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.     

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.     

Contrast adaptation and contrast masking in human vision.

After a preliminary study of visual evoked potentials (VEPS) to a test grating seen in the presence of masks at different orientations, psychophysical data are presented showing the effects of adaptation and of masking on thresholds for detecting the same test grating. The test is a vertical grating of spatial frequency 2 cycles per degree; adapting and masking gratings differ from the test either in orientation or in spatial frequency. The effects of adaptation and masking are explained by a single mechanism model that assumes: (i) adaptation and masking both alter the contrast response (or transducer) function of the mechanism that detects the test; (ii) masks, but not adaptors, stimulate the mechanism that detects the test; and (iii) a test is detectable when it raises response level by a constant amount. The model incorporates two distinct tuning functions, a broad adaptive contrast function and a narrow effective contrast function. It accounts adequately for all the data, including the location and size of the facilitative dip found in some masking functions, the constant slopes of the threshold elevation segments of adaptation functions and the varying slopes of masking functions. It also predicts the sometimes surprising joint effects of adaptation followed by masking and of two masks operating simultaneously.     

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.     

Psychophysical evidence of damaged active processing mechanisms in Belgian Waterslager Canaries

Belgian Waterslager canaries (BWC), bred for a distinct low-pitched song, have an inherited high-frequency hearing loss associated with hair cell abnormalities. Hair cells near the abneural edge of the papilla, which receive primarily efferent innervation in normal birds, are among the most severely affected. These cells are thought to support nonlinear active processing in the avian ear, though the mechanisms are poorly understood. Here we present psychophysical evidence that suggests degraded active processing in BWC compared to normal-hearing non-BWC. Critical ratios, psychophysical masking patterns and phase effects on masking by harmonic complexes were measured in BWC and non-BWC using operant conditioning procedures. Critical ratios were much larger in BWC than in non-BWC at high frequencies. Psychophysical tuning curves derived from the masking patterns for BWC were broadened at high frequencies. BWC also showed severely reduced phase effects on masking by harmonic complexes compared to non-BWC. As has been hypothesized previously for hearing-impaired humans, these results are consistent with a loss of active processing mechanisms in BWC.     

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.     

Masking release for gap detection.

In random noise, masking is influenced almost entirely by noise components in a narrow band around the signal frequency. However, when the noise is not random, but has a modulation pattern which is coherent across frequency, noise components relatively remote from the signal frequency can actually produce a release from masking. This masking release has been called comodulation masking release (CMR). The present research investigated whether a similar release from masking occurs in the analysis of a suprathreshold signal. Specifically, the ability to detect the presence of a temporal gap was investigated in conditions which do and do not result in CMR for detection threshold. Similar conditions were investigated for the masking level difference (a binaural masking release phenomenon). The results indicated that suprathreshold masking release for gap detection occurred for both the masking-level difference (MLD) and for CMR. However, masking release for gap detection was generally smaller than that obtained for detection threshold. The largest gap detection masking release effects obtained corresponded to relatively low levels of stimulation, where gap detection was relatively poor.     

Lesions of the Intergeniculate Leaflet Lead to a Reorganization in Circadian Regulation and a Reversal in Masking Responses to Photic Stimuli in the Nile Grass Rat

Light influences the daily patterning of behavior by entraining circadian rhythms and through its acute effects on activity levels (masking). Mechanisms of entrainment are quite similar across species, but masking can be very different. Specifically, in diurnal species, light generally increases locomotor activity (positive masking), and in nocturnal ones, it generally suppresses it (negative masking). The intergeniculate leaflet (IGL), a subdivision of the lateral geniculate complex, receives direct retinal input and is reciprocally connected with the primary circadian clock, the suprachiasmatic nucleus (SCN). Here, we evaluated the influence of the IGL on masking and the circadian system in a diurnal rodent, the Nile grass rat (Arvicanthis niloticus), by determining the effects of bilateral IGL lesions on general activity under different lighting conditions. To examine masking responses, light or dark pulses were delivered in the dark or light phase, respectively. Light pulses at Zeitgeber time (ZT) 14 increased activity in control animals but decreased it in animals with IGL lesions. Dark pulses had no effect on controls, but significantly increased activity in lesioned animals at ZT0. Lesions also significantly increased activity, primarily during the dark phase of a 12:12 light/dark cycle, and during the subjective night when animals were kept in constant conditions. Taken together, our results suggest that the IGL plays a vital role in the maintenance of both the species-typical masking responses to light, and the circadian contribution to diurnality in grass rats.     

