A clock for all seasons in the subterranean.

In 1976, Pittendrigh and Daan established a theoretical framework which has coordinated research on circadian clock entrainment and photoperiodism until today. The "wild clocks" approach, which concerns studying wild species in their natural habitats, has served to test their models, add new insights, and open new directions of research. Here, we review an integrated laboratory, field and modeling work conducted with subterranean rodents (Ctenomys sp.) living under an extreme pattern of natural daily light exposure. Tracking animal movement and light exposure with biologgers across seasons and performing laboratory experiments on running-wheel cages, we uncovered the mechanisms of day/night entrainment of the clock and of photoperiodic time measurement in this subterranean organism. We confirmed most of the features of Pittendrigh and Daan's models but highlighted the importance of integrating them with ecophysiological techniques, methodologies, and theories to get a full picture of the clock in the wild. This integration is essential to fully establish the importance of the temporal dimension in ecological studies and tackling relevant questions such as the role of the clock for all seasons in a changing planet.

Temporal Dissociation Between Activity and Body Temperature Rhythms of a Subterranean Rodent (Ctenomys famosus) in Field Enclosures.

Several wild rodents, such as the subterranean tuco-tucos (Ctenomys famosus), switch their time of activity from diurnal to nocturnal when they are transferred from field to the laboratory. Nevertheless, in most studies, different methods to measure activity in each of these conditions were used, which raised the question of whether the detected change in activity timing could be an artifact. Because locomotor activity and body temperature (Tb) rhythms in rodents are tightly synchronized and because abdominal Tb loggers can provide continuous measurements across field and laboratory, we monitored Tb as a proxy of activity in tuco-tucos transferred from a semi-field enclosure to constant lab conditions. In the first stage of this study ("Tb-only group," 2012-2016), we verified high incidence (55%, n = 20) of arrhythmicity, with no consistent diurnal Tb rhythms in tuco-tucos maintained under semi-field conditions. Because these results were discrepant from subsequent findings using miniature accelerometers (portable activity loggers), which showed diurnal activity patterns in natural conditions (n = 10, "Activity-only group," 2016-2017), we also investigated, in the present study, whether the tight association between activity and Tb would be sustained outside the lab. To verify this, we measured activity and Tb simultaneously across laboratory and semi-field deploying both accelerometers and Tb loggers to each animal. These measurements (n = 11, "Tb + activity group," 2019-2022) confirmed diurnality of locomotor activity and revealed an unexpected loosening of the temporal association between Tb and activity rhythms in the field enclosures, which is otherwise robustly tight in the laboratory.

Coupling Between Subregional Oscillators Within the Suprachiasmatic Nucleus Determines Free-Running Period in the Rat.

Rats housed in a 22-h light-dark cycle (11:11, T22) exhibit 2 distinct circadian locomotor activity (LMA) bouts simultaneously: one is entrained to the LD cycle and a second dissociated bout maintains a period greater than 24 h. These 2 activity bouts are associated with independent clock gene oscillations in the ventrolateral (vl-) and dorsomedial (dm-) suprachiasmatic nucleus (SCN), respectively. Previous results in our laboratory have shown that the vl- and dm-SCN oscillators are weakly coupled under T22 and that the period of the dissociated bout depends on coupling between the 2 subdivisions. Here, we sought to study the behavior of the T22 SCN pacemaker upon release into free-running conditions and compare it to the behavior of the system upon release from typical 24-h (12:12, T24) entrainment. T22-desynchronized rats or T24-entrained rats were released into constant darkness (DD). Activity rhythms in T22 rats rapidly resynchronized upon release into DD, and the free-running period (FRP) of the fused rhythm was longer than the FRP of T24 rats. We then asked whether the in vivo period changes were also present in the ex vivo SCN. Per1-luc rats were desynchronized in T22 for assessment of SCN Per1-luc ex vivo. Similar to behavioral FRP, the period of ex vivo SCN explanted from T22 rats was longer than that for T24 animals. Mathematical models supported the observed behavior of the dual oscillator system as the result of mutual coupling between the vl- and dm-SCN oscillators. This bidirectionally coupled model predicted both the FRP of the T22 system and its phase-shifting response to light. Together, these data support a model of pacemaker organization in which a light-sensitive vl-SCN oscillator is mutually coupled with a light-insensitive dm-SCN oscillator, and together they determine the period of the coupled system as a whole and its response to light pulses.

