In Praise of Masking: Behavioural Responses of Retinally Degenerate Mice to Dim Light

There are two ways in which animals compartmentalize their activities into day or night: they can have an endogenous clock which is synchronized each day by a light-dark cycle or there can be a direct response to light such as the decrease in activity by a nocturnal rodent. In the first case activity is said to be entrained by light. In the second case activity is said to be masked by light. Any demonstration of entrainment by periodic presentation of a stimulus must show that activity occurring in phase with that stimulus is not simply a direct response to the stimulus but represents control of phase by an endogenous clock. Thus masking has come to be something to be avoided and excluded in experiments on circadian rhythms. This has led chronobiologists into displaying a lack of interest or a negative attitude toward masking, though there are some exceptions (e.g., refs. 1-5).     

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. (Author correspondence: shin.yamazaki@vanderbilt.edu)     

Electrical Stimulation of Inner Retinal Neurons in Wild-Type and Retinally Degenerate (rd/rd) Mice

Electrical stimulation of the retina following photoreceptor degeneration in diseases such as retinitis pigmentosa and age-related macular degeneration has become a promising therapeutic strategy for the restoration of vision. Many retinal neurons remain functional following photoreceptor degeneration; however, the responses of the different classes of cells to electrical stimuli have not been fully investigated. Using whole-cell patch clamp electrophysiology in retinal slices we investigated the response to electrical stimulation of cells of the inner nuclear layer (INL), pre-synaptic to retinal ganglion cells, in wild-type and retinally degenerate (rd/rd) mice. The responses of these cells to electrical stimulation were extremely varied, with both extrinsic and intrinsic evoked responses observed. Further examination of the intrinsically evoked responses revealed direct activation of both voltage-gated Na⁺ channels and K⁺ channels. The expression of these channels, which is particularly varied between INL cells, and the stimulus intensity, appears to dictate the polarity of the eventual response. Retinally degenerate animals showed similar responses to electrical stimulation of the retina to those of the wild-type, but the relative representation of each response type differed. The most striking difference between genotypes was the existence of a large amplitude oscillation in the majority of INL cells in rd/rd mice (as previously reported) that impacted on the signal to noise ratio following electrical stimulation. This confounding oscillation may significantly reduce the efficacy of electrical stimulation of the degenerate retina, and a greater understanding of its origin will potentially enable it to be dampened or eliminated.     

Dim Light at Night Does Not Disrupt Timing or Quality of Sleep in Mice

Artificial nighttime illumination has recently become commonplace throughout the world; however, in common with other animals, humans have not evolved in the ecological context of chronic light at night. With prevailing evidence linking the circadian, endocrine, immune, and metabolic systems, understanding these relationships is important to understanding the etiology and progression of several diseases. To eliminate the covariate of sleep disruption in light at night studies, researchers often use nocturnal animals. However, the assumption that light at night does not affect sleep in nocturnal animals remains unspecified. To test the effects of light at night on sleep, we maintained Swiss-Webster mice in standard light/dark (LD) or dim light at night (DLAN) conditions for 8–10 wks and then measured electroencephalogram (EEG) and electromyogram (EMG) biopotentials via wireless telemetry over the course of two consecutive days to determine differences in sleep timing and homeostasis. Results show no statistical differences in total percent time, number of episodes, maximum or average episode durations in wake, slow-wave sleep (SWS), or rapid eye movement (REM) sleep. No differences were evident in SWS delta power, an index of sleep drive, between groups. Mice kept in DLAN conditions showed a relative increase in REM sleep during the first few hours after the dark/light transition. Both groups displayed normal 24-h circadian rhythms as measured by voluntary running wheel activity. Groups did not differ in body mass, but a marked negative correlation of body mass with percent time spent awake and a positive correlation of body mass with time spent in SWS was evident. Elevated body mass was also associated with shorter maximum wake episode durations, indicating heavier animals had more trouble remaining in the wake vigilance state for extended periods of time. Body mass did not correlate with activity levels, nor did activity levels correlate with time spent in different sleep states. These data indicate that heavier animals tend to sleep more, potentially contributing to further weight gain. We conclude that chronic DLAN exposure does not significantly affect sleep timing or homeostasis in mice, supporting the use of dim light with nocturnal rodents in chronobiology research to eliminate the possible covariate of sleep disruption.     

Mice Lacking the p75NGFR Receptor Exhibit Abnormal Responses to Light

A role for nerve growth factor (NGF) in the mammalian circadian system was assessed in mice using a line of gene targeted animals carrying a null mutation at the low affinity p75 NGFR receptor locus. Receptor-deficient mice exhibited positive phase angles of entrainment to 24 h light-dark cycles. In constant dark, circadian rhythms of behavior were normal but phase shifts induced by brief pulses of light were significantly decreased. These results support the hypothesis that NGF, through an action mediated at least in part by p75 NGFR receptors in the SCN, is involved in the regulation of circadian responses to light.     

