Chapter 146 Chronobiologic Monitoring Techniques

Abstract

This chapter serves as an introduction to fundamental methodologies in circadian rhythm research. Exciting advances have occurred in techniques for measuring circadian rhythms. We are on the threshold of being able to measure circadian rhythm period length from saliva gathered from a subject or patient; now able to measure circadian rhythm period length, phase, and amplitude from genes extracted from a subject or patient’s forearm. These are remarkable advances considering that the earliest studies of circadian rhythm properties required that the subject live in a cave for several weeks (and the results were grossly inaccurate). Although methods can be described to measure the circadian rhythm properties of single-celled organisms, worms, flies, or mammalian organs, this chapter concentrates exclusively on methodologies applicable to mammalian and, most specifically, human research. This chapter examines controversies and unresolved issues, and critically evaluates varying chronobiological protocols.

Major advances include standardized measurement of human circadian period length from melatonin and the wider dissemination of less invasive and more-standardized kits for measuring salivary melatonin. A melatonin phase-response curve similar to the well-accepted phase-response curve to light has been established. We now also have the capacity to estimate circadian rhythms from genes extracted from human fibroblast cells, leukocytes, or blood cells.

Basic Concepts and Terminology

The purpose of chronobiological monitoring techniques in humans is to measure biological and behavioral properties of circadian rhythms, to better understand normal and abnormal clocks that control human alertness and sleepiness. Chronobiology research is based on the assumption that a given organism contains within it a core mechanism or clock for generating rhythmic expression of physiologic parameters. The fluctuations in any biological or behavioral parameter that display an approximately 24-hour periodicity are assumed to be the direct or indirect consequence of an underlying pacemaker that has its own inherent and autonomous periodicity.

The internal clock has a variety of properties that are indirectly studied in circadian rhythm research by examining output variables. These include amplitude, or the quantity of change in a parameter that occurs from apogee to nadir by measuring variables such as temperature, cortisol, or cognitive problem solving; period length (or tau), or the temporal duration of one complete circadian cycle under free-running conditions; phase, or the relation of the internal clock to the environment, such as sunrise and morning awakening; rate of change, or the rapidity with which a circadian parameter switches from its active to dormant or dormant to active modes; and the duration of the active period relative to the duration of the dormant period. Chronobiological techniques have evolved to measure each of these variables, how they may be manipulated, and how they are interrelated.

The process by which the internal clock stays in synchrony with the environment is called entrainment. Stimuli, such as light, that bring about entrainment are called zeitgebers. The circadian mechanism is always subject to the influences of masking. Masking is the ability of an external variable, such as light, sound, or physical activity, to alter a behavioral or physiologic parameter without affecting the core pacemaker.

The distinction between entrainment and masking is not clearly defined. Exposure to daily sunlight, for example, entrains us to our local time zone and masks our internal clock from expressing its endogenous rhythm. In animals, masking influences may be removed by placing the animal in continuous light or dark conditions.

As simple and straightforward as it would seem to be to measure the period length of the human circadian rhythm, the field of circadian rhythms research has undergone continual upheaval as methodologic errors have been repeatedly revealed and new paradigms designed to correct them. In humans, for example, there have been continued corrections of the period length of the free-running circadian rhythm length, or period, which is now estimated at slightly greater than 24 hours, 24.18 hours being the most recent value.¹

In addition to a core pacemaker, each circadian system has input and output components. Input components are principally responsive to photic information but respond to a variety of stimuli, each of which is manipulated in circadian studies to examine the effect on output variables such as the sleep-wake cycle, temperature, or behaviors such as reaction time or problem-solving ability.

Methodologic Questions in the Field of Circadian Rhythms

During the preceding 5 decades, various estimates have placed the human circadian period length anywhere from 13 to 65 hours.² Also, greater interindividual inconstancy in circadian variables in humans had been observed than that observed in other mammals. These findings showed a disparity between humans and nonhuman mammals, in which circadian rhythms are remarkably stable from day to day. In humans, there have been continued corrections of the period length of the circadian rhythm of activity, which has been most recently estimated at slightly greater than 24 hours, with a precision not that different from our mammalian relatives.

Disagreements continue over which stimuli are capable of acting as zeitgebers to entrain or shift circadian rhythms. Initially, only bright light was considered to be capable of shifting the timing of circadian rhythms.³ Subsequently, light of moderate intensity was described as being capable of shifting circadian rhythms,⁴ and, most recently, studies now maintain that ordinary room light is capable of inducing a circadian shift.⁵^,⁶ Some authors have claimed that extraocular light is capable of influencing circadian rhythms. One study demonstrated that behind-the-knee (popliteal region) bright light is capable of inducing shifts in circadian timing,⁷ but there have been multiple failures to replicate this finding.⁸^–¹¹ As chronobiology research progressed, a series of modifications of experimental design successively eliminated some masking artifacts and external zeitgebers that previously introduced artifact.¹

