46; p < 0.01 – Figure 6).
For social synchrony in LL, the general correlation between activity profiles of experimental and control ani-mals was higher for adults (t = 6.37; p < 0.05 – Figure 7A). The LAG of the maximum correlation was positive for the adult and negative for the aged animals, indicating adults presented the activity profile with phase advancement relative to the control group, while the aged animals exhibited pronounced phase delay (t = 23.89; p < 0.05 –
Figure 7B). The maximum correlation for this LAG did not differ between groups (t = 0.69; p > 0.05 – Figure 7C).
After LL, resynchronization time of the CAR differed between the groups. Three of the aged animals required more than double the amount of time of the adults to resynchronize to the LD cycle (aged: 12.7 ± 5.5 days, adults: 5.0 ± 2.2 days, t = 2.59, p < 0.05), whereas the other (“Boris”) continuously free-ran.
Discussion
This study shows evidence of ontogenetic changes asso-ciated with aging on social synchronization of the CAR in a diurnal nonhuman primate, the common marmoset, mediated through the vocalizations of conspecifics. For most adult animals, who initially showed free-running CAR with τ < 24 h in LL in the presence of social cues, social synchronization occurred when the cues of outdoor conspecifics coincided with the final half of active phase. In contrast, for aged animals, social cues exerted a weaker effect, since two animals showed free-running with
prolongation of τ and the two others who showed relative coordination later became free-running.
Furthermore, the average values of the social phase angles (ψon and ψoff) and LAG, which indicate the phase
relations between the activity profiles, presented nega-tive values for aged animals, indicating a phase delayed relationship of the CAR compared to control animals. In addition, the social synchrony (indicator of the degree of the “in phase” relationships of the activity profiles) indi-cates the activity profiles of the adult animals were maintained in greater proximity and in a stronger phase correlation with those maintained outdoors.
Thus, the social synchronization of CAR weakens with age in marmosets, showing differences in phase- angle relationships with conspecifics compared with young adults. This phase-angle relationship is similar to that observed in a rare study of aged marmosets that showed a phase delay of 2 h in the offset of activity in a 10 yr-old male in relation to an 8-yr-old female
Figure 3. Double actograms of young adults (above) and aged (below) marmosets after transfer of environmental condition (photophase: ~5:15 to ~17:15 h) to experimental room (LD 12/12 – photophase 03:00 to 15:00 h). The first 10 days correspond to adaptation to experimental conditions. There were registry failures for “Cássio” from the 8th to 10th days.
(Hoffmann et al. 2012). Despite age, the gradual adjust-ment of the CAR of experiadjust-mental animals to that of conspecifics indicates social synchronization is mediated by entrainment. Moreover, physical contact was not necessary for social synchronization, differing from stu-dies that pointed to cohabitation as an important factor for promoting social synchrony in reproductive pairs of adult marmosets (Bessa et al. 2018), squirrels (Rajaratnam and Redman 1999) and hamsters (Paul and Schwartz 2007). In future studies, it will important to evaluate the effect of isolation and different degrees of
cohabitation on biological rhythms in aged marmosets, so as to understand the role of cohabitation in social synchronization during aging.
The response of the CAR to social cues in adults raises the possibility that conspecific vocalizations could be acting on the circadian timing system by an indirect way. The synchronization of free-running rhythm with τ < 24 h occurred when vocalizations coincided with the end of the activity phase. This response indicates that adjustment of the rhythm occurred by phase delay, and this is compa-tible with the photic responses observed on the phase
Figure 4. Double actograms of young adults (above) and aged (below) marmosets under LL with social cues (56 days). The protocol covers the previous 10 days on LD 12/12 and the 30 days during resynchronization to LD. The interruption of recording of “Boris” was due to the death of this animal. The gray box shows the duration of active phase of outdoor animals.
Table 1. Periods of the circadian activity rhythm of adult and aged marmosets during 50 days in LL under social vocalization cycle, obtained through the Sokolove-Bushell Periodogram method (El temps). The values are expressed in hours and the decades correspond to the interval of 10 days.
Group Animal Decade 1 Decade 2 Decade 3 Decade 4 Decade 5
ADULT Azarro 23.9 23.9 24.0 24.0 24.1 Genilson 23.6 23.9 23.8 24.2 23.9 Otelo 23.6 23.9 23.9 23.9 23.8 Zeringue 23.9 23.8 24.1 24.1 23.9 AGED Boris 24.1 24.2 24.3 24.2 24.4 Cássio 24.3 24.1 24.0 23.8 23.9 Chaves 24.3 24.3 24.1 24.1 24.7 Clarus 24.0 24.1 24.0 23.9 23.8
response curve for this species (Glass et al. 2001; Wechselberger and Erkert 1994). Thus, we suggest the arousal elicited by the nonphotic cues increases the photic input on the circadian timing system, as suggested by Wechselberger (1994), who evaluated the effect of playback calls from conspecifics in front of a mirror on the CAR of marmosets.
