Circadian Rhythms and Masking: An Overview

Masking, as is well known, enables an organism to act immediately and in an appropriate way to changes of the environment, integrating with internally produced rhythmicity. It now appears that masking can be used to cover a far wider range of problems than was originally intended. To separate masking effects from the effects due to an internal oscillator, several techniques have been used. Such protocols, however, like the constant routine protocol, often replace one form of masking by another. The situation becomes even more complex when one realizes that the output of an internal oscillator modifies the input. The question might be asked whether it is possible to study the properties of the internal oscillator in vivo at all. This article attempts to produce a framework for future discussions.     

Masking in Humans: The Problem and Some Attempts to Solve IT

Different types of masking are discussed together with an account of the masking effect that the sleep-wake cycle exerts upon the circadian rhythms of body temperature and urinary excretion. The relative importance to masking of the several components of differences between sleeping and wakefulness are then assessed.

Means to deal with the problem of masking fall into two major categories. These attempt to minimise masking effects by protocols such as constant routines or control days, and mathematical models which separate results obtained in the presence of masking influences into endogenous and exogenous components. (The problem of the extent to which masking influences can render the endogenous component of a rhythm an impure reflection of the internal oscillator is considered also.) These different techniques are compared with respect to their usefulness and assumptions.

Finally, a brief speculation is given of the usefulness of masking.     

The Expression of Locomotor Orcadian Rhythm in Female German Cockroach, Blattella Germanica (L.)

Fifteen percent of intact female German cockroaches (n = 13), Blattella germanica (L.) (Dictyoptera: Blattellidae), had weak free-running locomotor rhythmicity under 28°C and constant darkness conditions. However, 86% of ovariectomized females (n = 14) showed a strong free-running rhythm under the same conditions with a circadian period of 23.60 ± 0.15 h, similar to the male's period of 23.45±0.03 h. In addition, the locomotory activities occurred mainly during the subjective night under DD conditions as was the case in males. These results indicated that female locomotion was under the control of a circadian oscillator, which was masked by the existence of ovaries. This internal masking effect could be removed by the existence of males, but females had no effect on the locomotor pattern of another female. Since the male failed to entrain female locomotion, its role as a zeitgeber was excluded. That the locomotory pattern of the females still coincided with their reproductive cycle when exposed to male odor suggests that exposure to a male only partially removed the internal masking effects.     

Alternating Reducing and Enhancing Effect of Pregnancy on Breast Cancer Risk May Reveal Periodic Phenomena which Mask Circannual Rhythms

Although a relationship between reproductive factors and breast cancer risk has been known for long, there are many contradictions between different studies, and a connection with a biological mechanism has not yet been found. Recently, new details have been revealed about different effects of pregnancy on breast cancer risk. During pregnancy breast cancer risk is strongly reduced, while inversely,some years after giving birth the risk is increased. Furthermore, risk is strongly reduced in women whose consecutive births of four or more children occur after short intervals and are terminated before the age of 30. Recently, we have postulated rhythmicity in the occurrence of breast cancer in a subgroup of young women. By analogy with circadian rhythms it may be assumed that circannual rhythms too are influenced by synchronizers and masking effects. In circadian rhythms masking may be by external or internal periodic factors, such as the sleep/wake cycle, meal timing and activity. Over the year pregnancy may be such a periodic factor, with alternation of an empty uterus and one wich gradually enlarges as a consequence of a developing fetus. A masking factor is active only as long as present. Therefore the circannual rhythmicity of breast cancer is attenuated or nullified only during pregnancy. After giving birth, the temporal deficit of the rhythmic increase of tumor growth may be restored, leading to a rebound effect expressed as an increased clinical detection of malignancy some years later. When several consecutive pregnancies occur after short intervals the masking effect continues uninterruptedly, accompanied by a strong reduced breast cancer risk. The same biological mechanism may underly the remission during pregnancy in autoimmune disorders such as rheumatoid arthritis and Graves' disease. The immune system, crucial in autoimmune disorders, is build up of several constituents which are clearly rhythmic, e.g the lymphocytes. Masking of one or more of these rhythmicities may lead to disease attenuation, followed by relapses as soon as the masking factor has been eliminated.     