The field has also been inundated by controversy over what constitutes a zeitgeber, or a stimulus capable of entraining a circadian rhythm. There is evidence that nonphotic stimuli are capable of entraining individuals.¹² Exercise,¹³^,¹⁴ social activity,¹⁵ feeding schedule,¹⁶ ambient temperature,¹⁷ napping in darkness,¹⁸ administration of melatonin,¹⁹ and knowledge of time of day or night²⁰ each has the capacity to entrain human and nonhuman subjects. Some studies suggest a major role for such nonvisual zeitgebers,¹² whereas other studies imply a more modest role.²¹ The extent to which melatonin is capable of shifting circadian rhythms was controversial until recently.²²

Another area of research involves separating behavioral and biological properties of circadian rhythms from other physiologic processes. Many behavioral and biological phenomena are influenced by the sleep-wake cycle, or the duration of prior wakefulness. This is known as the homeostatic model, which describes an increasing pressure to sleep with increased duration of wakefulness and a decreased pressure to sleep with increased duration of sleep.²³ Circadian rhythm studies must separate variance in a biological or behavioral parameter into homeostatic and circadian components; that is, they must identify what portion of the parameter’s variance is circadian and what portion is homeostatic.²⁴^,²⁵ Neither the circadian nor the homeostatic models can explain in a compelling manner why humans are most wide awake in the hour or two preceding sleep and sleepiest in the hour after morning awakening.

An additional area of continued controversy is what biological metric is best used to approximate the properties of the underlying core pacemaker, such as period length. Core body temperature ²⁴ and melatonin²⁶ secretory profiles have been used in most studies. Some research uses dim-light melatonin onset as the marker of the beginning of a new circadian cycle, others use dim-light melatonin offset,²⁶ and many use the temperature nadir. However, there is no obvious point that can be identified as the minimum of temperature or the onset of melatonin secretion. Temperature fluctuates from moment to moment, and the minimum or maximum must be extracted mathematically. Melatonin rises gradually after sunset, and an arbitrary level must be selected to denote onset or offset. In addition, it must be emphasized that extracting circadian rhythm data relies on mathematical criteria and data analytic techniques selected in each experimental protocol.¹

The amount of information uncovered in the past few decades in the field of circadian rhythm research has been truly astounding and includes the identification of basic anatomy, novel photoreceptors, and genetic mechanisms. The field has not accepted specific definitions in the areas of contention listed previously, nor has it accepted a single chronobiological monitoring technique as being the gold standard. Various studies using different experimental paradigms have contributed to the turbulence in this field. This chapter briefly reviews the various paradigms recently used in circadian rhythm research and discusses the strengths and weaknesses of each. This chapter does not present an in-depth description of the details of each model, but the reader is directed to a few key references in which a fuller exposition is offered.

Paradigms

Fixed Light-Dark Schedules, Double Plotted

The most common model for animal studies of circadian variables and activity schedule consists of placing the animal in a cage with a running wheel and subjecting the animal to a fixed schedule of light and dark. The running wheel’s turns are counted continuously. Typically, the animal’s running behavior on the wheel is plotted as a vertical or horizontal line per unit time, or wheel turns per unit time. These counts are plotted successively for 24 hours, yielding a visual representation of when and how much the animal ran that day. Successive days are stacked, enabling the reader to visually appreciate changes in the timing of activity that occurred during the experiment. The entire plot is duplicated side by side, called double plotting, because seeing the image next to itself enables the reader to appreciate what happened in the experiment more readily. For example, if the animal’s principal expression of wheel-running activity drifts from before to after midnight, double plotting allows the reader to more easily follow the continuity of changed running wheel times. Drinking or feeding may be plotted in the same manner. Protocols are labeled as LL (constant light), LD (fixed light-dark schedule, most commonly 12 hours light and 12 hours dark), or DD (constant darkness). Constant dim light is sometimes used in such protocols, or light dim enough to prevent suppression of the rat’s or hamster’s secretion of melatonin. In some studies the LD period length may be longer than or shorter than 24 hours, and as short as 1 hour in some studies.

The DD paradigm has been successful in demonstrating the remarkable consistency of a given species’ free-running activity rhythm in constant darkness or dim light, successfully elucidating the period length of its biological clock.¹ Often, the timing of light and dark are abruptly changed to measure the animal’s reaction, which can take several days to adapt. Such paradigms are at the core of much genetic research, in which normal (wild-type) animals (+/+) are compared with heterozygous (+/−, −/+) or homozygous (−/−) knockouts. Studies reveal genetic phenotypes (behavioral expression) because knockout animals display altered periodicity, diminished rhythmicity, or lack of any circadian rhythm in running, feeding, or drinking behavior.

The obvious strength of the fixed light-dark schedule with activity monitoring is its ability to accurately and economically measure an animal’s rest-activity schedule. Alterations in rest-activity schedules after genetic knockouts have become the basis for a standard genotype–phenotype model. Weaknesses of this design include its inability to examine complex interactions that would occur in more natural circumstances that could have an impact on the circadian system. If only light, dark, or light versus dark are studied, then all effects appear to be a function of the animal’s illumination schedule. The paradigm is subject to overly simple interpretation, the implications of which are discussed later.