Mice Lacking the p75NGFR Receptor Exhibit Abnormal Responses to Light

A role for nerve growth factor (NGF) in the mammalian circadian system was assessed in mice using a line of gene targeted animals carrying a null mutation at the low affinity p75 NGFR receptor locus. Receptor-deficient mice exhibited positive phase angles of entrainment to 24 h light-dark cycles. In constant dark, circadian rhythms of behavior were normal but phase shifts induced by brief pulses of light were significantly decreased. These results support the hypothesis that NGF, through an action mediated at least in part by p75 NGFR receptors in the SCN, is involved in the regulation of circadian responses to light.     

Programming of Mice Circadian Photic Responses by Postnatal Light Environment

Early life programming has important consequences for future health and wellbeing. A key new aspect is the impact of perinatal light on the circadian system. Postnatal light environment will program circadian behavior, together with cell morphology and clock gene function within the suprachiasmatic nucleus (SCN) of the hypothalamus, the principal circadian clock in mammals. Nevertheless, it is still not clear whether the observed changes reflect a processing of an altered photic input from the retina, rather than an imprinting of the intrinsic molecular clock mechanisms. Here, we addressed the issue by systematically probing the mouse circadian system at various levels. Firstly, we used electroretinography, pupillometry and histology protocols to show that gross retinal function and morphology in the adult are largely independent of postnatal light experiences that modulate circadian photosensitivity. Secondly, we used circadian activity protocols to show that only the animal''s behavioral responses to chronic light exposure, but not to constant darkness or the acute responses to a light stimulus depend on postnatal light experience. Thirdly, we used real-time PER2::LUC rhythm recording to show long-term changes in clock gene expression in the SCN, but also heart, lung and spleen. The data showed that perinatal light mainly targets the long-term adaptive responses of the circadian clock to environmental light, rather than the retina or intrinsic clock mechanisms. Finally, we found long-term effects on circadian peripheral clocks, suggesting far-reaching consequences for the animal''s overall physiology.     

Fetal Behavioural Responses to Maternal Voice and Touch

Background

Although there is data on the spontaneous behavioural repertoire of the fetus, studies on their behavioural responses to external stimulation are scarce.

Aim, Methods

The aim of the current study was to measure fetal behavioural responses in reaction to maternal voice; to maternal touch of the abdomen compared to a control condition, utilizing 3D real-time (4D) sonography. Behavioural responses of 23 fetuses (21st to 33rd week of gestation; N = 10 in the 2nd and N = 13 in the 3rd trimester) were frame-by-frame coded and analyzed in the three conditions.

Results

Results showed that fetuses displayed more arm, head, and mouth movements when the mother touched her abdomen and decreased their arm and head movements to maternal voice. Fetuses in the 3rd trimester showed increased regulatory (yawning), resting (arms crossed) and self-touch (hands touching the body) responses to the stimuli when compared to fetuses in the 2nd trimester.

Conclusion

In summary, the results from this study suggest that fetuses selectively respond to external stimulation earlier than previously reported, fetuses actively regulated their behaviours as a response to the external stimulation, and that fetal maturation affected the emergence of these differential responses to the environment.     

Light Masking in the Field: An Experiment with Nocturnal and Diurnal Spiny Mice Under Semi-natural Field Conditions

Light masking has been studied almost exclusively in the laboratory. The authors populated four field enclosures with locally coexisting nocturnal Acomys cahirinus and diurnal A. russatus, and monitored their body temperatures (T_b) using implanted temperature-sensitive radio transmitters. A 3-h light pulse was initiated at the beginning of two consecutive nights; preceding nights were controls. A. cahirinus T_b and calculated activity levels decreased significantly during the light pulse, demonstrating a negative light masking response (light effect on T_b: ?0.32°C?±?0.15°C; average calculated activity records during the light pulse: 7?±?1.53, control: 9.8?±?1.62). Diurnal A. russatus did not respond to the light pulse. We conclude that light masking is not an artifact of laboratory conditions but represents a natural adaptive response in free-living populations. (Author correspondence: Shayroti@post.tau.ac.il)     

Heterotrophic Growth of Blue-Green Algae in Dim Light

A unicellular blue-green alga, Agmenellum quadruplicatum, and a filamentous blue-green alga, Lyngbya lagerheimíi, were grown heterotrophically in dim light with glucose as major source of carbon and possibly energy. The dim-light conditions did not support autotrophic growth. The two blue-green algae appeared to have the same metabolic block, namely an incomplete tricarboxylic acid cycle, as has been found in other obligately phototrophic blue-green algae. Under dim-light conditions, glucose made a greater contribution to cell constituents (amino acids) of A. quadruplicatum and L. lagerheimii than under high-light conditions.