Photic phase response curve in Octodon degus: assessment as a function of activity phase preference

Light exposure during the early and late subjective night generally phase delays and advances circadian rhythms, respectively. However, this generality was recently questioned in a photic entrainment study in Octodon degus. Because degus can invert their activity phase preference from diurnal to nocturnal as a function of activity level, assessment of phase preference is critical for computations of phase reference [circadian time (CT) 0] toward the development of a photic phase response curve. After determining activity phase preference in a 24-h light-dark cycle (LD 12:12), degus were released in constant darkness. In this study, diurnal (n = 5) and nocturnal (n = 7) degus were randomly subjected to 1-h light pulses (30-35 lx) at many circadian phases (CT 1-6: n = 7; CT 7-12: n = 8; CT 13-18: n = 8; and CT 19-24: n = 7). The circadian phase of body temperature (Tb) onset was defined as CT 12 in nocturnal animals. In diurnal animals, CT 0 was determined as Tb onset + 1 h. Light phase delayed and advanced circadian rhythms when delivered during the early (CT 13-16) and late (CT 20-23) subjective night, respectively. No significant phase shifts were observed during the middle of the subjective day (CT 3-10). Thus, regardless of activity phase preference, photic entrainment of the circadian pacemaker in Octodon degus is similar to most other diurnal and nocturnal species, suggesting that entrainment mechanisms do not determine overt diurnal and nocturnal behavior.     

Circadian Phase Shifting Associated with Routine Cage Maintenance: A Retrospective Analysis

Stimuli that evoke behavioral activation can phase-shift free-running circadian activity rhythms in Syrian hamsters. Activation-induced phase shifting is characterized by a phase-response curve (PRC) that is dissimilar to the PRC for photic phase shifting, and recent studies indicate that complex interactions may occur between photic and non-photic phase shifting. Since animals in the laboratory may be exposed to both photic and behaviorally activating stimulation during routine cage maintenance procedures, we performed a retrospective analysis of possible phase shifts associated with cage cleaning in individually housed hamsters maintained in either constant darkness (DD) or dim red light (RR) during the course of an ongoing study of drug-induced phase shifting. All cage cleanings were conducted under RR and were separated from drug treatments by at least one week. The results indicated that both photic and non-photic phase shifts could be induced by routine cage maintenance procedures, depending on the circadian timing of the procedure, on lighting conditions, and on the degree of evoked activity.     

Circadian Phase Shifting Associated with Routine Cage Maintenance: A Retrospective Analysis

Stimuli that evoke behavioral activation can phase-shift free-running circadian activity rhythms in Syrian hamsters. Activation-induced phase shifting is characterized by a phase-response curve (PRC) that is dissimilar to the PRC for photic phase shifting, and recent studies indicate that complex interactions may occur between photic and non-photic phase shifting. Since animals in the laboratory may be exposed to both photic and behaviorally activating stimulation during routine cage maintenance procedures, we performed a retrospective analysis of possible phase shifts associated with cage cleaning in individually housed hamsters maintained in either constant darkness (DD) or dim red light (RR) during the course of an ongoing study of drug-induced phase shifting. All cage cleanings were conducted under RR and were separated from drug treatments by at least one week. The results indicated that both photic and non-photic phase shifts could be induced by routine cage maintenance procedures, depending on the circadian timing of the procedure, on lighting conditions, and on the degree of evoked activity.     

Behavioral feedback regulation of circadian rhythm phase angle in light-dark entrained mice

Induced and spontaneous wheel running can alter the phase and period (tau) of circadian rhythms in rodents. The relationship between spontaneous running and the phase angle (psi) of entrainment to 24-h light-dark (LD) cycles was evaluated in C57BL/6j mice. With a wheel freely available, psi was significantly correlated with the absolute (r = 0.32) and relative (r = 0.44) amount of activity during the first 2 h of the activity period. When wheels were locked during the first half of the night in LD and then unlocked in constant dark (DD), mice exhibited a delayed psi and lengthened tau compared with mice that had wheels locked during the second half of the night. In DD, tau correlated negatively with total daily activity. To evaluate if wheel running modulates the phase-resetting actions of LD, phase shifts to light pulses were measured at two time points in DD, when daily activity levels differed by 40%. Phase delays to light were 56% greater when activity levels were lower. However, in a counterbalanced follow-up experiment, phase advances and delays to light pulses were not affected by the availability of wheels, although an effect of time in DD was replicated. Spontaneous activity can regulate psi and tau without altering the response of the pacemaker to light.     