For aged animals, this effect is weaker and the τ ≥ 24 h expressed in LL might result from a different endogenous pattern characteristic of aging added to the synchronizing
effects exerted concomitantly by nonphotic and photic cues (Mistlberger and Skene 2004). Future studies isolating pho-tic and nonphopho-tic cues are necessary to understand the mechanisms involved in the observed aging-related changes of marmosets in the CAR in response to social cues. Independent of mechanism, social cues from conspe-cific vocalizations are considered of great importance for the synchronization of the CAR, either acting alone or as a coadjuvant factor, as observed in previous studies in our laboratory (Gonçalves et al. 2009; Melo et al. 2013; Mendes
Figure 5. (A) Intradaily variability (IV) (mean ± SE) and (B) interdaily stability (IS) of circadian activity rhythm of young adults and aged animals under LD cycle (12/12), constant light (LL) and resynchronization to LD (LDr). Values are expressed in arbitrary units (a.u.).
Figure 6. Social phase angles between the beginning of activity of animals in LL (50 days) in relation to the beginning of active phase of conspecifics maintained outdoors (ψon) of aged (A) and young adults (C); and for the end activity (ψoff) of aged (B) and young adults (D).
et al. 2008; Silva et al. 2014). Future studies are also neces-sary to evaluate the effect of other social-related cues on circadian rhythms in this species.
The differences in social synchronization of CAR between aged and young adults may be associated with anatomical-physiological modifications of the circadian timing system (CTS). Studies on humans indicate an association between natural aging and reduction of envir-onmental inputs to the CTS (Revell and Skene 2010). Furthermore, changes in the central oscillator could weaken the signal sent to peripheral oscillators due to the reduction of neurotransmitters and the coupling of cells (Farajnia et al. 2013). In marmosets, aging is corre-lated with decreased body cell diameter, increased num-ber of astrocytes and reduction of neurotransmitters VIP, VP, 5-HT (Engelberth 2013) and NPY (Engelberth et al.
2014) in the SCN. Aged rodents show lower responsive-ness of the SCN to NPY, neurotransmitter that sends nonphotic information to the SCN via the geniculo- hypothalamic tract (Van Reeth et al. 1993), which could reduce nonphotic responses. In addition, there are changes on effectors associated with the musculoskeletal system, such as a reduction of lean mass/increase of fat mass (Rhesus monkey, Black et al. 2001) and/or bone demineralization (humans and nonhuman primates, Roth et al. 2004). Motor activity can act directly on the CTS as a nonphotic zeitgeber by increasing coupling and favoring robust expression of rhythm, but also, indirectly, closing a feedback loop that reinforces environmental inputs that reach the CTS (Mistlberger and Skene 2004).
Studies have shown properties of the circadian timing system can change differently during aging, such that defi-ciency in photic synchronization capacity may precede loss of rhythmic generation of the CAR, as evidenced in the study carried out on middle-aged rats until their death (Weinert and Weinert 1998). Thus, in the final stages of life the ontogenetic process would lead to reversal of what
is observed at the beginning of life, with the appearance of rhythmic generation at first and, later, of the process of synchronization (Weinert 2005). Future longitudinal stu-dies are necessary to verify this process in marmosets.
In this context, the reinforcement of social cues during aging is of great importance. This could contribute to the maintenance of stability of biological rhythms favoring health (Kondratova and Kondratov 2012). Fragmentation and loss of rhythmicity (Menna-Barreto and Wey 2007; Weinert and Weinert 1998; Yoon et al. 2003) toward the end of life can accelerate the aging process and impair quality of life (Rana and Mahmood 2010; Tranah et al.
2010). For marmosets, the social habit could act as an important factor for synchronization.
In addition to the social effects on CAR, aging was associated with changes in photic synchronization and the free-running rhythm in LL. Under the LD cycle, adults showed a strictly diurnal pattern, whereas aged marmosets exhibited more episodes of activity during the dark phase. In LL, adults showed τ < 24 h during the first days, similar to previous studies in adult marmosets maintained in LL without social cues (Erkert 1989; Gonçalves et al. 2009). In contrast, aged animals showed a prolongation of τ and positive correlation was observed between age and τ during the first 20 days in LL. In a previous study (Erkert 1989), adult marmosets showed lengthening of τ with age when animals of 3 yrs old (τ = 23.2 ± 0.3 h) were compared with those of 4 yrs old of age (τ = 23.4 ± 0.3 h). Our data indicate prolongation of the endogenous τ with aging with values >24 h, similar to that observed in a case study using a long-itudinal methodology carried out in our laboratory (Gonçalves et al. 2016). Future studies in constant light (LL) in the absence of social cues are necessary to confirm this change in marmosets.
