The most common model of photo-biological rhythmicity involves the initial intake of periodic environmental signals through photoreceptive organs. These external stimuli are then relayed to and integrated in the central pacemaker of the organism, which thus, in a multi-oscillator system, controls the oscillations of other biological clocks. These secondary oscillators account for the overt and quantifiable rhythms that are measured by researchers with behavioral, electrical or immunochemical assays. In most mammals, it seems that this once-hypothetical “central pacemaker” is the suprachiasmatic nucleus, or SCN, of which mammals have two that are bilaterally symmetrical and located in a part of the hypothalamus just above the optic chiasm. A small bundle of white matter known as the retinal-hypothalamic tract (RHT) synapses onto the SCN, feeding environmental light cycle information directly to the nuclei. The RHT, along with other retinal projections to the hypothalamus, account for the SCN’s ability to entrain to light-dark cycles on Earth (Rusak & Boulos, 1981). It is important to note that, as the term “pacemaker” implies, the SCN still produces electrical and chemical rhythms without environmental or neuronal input. For example, radioactively labeled glucose was used to visualize glucose intake in the SCN, which shows a clear daily rhythm (Schwartz & Gainer, 1977).
In order to be called the central pacemaker, however, the SCN had to meet many more criteria to prove that it was indeed responsible for maintaining its own rhythms and keeping other biological rhythms under control. The SCN passed many of these tests, which began as early as the mid-to-late 1970s. Among these, brain lesioning and in vivo isolation experiments are some of the most conceptually simple protocols for determining function of certain brain nuclei. Ablation of the SCN by lesioning techniques, for example, abolishes rhythmicity in corticosterone release from the adrenal glands (Moore & Eichler, 1972). In addition, surgical synaptic isolation of the SCN in an “island” of hypothalamic tissue eradicates electrical rhythmicity in brain regions outside of the island. These action potential rhythms were retained in the SCN and the surrounding brain tissue within the island, suggesting that some region inside the island is responsible for maintaining other neural rhythms and is not dependent on other brain regions for the generation of its own rhythms (Inouye & Kawamura, 1979). In vitro rhythms of SCN cells further legitimize the central pacemaker hypothesis. In one such experiment, rats were raised on different light cycles and then sacrificed. After extracting SCN cultures and measuring the electrical activity of these cells in vitro, rhythms persisted. What’s more, these rhythms were in phase with the rhythms that would be observed in vivo (Green & Gillette, 1982). Finally, transplantation or reintroduction experiments cement the observation that the SCN is the central pacemaker in mammalian systems. For example, ablating the neonatal SCN tissue in golden hamsters (Mesocricetus auratus) leads to arrhythmicity in drinking habits and locomotor activity in adult hamsters. Transplantation of developing SCN tissue to the lesioned SCN area restores these rhythms completely (Lehman, et. al., 1987).
Though reintroduction of SCN tissue reinstates the rhythms that were lost, many researchers wondered what properties these rhythms exhibited. If these behavioral rhythms had the same phase relationship and free-running period length as the tissue donor, then this would be stronger proof that the SCN is the master regulator of the circadian system in mammals. Martin R. Ralph and colleagues pursued this knowledge in their 1990 Science publication involving SCN transplantation and the properties of the reinstated rhythms. The hypothesis was, as stated above: after transplantation, the restored rhythm “should reflect the genotype of the donor tissue and not that of the lesioned host” (Ralph, Foster, Davis, & Menaker, 1990). To examine this, they utilized a novel genetic variant of golden hamsters: the tau mutant. While wild-type hamsters have a free-running period of approximately 24 hours, tau homozygotes have a period of about 20 hours, and heterozygotes have a period of around 22 hours. Since the tau gene seemed to follow simple Mendelian genetic patterns, breeding desired mutants was simple after deducing the period of each hamster in constant dim light or dark.
Only male hamsters were kept in these dim light or dark conditions when they reached eight years old. When the animals established a constant free-running period, they were anesthetized before SCN lesioning by applying a current to the area. To check if these lesions were correctly carried out, behavioral assays were conducted: that is, when the animal showed no apparent rhythm for greater than 24 hours, the procedure was deemed successful. Later, when the animals were sacrificed, histological assays helped to determine the validity of the surgical procedure. Most SCN transplantation occurred three to four weeks after the lesioning procedure, but some variation was planned to make sure that timing of the transplantation relative to the ablation had no significant effect on the results. The tissue that was transplanted was fetal tissue from hamsters of known tau genotype. This tissue was either from the SCN or from the cortex of the brain, which was used as a control. During the procedure, the transplant was taken up by a micropipette and implanted in the third ventricle next to the ablation site (Ralph, Foster, Davis, & Menaker, 1990).
