Matthew P. Butler

Huddling, locomotor, and nest-building behaviors of furred and furless Siberian hamsters

Rodents living in the cold employ both behavioral and physiological mechanisms to achieve thermoregulation. We examined the impact of fur loss on behavioral thermoregulation in cold-challenged Siberian hamsters (Phodopus sungorus). Intact female hamsters exposed to an ambient temperature (T(a)) of 5 degrees C increased their general locomotor activity by 50% relative to animals maintained at 23 degrees C. At both T(a)s, fur removal resulted in substantial increases in daily food intake (37% and 22% at 5 and 23 degrees C, respectively) but did not affect the amount of locomotor activity; increased food intake after fur loss evidently is not caused by increases in locomotor activity. Furred hamsters housed in groups of three at 5 degrees C consumed 16% less food per day than did singly housed individuals. Fur removal resulted in identical 39% increases in food intake in group- or singly housed animals. Energy savings that accrued from huddling were identical in furred and furless animals; this behavior conserves energy even in the absence of an insulative pelage. The availability of nesting material resulted in an 18% reduction in food consumption in intact animals kept at 5 degrees C. The increase in food intake produced by fur removal was attenuated by approximately 80% when furless animals had access to nesting material. Huddling and nest-building behaviors each ameliorate energetic challenges posed by absence of fur; animals that concurrently employ several modes of thermoregulation realize substantial energy savings in the cold.

Androgens Modulate Structure and Function of the Suprachiasmatic Nucleus Brain Clock

Gonadal hormones can modulate circadian rhythms in rodents and humans, and androgen receptors are highly localized within the core region of the mouse suprachiasmatic nucleus (SCN) brain clock. Although androgens are known to modulate neural plasticity in other CNS compartments, the role of androgens and their receptors on plasticity in the SCN is unexplored. In the present study, we ask whether androgens influence the structure and function of the mouse SCN by examining the effects of gonadectomy (GDX) on the structure of the SCN circuit and its responses to light, including induction of clock genes and behavioral phase shifting. We found that after GDX, glial fibrillary acidic protein increased with concomitant decreases in the expression of the synaptic proteins synaptophysin and postsynaptic density 95. We also found that GDX exerts effects on the molecular and behavioral responses to light that are phase dependent. In late night [circadian time (CT)21], GDX increased light-induced mPer1 but not mPer2 expression compared with intact (INT) controls. In contrast, in early night (CT13.5), GDX decreased light induced mPer2 but had no effect on mPer1. At CT13.5, GDX animals also showed larger phase delays than did INT. Treatment of GDX animals with the nonaromatizable androgen dihydrotestosterone restored glial fibrillary acidic protein, postsynaptic density 95, and synaptophysin in the SCN and reinstated the INT pattern of molecular and behavioral responses to light. Together, the results reveal a role for androgens in regulating circuitry in the mouse SCN, with functional consequences for clock gene expression and behavioral responses to photic phase resetting stimuli.

Basis of Robustness and Resilience in the Suprachiasmatic Nucleus: Individual Neurons Form Nodes in Circuits that Cycle Daily

How the cellular elements of the SCN are synchronized to each other is not well understood. We explore circadian oscillations manifest at the level of the cell, the tissue, and the whole animal to better understand intra-SCN synchrony and master clock function of the nucleus. At each level of analysis, responses to variations in operating environment (robustness), and following damage to components of the system (resilience), provide insight into the mechanisms whereby the SCN orchestrates circadian timing. Tissue level rhythmicity reveals circuits associated with an orderly spatiotemporal daily pattern of activity that is not predictable from their cellular elements. Specifically, in stable state, some SCN regions express low amplitude or undetectable rhythms in clock gene expression while others produce high amplitude oscillations. Within the SCN, clock gene expression follows a spatially ordered, repeated pattern of activation and inactivation. This pattern of activation is plastic and subserves responses to changes in external and internal conditions. Just as daily rhythms at the cellular level depend on sequential expression and interaction of clock genes, so too do rhythms at the SCN tissue level depend on sequential activation of local nodes. We hypothesize that individual neurons are organized into nodes that are themselves sequentially activated across the volume of the SCN in a cycle that repeats on a daily basis. We further propose that robustness is expressed in the ability of the SCN to sustain rhythmicity over a wide range of internal and external conditions, and that this reflects plasticity of the underlying nodes and circuits. Resilience is expressed in the ability of SCN cells to oscillate and to sustain activity-related rhythms at the behavioral level. Importantly, other aspects of pacemaker function remain to be examined.