There is no consensus about the characteristics of the aging-related changes on τ in other mammals. Shortening of τ was observed in rodents [hamsters (Morin 1988;
Figure 7. Correlations between the activity profiles of experimental animals (young adults and aged) and control animals for 50 days under constant light conditions (LL): (A) correlation, (B) LAG and (C) maximum correlation (average ± SE). (* independent t-test,
Rosenberg et al. 1991); deermice (Pittendrigh and Daan
1974)] and a nocturnal primate [Microcebus murinus (Aujard et al. 2006)]. In contrast, prolongation was seen in mice (Gutman et al. 2011) and no differences in the diurnal primate, Rhesus monkey, Macaca mulatta (Zhdanova et al. 2011). In rats and humans, studies show different results. In rats, shortening (Rietveld et al. 1985; Van Gool et al. 1987) and lengthening of τ (Mayeda et al.
1997; Possidente et al. 1995) was observed. In humans, studies show longer (Kendall et al. 2001), shorter (Weitzman et al. 1982) or the same τ (Czeisler et al. 1999) with aging. These differences might be associated with species-related differences in τ or in the case of rats and humans, methodological differences. Thus, further studies are needed to analyze changes associated with age in τ in mammals (Kendall et al. 2001). Such ontogenetic changes could be associated with adaptation to changes in the environment during development with consequences upon biological fitness (Weinert 2005).
During photic resynchronization, aged animals required more time to resynchronize and showed higher IV values. Higher values of this index of rhythm fragmentation during aging are observed in another nonhuman primate (Microcebus murinus) (Aujard et al. 2007; Cayetanot et al.
2005) and in humans, where the increase on IV is accom-panied by an elevation in IS (Huang et al. 2002; Witting et al. 1990). However, IV did not show age-related differ-ences during the first LD, perhaps related to the small size of our sample. The loss of resynchronization capacity increased number of transients and a higher rhythm frag-mentation may be indicative of circadian timing system weaknesses in aged marmosets.
In addition to the characterization of a primate model to understand aging-related changes on social synchro-nization in diurnal mammals, the use of actiwatches to record activity supports the perspectives launched about social synchronization in marmosets at the beginning of the 90s (Erkert and Schardt 1991), and it extends the discussion about the importance of the adequacy of methods of recording and use of parameters of activity as significant biomarkers for changes with aging. This aspect is of great importance for the ontogenetic inves-tigation of CAR and other physiological variables in primates, since the use of this methodology enables observation of changes in activity pattern with aging that are more easily investigated than other markers, making it more appropriate for studies in animal models and in humans for the prognosis of pathologies and mortality.
In summary, this study stands out as pioneering in describing aging-related changes on social synchronization in a nonhuman diurnal primate using actiwatches for activity recording. For marmosets, aging may be associated
with a lower capacity for nonphotic synchronization by auditory cues from conspecifics, possibly due to reducing the rhythm generation capacity, sensory uptake and response of circadian oscillators to auditory cues. This can make aged marmosets more vulnerable, since social cues can act as an important coadjuvant factor for photic syn-chronization. Since there was variability in the response to conspecific stimuli, the small number of animals used pro-vided only limited evaluation of the effect of social synchro-nization. In addition, the synchronization mediated by nonphotic cues was not studied separately from the photic stimuli, requiring additional studies with a larger sample testing only nonphotic cues. Besides, the use of experimen-tal protocols that evaluate the endogenous τ in LL without nonphotic cues and studies under conditions close to nat-ural is needed. These approaches are essential for under-standing the mechanisms involved in age-related changes in the CAR of marmosets in response to photic and non-photic cues.
Acknowledgements
This article is dedicated to Prof. Dr. Alexandre Menezes for the valuable contribution to the chronobiological studies in mar-mosets. We would like to thank Antônio Barbosa, Luís do Nascimento, Geniberto dos Santos, José Rubens de Souza and Edinólia Rodrigues for technical support at the Primatology Colony of UFRN. We also thank the support on the care of animals to our esteemed colleagues Galileu Borges, Maria Luiza Cruz, Rosane Lampert e Sabinne Galina. Our thanks extend to the Postgraduate Program in Psychobiology at UFRN.
Funding
This work was supported by Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq);
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Universidade Federal do Rio Grande do Norte (UFRN).
ORCID
Carolina V. M. Azevedo http://orcid.org/0000-0003-4985-
4755
Declaration of interest statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References
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