WILLIAM BECHTEL FROM MOLECULES TO NETWORKS: ADOPTION OF SYSTEMS APPROACHES IN CIRCADIAN RHYTHM RESEARCH ABSTRACT In the 1990s circadian rhythm researchers made enormous progress in identifying the components and operations within the responsible mechanism in various spe- cies using the tools of molecular biology. In the past decade it has proven essential to supplement these with the tools of systems biology both to identify additional components but especially to understand how the mechanism can generate circa- dian phenomena. This has proven especially important since research has shown that individual neurons in the mammalian mechanism are highly variable in their behavior and that the way they are organized in networks is crucial to generating circadian behavior. 1. INTRODUCTION From its roots in the study of circadian rhythms observed in physiology and be- havior, circadian rhythm research rapidly adopted and energetically pursued a mo- lecular biological approach in the last decades of the 20th century. This research has been highly productive in revealing many of the components of the circadian mechanisms in each of the major model systems: cyanobacteria, fungi, plants, and various animals (especially fruit fl ies and mice). But success in decompos- ing the mechanisms has also generated challenges in recomposing them, a crucial step in understanding how they work. Although in some fi elds it is possible for researchers to literally recompose mechanisms (e.g., by reconstituting a chemical reaction in vitro), in other cases researchers must do so more indirectly, either by imagining the interactions of the components performing their various operations or by constructing computational models that demonstrate how the hypothesized set of components would interact if they operated in the manner characterized. Im- agination suffi ces when mechanisms are relatively simple, involving components performing linear operations and organized sequentially. But when the parts iden- tifi ed operate non-linearly and are organized non-sequentially, such an approach 2 William Bechtel fails. The alternative, increasingly being pursued in circadian rhythm research, is to turn to computational modeling and dynamical systems analysis.1 A further challenge stems from the fact that underlying the strategy of de- composing mechanisms is the assumption that the mechanism itself and each of its components operate largely in isolation from other mechanisms or components so that the whole system exhibits what Herbert Simon referred to as near decom- posability.2 Assuming near decomposability is a heuristic, and a characteristic of heuristics is that they can fail. Increasingly biologists are learning that the mecha- nisms they study are less decomposable then they thought, and circadian mecha- nisms are no exceptions. The challenge is to relax the decomposability assumption and incorporate the infl uences from other components that alter the behavior of the components into one’s account without losing the ability to explain the operation of the mechanism in terms of its components. Once again, this is leading circadian researchers to turn to computational modeling, which has the resources to char- acterize multiple interactions affecting individual components while they operate within a mechanism. My focus in this paper will be on the steps in recomposing circadian mecha- nisms in the last decade that has led to a focus on networks at various levels of organization, including ones at which clock mechanisms interact with other biological mechanisms. This has resulted in an increased focus on networks as opposed to individual components and on the employment of tools from systems biology to understanding the responsible mechanisms. Before examining these developments, though, I will set the stage by introducing circadian rhythms re- search and briefl y describing the results of the more traditional mechanist project of decomposing circadian mechanisms. 1 Mechanisms and mechanistic explanation has been the focus of considerable discus- sion in recent philosophy of science. See, for example, William Bechtel and Robert C. Richardson, Discovering Complexity: Decomposition and Localization as Strate- gies in Scientifi c Research. Cambridge (Mass.): MIT Press. 1993 edition published by Princeton University Press 1993/2010; Peter Machamer, Lindley Darden, and Carl F. Craver, “Thinking About Mechanisms”, in: Philosophy of Science 67, 2000, pp. 1-25. In recent papers I have distinguished basic mechanistic explanation, which focuses on recomposing mechanisms through mental simulation, and dynamic mechanistic ex- planation, which appeals to computational model and dynamical systems theory to recompose mechanisms and explain how they function. See William Bechtel, “Mecha- nism and Biological Explanation”, in: Philosophy of Science 78, 4, 2011, pp. 533-57; William Bechtel and Adele Abrahamsen, “Dynamic Mechanistic Explanation: Com- putational Modeling of Circadian Rhythms as an Exemplar for Cognitive Science”, in: Studies in History and Philosophy of Science Part A 41, 3, 2010, pp. 321-33. 