Divergent photic thresholds in the non-image-forming visual system: entrainment, masking and pupillary light reflex

Light is the principal cue that entrains the circadian timing system, but the threshold of entrainment and the relative contributions of the retinal photoreceptors—rods, cones and intrinsically photosensitive retinal ganglion cells—are not known. We measured thresholds of entrainment of wheel-running rhythms at three wavelengths, and compared these to thresholds of two other non-image-forming visual system functions: masking and the pupillary light reflex (PLR). At the entrainment threshold, the relative spectral sensitivity and absolute photon flux suggest that this threshold is determined by rods. Dim light that entrained mice failed to elicit either masking or PLR; in general, circadian entrainment is more sensitive by 1–2 log units than other measures of the non-image-forming visual system. Importantly, the results indicate that dim light can entrain circadian rhythms even when it fails to produce more easily measurable acute responses to light such as phase shifting and melatonin suppression. Photosensitivity to one response, therefore, cannot be generalized to other non-image-forming functions. These results also impact practical problems in selecting appropriate lighting in laboratory animal husbandry.

The role of the circadian system in fractal neurophysiological control

Many neurophysiological variables such as heart rate, motor activity, and neural activity are known to exhibit intrinsic fractal fluctuations – similar temporal fluctuation patterns at different time scales. These fractal patterns contain information about health, as many pathological conditions are accompanied by their alteration or absence. In physical systems, such fluctuations are characteristic of critical states on the border between randomness and order, frequently arising from nonlinear feedback interactions between mechanisms operating on multiple scales. Thus, the existence of fractal fluctuations in physiology challenges traditional conceptions of health and disease, suggesting that high levels of integrity and adaptability are marked by complex variability, not constancy, and are properties of a neurophysiological network, not individual components. Despite the subjects theoretical and clinical interest, the neurophysiological mechanisms underlying fractal regulation remain largely unknown. The recent discovery that the circadian pacemaker (suprachiasmatic nucleus) plays a crucial role in generating fractal patterns in motor activity and heart rate sheds an entirely new light on both fractal control networks and the function of this master circadian clock, and builds a bridge between the fields of circadian biology and fractal physiology. In this review, we sketch the emerging picture of the developing interdisciplinary field of fractal neurophysiology by examining the circadian systems role in fractal regulation.

Food anticipation depends on oscillators and memories in both body and brain

Despite the importance of learning and circadian rhythms to feeding, there has been relatively little effort to integrate these separate lines of research. In this review, we focus on how light and food entrainable oscillators contribute to the anticipation of food. In particular, we examine the evidence for temporal conditioning of food entrainable oscillators throughout the body. The evidence suggests a shift away from previous notions of a single locus or neural network of food entrainable oscillators to a distributed system involving dynamic feedback among cells of the body and brain. Several recent advances, including documentation of peroxiredoxin metabolic circadian oscillation and anticipatory behavior in the absence of a central nervous system, support the possibility of conditioned signals from the periphery in determining anticipatory behavior. Individuals learn to detect changes in internal and external signals that occur as a consequence of the brain and body preparing for an impending meal. Cues temporally near and far from actual energy content can then be used to optimize responses to temporally predictable and unpredictable cues in the environment.

Dose-Dependent Effects of Androgens on the Circadian Timing System and Its Response to Light

The hypothalamic suprachiasmatic nucleus (SCN) is the locus of a master clock that regulates circadian rhythms in physiology and behavior. Gonadectomy in male mice lengthens the period of circadian rhythms and increases the day-to-day variability of activity onset time. Both of these responses are rescued by the nonaromatizable androgen dihydrotestosterone. Androgen receptors (AR) are localized in SCN neurons that receive direct retinal input. To explore how androgens affect circadian clock function and its responsiveness to photic cues, we measured wheel-running behavior and SCN AR expression in intact, gonadectomized, and testosterone-replaced mice, held under various photic conditions. Gonadectomy lengthened circadian period in constant dim light but not in constant darkness. Increasing intensities of constant light parametrically increased circadian period, and this was potentiated at all intensities by gonadectomy. In contrast, gonadectomy did not alter light-induced pupil constriction, suggesting a nonretinal locus of hormone action. In hormone-replaced animals housed in constant darkness, T concentration was positively correlated with precision of activity onset and with SCN AR expression and negatively correlated with duration of activity. We infer the existence of two androgenic mechanisms: one modulates SCN responsiveness to light, and the second modulates SCN timekeeping and locomotor activity in a dose-dependent manner. Finally, the effects of androgens on period are a result of hormonal modulation of the SCNs response to photic input rather than to a change in the inherent period of oscillators in the absence of light.

