The Study Of Circadian Rhythms In The Laboratory
While circadian rhythms are endogenously (internally) generated, they can also be directly affected by changes in the environment or by changes in behavior. For example, nocturnal rodents typically are inactive during the light portion of the light-dark cycle. If bright lights were turned on during the animal's normal dark time (when it would typically be active), the animal may cease activity. Thus, the endogenous component of the animal's circadian activity rhythm is acutely altered by exposure to light. This can also occur when human circadian rhythms are studied, and the endogenous circadian component of many physiologic and behavioral rhythms can be affected by things such as ambient light, activity, sleep-wake state, food intake, postural change, and emotional state. Thus, it is important that studies of circadian rhythmicity be conducted under controlled conditions in which the endogenous component of circadian rhythms can be measured.
Studies of circadian rhythms in humans began as early as the 1930s. Nathaniel Kleitman studied human subjects in Mammoth Cave in Kentucky, an environment where temperature, humidity, and darkness were constant. While the experimental subjects in those studies were allowed access to artificial lighting, Kleitman's studies revealed that humans, like other organisms, continue to exhibit near-24-hour physiological rhythms even when living in constant conditions. In the 1960s, JÎrgen Aschoff and colleagues began a series of circadian rhythm studies in Germany. They studied their subjects in underground bunkers, which, like the cave used by Kleitman, were shielded from information from the external environment. In the 1970s and later, special laboratories were developed for the study of circadian rhythms in humans. Those laboratory study rooms were typically shielded from outdoor light, were soundproof, and contained no obvious means of telling the time of day (e.g., they did not have clocks, radios, televisions).
Results of studies from humans living in those special laboratory conditions have revealed that there are circadian rhythms in many aspects of human behavior and physiology. Those rhythms include daily oscillations of hormone levels (including such hormones as cortisol, melatonin, thyroid stimulating hormone, and prolactin); core body temperature; EEG activity; alertness and vigilance; sleep tendency; and many aspects of performance. Neuroanatomical studies have also found that the same structures that comprise the circadian timing system in mammals, the SCN and RHT, are present in the human brain.
The particular methods used for studying human circadian rhythms depend on the aspect of circadian physiology that is of interest. The constant routine is an effective protocol to assess phase, amplitude, or the effect of a particular stimulus on the endogenous output of the human circadian system. In this protocol, subjects' circadian rhythms are measured for at least one complete circadian cycle while they remain awake, in a constant posture, in constant dim light, and with food and fluid intake distributed across day and night. In constant routine studies, often multiple variables controlled by the circadian timing system are measured simultaneously, so the phase and amplitude of each of those rhythms can be assessed. In studies in which the influence on the circadian timing system of a particular stimulus is of interest, an initial assessment of circadian phase and amplitude is made, the stimulus is applied, and then a reassessment of phase is done. Thus, the change in phase and amplitude as a result of the stimulus can be estimated.
Initial studies attempting to measure the period of the human circadian system used the free-running protocol. This protocol required subjects to live in an environment without time-of-day information, but allowed them to self-select their light exposure. As described above, it is now understood that light is the primary signal from the environment that affects the human circadian system, and light has phase-dependent effects on circadian timing. Experts also know that when subjects are allowed to choose when to go to sleep and wake without knowledge of what time it is, they prefer to go to sleep several hours later than they do under normal, entrained conditions. In doing so, they remain awake, exposed to light, throughout much of the time in the circadian cycle when light causes phase delay shifts. In addition, they remain asleep several hours beyond when they would wake up under normal entrained conditions, shielding themselves from light exposure during the time when light causes phase advance shifts. Thus, in free-running studies, allowing subjects to self-select their own light-dark exposure leads to cumulative phase delay shifts each day, and an observed free-running period that is consequently longer. It was widely reported based on results from free-running studies that the period of the human circadian system was near 25 hours, substantially longer and much more variable than the periods reported from most other species.
In more recent studies carried out in the 1990s, the forced desynchrony protocol was used to assess circadian period in humans under conditions that minimizes the influence of the light-dark cycle on the observed period. In this protocol, subjects are scheduled to live on a sleep-wake cycle length that is much shorter (typically ü20 hours) or longer (ü28 hours) than 24 hours. Furthermore, ambient light levels during the entire time one is awake are kept to a low level to minimize the phase-shifting effect of such light exposure. Using such protocols, it has been reported that the period emanating from the human circadian pacemaker is very close to 24 hours, with much less interindividual variability, similar to that found in most other mammalian species.