Telling the Seasons Underground: The Circadian Clock and Ambient Temperature Shape Light Exposure and Photoperiodism in a Subterranean Rodent.

Living organisms anticipate the seasons by tracking the proportion of light and darkness hours within a day-photoperiod. The limits of photoperiod measurement can be investigated in the subterranean rodents tuco-tucos (Ctenomys aff. knighti), which inhabit dark underground tunnels. Their exposure to light is sporadic and, remarkably, results from their own behavior of surface emergence. Thus, we investigated the endogenous and exogenous regulation of this behavior and its consequences to photoperiod measurement. In the field, animals carrying biologgers displayed seasonal patterns of daily surface emergence, exogenously modulated by temperature. In the laboratory, experiments with constant lighting conditions revealed the endogenous regulation of seasonal activity by the circadian clock, which has a multi-oscillatory structure. Finally, mathematical modeling corroborated that tuco-tuco's light exposure across the seasons is sufficient for photoperiod encoding. Together, our results elucidate the interrelationship between the circadian clock and temperature in shaping seasonal light exposure patterns that convey photoperiod information in an extreme photic environment.

Quantitative Study of Dual Circadian Oscillator Models under Different Skeleton Photoperiods.

The daily proportion of light and dark hours (photoperiod) changes annually and plays an important role in the synchronization of seasonal biological phenomena, such as reproduction, hibernation, and migration. In mammals, the first step of photoperiod transduction occurs in the suprachiasmatic nuclei (SCN), the circadian pacemaker that also coordinates 24-h activity rhythms. Thus, in parallel with its role in annual synchronization, photoperiod variation acutely shapes day/night activity patterns, which vary throughout the year. Systematic studies of this behavioral modulation help understand the mechanisms behind its transduction at the SCN level. To explain how entrainment mechanisms could account for daily activity patterns under different photoperiods, Colin Pittendrigh and Serge Daan proposed a conceptual model in which the pacemaker would be composed of 2 coupled, evening (E) and morning (M), oscillators. Although the E-M model has existed for more than 40 years now, its physiological bases are still not fully resolved, and it has not been tested quantitatively under different photoperiods. To better explore the implications of the E-M model, we performed computer simulations of 2 coupled limit-cycle oscillators. Four model configurations were exposed to systematic variation of skeleton photoperiods, and the resulting daily activity patterns were assessed. The criterion for evaluating different model configurations was the successful reproduction of 2 key behavioral phenomena observed experimentally: activity psi-jumps and photoperiod-induced changes in activity phase duration. We compared configurations with either separate light inputs to E and M or the same light inputs to both oscillators. The former replicated experimental results closely, indicating that the configuration with separate E and M light inputs is the mechanism that best reproduces the effects of different skeleton photoperiods on day/night activity patterns. We hope this model can contribute to the search for E and M and their light input organization in the SCN.

Acute effects of light and darkness on the activity and temperature rhythms of a subterranean rodent, the Anillaco tuco-tuco.

Tuco-tucos from Anillaco (Ctenomys aff. knighti), are subterranean rodents that run vigorously on laboratory wheels during the night but are active during the day in semi-natural enclosures, where they surface for foraging and burrow maintenance, under intense sunlight. Several studies have shown that light causes opposite, inhibitory and stimulatory, "masking" effects on the activity levels between nocturnal and diurnal species, respectively. Because of the alternating subterranean/surface activity of tuco-tucos in nature during the day and their ability to shift from diurnal to nocturnal patterns in field-to-lab transitions, we assessed the acute effects of light and darkness on running wheel activity, general activity and body temperature. Adult males and females were kept in a LD 12:12 h regimen and exposed to light and dark pulses to verify masking effects in their rhythms. A first experiment consisted in submitting animals to light pulses of different illuminance during the dark phase. Clear inhibition of wheel-running activity occurred, being the response more pronounced as illuminance of the pulse increased, a response typically seen in nocturnal rodents. A second experiment consisted in submitting animals to light pulses during the dark phase, and later to dark pulses during the light phase. This protocol occurred three times in the conditions: 1) without a wheel, 2) with free access to a wheel, and 3) with a blocked wheel. Wheel running was inhibited and body temperature decreased in most animals during the light pulse, with little to no inhibition on general activity. Dark pulses during the light phase had no effect on wheel-running activity nor on general activity but did affect body temperature. Interestingly, there was a single individual that switched from nocturnal to diurnal when kept without a running wheel, offering an opportunity to test nocturnal and diurnal masking patterns to light in the same individual.