Recognition of forward-masked complex and simple images

The accuracy and time of motor response in tests for visual recognition of simple (sets of horizontal and vertical lines, angles, and crosses) and complex (contour images of animate and inanimate objects) images forward-masked with stimuli of varying complexity were estimated in psychophysiological experiments. It was found that forward masking impaired recognition, the mask whose complexity was close to that of the given test image being the most efficient in each particular case. The masking effect was the strongest in the experiments where the sets of test and masking images coincided. Recognition of complex images proved to be more resistant to masking than recognition of simple images. The observed effects of forward masking are assumed to be determined by lateral inhibition and changes in the excitability cycle. The relative contributions of these mechanisms depend on the degree of similarity between the test and masking stimuli, as well as the hierarchical level of the visual cortex at which the test stimulus is “targeted.”     

Binaural masking and sensitivity to interaural delay in the inferior colliculus.

The binaural masking level difference (BMLD) is a psychophysical effect whereby signals masked by a noise at one ear become unmasked by sounds reaching the other. BMLD effects are largest at low frequencies where they depend on signal phase, suggesting that part of the physiological mechanism responsible for the BMLD resides in cells that are sensitive to interaural time disparities. We have investigated a physiological basis for unmasking in the responses of delay-sensitive cells in the central nucleus of the inferior colliculus in anaesthetized guinea pigs. The masking effects of a binaurally presented noise, as a function of the masker delay, were quantified by measuring the number of discharges synchronized to the signal, and by measuring the masked threshold. The noise level for masking was lowest at the best delay for the noise. The mean magnitude of the unmasking across our neural population was similar to the human psychophysical BMLD under the same signal and masker conditions.     

PACEMAKER PHASE CONTROL VERSUS MASKING BY LIGHT: SETTING THE CIRCADIAN CHRONOTYPE IN DUAL OCTODON DEGUS

There are two main processes involved in the expression of circadian rhythmicity: entrainment and masking. Whereas the first operates via the central pacemaker to anticipate predictable environmental conditions, masking (mainly induced by light) functions as a direct modulator of the circadian output signal induced by nonpredictable events. The Chilean rodent Octodon degus presents both diurnal and nocturnal chronotypes when given free access to an exercise wheel. Two steady-entrainment phases and graded masking by light seem to generate the wide variability of chronotypes in this species. The aim of this study was to characterize the differential masking by light according to the individual chronotypes, their stability over time, and the influence of wheel running availability and ambient temperature upon the degus' nocturnality. To this end, diurnal and nocturnal degus were subjected to ultradian cycles (1:1-h light-dark [LD]), with and without wheel running availability, and under both normal and high diurnal ambient temperature cycles. The present results show that diurnal and nocturnal degus present a stable masking by light, each according to its respective chronotype. Thus, whereas diurnal animals increased their activity with light, in nocturnal degus light induced a sharp drop in wheel running activity. These two types of masking responses appeared not only when the animals were synchronized to the 12:12-h LD cycle, but also under ultradian cycles. Different masking effects persisted when wheel running was made unavailable and when the animals shifted their circadian activity patterns in response to ultradian cycles or to diurnal exposure to high temperatures. In conclusion, our results show that the positive and negative masking effects of light on diurnal and nocturnal degus, respectively, seem to occur independently of relative phase control by the central pacemaker or the negative masking induced by high environmental temperatures. (Author correspondence: angerol@um.es)     

Regression Models for the Estimation of Orcadian Rhythms in the Presence of Effects Due to Masking

