In the field of chronobiology (the study of circadian rhythms), the dual circadian oscillator model refers to a model of entrainment (where rhythmic events in an organism match oscillation in the environment) initially proposed by Colin Pittendrigh and Serge Daan. The dual oscillator model suggests the presence of two coupled circadian oscillators: E (evening) and M (morning). The E oscillator is responsible for entraining the organism's evening activity (activity offset) to dusk cues when the daylight fades, while the M oscillator is responsible for entraining the organism's morning activity (activity onset) to dawn cues, when daylight increases. The E and M oscillators operate in an antiphase relationship. As the timing of the sun's position fluctuates over the course of the year, the oscillators' periods adjust accordingly. Other oscillators, including seasonal oscillators, have been found to work in conjunction with circadian oscillators in order to time different behaviors in organisms such as fruit flies.
Discovery In 1966, Jürgen Aschoff, a German chronobiologist, observed that some animals exhibited two bouts of activity per day, one in the morning and one in the evening. These bouts of activity are defined by the animals' anticipation of the lights turning on or off. In 1976, Colin Pittendrigh and Serge Daan, two chronobiologists, first proposed a dual-oscillator model for nocturnal rodents as the mechanism for these E and M bouts of activity. The model hypothesized the presence of two separate oscillators that have opposite dependence on light intensity. Pittendrigh and Daan found that the M oscillator is synchronized to dawn and experiences acceleration from light, meaning that the period decreases with each subsequent cycle. The E oscillator, on the other hand, is synchronized to dusk and experiences deceleration from light, meaning that the period increases for each subsequent cycle. They postulated that the E&M model had an enhanced ability to adjust the circadian rhythm to the season and changes in day length. Pittendrigh and Daan found several limitations in the model of a single oscillator controlling sleep/wake behavior that led them to develop the dual oscillator model. The first key finding was the splitting behavior in the locomotor activity of hamsters under constant high intensity light conditions. The two separate bouts of activity indicated that more than one oscillator may be controlling locomotor activity. They also observed transient changes in the phase of sleep/wake behavior in Drosophila melanogaster following temperature changes. The lack of a new steady state rhythm suggested the presence of another temperature sensitive oscillator downstream of the known oscillator. These findings and others led Pittendrigh and Daan to propose the dual circadian oscillator model.
They examine slices of the hamster hypothalamus sectioned horizontally through the optic chiasm, in addition to the standard vertical (coronal) sections (Fig. 1). Whereas the coronal sections always gave a single plateau of increased activity that lasted 7 hours and occurred once a day, the horizontal sections generated a completely unexpected output: two peaks, each lasting about 4 hours, that were clearly separable.
Following Pittendrigh and Daan's behavioral characterization of the dual oscillator model, it took several years before scientists discovered the mechanistic basis of it. In 2000, Anita Jagota, Horacio de la Iglesia, and William J. Schwartz were the first group to show two distinct peaks of electrical activity in the mammalian suprachiasmatic nucleus (SCN) after studying horizontally sectioned hamster hypothalamus. Further experiments need to be conducted to validate that the two peaks represent the E and M oscillators. In 2004, Brigette Grima and Dan Stoleru independently investigated E and M activity peaks in Drosophila melanogaster (fruit flies) using different gene expression manipulations. They found two separate circadian neuron groups control the E and M peaks of activity in Drosophila melanogaster. They also found the lateral neurons are responsible for the morning and evening peaks. In 2007, Stoleru found that the M cells dominate the circadian rhythm on short days while the E cells dominate the rhythm on long days. This alternating domination pattern allows the circadian rhythms in animals to persist on both short and long days, respectively. Stoleru's work significantly contributed to the field of chronobiology by revealing the mechanism through which animals are able to adapt to environmental changes such as seasonal variations in daylight length. His research has also provided insights into the circadian clock's role in Seasonal Affective Disorder (SAD) and other related mood disorders that are responsive to light therapy.
Background
Distinctive features of the E&M dual oscillator model include alpha compression and the presence of an intermediate τ value. Each oscillator has a unique τ (tau) which is the period of an organism's sleep/wake cycle when they are in constant conditions with no environmental cues, also known as free-running. When coupled, these oscillators produce a distinct, observed free-running period known as an intermediate τ, which is a function of the E and M oscillator's respective τ values. Alpha compression, a term coined by Jürgen Aschoff, refers to the observation that under constant light conditions, the length of activity of nocturnal organisms shortens. The organism's duration of activity is called the alpha phase and typically measured in terms of locomotor activity. Alternatively, the alpha phase of diurnal organisms lengthens under constant light; this phenomenon is known as alpha expansion. Furthermore, alpha compression refers to the decreasing duration of activity before splitting occurs. Splitting is defined as the process by which one bout of activity separates into two, distinct bouts of activity, each free-running at a τ independent of the other. Pittendrigh observed that the M oscillator ran shorter after splitting compared to the E oscillator's relatively longer intrinsic period as indicated in the actogram, a diagram of the organism's daily rest and activity phases. After splitting occurs, the oscillators responsible for the two distinct activity bouts either recouple into one with an intermediate τ or stabilize at their new, separate τ values.