Circadian and photoperiodic effects of brief light pulses in male Djungarian hamsters

The effects of brief light pulses (1-60 min in duration) on the circadian rhythm of locomotor activity and/or the neuroendocrine-gonadal axis was investigated in male Djungarian hamsters. Exposure of hamsters free-running in constant darkness to a single 1-h pulse of light induced phase-dependent phase shifts in the rhythm of locomotor activity. The general shape of the "phase-response curve" was similar to that observed in other animals; phase-delays and phase-advances were induced by light pulses delivered in the early and late subjective night, respectively, while light pulses during the subjective day induced little or no phase-shift in the activity rhythm. Animals exposed for 7 days to 1-min of light during the night in animals otherwise exposed to 6L:18D resulted in increased levels of serum FSH and testicular weight. Daily exposure to two 1-h or two 10-min pulses of light (but not two 1-min pulses) for 10 days resulted in stable entrainment of the activity rhythm as well as testicular weight gains and serum FSH increases. When two 10-min pulses of light were presented 8 and 16 h apart, some animals showed a short-day entrainment pattern (i.e., locomotor activity confined to the long period of darkness) while other animals showed a long-day entrainment pattern (i.e., locomotor activity confined to the short period of darkness). Importantly, the stimulatory effects of light on neuroendocrine-gonadal activity were clearly dependent on the phase-relationship between the light pulses and the circadian rhythm of locomotor activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resetting of the hamster circadian system by dark pulses

Circadian rhythms of animals are reset by exposure to light as well as dark; however, although the parameters of photic entrainment are well characterized, the phase-shifting actions of dark pulses are poorly understood. Here, we determined the tonic and phasic effects of short (0.25 h), moderate (3 h), and long (6-9 h) duration dark pulses on the wheel-running rhythms of hamsters in constant light. Moderate- and long-duration dark pulses phase dependently reset behavioral rhythms, and the magnitude of these phase shifts increased as a function of the duration of the dark pulse. In contrast, the 0.25-h dark pulses failed to evoke consistent effects at any circadian phase tested. Interestingly, moderate- and long-dark pulses elevated locomotor activity (wheel-running) on the day of treatment. This induced wheel-running was highly correlated with phase shift magnitude when the pulse was given during the subjective day. This, together with the finding that animals pulsed during the subjective day are behaviorally active throughout the pulse, suggests that both locomotor activity and behavioral activation play an important role in the phase-resetting actions of dark pulses. We also found that the robustness of the wheel-running rhythm was weakened, and the amount of wheel-running decreased on the days after exposure to dark pulses; these effects were dependent on pulse duration. In summary, similarly to light, the resetting actions of dark pulses are dependent on both circadian phase and stimulus duration. However, dark pulses appear more complex stimuli, with both photic and nonphotic resetting properties.     

Non-parametric photic entrainment of Djungarian hamsters with different rhythmic phenotypes

