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Of Swiss and Huguenot descent, Du Bois-Reymond was born in Berlin and educated at the university there and in Neuchâtel (Switzerland). He is famous as the first to demonstrate how electrical currents in nerve and muscle fibers are generated. He began his studies under the eminent physiologist Johannes Müller at Berlin with work on fish capable of discharging electric currents as an external shock (e.g. eels). Turning his attention to nerve and muscle activity he then showed (1843) that applying a stimulus to the nerve brings about a drop in the electrical potential at the point of stimulus. This reduction in potential is the impulse, which travels along the nerve as “waves of relative negativity.” This variation in negativity is the main cause of muscle contraction. Du Bois-Reymond's pioneering research, for which he devised a specially sensitive galvanometer capable of measuring the small amounts of electricity involved, was published as Untersuchungen über tierische Elektricität (2 vols. 1848–84; Researches on Animal Electricity): a landmark in electrophysiology, although subject to later elaboration. Du Bois-Reymond's collaboration with fellow physiologists Hermann von Helmholtz, Carl Ludwig, and Ernst von Brücke was of great significance in linking animal physiology with physical and chemical laws.

Du Bois-Reymond was elected a member of the Berlin Academy of Sciences in 1851 and succeeded Müller as professor of physiology at Berlin in 1858. He was also instrumental in founding the Berlin Physiological Institute, opened in 1877, then the finest establishment of its kind.