E and M cells possess different capabilities to control behavior and respond to light through either accelerating or decelerating their individual, internal circadian clock speeds. The difference in the phase angle of entrainment, or the relationship between the timing of the biological clock and the timing of the external time cue, of each cell varies depending on the amount of light in the environment. Greater amounts of light lead to a greater phase angle of entrainment. The amount of light and pigment dispersing factor (PDF) control the acceleration and deceleration of the speed of M and E cells, respectively. Furthermore, the coupling of the E and M oscillators increases as the phase angle of entrainment decreases, displaying an inverse relationship between the duration of light and the coupling of the two oscillators. This phenomenon also shows the importance of E and M cells for adapting the activity of an organism different photoperiods.
Evidence in single-celled organisms In single-celled dinoflagellates such as Gonyaulax polyedra, researchers have found evidence of circadian rhythms in bioluminescence, photosynthetic capacity, time of cell division, and enzyme synthesis rates. Bioluminescence can be expressed through either independent flashing or a continual glow. Both modes of bioluminescent expression are rhythmic and peak at different times. Researchers have hypothesized that the two rhythms operate under distinct pacemakers, as they appear to peak at different times under varying conditions (such as long vs. short days or a 23-hour vs. a 24-hour entraining period). Under constant conditions, the two rhythms in bioluminescence free-run with different periods, suggesting a dual-oscillator model, and they also appear to be coupled. However, the molecular mechanism of that coupling is not yet known.
Evidence in Drosophila melanogaster
Drosophila melanogaster, or fruit flies, show diurnal rhythms in locomotor activity that are corpuscular, meaning they exhibit both morning and evening peaks in activity that align with dawn and dusk. Both these bouts of activity are intrinsic and observed in constant darkness, although the morning peak is more pronounced during a light-dark cycle. Groups of lateral clock neurons in the Drosophila brain have been found to contain neurons responsible for these morning and evening peaks, indicating they could be the source for the M and E oscillators. Independent studies have found that the ventrolateral neurons anticipate lights-on while the dorsolateral neurons anticipate lights-off. Further studies have narrowed down the morning anticipation to four small ventrolateral neurons, which are the master clock during constant darkness and express pigment-dispersing factor (PDF). PDF is involved in the molecular coupling mechanism of M and E oscillators: M oscillator cells express PDF and entrain to dawn, while E cells receive PDF and become phase delayed, entraining to dusk.
However, other studies have shown that flies lacking lateral neurons still show residual morning and evening peaks, indicating that dorsal neurons play a role in evening and morning oscillations. Researchers have shown that rhythms of core clock proteins such as PER are the same in both morning and evening cells. During long days and high temperatures, scientists have observed phase advances in morning cells and phase delays in evening cells of molecular rhythms, potentially explaining how these cells determine different bouts of activity. However, there are other clock neurons found in fruit flies that do not function as E or M cells. In addition, other studies have found results inconsistent with the traditional dual oscillator model, suggesting a network of oscillators instead. These results have led some researchers to propose a plastic oscillator model in which different neurons can assume the role of E or M when needed.
Clock neurons in the fly brain entrain to external light stimuli via a cryptochrome (CRY) response pathway. In response to light exposure, CRY binds to the timeless (TIM) protein, ultimately leading to the degradation of TIM within the clock neuron and delaying the internal circadian oscillation of period (PER) and TIM proteins, meaning their onset and offset of activity occur later in the day.
The E and M oscillators in Drosophila are also theorized to have different temperature sensitivities. A group of chronobiologists found that the Drosophila morning activity peak synchronized to temperature increases in the morning, whereas the evening activity peak synchronized to the decrease in evening temperature. They also showed that the phase of the evening peak depended on temperature level, as the evening peak in activity was delayed at high temperatures. However, the morning peak was not influenced significantly by changes in temperature, suggesting that the E and M oscillators have different sensitivities to changes in temperature levels.
Circadian oscillators also work with seasonal oscillators to time behaviors such as daily activity throughout the year. For example, the expression of dper (the Drosophila per gene) and tim (Timeless (gene)) varies with temperature and length of day. Colder, shorter days increase the accumulation of mRNA transcripts for dper and tim, affecting the timing of evening activity and midday inactivity.
Lateral neurons