To investigate the role of non-parametric light effects in entrainment, Djungarian hamsters of two different circadian phenotypes were exposed to skeleton photoperiods, or to light pulses at different circadian times, to compile phase response curves (PRCs). Wild-type (WT) hamsters show daily rhythms of locomotor activity in accord with the ambient light/dark conditions, with activity onset and offset strongly coupled to light-off and light-on, respectively. Hamsters of the delayed activity onset (DAO) phenotype, in contrast, progressively delay their activity onset, whereas activity offset remains coupled to light-on. The present study was performed to better understand the underlying mechanisms of this phenomenon. Hamsters of DAO and WT phenotypes were kept first under standard housing conditions with a 14:10 h light–dark cycle, and then exposed to skeleton photoperiods (one or two 15-min light pulses of 100 lx at the times of the former light–dark and/or dark–light transitions). In a second experiment, hamsters of both phenotypes were transferred to constant darkness and allowed to free-run until the lengths of the active (α) and resting (ρ) periods were equal (α:ρ = 1). At this point, animals were then exposed to light pulses (100 lx, 15 min) at different circadian times (CTs). Phase and period changes were estimated separately for activity onset and offset. When exposed to skeleton-photoperiods with one or two light pulses, the daily activity patterns of DAO and WT hamsters were similar to those obtained under conditions of a complete 14:10 h light–dark cycle. However, in the case of giving only one light pulse at the time of the former light–dark transition, animals temporarily free-ran until activity offset coincided with the light pulse. These results show that photic entrainment of the circadian activity rhythm is attained primarily via non-parametric mechanisms, with the “morning” light pulse being the essential cue. In the second experiment, typical photic PRCs were obtained with phase delays in the first half of the subjective night, phase advances in the second half, and a dead zone during the subjective day. ANOVA indicated no significant differences between WT and DAO animals despite a significantly longer free-running period (tau) in DAO hamsters. Considering the phase shifts induced around CT0 and the different period lengths, it was possible to model the entrainment patterns of both phenotypes. It was shown that light-induced phase shifts of activity offset were sufficient to compensate for the long tau in WT and DAO hamsters, thus enabling a stable entrainment of their activity offsets to be achieved. With respect to activity onsets, phase shifts were sufficient only in WT animals; in DAO hamsters, activity onset showed increasing delays. The results of the present paper clearly demonstrate that, under laboratory conditions, the non-parametric component of light and dark leads to circadian entrainment in Djungarian hamsters. However, a stable entrainment of activity onset can be achieved only if the free-running period does not exceed a certain value. With longer tau values, hamsters reveal a DAO phenotype. Under field conditions, therefore, non-photic cues/zeitgebers must obviously be involved to enable a proper circadian entrainment.     

No evidence for extraocular photoreceptors in the circadian system of the Syrian hamster.

Campbell and Murphy reported recently that 3 h of bright light (13,000 lux) exposure to the area behind the knee caused phase shifts of the circadian rhythms of both body temperature and saliva melatonin in humans. The authors tested the hypothesis that extraocular photoreception is also involved in the circadian system of the Syrian hamster. Hamsters were bilaterally enucleated (eyes removed), and their backs were shaved. Hamsters with stable free-running rhythms in constant darkness were exposed to direct sunlight for 1 or 3 hours during their subjective night. Intact (control) animals showed phase shifts as expected, but the locomotor activity of enucleated animals was unaffected by the exposure to sunlight. The authors also measured the pineal melatonin content after exposure to sunlight. Pineal melatonin content in intact animals declined markedly as expected, but no decline was observed in the enucleated hamsters. The authors conclude that extraocular phototransduction is not capable of shifting the phase of the hamster's locomotor activity rhythm or of suppressing pineal melatonin synthesis.     

Photic and nonphotic inputs to the diurnal circadian clock

Diurnal animals occupy a different temporal niche from nocturnal animals and are consequently exposed to different amounts of light as well as different dangers. Accordingly, some variation exists in the way that diurnal animals synchronize their internal circadian clock to match the external 24-hour daily cycle. First, though the brain mechanisms underlying photic entrainment are very similar among species with different daily activity patterns, there is evidence that diurnal animals are less sensitive to photic stimuli compared to nocturnal animals. Second, stimuli other than light that synchronize rhythms (i.e. nonphotic stimuli) can also entrain and phase shift daily rhythms. Some of the rules that govern nonphotic entrainment in nocturnal animals as well as the brain mechanisms that control nonphotic influences on rhythms do not appear to apply to diurnal animals, however. Some evidence supports the idea that arousal or activity plays an important role in entraining rhythms in diurnal animals, either during the light (active) or dark (inactive) phases, though no consistent pattern is seen. GABAergic stimulation induces phase shifts during the subjective day in both diurnal and nocturnal animals. In diurnal Arvicanthis niloticus (Nile grass rats), SCN GABA_A receptor activation at this time results in phase delays while in nocturnal animals phase advances are induced. It appears that the effect of GABA at this circadian phase results from the inhibition of period gene expression in both diurnal and nocturnal animals. Nonetheless, the resulting phase shifts are in opposite directions. It is not known what stimuli or behaviours ultimately induce changes in GABA activity in the SCN that result in alterations of circadian phase in diurnal grass rats. Taken together, studies such as these suggest that it may be problematic to apply the principles governing nocturnal nonphotic entrainment and its underlying mechanisms to diurnal species including humans.     