Alternating Reducing and Enhancing Effect of Pregnancy on Breast Cancer Risk May Reveal Periodic Phenomena which Mask Circannual Rhythms

Although a relationship between reproductive factors and breast cancer risk has been known for long, there are many contradictions between different studies, and a connection with a biological mechanism has not yet been found. Recently, new details have been revealed about different effects of pregnancy on breast cancer risk. During pregnancy breast cancer risk is strongly reduced, while inversely,some years after giving birth the risk is increased. Furthermore, risk is strongly reduced in women whose consecutive births of four or more children occur after short intervals and are terminated before the age of 30. Recently, we have postulated rhythmicity in the occurrence of breast cancer in a subgroup of young women. By analogy with circadian rhythms it may be assumed that circannual rhythms too are influenced by synchronizers and masking effects. In circadian rhythms masking may be by external or internal periodic factors, such as the sleep/wake cycle, meal timing and activity. Over the year pregnancy may be such a periodic factor, with alternation of an empty uterus and one wich gradually enlarges as a consequence of a developing fetus. A masking factor is active only as long as present. Therefore the circannual rhythmicity of breast cancer is attenuated or nullified only during pregnancy. After giving birth, the temporal deficit of the rhythmic increase of tumor growth may be restored, leading to a rebound effect expressed as an increased clinical detection of malignancy some years later. When several consecutive pregnancies occur after short intervals the masking effect continues uninterruptedly, accompanied by a strong reduced breast cancer risk. The same biological mechanism may underly the remission during pregnancy in autoimmune disorders such as rheumatoid arthritis and Graves’ disease. The immune system, crucial in autoimmune disorders, is build up of several constituents which are clearly rhythmic, e.g the lymphocytes. Masking of one or more of these rhythmicities may lead to disease attenuation, followed by relapses as soon as the masking factor has been eliminated.     

Masking responses to light in period mutant mice

Masking is an acute effect of an external signal on an overt rhythm and is distinct from the process of entrainment. In the current study, we investigated the phase dependence and molecular mechanisms regulating masking effects of light pulses on spontaneous locomotor activity in mice. The circadian genes, Period1 (Per1) and Per2, are necessary components of the timekeeping machinery and entrainment by light appears to involve the induction of the expression of Per1 and Per2 mRNAs in the suprachiasmatic nuclei (SCN). We assessed the roles of the Per genes in regulating masking by assessing the effects of light pulses on nocturnal locomotor activity in C57BL/6J Per mutant mice. We found that Per1(-/-) and Per2(-/-) mice had robust negative masking responses to light. In addition, the locomotor activity of Per1(-/-)/Per2(-/-) mice appeared to be rhythmic in the light-dark (LD) cycle, and the phase of activity onset was advanced (but varied among individual mice) relative to lights off. This rhythm persisted for 1 to 2 days in constant darkness in some Per1(-/-)/Per2(-/-) mice. Furthermore, Per1(-/-)/Per2(-/-) mice exhibited robust negative masking responses to light. Negative masking was phase dependent in wild-type mice such that maximal suppression was induced by light pulses at zeitgeber time 14 (ZT14) and gradually weaker suppression occurred during light pulses at ZT16 and ZT18. By measuring the phase shifts induced by the masking protocol (light pulses were administered to mice maintained in the LD cycle), we found that the phase responsiveness of Per mutant mice was altered compared to wild-types. Together, our data suggest that negative masking responses to light are robust in Per mutant mice and that the Per1(-/-)/Per2(-/-) SCN may be a light-driven, weak/damping oscillator.     