Dec1 and Dec2 Expression is Disrupted in the Suprachiasmatic Nuclei of Clock Mutant Mice

DEC1 and DEC2 are basic helix-loop-helix transcription factors that functionally resemble negative feedback components of the mammalian circadian clock. The genes Dec1 and Dec2 are expressed rhythmically in the rat suprachiasmatic nuclei, and Dec1 expression is stimulated by light in a timedependent manner with the kinetics of an immediate early gene. DEC1 and DEC2 can inhibit CLOCK:BMAL1 transactivation of the clock gene Per1, suggesting that these transcription factors may help regulate circadian timing. The authors present data on the expression pattern of Dec1 and Dec2 in wild-type and homozygous Clock mutant mice. In the suprachiasmatic nuclei, the Clock mutation significantly reduces the expression of Dec1 and Dec2. Dec1 becomes arrhythmic; Dec2 remains weakly rhythmic in a 12L:12D light-dark cycle but is arrhythmic in constant darkness. A robust attenuation of the Dec1 and Dec2 signals in Clock mutant mice was detected in all brain areas examined. These data point to up-regulation of Dec1 and Dec2 by Clock in vivo.

Seasonal regulation of reproduction: altered role of melatonin under naturalistic conditions in hamsters

The seasonal reproductive cycle of photoperiodic rodents is conceptualized as a series of discrete melatonin-dependent neuroendocrine transitions. Least understood is the springtime restoration of responsiveness to winter-like melatonin signals (breaking of refractoriness) that enables animals to once again respond appropriately to winter photoperiods the following year. This has been posited to require many weeks of long days based on studies employing static photoperiods instead of the annual pattern of continually changing photoperiods under which these mechanisms evolved. Maintaining Siberian hamsters under simulated natural photoperiods, we demonstrate that winter refractoriness is broken within six weeks after the spring equinox. We then test whether a history of natural photoperiod exposure can eliminate the requirement for long-day melatonin signalling. Hamsters pinealectomized at the spring equinox and challenged 10 weeks later with winter melatonin infusions exhibited gonadal regression, indicating that refractoriness was broken. A photostimulatory effect on body weight is first observed in the last four weeks of winter. Thus, the seasonal transition to the summer photosensitive phenotype is triggered prior to the equinox without exposure to long days and is thereafter melatonin-independent. Distinctions between photoperiodic and circannual seasonal organization erode with the incorporation in the laboratory of ecologically relevant day length conditions.

Twelve-hour days in the brain and behavior of split hamsters.

Hamsters will spontaneously ‘split’ and exhibit two rest–activity cycles each day when housed in constant light (LL). The suprachiasmatic nucleus (SCN) is the locus of a brain clock organizing circadian rhythmicity. In split hamsters, the right and left SCN oscillate 12 h out of phase with each other, and the twice‐daily locomotor bouts alternately correspond to one or the other. This unique configuration of the circadian system is useful for investigation of SCN communication to efferent targets. To track phase and period in the SCN and its targets, we measured wheel‐running and FOS expression in the brains of split and unsplit hamsters housed in LL or light–dark cycles. The amount and duration of activity before splitting were correlated with latency to split, suggesting behavioral feedback to circadian organization. LL induced a robust rhythm in the SCN core, regardless of splitting. The split hamsters’ SCN exhibited 24‐h rhythms of FOS that cycled in antiphase between left and right sides and between core and shell subregions. In contrast, the medial preoptic area, paraventricular nucleus of the hypothalamus, dorsomedial hypothalamus and orexin‐A neurons all exhibited 12‐h rhythms of FOS expression, in‐phase between hemispheres, with some detectable right–left differences in amplitude. Importantly, in all conditions studied, the onset of FOS expression in targets occurred at a common phase reference point of the SCN oscillation, suggesting that each SCN may signal these targets once daily. Finally, the transduction of 24‐h SCN rhythms to 12‐h extra‐SCN rhythms indicates that each SCN signals both ipsilateral and contralateral targets.