Nocturnal activity in a diurnal rodent (Arvicanthis niloticus): the importance of masking

It is known that day-active Nile grass rats, Arvicanthis niloticus, increase the amount of activity in the night relative to that in the day when provided with running wheels. This was confirmed in the present study. Animals without a wheel displayed 69.0% of their general activity in the L phase of a 12:12 h light-dark cycle; animals provided with wheels had only 48.6% of their wheel revolutions in the light. The contribution of direct (masking) responses to light to the increased nocturnality of animals with wheels was examined in two experiments. In experiment 1, masking was tested by exposing the animals to repeated cycles of 30 min of entraining light and 30 min of a different, usually dimmer light, during the L phase of a 12:12 h light-dark cycle. For animals with wheels, there was more running during the 30-min pulses of dim light or darkness than during the 30-min periods of entraining light. In contrast, for animals without wheels, there was more general activity during the 30-min periods of entraining light than during the 30-min pulses of dim light or darkness. In experiment 2, the animals were first exposed to a 12:12 h light-dark cycle and then put on a 1:10:1:12 h LDLD skeleton photoperiod. Animals with wheels increased their running during the subjective day of the skeleton photoperiod compared to that in the actual day of the 12:12 h light-dark cycle. Animals without wheels showed similar levels of general activity during the subjective day of the skeleton photoperiod and the actual day of the 12:12 h cycle. These experiments demonstrate that when Nile rats have running wheels, their increased nocturnal activity is associated with an increased suppression of locomotion in direct response to light. It is possible that changes in masking responses to light may be an essential and integral component of switching between diurnal and nocturnal activity profiles.     

Lesions of the thalamic intergeniculate leaflet alter hamster circadian rhythms

We have investigated the effects of destruction of the geniculo-hypothalamic tract (GHT) on the circadian system of golden hamsters. In the first experiment, intact hamsters were housed in constant darkness, and phase shifts in running-wheel activity rhythms were assessed following 15-min light pulses administered at circadian time (CT) 12 (defined as the beginning of activity), CT 14, CT 18, and CT 20. Responses to light pulses at the same CTs were then reassessed after GHT lesions. Hamsters with complete lesions showed decreases in phase advances caused by light pulses at CT 18 and CT 20. Phase delays elicited by light at CT 12 and CT 14 were not altered. In a second study, intact and GHT-ablated hamsters housed in constant light received 6-hr dark pulses at various CTs. Hamsters with complete GHT ablation showed smaller advances than controls to dark pulses centered on CT 8-10. After 110 days in constant light, 7 of 10 intact hamsters showed splitting of their activity rhythms into two components, while only 1 of the 8 similarly treated ablated hamsters displayed dissociated activity components. Ablated hamsters had significantly shorter free-running periods during the first 35 days of exposure to constant light than did the intact hamsters. These results demonstrate that destruction of the GHT in the hamster alters phase shifting in response to periods of light or dark, and they indicate a role for the GHT in mediating several photic effects on the circadian system.     

Circadian organization in male mice lacking the gene for endothelial nitric oxide synthase (eNOS-/-)

Circadian (approximately 24 h) rhythms in physiology and behavior are generated by the bilateral suprachiasmatic nucleus (SCN) of the anterior hypothalamus. For these endogenous rhythms to be synchronized with the external environment, light information must be transmitted to pacemaker cells within the SCN. This transmission of light information is accomplished via a direct retino-hypothalamic tract (RHT). Nitric oxide (NO), an endogenous gas that functions as a neurotransmitter, has been implicated as a messenger necessary for photic entrainment. Three isoforms of the enzyme that form NO, NO synthase, have been identified (a) in neurons (nNOS), (b) in the endothelial lining of blood vessels (eNOS), and (c) as an inducible form in macrophages (iNOS). The present study was undertaken to determine the specific role of eNOS in circadian organization and photic entrainment. Wild-type (WT) and eNOS-/- mice were initially entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was phase advanced. After an additional 3 weeks, animals were held in constant darkness (DD). eNOS-/- animals did not exhibit a deficit in the ability to entrain to the LD cycle, phase-shift locomotor activity, or free-run in constant conditions. Animals held in DD were killed after light exposure during either the subjective day or the subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos protein expression in the SCN of both WT and eNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that endothelial isoform of NOS may not be necessary for photic entrainment in mice.     

