Exorcizing animal spirits

The ancient theory of 'animal spirits' (pneuma psychikon in Greek; spiritus animalis in Latin) was first proposed by Alexandrian physicians in the third century BCE. Animal spirits were thought to be weightless, invisible entities that flowed through the hollow nerves to mediate the functioning of the body. The animal spirits theory was related to the notion of the four humours (blood, phlegm, and yellow and black bile), and was popularised by the Roman physician Galen (c. 129 -216) in the second century AD. Because of Galen, animal spirits dominated thinking about the nervous system for 1,500 years; they were exorcised very recently - it was only during the latter part of the 18th century that investigators began to decipher the electrochemical language of the nervous system.

Galen believed that nutrients were absorbed by the liver, which produced 'natural spirits'. These were transported to the left ventricle of the heart, which transformed them into vital spirits; these in turn were carried to the brain by the carotid arteries, and again transformed into animal spirits when mixed with inhaled air ("pneuma") in the cerebral ventricles, or in a plexus of blood vessels at the base of the brain which Galen called the rete mirable ('wonderful net'). Animal spirits were thought to be stored in the ventricles of the brain until needed, and then transported through the hollow nerves to force the muscles into action, or to carry sensory impressions. Animal spirits were also believed to flow into the brain, which was by then considered the seat of intellect (which consisted of the imagination, cognition and memory).

Despite the publication of De humani, and the refutation of Galen's ideas contained within it, the theory of animal spirits remained dominant throughout the ages, and was still being propounded in the seventeenth century by the French mathematician and philosopher Rene Descartes (1596-1650).

Now as these spirits thus enter into the cavity of the brain, so they pass from there into the pores of its substance, and from these pores into the nerves; where, as they enter...now into some, now into other pores, they have the power to change the shape of the muscles in which the nerves are inserted, and by this means to cause motion in all the parts. Just as you may have seen that the power of moving water...is alone sufficient to move the different machines in the grottos and fountains of our kings' gardens, according to the various arrangements of the pipes conducting it.

For Descartes, the only differences between humans and animals were that the human body was more complex than that of animals, and possessed a soul; otherwise, humans and animals functioned in exactly the same way:

Finally, there is a reasoning soul in this machine; it has its principle site in the brain, where it is like the fountaineer who must be at the reservoir, whither all the pipes of the machine are extended, when he wishes to start, stop, or in some way alter their actions.

According to Descartes, sleep and waking were dependent on the flow of spirits in the brain. When one was awake, more spirits flowed through brain, causing it to distend, while, when we sleep, the amount of spirits flowing through the brain was reduced, causing the brain to shrink. (Descartes illustrates this in the diagram above; the state of the brain during sleep and in waking hours are depicted at the top and bottom of the image, respectively). Descartes also believed that the flow of spirits around the brain was controlled by the pineal gland, which was also the seat of the human soul.

...it is necessary to believe that the spirits, flowing through the nerves into the muscles, and inflating them sometimes more and sometimes less, now some, now others according to the different ways in which the brain distributes them, cause the movements of all the limbs; and that the little threads of which the internal substance of the nerves is composed serve the senses.

Whereas the ancients regarded animal spirits to be weightless and invisible, Descartes argued that they were actually liquids that flowed through the nerve. In the seventeenth century, mechanics was cutting-edge technology. Hence, Descartes described human behaviour as little different from the mechanisms governing the movements of automata; he regarded the human body as a machine, and described the function of the human body, including the nerves, in terms of hydraulic-powered mechanics:

If the fire A is close to the foot B, the small parts of this fire, which, as you know, move very quickly, have the force to move the part of the skin of the foot that they touch, and by this means pull the small thread C, which you can see is attached, simultaneously opening the entrance of the pore d, e, where this small thread ends...the entrance of the pore or small passage d, e, being thus opened, the animal spirits in the concavity F enter the thread and are carried by it to the muscles that are used to withdraw the foot from the fire.

Descartes expounded his views on human and animal behaviour in De Homine ('The Treatise on Man'), which was published in the early 1630s. Despite his gross errors regarding the function of the nervous system, De Homine was highly influential; it provided the first mechanistic interpretation of human physiology, and, in this sense, laid the foundation for the biological study of behaviour. Descartes can also be credited with the first description of the reflex arc (above).