Day and night in the subterranean: measuring daily activity patterns of subterranean rodents (Ctenomys aff. knighti) using bio-logging.

While most studies of the impacts of climate change have investigated shifts in the spatial distribution of organisms, temporal shifts in the time of activity is another important adjustment made by animals in a changing world. Due to the importance of light and temperature cycles in shaping activity patterns, studies of activity patterns of organisms that inhabit extreme environments with respect to the 24-hour cyclicity of Earth have the potential to provide important insights into the interrelationships among abiotic variables, behaviour and physiology. Our previous laboratory studies with Argentinean tuco-tucos from the Monte desert (Ctenomys aff. knighti) show that these subterranean rodents display circadian activity/rest rhythms that can be synchronized by artificial light/dark cycles. Direct observations indicate that tuco-tucos emerge mainly for foraging and for removal of soil from their burrows. Here we used bio-logging devices for individual, long-term recording of daily activity/rest (accelerometry) and time on surface (light-loggers) of six tuco-tucos maintained in outdoor semi-natural enclosures. Environmental variables were measured simultaneously. Activity bouts were detected both during day and night but 77% of the highest values happened during the daytime and 47% of them coincided with time on surface. Statistical analyses indicate time of day and temperature as the main environmental factors modulating time on surface. In this context, the total duration that these subterranean animals spent on surface was high during the winter, averaging 3 h per day and time on surface occurred when underground temperature was lowest. Finally, transport of these animals to the indoor laboratory and subsequent assessment of their activity rhythms under constant darkness revealed a switch in the timing of activity. Plasticity of activity timing is not uncommon among desert rodents and may be adaptive in changing environments, such as the desert where this species lives.

Novel Light/Dark Regimens with Minimum Light Promote Circadian Disruption: Simulations with a Model Oscillator.

Artificial lab manipulation of LD cycles has enabled simulations of the disruptive conditions found in modern human societies, such as jet-lag, night-work and light at night. New techniques using animal models have been developed, and these can greatly improve our understanding of circadian disruption. Some of these techniques, such as in vivo bioluminescence assays, require minimum external light. This requirement is challenging because the usual lighting protocols applied in circadian desynchronization experiments rely on considerable light input. Here, we present a novel LD regimen that can disrupt circadian rhythms with little light per day, based on computer simulations of a model limit-cycle oscillator. The model predicts that a single light pulse per day has the potential to disturb rhythmicity when pulse times are randomly distributed within an interval. Counterintuitively, the rhythm still preserves an underlying 24-h periodicity when this interval is as large as 14 h, indicating that day/night cues are still detectable. Only when pulses are spread throughout the whole 24-h day does the rhythm lose any day-to-day period correlation. In addition, the model also reveals that stronger pulses of brighter light should exacerbate the disrupting effects. We propose the use of this LD schedule-which would be compatible with the requirements of in vivo bioluminescence assays-to help understand circadian disruption and associated illnesses.

The Interplay of Energy Balance and Daily Timing of Activity in a Subterranean Rodent: A Laboratory and Field Approach.

The tuco-tuco (Ctenomys aff. knighti) is among the rodent species known to be nocturnal under standard laboratory conditions and diurnal under natural conditions. The circadian thermoenergetics (CTE) hypothesis postulates that switches in activity timing are a response to energetic challenges; daytime activity reduces thermoregulatory costs by consolidating activity to the warmest part of the day. Studying wild animals under both captive and natural conditions can increase understanding of how temporal activity patterns are shaped by the environment and could serve as a test of the CTE hypothesis. We estimated the effects of activity timing on energy expenditure for the tuco-tuco by combining laboratory measurements of metabolic rate with environmental temperature records in both winter and summer. We showed that, in winter, there would be considerable energy savings if activity is allocated at least partially during daylight, lending support to the CTE hypothesis. In summer, the impact of activity timing on energy expenditure is small, suggesting that during this season other factors, such as predation risk, water balance, and social interaction, may have more important roles than energetics in the determination of activity time.