Effects of prolonged exposure to darkness on circadian photic responsiveness in the mouse

Circadian rhythms in mammals are generated by an endogenous pacemaker but are modulated by environmental cycles, principally the alternation of light and darkness. Although much is known about nonparametric effects of light on the circadian system, little is known about other effects of photic stimulation. In the present study, which consists of a series of five experiments in mice, various manipulations of photic stimulation were used to dissect the mechanisms responsible for a variation in the magnitude of light-induced phase-shifts that results from prolonged exposure to darkness. The results confirmed previous observations that prolonged exposure to darkness causes an increase in the magnitude of phase shifts (both phase advances and phase delays) evoked by discrete light pulses. The results also indicated that the increase in responsiveness results from the lack of exposure to light per se and not from collateral effects of exposure to constant darkness such as the lack of previous entrainment. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system but with a much slower temporal course. The results suggest that circadian dark adaptation may take place at the retinal level, but it is not clear whether it involves a change in the sensitivity or maximal responsiveness of the system.     

Effects of Prolonged Exposure to Darkness on Circadian Photic Responsiveness in the Mouse

Circadian rhythms in mammals are generated by an endogenous pacemaker but are modulated by environmental cycles, principally the alternation of light and darkness. Although much is known about nonparametric effects of light on the circadian system, little is known about other effects of photic stimulation. In the present study, which consists of a series of five experiments in mice, various manipulations of photic stimulation were used to dissect the mechanisms responsible for a variation in the magnitude of light-induced phase-shifts that results from prolonged exposure to darkness. The results confirmed previous observations that prolonged exposure to darkness causes an increase in the magnitude of phase shifts (both phase advances and phase delays) evoked by discrete light pulses. The results also indicated that the increase in responsiveness results from the lack of exposure to light per se and not from collateral effects of exposure to constant darkness such as the lack of previous entrainment. The lack of exposure to light causes the circadian system to undergo a process of dark adaptation similar to dark adaptation in the visual system but with a much slower temporal course. The results suggest that circadian dark adaptation may take place at the retinal level, but it is not clear whether it involves a change in the sensitivity or maximal responsiveness of the system.     

Djungarian hamsters: a species with a labile circadian pacemaker? Arrhythmicity under a light-dark cycle induced by short light pulses

In most cases, phase-shifting effects of light pulses are studied in animals kept in constant darkness (DD) or in animals released into DD following the stimulus. In this study, the authors exposed Djungarian hamsters (Phodopus sungorus) to short light pulses during the dark phase of a 16:8 light-dark (LD) cycle and thus obtained a type VI phase response curve. Light pulses early in the night caused phase delays of the activity onset as well as phase advances of the activity offset, whereas light pulses later in the night resulted in phase advances of the activity offset only. A combination of two 15-min light pulses-the first one given late in the scotophase and the second given early in the dark phase of the following night-led to a strong compression of the activity phase alpha. In 75% of all animals, daily rhythms were no longer visible after complete alpha compression, and long-term arrhythmicity (up to 145 days) persisted despite continued exposure to an LD cycle. Because three independent output rhythms of the clock (i.e., activity, body temperature, and melatonin rhythms) were equally affected, the authors conclude that overt arrhythmicity was due not merely to disrupted output pathways but to an altered state of the central pacemaker. The authors suggest a qualitative two-oscillator model to explain this phenomenon. Their hypothesis assumes that, due to loose coupling, the pacemaker of Djungarian hamsters can be driven to a state of zero phase difference between the two oscillators, with zero amplitude of their outputs.     