2 Herbert A. Simon, “The Architecture of Complexity: Hierarchic Systems”, in: Pro- ceedings of the American Philosophical Society 106, 1962, pp. 467-82. From Molecules to Networks 3 2. FROM CIRCADIAN RHYTHMS TO CLOCK MECHANISMS Circadian rhythms involve endogenously generated oscillations of approximately 24 hours (hence the term circadian from circa [about] + dies [day]) that affect a wide variety of physiological processes and behaviors. For example, human body temperature is lower during the night and raises during the day, varying by nearly a degree Celsius. These rhythms are entrainable to the local day-night cycle; when entrainment cues such as daylight are lacking, they free-run and thereby reveal that their period is not exactly 24 hours. This was one of the crucial features of circadian rhythms that convinced the pioneer circadian researchers in the mid- dle of the 20th century that these rhythms were endogenously maintained and not responses to external cues. The evidence presented at the 1960 Symposium on Biological Clocks at Cold Springs Harbor largely settled the question of endog- enous origin of circadian rhythms.3 While the mechanistic metaphor of a clock was widely embraced by many researchers and employed in the title of the 1960 symposium, the tools for actually investigating the clock mechanism were indi- rect, relying on such approaches as varying the period of the light-dark cycle or restricting light exposure to pulses at different parts of the cycle to see how they affected the mechanism. In the two decades after 1960 a variety of researchers identifi ed the locus and began decomposing the hypothesized clock. Although in single-cell organ- isms and in plants researchers assumed the mechanism was found in each cell, animal researchers assumed that the clock was localized within the brain. Richter discovered that lesions to the hypothalamus disrupted circadian behavior and con- cluded that circadian rhythms were generated “somewhere in the hypothalamus.” 4 In 1972 two research groups further narrowed the locus to the suprachiasmatic nucleus (SCN), a bilateral nucleus located just above the optic chiasm that in the mouse consists of approximately 20,000 neurons. It was the target of projections from the retina, allowing for entrainment by light,5 and lesions to it rendered ani- mals arrhythmic.6 Inouye and Kawamura showed, using multi-electrode record- 3 This conference in many respects marks the founding of circadian rhythm research as a distinct research fi eld. The papers and some of the discussion were published in Cold Spring Harbor Symposia on Quantitative Biology 25, 1960. 4 Curt P. Richter, Biological Clocks in Medicine and Psychiatry . Springfi eld, IL: Charles C. Thomas 1965. 5 Robert Y. Moore and Nicholas J. Lenn, “A Retinohypothalamic Projection in the Rat”, in: The Journal of Comparative Neurology 146, 1, 1972, pp. 1-14. 6 Friedrich K. Stephan and Irving Zucker, “Circadian Rhythms in Drinking Behavior and Locomotor Activity of Rats Are Eliminated by Hypothalamic Lesions”, in: Pro- ceedings of the National Academy of Sciences (USA) 69, 1972, pp. 1583-86; Robert Y. Moore and Victor B. Eichler, “Loss of a Circadian Adrenal Corticosterone Rhythm Following Suprachiasmatic Lesions in the Rat”, in: Brain Research 42, 1972, pp. 201- 06. 4 William Bechtel ing, that isolated SCN tissue alone remained rhythmic.7 The case for this locus was made more compelling when in 1990 Ralph, Foster, Davis, and Menaker denib- strated that transplanting the SCN from a mutant hamster with a shortened rhythm into ventricles of a SCN-lesioned host restored rhythms in the recipient that cor- responded to those of the donor.8 To explain how a localized mechanism could function as a clock, research- ers needed to decompose it to identify its component parts and the operations they performed. This research proceeded independently using fruit fl ies during the same period as mammalian researchers were localizing the mammalian clock in the SCN. Since investigators beginning with Darwin viewed circadian rhythms as inherited, a natural strategy was to try to identify responsible genes. Seymour Ben- zer developed a strategy for identifying genes responsible for traits by exposing fruit fl ies to mutagenic agents and linking resulting aberrant traits to the mutated gene. In 1971, as a graduate student with Benzer, Konopka pursued this approach to circadian rhythms in fruit fl ies, creating