Entrained 24-Hour Studies

In entrained 24-hour studies, subjects live in laboratory conditions with constant bedtime, wakeup time, meal time, and timed activities. Subjects are aware of time of day and are not isolated from normal zeitgebers such as light from windows. This protocol is used to study biological or behavioral parameters such as hormone secretory profiles or cognitive processes under constant conditions. Often, indwelling catheters with recurrent blood draws are used in such studies.

Phase-Shifting Protocols

One of the most experimentally robust findings in the area of circadian rhythm research is the capacity of zeitgebers such as bright light to change the time of day at which the circadian system switches from its active or day mode to its dormant or night mode. This is referred to as a phase shift. It is similar to jet lag or shift work in the real world.

Many studies have examined the capacity of light of varying intensities and durations to alter the timing of sleep, melatonin secretion, or the temperature nadir. These studies share in common a baseline in which environmental light is controlled but typically is similar to the timing of natural environmental light. Often the baseline consists of dim light (ranging from about 70 lux, equivalent to romantic restaurant lighting, to as low as 1.5 lux, similar to the light from a candle) in different studies. In the experimental condition, light is then delivered to the subject in what would normally be the dark or night portion of the circadian rhythm.²⁷ Light is delivered for the same duration but at a different time during this experimental condition for 1 or several days. In some studies there is then a subsequent condition in which the subject is in constant dim light or in a constant routine (described later).

Circadian variables such as wheel running in rodents and hamsters, the timing of sleep, or the timing of melatonin secretion are monitored during each condition.¹^–³ The magnitude of the change in a circadian variable after the altered timing of light is measured. A phase response curve (see Fig. 146-1) shows the magnitude of response to light exposure (typically 1 to 3 hours) at different times throughout the circadian cycle in changing the timing of a circadian output variable.²⁸ Most studies show circadian rhythm shifts in response to light occurring only if the light is delivered at specific times, typically at or near normal hours of darkness, and indicate that mammals are not responsive to bright light administered during normal hours of daylight.²⁹ Other studies claim there is no dead zone and that bright light delivered at any hour, including normal daylight hours, has a phase-shifting effect.³⁰

Figure 146-1 Accurately identifying the effect that light and melatonin have on advancing or delaying circadian timing is at the core of much circadian rhythm research. The magnitude of the phase advances (falling asleep and awakening earlier, represented by positive numbers) and delays (falling asleep and awakening later, represented by negative numbers) are plotted against the center of the period of light exposure relative to the core body temperature minimum at 0 h. The filled circles represent data from plasma melatonin, and the open circle represents data from salivary melatonin in one subject from whom blood samples were not acquired. The solid curve is a harmonic function fitted through all of the data points. Light’s phase-delaying effect increases as it approaches the temperature minimum, then it flip-flops in the 6-hour period surrounding the temperature minimum to a maximum phase-advancing effect 6 hours after the temperature minimum.

(From Khalsa SB, Jewett ME, Cajochen C, Czeisler CA. A phase response curve to single bright light pulses in human subjects. J Physiol 2003;549(Pt 3):945-52.)

Time-Isolation Protocols

Animals may be kept in total darkness or in light dim enough to have no effect on their circadian timing mechanism. Humans have typically been isolated in an environment free of time cues, such as an underground facility or an internal suite of rooms in an enclosed bunker. Great pains are taken not to give subjects any clues as to time, including double-door entry chambers and randomized schedules for technicians interacting with subjects. In such studies, for many years, subjects were permitted to turn off their room lights and retire when they wished and awaken when they wished. Subjects were instructed not to nap.

One of the more unexpected findings in circadian rhythm research is that the subjective sense of sleepiness, or the wish to sleep, is at its minimum at the normal time of sleep initiation and that sleepiness is greatest at the normal time of awakening.³¹^–³⁵ This propensity to be alert at the normal hour of onset of sleep has bedeviled studies attempting to assess the period length of the human circadian clock using time-isolation protocols. Under conditions of time isolation, a variety of studies showed great variability in human sleep-wake cycle length, unlike studies in any other mammal. Studies showed sleep-wake cycles as long as 36 hours and great day-to-day variability in both sleep and wake duration.³⁶ Subjects who remained in time-isolation conditions for a sufficient interval developed desynchrony between their temperature rhythm and their sleep-wake cycle. Under normal conditions, the temperature minimum occurs somewhat after the middle of the sleep period. In subjects in temporal isolation protocols, the interval from one temperature minimum to the next has a cycle length of 24 to 25 hours, but the interval between sleep initiation on successive nights would be longer, meaning that the temperature minimum can stray elsewhere in the sleep-wake schedule.³⁷

The time-isolation protocol is no longer employed because results from these studies are widely disparate from those of circadian rhythm studies of all other mammalian species. In disregarding this experimental approach, a basic attribute of human behavior is being ignored: Most persons appear to prefer a sleep-wake schedule longer than 24 hours, when allowed. This perhaps unique human preference for longer wake and sleep episodes has been overlooked in the circadian rhythm field’s fervor to produce human data as similar as possible to that from other mammals, invertebrates, and plants.

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