Entrainment of circadian rhythms to irregular light/dark cycles: a subterranean perspective.

Synchronization of biological rhythms to the 24-hour day/night has long been studied with model organisms, under artificial light/dark cycles in the laboratory. The commonly used rectangular light/dark cycles, comprising hours of continuous light and darkness, may not be representative of the natural light exposure for most species, including humans. Subterranean rodents live in dark underground tunnels and offer a unique opportunity to investigate extreme mechanisms of photic entrainment in the wild. Here, we show automated field recordings of the daily light exposure patterns in a South American subterranean rodent, the tuco-tuco (Ctenomys aff. knighti ). In the laboratory, we exposed tuco-tucos to a simplified version of this natural light exposure pattern, to determine the minimum light timing information that is necessary for synchronization. As predicted from our previous studies using mathematical modeling, the activity rhythm of tuco-tucos synchronized to this mostly simplified light/dark regimen consisting of a single light pulse per day, occurring at randomly scattered times within a day length interval. Our integrated semi-natural, lab and computer simulation findings indicate that photic entrainment of circadian oscillators is robust, even in face of artificially reduced exposure and increased phase instability of the synchronizing stimuli.

Nocturnal to Diurnal Switches with Spontaneous Suppression of Wheel-Running Behavior in a Subterranean Rodent.

Several rodent species that are diurnal in the field become nocturnal in the lab. It has been suggested that the use of running-wheels in the lab might contribute to this timing switch. This proposition is based on studies that indicate feed-back of vigorous wheel-running on the period and phase of circadian clocks that time daily activity rhythms. Tuco-tucos (Ctenomys aff. knighti) are subterranean rodents that are diurnal in the field but are robustly nocturnal in laboratory, with or without access to running wheels. We assessed their energy metabolism by continuously and simultaneously monitoring rates of oxygen consumption, body temperature, general motor and wheel running activity for several days in the presence and absence of wheels. Surprisingly, some individuals spontaneously suppressed running-wheel activity and switched to diurnality in the respirometry chamber, whereas the remaining animals continued to be nocturnal even after wheel removal. This is the first report of timing switches that occur with spontaneous wheel-running suppression and which are not replicated by removal of the wheel.

Modeling natural photic entrainment in a subterranean rodent (Ctenomys aff. knighti), the Tuco-Tuco.

Subterranean rodents spend most of the day inside underground tunnels, where there is little daily change in environmental variables. Our observations of tuco-tucos (Ctenomys aff. knighti) in a field enclosure indicated that these animals perceive the aboveground light-dark cycle by several bouts of light-exposure at irregular times during the light hours of the day. To assess whether such light-dark pattern acts as an entraining agent of the circadian clock, we first constructed in laboratory the Phase Response Curve for 1 h light-pulses (1000lux). Its shape is qualitatively similar to other curves reported in the literature and to our knowledge it is the first Phase Response Curve of a subterranean rodent. Computer simulations were performed with a non-linear limit-cycle oscillator subjected to a simple model of the light regimen experienced by tuco-tucos. Results showed that synchronization is achieved even by a simple regimen of a single daily light pulse scattered uniformly along the light hours of the day. Natural entrainment studies benefit from integrated laboratory, field and computational approaches.

Period determination in the food-entrainable and methamphetamine-sensitive circadian oscillator(s).

Daily rhythmic processes are coordinated by circadian clocks, which are present in numerous central and peripheral tissues. In mammals, two circadian clocks, the food-entrainable oscillator (FEO) and methamphetamine-sensitive circadian oscillator (MASCO), are "black box" mysteries because their anatomical loci are unknown and their outputs are not expressed under normal physiological conditions. In the current study, the investigation of the timekeeping mechanisms of the FEO and MASCO in mice with disruption of all three paralogs of the canonical clock gene, Period, revealed unique and convergent findings. We found that both the MASCO and FEO in Per1(-/-)/Per2(-/-)/Per3(-/-) mice are circadian oscillators with unusually short (∼21 h) periods. These data demonstrate that the canonical Period genes are involved in period determination in the FEO and MASCO, and computational modeling supports the hypothesis that the FEO and MASCO use the same timekeeping mechanism or are the same circadian oscillator. Finally, these studies identify Per1(-/-)/Per2(-/-)/Per3(-/-) mice as a unique tool critical to the search for the elusive anatomical location(s) of the FEO and MASCO.