The experiments of Dutch microscopist Jan Swammerdam (1637-1680) would soon prove Descartes wrong; they would also pave the way for the modern understanding of nerve function. Swammerdam carried out experiments in simple preparations of nerves and muscle from the frog:

if...you take hold, aa, of each tendon with your hand and then irritate b the propending nerve with scissors, or any other instrument, the muscle will recover its former motion, which it had lost. You will see that it is immediately contracted, and draws together, as it were, both the hands, which hold the tendons.

This showed unequivocally that animal spirits originating in the brain were not required for muscular contractions. Swammerdam continued to perfect his experimental procedure, and devised a method to measure precisely the change in the volume of muscle as it contracted. This involved placing the nerve-muscle preparation into an air-tight syringe, and observing the movement of an air bubble in the end of the syringe in response to the muscle contraction.

If we have a mind to observe, very exactly, in what degree the muscle thickens in its contraction, and how far its tendons approach towards each other, we must put the muscle into a glass tube, a, and run two fine needles through its tendons, where they had been held before by the fingers; and then fix the points of those needles, neither too loose nor too firmly, in a piece of cork. If afterwards you irritate, c, the nerves, you will see the muscles drawing, d, the heads of the needles together out of the paces; and that the belly of the muscle itself becomes considerably thicker in the cavity of the glass tube, and stops up the whole tube, after expelling the air. This continues till the contraction ceases, and the needles then move back to their former places.

This experiment was very significant: not only did Swammerdam inadvertently invent the plethymograph, but he also provided a basis for the mathematical measurement of biological phenomena, a hallmark of the scientific revolution that would follow. More importantly, it showed that Descartes was incorrect in his prediction that muscles do not change in size when they contract. Swammerdam also clearly emphasises that his findings are not specific to the frog, but instead are generally applicable.

From these experiments, therefore, it may, I think, be fairly concluded, that a simple and natural motion or irritation of the nerve alone is necessary to produce muscular motion, whether it has its origins in the brain, in the marrow, or elsewhere...Experiments on the particular motion of the muscles in the frog; which may be also, in general, applied to all the motions of the muscles in Men and Brutes.

Although the work of Swammerdam would mark the beginning of the end for animal spirits, various researchers would continue to explain nerve function in those terms, and it was not until the end of the eighteenth century that animal spirits would finally be exorcised. Instrumental in the exorcism was Luigi Galvani (1737-1798), the Italian biologist and physician who discovered bioelectricity; in the process, he also provided the basis for neurophysiology.

Galvani's inspiration came to him as he sat in the kitchen while his wife prepared frog soup. Galvani noticed that muscular contractions occurred when the nerves of the frog were accidentally touched with a knife. In his laboratory at the University of Bologna, Galvani left the lower half of a dissected frog left near an electrical machine, and again noticed that the legs twitched even when they were not in contact with the machine. Then, as Galvani's assistant was drawing sparks from the machine's brass conductor, he touched one of the nerves in the frog's leg with a knife he was holding, causing the muscles to twitch and the legs to kick:

While one of those who were assisting me touched lightly, and by chance, the point of his scalpel to the internal crural nerves of the frog, suddenly all the muscles of its limbs were seen to be so contracted that they seem to have fallen into tonic convulsions.

Galvani concluded that muscular contractions were caused by the flow of electricity from nerve to muscle, and began to think of "animal electricity" as a fluid, produced in and secreted by the brain, which flowed along the nerves to activate the muscles. Thus, nerves eventually came to be viewed not as hollow tubes but as conductors of electricity. However, lacking the technology to measure the electrical currents generated by the nerves, Galvani was not able to obtain any direct evidence. But the scene was set for the discovery of nerve function.

From the mid-nineteenth century onwards, real progress was made in the understanding of the functioning the nervous system. Emil du Bois-Reymond (1818-1896) made great advances in our understanding of the electrical activity of the nervous system, and is considered to be the father of modern electrophysiology. Working at the University of Berlin, where he would be appointed to the Chair in Physiology, du Bois-Reymond invented a highly sensitive galvanometer, with which he could measure the electrical impulses produced by nerves, and an induction coil with which he electrically stimulated nerve fibres. With this apparatus, du Bois-Reymond discovered that the voltage across a nerve cell membrane (what we now call the resting potential) diminished upon stimulation of the nerve. With his galvanometer, he also detected what he called the "action current" - the change in transmembrane current that occurred when a nerve was stimulated - and noticed that this was propagated along the length of the nerve fibre, and in muscle:

The law of muscular current may be shortly expressed as follows: Any point of the natural or artificial longitudinal section of muscle is positive relation to any point of the natural or artificial transverse section...every particle of a muscle, however minute, ought to produce a current in the same manner as the whole muscle...As to the nerves...they are possessed of an electromotive power, which acts according to the same law as muscles.

