**2. Otto Loewi and his famous experiments**

At the beginning of the 20th century, Loewi had worked on cardiac physiology for several years and published a continuous stream of papers in Pflügers Archiv (Pflüger's Archiv für die gesamte Physiologie des Menschen und der Tiere; today: Pflügers Archiv: European Journal of Physiology), the oldest physiological journal [3, 4]. The question that concerned scientists at the time was the mechanism that changed the beating frequency of the heart. Two alternatives presented themselves. In one of them, the heart itself released a substance that changed its beating frequency, whereas the other one postulated that some substance was released by nerve fibers, i.e., a form of chemical transmission existed [5]. In 1921, Loewi published a short paper [6] in this regard that described the key experiment carried out by him. It was a series of fortunate circumstances that led to his discovery of chemical transmission with acetylcholine as the messenger [7].

Loewi has recounted the nights before the critical experiment as he dreamt about it in his sleep [5]. The experiment used one frog heart where the vagus nerve was stimulated with electrical impulses. Electrical stimulation of the vagus nerve slowed

down the heartbeat. The liquid from that heart (saline solution, i.e., Ringer's solution) was collected and applied to a second heart. The application resulted in a reduction in the beating frequency of the second heart. Loewi interpreted the slowing down of the second heart to mean that the vagus nerve had released a substance, the 'Vagusstoff' or vagus substance (acetylcholine) and proved that a soluble chemical released by the vagus nerve was controlling the heart rate. The chemical substance affected the heart rate through a process of chemical transmission [8]. The solution from the first heart slowed the second heart, and its beats diminished exactly as if its vagus nerve had been stimulated. In an additional experiment, Loewi stimulated the accelerator nerve of the first heart. When the nerve was stimulated and the Ringer solution from this heart was transferred, the second heart accelerated, and its beats increased [9]. Through these experiments, Loewi had solved the problem of determining whether any substance originated in the heart muscle to inhibit or activate it or whether the substance originated from the innervating nerve. In 1922, Loewi published a companion paper in which he ruled out the possibility that the chemical substances came from the heart [10]. In these experiments, he used high doses of nicotine which paralyzed the donor heart. Nevertheless, it was still possible to collect and transfer the Vagusstoff or the Acceleransstoff (adrenaline or epinephrine) from the paralyzed heart after stimulating the nerve. The idea for the initial experiment came to Loewi in a dream which prompted him to immediately go the lab after he woke up and carry out the experiment at 3 am in the morning [5, 11, 12]. His hypothesis of chemical transmission that he had postulated years earlier was proved by 5 am in the morning [11]. Loewi was lucky to carry out the experiment in a specific frog species at the time of day (diurnal cycle of the frog) and year that he did. The enzyme acetylcholinesterase rapidly metabolizes acetylcholine and could have prevented its effect on the heart. It was no surprise that chemical transmission was discovered in the peripheral nervous system as it affected visceral organs and skeletal muscles [5]. The experimental preparations were more accessible, and it was feasible to study drug effects. Moreover, chemical transmission was realized because certain drugs mimicked the stimulation of the autonomic nervous system that innervated visceral organs.