The use of Constant Routines in Unmasking the Endogenous Component of Human Circadian Rhythms

A major problem in the study of the internal clock(s) that drives human circadian rhythms is that due to the effect produced by rhythmicity of habits and external influences (‘masking’). A particularly potent factor in this respect is the sleep-wake cycle. It is anomalous that, even though this masking influence is widely accepted, most studies of circadian rhythmicity have been performed in the presence of such interferences.

A protocol is described, the constant routine, by which these exogenous influences can be minimized, thereby enabling a closer scrutiny of the internal clock(s) to be made. An account is given of the different circumstances in which the constant routines have been used together with the results derived from such studies. Briefly, they indicate that nychthemeral studies can give misleading information about the rate of adjustment of the internal clock to various manipulations, e.g. time-zone transition, shift work.

In addition, future studies making use of constant routines are described, in particular those which might enable the presence of more than one internal clock to be established.     

Critical Interval Hypotheses for Depression

Two chronobiologic models are presented for the etiology of depression. The internal coincidence model suggests that a phase advance of the strong oscillator in reference to the weak oscillator causes depression. An external coincidence model suggests that depression is caused when the light/dark cycle or photoperiod provides too little illumination during a critical photosensitive interval, which might in turn occur early due to a phase advance. Thus, depression might be treated by drugs or other manipulations which delay the phase of internal circadian rhythms. Depression might also be treated with bright illumination during the critical photosensitive interval. Preliminary experiments suggest that bright artificial light does have antidepressant effects. The optimal times for light exposure and the most responsive patient groups have not yet been identified.     

What Do Contrast Threshold Equivalent Noise Studies Actually Measure? Noise vs. Nonlinearity in Different Masking Paradigms

The internal noise present in a linear system can be quantified by the equivalent noise method. By measuring the effect that applying external noise to the system’s input has on its output one can estimate the variance of this internal noise. By applying this simple “linear amplifier” model to the human visual system, one can entirely explain an observer’s detection performance by a combination of the internal noise variance and their efficiency relative to an ideal observer. Studies using this method rely on two crucial factors: firstly that the external noise in their stimuli behaves like the visual system’s internal noise in the dimension of interest, and secondly that the assumptions underlying their model are correct (e.g. linearity). Here we explore the effects of these two factors while applying the equivalent noise method to investigate the contrast sensitivity function (CSF). We compare the results at 0.5 and 6 c/deg from the equivalent noise method against those we would expect based on pedestal masking data collected from the same observers. We find that the loss of sensitivity with increasing spatial frequency results from changes in the saturation constant of the gain control nonlinearity, and that this only masquerades as a change in internal noise under the equivalent noise method. Part of the effect we find can be attributed to the optical transfer function of the eye. The remainder can be explained by either changes in effective input gain, divisive suppression, or a combination of the two. Given these effects the efficiency of our observers approaches the ideal level. We show the importance of considering these factors in equivalent noise studies.     

Effect of harmonicity on the detection of a signal in a complex masker and on spatial release from masking

The amount of masking of sounds from one source (signals) by sounds from a competing source (maskers) heavily depends on the sound characteristics of the masker and the signal and on their relative spatial location. Numerous studies investigated the ability to detect a signal in a speech or a noise masker or the effect of spatial separation of signal and masker on the amount of masking, but there is a lack of studies investigating the combined effects of many cues on the masking as is typical for natural listening situations. The current study using free-field listening systematically evaluates the combined effects of harmonicity and inharmonicity cues in multi-tone maskers and cues resulting from spatial separation of target signal and masker on the detection of a pure tone in a multi-tone or a noise masker. A linear binaural processing model was implemented to predict the masked thresholds in order to estimate whether the observed thresholds can be accounted for by energetic masking in the auditory periphery or whether other effects are involved. Thresholds were determined for combinations of two target frequencies (1 and 8 kHz), two spatial configurations (masker and target either co-located or spatially separated by 90 degrees azimuth), and five different masker types (four complex multi-tone stimuli, one noise masker). A spatial separation of target and masker resulted in a release from masking for all masker types. The amount of masking significantly depended on the masker type and frequency range. The various harmonic and inharmonic relations between target and masker or between components of the masker resulted in a complex pattern of increased or decreased masked thresholds in comparison to the predicted energetic masking. The results indicate that harmonicity cues affect the detectability of a tonal target in a complex masker.     