As expected, control cortical tissue transplants showed no rhythms. Rhythms returned in 80% of the hosts that received donor SCN tissue. In these hamsters, “the period of restored rhythms always matched that predicted by the genotype of the donor tissue,” corresponding with the posited hypothesis. For example, imagine a wild-type hamster that had its SCN ablated; three weeks after lesioning, fetal SCN tissue from a tau heterozygote was transplanted in the third ventricle near the site of ablation. After the procedure, this hamster showed a free-running period of approximately 22 hours in the same constant conditions it was held in before transplantation. The researchers concluded from these behavioral data that the SCN has a role in the determination of the properties of behavioral rhythms in mammals.
As stated before, the researchers also performed histological stains on the brains of the hamsters after obtaining the behavioral data to make certain their transplants had made synaptic connections with the host brain. In the hamsters that regained the rhythmicity of the donor, the ablation and transplant were identified, and staining for vasoactive polypeptide (VIP) was obvious in some sections of the hamster brain tissue between the host and donor tissue, suggesting communication between the two tissue areas. In addition, neuropeptide Y (NPY) axons were visualized in the stain extending into the implant. However, no NPY cell bodies were seen in the SCN transplants, which was expected since NPY cells are not found there. In the hamsters that failed to regain rhythmicity in locomotor activity, VIP axons did not extend into the implant, suggesting that there was no communication between donor and host tissue, resulting in arrhythmic behavior. In all cortical implants, axons extended into the implants. Finally, it should be noted that it was not possible to determine from which tissue, host or donor, these projections originated. (Ralph, Foster, Davis, & Menaker, 1990)
Upon analyzing the data, the researchers were surprised that the host genotype had no effect on the rhythms of the animals especially because there was “evidence for the existence of oscillators outside the SCN in the mammalian brain.” They concluded that “the occupies a position at the top of the circadian hierarchy in mammals.” (Ralph, Foster, Davis, & Menaker, 1990) In other words, the fact that the period of the donor’s rhythm was observed after SCN transplantation no matter what the host’s tau genotype was is sound evidence that the SCN is the central pacemaker in an M. auratus model.
This study has been cited more than 1400 times since its publication in 1990, most likely for two reasons. First, its use of tau mutant hamsters revolutionized the ability of chronobiologists to breed rodents for certain free-running periods using simple Mendelian heritability. The tau allele would be used in countless studies across different rodent models and for other kinds of research besides transplantation studies. Second, this paper marks the final step toward proving that the SCN is the central pacemaker in mammals. By demonstrating that only the donor’s free-running period of locomotor activity is preserved after transplantation of the SCN, the researchers gave strong evidence that the SCN is the master oscillator that drives other secondary biological clocks. Undoubtedly, other chronobiologists were quick to replicate the results of this study.
Another aspect of this publication can be seen as both a strength and a weakness: its length. The Science article takes up little more than three pages and is dominated by three large figures. There is also no division of the text into sections such as “Introduction” or “Conclusion.” Rather, conclusions from the data are scattered throughout the paper and there is little discussion of sources of error or further considerations for research. This does streamline the paper, however, and the description of the methods is sound and innovative in the case of the tau mutant hamsters. However, certain aspects of the paper seem skeletal, especially when discussing the histological analysis of the brain slices. Although the researchers may have discussed this among themselves, they fail to consider any possible reasons for why some SCN transplants failed to innervate with host tissue. While 80% of the hamsters with SCN implants regained rhythmicity, 20% did not and histological examination shows that axonal connections failed to grow between the tissue. They do not offer much of an explanation of this in the final publication.
Despite its short length and lack of organization, the methods and results of this paper have had a major impact on the search for the mammalian pacemaker, solidifying the suprachiasmatic nucleus as the master oscillator. In addition, the novel use of tau mutant hamsters has revolutionized studying the effect of differing free-running periods on diverse dependent variables across the field of chronobiology. While it was one of many publications investigating pacemaker properties of the SCN, it marks a point in the history of the field where research on scouring to localize the pacemaker could end, and in-depth studies on the morphological, chemical, and electrical properties of the SCN could begin.