At around the same time, Herman von Helmholtz stimulated nerve fibres at different distances from muscle, and compared the delay in muscle contraction to calculate the velocity at which the nervous impulse was propagated along the fibre. His estimate of the conduction velocity was 27 metres per second (myelinated fibres are now known to have a conduction velocity of 70-120 m/s, while unmyelinated fibres conduct action potentials at velocities of up to 2 m/s).

By the turn of the century, then, it had been established that the nervous system is composed of cells, and that these cells could generate electrical signals. However, the question of how the signals are transmitted from one cell to another, or from nerve cell to muscle, remained unanswered. This situation would not last for long, however, because, in the first half of the 20th Century, with the development of new techniques, the understanding of nerve cell function began to progress in leaps and bounds.

The German pharmacologist Otto Loewi (1873-1961) was one of the key figures involved in determining that the electrical impulses generated by nerve cells are transmitted to other cells via a chemical messenger. Loewi devised a simple experiment which demonstrated that nerve fibres release a chemical when stimulated. He removed the hearts from two frogs, and placed them in separate chambers containing warm Ringer's solution, so that they continued to beat. Upon stimulation of its vagus nerve (which innervates the organ), the frequency of beats of one of the hearts was reduced. From the chamber containing the heart, Loewi took some of the solution in which the organ was immersed, and transferred it to the chamber containing the other heart. This caused the beating of the second heart to slow down, and the only conclusion that could be drawn was that stimulation of the vagus nerve innervating the first heart caused the release of a substance into the solution. Loewi had, for the first time, identified a neurochemical tramsmitter, and he called it vagusstoff (meaning vagal substance), because it was released from the vagus when the nerve was electrically stimulated. Later, when the chemical structure of that chemical was determined, it would be given the name acetylcholine.

Loewi's work was extended by Henry Dale, and the two shared the Nobel Prize for Physiology or Medicine in 1936. Loewi had conducted his experiments on the parasympathetic branch of the autonomic nervous system, and doubted that acetylcholine was released by the parts of the nervous system that controlled voluntary actions. Dale, who had identified acetylcholine as a putative neurotransmitter in 1914, showed that it was released at the neuromuscular junction, and isolated it from mammalian organs. He also showed that all preganglionic nerve fibres in the autonomic nervous system released acetylcholine; formulated 'Dale's Principle', which states that a nerve fibre releases only one type of neurotransmitter (this is now known not to be the case); and coined the terms 'cholinergic' and 'adrenergic', to describe nerve fibres which release acetylcholine and adrenaline, respectively.

In the early 1950s, Alan Hodgkin and Andrew Huxley carried out a series of 5 experiments which would determine how the electrical impulse (or action potential) was generated. By poking newly-developed, very fine microelectrodes directly into the giant axon of the squid, Hodgkin and Huxley measured the changes in membrane voltage that occurred during the action potential. They thus established precisely the mechanism of the action potential: it was generated by the movements of ions across the nerve cell membrane, in both directions. Like Loewi and Dale before them, Hodgkin and Huxley would share a Nobel prize for their.

Also in the 1950s, the use of microelectrodes and the invention of the electron microscope would allow researchers to study the function of the synapse in greater detail. In 1951, John Eccles succeeded for the first time to insert microelectrodes into neurons in the central nervous system, and measured both types of responses that could occur in response to neurotransmitter release (the excitatory and inhibitory postsynaptic potentials). A little later, Bernard Katz used microelectrodes to investigate the neuromuscular junction, and determined that the fluctuations in the membrane voltage of the nerve were due to the release of acetylcholine from synaptic vesicles, and that this release was quantized. Eccles and Katz, too, would win Nobel Prizes for their contributions to neuroscience.

Today, researchers have many powerful techniques at their disposal, allowing them to investigate the function of the nervous system at the sub-cellular level. We now know that the generation of action potentials relies on membrane-spanning proteins called voltage-gated ion channels. These barrel-shaped molecules contain pores which open and close in response to changes in the voltage across the nerve cell membrane, and control the movements of ions elucidated by Hodgkin and Huxley. Individual amino acid residues within the central pore of the ion channels act as voltage sensors and specify which species of ion can pass through the pore, and the channels are able to adopt millions of different conformations to control the flow ions across the nerve cell membrane. However, the brain is the most complex structure in the known universe, and with every question about its function that is answered, many new ones are raised.