The use of Constant Routines in Unmasking the Endogenous Component of Human Circadian Rhythms

A major problem in the study of the internal clock(s) that drives human circadian rhythms is that due to the effect produced by rhythmicity of habits and external influences ('masking'). A particularly potent factor in this respect is the sleep-wake cycle. It is anomalous that, even though this masking influence is widely accepted, most studies of circadian rhythmicity have been performed in the presence of such interferences.

A protocol is described, the constant routine, by which these exogenous influences can be minimized, thereby enabling a closer scrutiny of the internal clock(s) to be made. An account is given of the different circumstances in which the constant routines have been used together with the results derived from such studies. Briefly, they indicate that nychthemeral studies can give misleading information about the rate of adjustment of the internal clock to various manipulations, e.g. time-zone transition, shift work.

In addition, future studies making use of constant routines are described, in particular those which might enable the presence of more than one internal clock to be established.     

Contralateral Masking in Bilateral Cochlear Implant Patients: A Model of Medial Olivocochlear Function Loss

Contralateral masking is the phenomenon where a masker presented to one ear affects the ability to detect a signal in the opposite ear. For normal hearing listeners, contralateral masking results in masking patterns that are both sharper and dramatically smaller in magnitude than ipsilateral masking. The goal of this study was to investigate whether medial olivocochlear (MOC) efferents are needed for the sharpness and relatively small magnitude of the contralateral masking function. To do this, bilateral cochlear implant patients were tested because, by directly stimulating the auditory nerve, cochlear implants circumvent the effects of the MOC efferents. The results indicated that, as with normal hearing listeners, the contralateral masking function was sharper than the ipsilateral masking function. However, although there was a reduction in the magnitude of the contralateral masking function compared to the ipsilateral masking function, it was relatively modest. This is in sharp contrast to the results of normal hearing listeners where the magnitude of the contralateral masking function is greatly reduced. These results suggest that MOC function may not play a large role in the sharpness of the contralateral masking function but may play a considerable role in the magnitude of the contralateral masking function.     

Factors Limiting Performance in a Multitone Intensity-Discrimination Task: Disentangling Non-Optimal Decision Weights and Increased Internal Noise

To identify factors limiting performance in multitone intensity discrimination, we presented sequences of five pure tones alternating in level between loud (85 dB SPL) and soft (30, 55, or 80 dB SPL). In the “overall-intensity task”, listeners detected a level increment on all of the five tones. In the “masking task”, the level increment was imposed only on the soft tones, rendering the soft tones targets and loud tones task-irrelevant maskers. Decision weights quantifying the importance of the five tone levels for the decision were estimated using methods of molecular psychophysics. Compatible with previous studies, listeners placed higher weights on the loud tones than on the soft tones in the overall-intensity condition. In the masking task, the decisions were systematically influenced by the to-be-ignored loud tones (maskers). Using a maximum-likelihood technique, we estimated the internal noise variance and tested whether the internal noise was higher in the alternating-level five-tone sequences than in sequences presenting only the soft or only the loud tones. For the overall-intensity task, we found no evidence for increased internal noise, but listeners applied suboptimal decision weights. These results are compatible with the hypothesis that the presence of the loud tones does not impair the precision of the representation of the intensity of the soft tones available at the decision stage, but that this information is not used in an optimal fashion due to a difficulty in attending to the soft tones. For the masking task, in some cases our data indicated an increase in internal noise. Additionally, listeners applied suboptimal decision weights. The maximum-likelihood analyses we developed should also be useful for other tasks or other sensory modalities.     