Field and laboratory studies provide insights into the meaning of day-time activity in a subterranean rodent (Ctenomys aff. knighti), the tuco-tuco.

South American subterranean rodents (Ctenomys aff. knighti), commonly known as tuco-tucos, display nocturnal, wheel-running behavior under light-dark (LD) conditions, and free-running periods >24 h in constant darkness (DD). However, several reports in the field suggested that a substantial amount of activity occurs during daylight hours, leading us to question whether circadian entrainment in the laboratory accurately reflects behavior in natural conditions. We compared circadian patterns of locomotor activity in DD of animals previously entrained to full laboratory LD cycles (LD12:12) with those of animals that were trapped directly from the field. In both cases, activity onsets in DD immediately reflected the previous dark onset or sundown. Furthermore, freerunning periods upon release into DD were close to 24 h indicating aftereffects of prior entrainment, similarly in both conditions. No difference was detected in the phase of activity measured with and without access to a running wheel. However, when individuals were observed continuously during daylight hours in a semi-natural enclosure, they emerged above-ground on a daily basis. These day-time activities consisted of foraging and burrow maintenance, suggesting that the designation of this species as nocturnal might be inaccurate in the field. Our study of a solitary subterranean species suggests that the circadian clock is entrained similarly under field and laboratory conditions and that day-time activity expressed only in the field is required for foraging and may not be time-dictated by the circadian pacemaker.

Forced desynchronization of activity rhythms in a model of chronic jet lag in mice.

We studied locomotor activity rhythms of C57/Bl6 mice under a chronic jet lag (CJL) protocol (ChrA(6/2) ), which consisted of 6-hour phase advances of the light-dark schedule (LD) every 2 days. Through periodogram analysis, we found 2 components of the activity rhythm: a short-period component (21.01 ± 0.04 h) that was entrained by the LD schedule and a long-period component (24.68 ± 0.26 h). We developed a mathematical model comprising 2 coupled circadian oscillators that was tested experimentally with different CJL schedules. Our simulations suggested that under CJL, the system behaves as if it were under a zeitgeber with a period determined by (24 - [phase shift size/days between shifts]). Desynchronization within the system arises according to whether this effective zeitgeber is inside or outside the range of entrainment of the oscillators. In this sense, ChrA(6/2) is interpreted as a (24 - 6/2 = 21 h) zeitgeber, and simulations predicted the behavior of mice under other CJL schedules with an effective 21-hour zeitgeber. Animals studied under an asymmetric T = 21 h zeitgeber (carried out by a 3-hour shortening of every dark phase) showed 2 activity components as observed under ChrA(6/2): an entrained short-period (21.01 ± 0.03 h) and a long-period component (23.93 ± 0.31 h). Internal desynchronization was lost when mice were subjected to 9-hour advances every 3 days, a possibility also contemplated by the simulations. Simulations also predicted that desynchronization should be less prevalent under delaying than under advancing CJL. Indeed, most mice subjected to 6-hour delay shifts every 2 days (an effective 27-hour zeitgeber) displayed a single entrained activity component (26.92 ± 0.11 h). Our results demonstrate that the disruption provoked by CJL schedules is not dependent on the phase-shift magnitude or the frequency of the shifts separately but on the combination of both, through its ratio and additionally on their absolute values. In this study, we present a novel model of forced desynchronization in mice under a specific CJL schedule; in addition, our model provides theoretical tools for the evaluation of circadian disruption under CJL conditions that are currently used in circadian research.

Modeling two-oscillator circadian systems entrained by two environmental cycles.