Circadian locomotor analysis of male mice lacking the gene for neuronal nitric oxide synthase (nNOS-/-)

Nitric oxide (NO) is an endogenous gas that functions as a neurotransmitter. Because NO is very labile with a half-life of less than 5 sec, most functional studies of NO have manipulated its synthetic enzyme, NO synthase (NOS). Three isoforms of NOS have been identified: (1) in the endothelial lining of blood vessels (eNOS), (2) an inducible form found in macrophages (iNOS), and (3) in neurons (nNOS). Most pharmacological studies to date have blocked all three isoforms of NOS. Previous studies using such agents have revealed that NO might be necessary for photic entrainment of circadian rhythms; general NOS inhibitors attenuate phase shifts of free-running behavior, light-induced c-fos expression in the suprachiasmatic nucleus (SCN), and phase shifts of neural firing activity in SCN maintained in vitro. To assess the specific role of nNOS in mediating entrainment of circadian rhythms, mice with targeted deletion of the gene encoding the neuronal isoform of NOS (nNOS-/-) were used. Wild-type (WT) and nNOS-/- mice initially were entrained to a 14:10 light:dark (LD) cycle. After 3 weeks, the LD cycle was either phase advanced or phase delayed. After an additional 3 weeks, animals were held in either constant dim light or constant dark. WT and nNOS-/- animals did not differ in their ability to entrain to the LD cycle, phase shift locomotor activity, or free run in constant conditions. Animals held in constant dark were killed after light exposure during either the subjective day or subjective night to assess c-fos induction in the SCN. Light exposure during the subjective night increased c-fos expression in the SCN of both WT and nNOS-/- mice relative to animals killed after light exposure during the subjective day. Taken together, these findings suggest that NO from neurons might not be necessary for photic entrainment.     

Light pulses do not induce c-fos or per1 in the SCN of hamsters that fail to reentrain to the photocycle

Circadian activity rhythms of most Siberian hamsters (Phodopus sungorus sungorus) fail to reentrain to a 5-h phase shift of the light-dark (LD) cycle. Instead, their rhythms free-run at periods close to 25 h despite the continued presence of the LD cycle. This lack of behavioral reentrainment necessarily means that molecular oscillators in the master circadian pacemaker, the SCN, were unable to reentrain as well. The authors tested the hypothesis that a phase shift of the LD cycle rendered the SCN incapable of responding to photic input. Animals were exposed to a 5-h phase delay of the photocycle, and activity rhythms were monitored until a lack of reentrainment was confirmed. Hamsters were then housed in constant darkness for 24 h and administered a 30-min light pulse 2 circadian hours after activity onset. Brains were then removed, and tissue sections containing the SCN were processed for in situ hybridization. Sections were probed with Siberian hamster c-fos and per1 mRNA probes because light rapidly induces these 2 genes in the SCN during subjective night but not at other circadian phases. Light pulses induced robust expression of both genes in all animals that reentrained to the LD cycle, but no expression was observed in any animal that failed to reentrain. None of the animals exhibited an intermediate response. This finding is the first report of acute shift in a photocycle eliminating photosensitivity in the SCN and suggests that a specific pattern of light exposure may desensitize the SCN to subsequent photic input.     

Circadian phase shifting induced by clonidine injections in Syrian hamsters

Abstract

The mammalian circadian pacemaker can be phase shifted by photic, pharmacological, and behaviorally‐derived stimuli. The phase‐response curves (PRCs) characterizing these diverse stimuli may comprise two distinct families; a photic PRC typified by the response to brief light pulses, and a non‐photic PRC, typified by the response to dark pulses and to behavioral activation. The present study examined the phase shifting effects of acute systemic treatment with the alpha2‐adrenoceptor agonist, clonidine, in Syrian hamsters. Clonidine injections (0.25 mg/kg, ip) delivered during subjective night mimicked the phase shifting effects of light pulses in animals housed in both constant darkness (DD) and constant red light (RR), but similar effects were not seen in saline‐treated controls. Both clonidine and saline injections resulted in phase advances during subjective day, but only in RR‐housed animals. Clonidine‐induced phase shifting was dose‐dependent, but rather high doses were required to induce phase shifts. Pretreatment with the selective noradrenergic neurotoxin, DSP‐4, blocked clonidine‐induced phase shifting. These results suggest that clonidine acts at presynaptic alpha2‐adrenergic autoreceptors to disinhibit spontaneous and/or evoked activity in the photic entrainment pathway.