The choice of a masking agent in the histochemical staining of metals

Summary The masking effects of standard masking agents (aminopolycarboxylic acids, carboxylic acids and phosphates) have been investigated in both test-tube experiments and tissue sections in order to ascertain the factors which must be considered when choosing a masking agent for the histochemical staining of a metal. The masking effectsin vitro were determined by spectrophotometry through the complexing of the dye Chrome Azurol S with aluminium, beryllium, and iron at pH 5 and 7. The effects were also examined by staining metal-containing tissue sections in a Chrome Azurol S masking agent system at the same pH values. In many cases, the masking effects observed in sections did not agree with those obtained in the test-tube experiments. This means that the published values of stability constants are not a sufficient guide for choosing a suitable masking agent for the staining of metals. The discrepancy is mainly attributable to the presence of protein in a solid state when metals are stained in sections. Therefore, in the future, consideration should be given to a metal-protein or masking agent-protein interaction using a model compound such as a chelate resin. The polyphosphates are among the most useful masking agents for metal staining in acidic solutions from a practical standpoint.     

Proteins found in a CikA interaction assay link the circadian clock, metabolism, and cell division in Synechococcus elongatus

Diverse organisms time their cellular activities to occur at distinct phases of Earth's solar day, not through the direct regulation of these processes by light and darkness but rather through the use of an internal biological (circadian) clock that is synchronized with the external cycle. Input pathways serve as mechanisms to transduce external cues to a circadian oscillator to maintain synchrony between this internal oscillation and the environment. The circadian input pathway in the cyanobacterium Synechococcus elongatus PCC 7942 requires the kinase CikA. A cikA null mutant exhibits a short circadian period, the inability to reset its clock in response to pulses of darkness, and a defect in cell division. Although CikA is copurified with the Kai proteins that constitute the circadian central oscillator, no direct interaction between CikA and either KaiA, KaiB, or KaiC has been demonstrated. Here, we identify four proteins that may help connect CikA with the oscillator. Phenotypic analyses of null and overexpression alleles demonstrate that these proteins are involved in at least one of the functions--circadian period regulation, phase resetting, and cell division--attributed to CikA. Predictions based on sequence similarity suggest that these proteins function through protein phosphorylation, iron-sulfur cluster biosynthesis, and redox regulation. Collectively, these results suggest a model for circadian input that incorporates proteins that link the circadian clock, metabolism, and cell division.     

Acoustic signal perception in a noisy habitat: lessons from synchronising insects

Acoustically communicating animals often have to cope with ambient noise that has the potential to interfere with the perception of conspecific signals. Here we use the synchronous display of mating signals in males of the tropical katydid Mecopoda elongata in order to assess the influence of nocturnal rainforest noise on signal perception. Loud background noise may disturb chorus synchrony either by masking the signals of males or by interaction of noisy events with the song oscillator. Phase-locked synchrony of males was studied under various signal-to-noise ratios (SNRs) using either native noise or the audio component of noise (<9 kHz). Synchronous entrainment was lost at a SNR of -3 dB when native noise was used, whereas with the audio component still 50% of chirp periods matched the pacer period at a SNR of -7 dB. Since the chirp period of solo singing males remained almost unaffected by noise, our results suggest that masking interference limits chorus synchrony by rendering conspecific signals ambiguous. Further, entrainment with periodic artificial signals indicates that synchrony is achieved by ignoring heterospecific signals and attending to a conspecific signal period. Additionally, the encoding of conspecific chirps was studied in an auditory neuron under the same background noise regimes.     