Several experimental studies have altered the phase relationship between photic and non-photic environmental, 24 h cycles (zeitgebers) in order to assess their role in the synchronization of circadian rhythms. To assist in the interpretation of the complex activity patterns that emerge from these "conflicting zeitgeber" protocols, we present computer simulations of coupled circadian oscillators forced by two independent zeitgebers. This circadian system configuration was first employed by Pittendrigh and Bruce (1959), to model their studies of the light and temperature entrainment of the eclosion oscillator in Drosophila. Whereas most of the recent experiments have restricted conflicting zeitgeber experiments to two experimental conditions, by comparing circadian oscillator phases under two distinct phase relationships between zeitgebers (usually 0 and 12 h), Pittendrigh and Bruce compared eclosion phase under 12 distinct phase relationships, spanning the 24 h interval. Our simulations using non-linear differential equations replicated complex non-linear phenomena, such as "phase jumps" and sudden switches in zeitgeber preferences, which had previously been difficult to interpret. Our simulations reveal that these phenomena generally arise when inter-oscillator coupling is high in relation to the zeitgeber strength. Manipulations in the structural symmetry of the model indicated that these results can be expected to apply to a wide range of system configurations. Finally, our studies recommend the use of the complete protocol employed by Pittendrigh and Bruce, because different system configurations can generate similar results when a "conflicting zeitgeber experiment" incorporates only two phase relationships between zeitgebers.

Dissociation of circadian and light inhibition of melatonin release through forced desynchronization in the rat.

Pineal melatonin release exhibits a circadian rhythm with a tight nocturnal pattern. Melatonin synthesis is regulated by the master circadian clock within the hypothalamic suprachiasmatic nucleus (SCN) and is also directly inhibited by light. The SCN is necessary for both circadian regulation and light inhibition of melatonin synthesis and thus it has been difficult to isolate these two regulatory limbs to define the output pathways by which the SCN conveys circadian and light phase information to the pineal. A 22-h light-dark (LD) cycle forced desynchrony protocol leads to the stable dissociation of rhythmic clock gene expression within the ventrolateral SCN (vlSCN) and the dorsomedial SCN (dmSCN). In the present study, we have used this protocol to assess the pattern of melatonin release under forced desynchronization of these SCN subregions. In light of our reported patterns of clock gene expression in the forced desynchronized rat, we propose that the vlSCN oscillator entrains to the 22-h LD cycle whereas the dmSCN shows relative coordination to the light-entrained vlSCN, and that this dual-oscillator configuration accounts for the pattern of melatonin release. We present a simple mathematical model in which the relative coordination of a single oscillator within the dmSCN to a single light-entrained oscillator within the vlSCN faithfully portrays the circadian phase, duration and amplitude of melatonin release under forced desynchronization. Our results underscore the importance of the SCN's subregional organization to both photic input processing and rhythmic output control.

Daily rhythms related to distinct social tasks inside an eusocial bee colony.

A bee colony is often compared to a multicellular organism, mainly because of its spatial organization. We propose that a temporal organization of equal importance is also present. To support this view, we studied the reproductive processes of two closely related species of stingless bees. Stingless bees enable observations of daily rhythms that are performed by distinct social classes. The emergent process, POP, is cyclic and consists of the building and provisioning of brood cells by the worker bees and egg-laying by the queen. Colonies were kept in the laboratory under constant conditions with the exit tube opening to the environment; thus, foragers had direct access to environmental cycles. At a later stage of the experiment, the exit tube was closed by a sieve; in this case, bees had their own stock of food, but the environmental LD cycle could still be detected when they were inside the exit tube. Daily POP rhythms were present and showed distinct temporal patterns in each species. A third condition was imposed on one of the species only: the exit tube was closed by a sieve and maintained inside a box that was provided with constant illumination. In this colony, the POP rhythm was perturbed by the destruction of the brood cells. Restoration of POP consisted of a rapid reconstruction of cells followed by a late oviposition in the same day. As different rhythmic patterns were detected, but showed regular timings with respect to one another, an interpretation based upon the concept of an internal temporal order is suggested.

A model for "splitting" of running-wheel activity in hamsters.

Splitting of locomotor activity rhythm in hamsters occurs when the animals are exposed for several weeks to constant light. The authors propose a mathematical model that explains splitting in terms of a switch in the sign of coupling of two oscillators, from positive to negative, due to long-term exposure to constant light. The model assumes that the two oscillators are not identical and that the negative coupling strengths achieved by each individual animal are variable. With these assumptions, the model provides a unified picture of all different splitting patterns presented by the hamsters, provides an explanation for why the two activity components cross each other during many patterns, and explains why the phase difference achieved by the split components is often near 180 degrees.