The estimation of human circadian rhythms from experimental data is complicated by the presence of “masking” effects associated with the sleep-wake cycle. The observed rhythm may include a component due to masking, as well as the endogenous component linked to a circadian pacemaker. In situations where the relationship between the sleep-wake cycle and the circadian rhythm is not constant, it may be possible to obtain individual estimates of these two components, but methods commonly used for the estimation of circadian rhythms, such as the cosinor analysis, spectral analysis, average waveforms and complex demodulation, have not generally been adapted to identify the modulations that arise from masking. The estimates relate to the observed rhythms, and the amplitudes and acrophases do not necessarily refer to the endogenous rhythm.

In this paper methods are discussed for the separation of circadian and masking effects using regression models that incorporate a sinusoidal circadian variation together with functions of time since sleep and time during sleep. The basic model can be extended to include a time-varying circadian rhythm and estimates are available for the amplitude and phase at a given time, together with their joint confidence intervals and tests for changes in amplitude and acrophase between any two selected times. Modifications of these procedures are discussed to allow for non-sinusoidal circadian rhythms, non-additivity of the circadian and time-since-sleep effects and the breakdown of the usual assumptions concerning the residual errors.

This approach enables systematic masking effects associated with the sleep-wake cycle to be separated from the circadian rhythm, and it has applications to the analysis of data from experiments where the sleep-wake cycle is not synchronized with the circadian rhythm, for example after time-zone transitions or during irregular schedules of work and rest.     

Ultradian Rhythms in Arousal-The Problem of Masking

Studies utilizing widely different experimental techniques provided evidence that there are spontaneous ultradian cycles in arousal during the waking state. These comprised of cyclic fluctuations between increased and decreased sleep propensity with a periodicity of about 1.5 hr. Being of relatively low amplitude, these cycles are vulnerable to masking effects by a variety of experimental conditions. Masking can be exerted by varying the tonic level of arousal, by coexisting slow ultradian components which are particularly prominent during the second half of the day, or by some specific experimental conditions. Furthermore, increased sleepiness was shown to enhance the slow ultradian components and suppress the 1.5-hr cycles in EEG indices of arousal on the one hand, and to emphasize the 1.5-hr cycles in motor activity and reaction time performance on the other hand. Much more attention should be paid to the problem of masking of ultradian cycles in arousal. Recognizing the sources and reasons for masking will ad vance our knowledge of the characteristics of these cycles and their function.     

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.     

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.     

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.     

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.     

The development of new purification methods to assess the circadian rhythm of body temperature in Mongolian gerbils

Six Mongolian gerbils were studied for 8-10d while housed in separate cages in a 12:12h light-dark (L-D) cycle (lights on at 07:00h). Recordings of body temperature, heart rate, and spontaneous activity were made throughout. The temperature and heart rate rhythms were “purified” to take into account the effects of activity, and then the rhythm of temperature was further purified to take into account other masking influences (“non-activity masking effects” or NAME,). The methods employed in the purification processes involved linear regression analysis or analysis of covariance, the latter using functions of activity and NAME as covariates. From these methods, it was possible to obtain not only an estimate of the endogenous component of the temperature rhythm but also a measure of circadian changes in the sensitivity of temperature to masking effects.

Even though all purification methods removed many of the effects of spontaneous activity from the temperature record, there remained temperature fluctuations at the L-D and D-L transitions that appeared to be independent of activity. The NAME was of only very marginal value in the purification process. Comparison of the purification methods indicated that the linear methods were inferior (both from a biological viewpoint and when the results were compared mathematically) to those that allowed the rate of rise of temperature due to increasing amounts of activity to become progressively less. The sensitivity of temperature and heart rate to the masking effects of activity showed a circadian rhythm, with sensitivities in the resting phase being greater than those in the active phase. These findings are compatible with the view that thermoregulatory reflexes are induced by spontaneous activity of sufficient amount, and that there is a circadian rhythm in the body temperature at which these reflexes are initiated and in their effectiveness.     

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.     

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.