Diurnality and nocturnality in nonhuman primates: comparative chronobiological studies in laboratory and nature

Looking for differences in circadian clock characteristics of diurnal and nocturnal nonhuman primates, this article summarizes results of chronobiological studies carried out in various nocturnal, diurnal, and cathemeral prosimian and anthropoid primate species under controlled laboratory conditions, under seminatural conditions, and in the wild. In almost all circadian parameters investigated, no differences were discernible between the two main chrono-ecotypes, either in circadian period length and the influence upon it of after-effects, of light intensity, and ambient temperature, or in the PRC, re-entrainment behavior, rhythm splitting, and internal desynchronization. Diurnal and nocturnal or cathemeral species differed only in the phase of artificial or natural LDs to which their circadian activity phase was adjusted as well as in the characteristics of masking activity upon the rhythms produced by the direct inhibiting or enhancing effects of light. Pronounced lunar periodicity—observed in the activity rhythm of nocturnal neotropical owl monkeys, genus Aotus, in seminatural and natural environments as well as in wild cathemeral Malagasy lemurs, genus Eulemur—is shown to result from masking effects of moonlight. In captive Eulemur fulvus albifrons, a change from dark-active over cathemeral to light-active behavior, without concurrently changing the circadian phase-setting of activity to D, was produced by direct masking effects of a stepwise reduction of darktime luminosity on an LD 12:12 cycle. Long-term activity recordings carried out in wild diurnal Malagasy sifakas (Propithecus verreauxi) and cathemeral redfronted lemurs (Eulemur fulvus rufus), as well as in wild nocturnal owl monkeys (Aotus a. azarai) of the North Argentinean Chaco, yielded in all species distinct bimodal long- and short-day activity patterns with pronounced peaks during dusk and dawn. Applying Pittendrigh's two-oscillator concept to these results, it is hypothesized that the differences in chrono-ecotype behavior may result from variations in internal coupling and external phase-setting of morning and evening oscillators (m, e) to dawn and dusk, interacting with direct masking effects of light.     

Detection of a Target Taste in a Complex Masker

Detection thresholds for sodium chloride were compared in aqueoussolution, in mixture with a sucrose masker, in mixture witha citric acid masker, and in mixture with both of these maskerstogether. Separately the two maskers raised the threshold ofsodium chloride by three to four times, and together by overnine times, a result consistent with independence (additivity)of the two masking effects. To achieve comparable masking witheither sucrose alone or with citric acid alone would requireincreasing their masking concentrations by about ten times.Hence multiple masking can be a far more efficient means ofconcealing a taste, whether an unpleasant one (e.g. the bittertaste of medicine) or a pleasant one (e.g. a salty or sweetcondiment). Multiple masking has dietary and culinary significance,especially for middle aged and elderly persons concerned aboutsalt intake, because their thresholds for NaCl, whether withor without maskers, are typically two or three times higherthan those of youthful persons. Chem. Senses 22: 529–534,1997.     

Internal desynchronization of the circadian activity and feeding rhythm in an owl monkey (Aotus lemurinus griseimembra): a case study

In the free-running circadian locomotor activity rhythm of a 7-year-old male owl monkey (Aotus lemurinus griseimembra) kept under constant light and climatic conditions (LL 0.2 lux, 25°C ± 1°C, 60 ± 5% relative humidity [RH]), a second rhythm component developed that showed strong relative coordination with the free-running activity rhythm of 24.4h and a 24h rhythm. The simultaneously recorded feeding activity rhythm strongly resembled this rhythm component. Therefore, it seems justified to infer that there was an internal desynchronization between the two behavioral rhythms or their circadian pacemakers, that is, between the light-entrainable oscillator located in the suprachiasmatic nuclei (SCN) and a food-entrainable oscillator located outside the SCN. This internal desynchronization may have been induced and/or maintained by a zeitgeber effect of the (irregular) 24h feeding schedule on the food-entrainable oscillator. The weak relative coordination shown by the activity rhythm indicates a much weaker coupling of the light-entrainable oscillator to the food-entrainable oscillator than vice versa. (Chronobiology International, 17(2), 147-153, 2000)