Bengt Falck – The Lund Histochemist… Research by Richard Burack

 

Bengt Falck – The Lund Histochemist Who Perfected the Fluorescence Method – He made visible what had been invisible.

By Richard Burack

During the first half of the 1940s a Doctoral Candidate at the University of Lund, Nils-Åke Hillarp, studied stained terminal threads of nerves (terminal axons) of the autonomic nervous system of frogs and rats microscopically. He confirmed an 1888 observation of Santiago Ramon y Cajal, who described “varicose terminal axons” while studying embryonic chick cerebellum in his cloistered Barcelona laboratory. The varicosities were closely juxtaposed and regularly spaced, making the fibrils look like strings of beads. Hillarp also proposed functional interpretations that materially elevated his reputation in the 1950s and early 60s, when neurohistology was a small, esoteric specialty. (He suffered an untimely death from cancer in 1965.)

Hillarp defended his thesis in 1946. Three years later a freshman medical student, Bengt Falck, knocked at his office door and respectfully asked permission to work in his laboratory.

In the same year that Hillarp received his Doctoral Degree Professor Ulf von Euler, of the Karolinska Institute, proposed that certain stimulatory nerves store and release noradrenaline, a substance of the “monoamine” category. By 1955, and surely by 1961, the world of physiology generally viewed noradrenaline as a major chemical transmitter of nerve signals. However, as late as 1961 he was unable to show conclusively that the noradrenaline was located in terminal axons. A few eminent scientists were therefore skeptical to the point of rejecting his proposal.

In August 1961 Bengt Falck, who had become an untenured Research Fellow in Lund, telephoned von Euler to reassure him that noradrenaline is stored in terminal axons. He based his assertion on irrefutable histochemical studies of axons in various organs from various animals. In 1970 Euler received a Nobel Prize and until his death in 1983 remained Falck’s grateful admirer.

But why was it Bengt Falck who called Professor von Euler? Why was it not Hillarp? Was he not still Falck’s senior? There is a disconcerting story here of enormous scientific success followed by widely distributed dubious allegations, charges and countercharges.

Hillarp’s Unanticipated Change of Address

In an unanticipated move, Associate Professor Hillarp announced in mid-1959 that he would take indefinite leave of absence from his tenured post in Lund at year’s end. He would join Professor Arvid Carlsson, a Lund pharmacologist, in Gothenburg 125 miles to the north. The University of Gothenburg had invited Carlsson to chair a new pharmacology department and Carlsson invited Hillarp to join him as a Fellow to devote himself solely to research.

When Bengt Falck heard the news from Hillarp he felt devastated and abandoned. Hillarp, his closest friend, seemed intent during 1958 and in early 1959 to work closely with Falck to find a way – any way – to make noradrenaline and related monoamine substances – adrenaline, dopamine and serotonin – visible within tissues. They were bent on locating them precisely within nerve cells that store them because they were known to be present in nerve tissues and to be biologically active, although exact intracellular locations were unknown. It was therefore almost impossible to understand their potential functions.

Attempts to convert noradrenaline to a colored substance within intact cells had never been successful in any laboratory. Hillarp was forced to opt out of this most preliminary work. He needed time to find a new home in Gothenburg, arrange the conditions of his indefinite leave and, most importantly, to see if the Swedish Medical Research Council would support his independent work in Gothenburg. The Council was accommodating; it provided him a grant for, among other ends, histochemical study of the localization of transmitter substances. As for Falck, he continued throughout 1959 to give as much time as he could to their ambitious joint project but was also significantly restrained because he had to present and defend his Doctoral Dissertation in 1959.

Unfortunately, Hillarp’s opportunity to contribute to the fluorescence project in Gothenburg was critically limited. Except for a microscope fitted with filters to transmit fluorescent wavelengths, Carlsson’s department had no histochemistry equipment whatever. Hillarp was marooned in a place with no equipment to work personally on the project he had begun with Falck. There was, then, mystery inherent in his determination to move far from the apparatus he would need. No doubt, Carlsson badly wanted him in his department; Hillarp was a scientist who could generate ideas. Carlsson needed a replacement for his two creative Research Fellows Åke Bertler and Evald Rosengren, whom he left behind in Lund.

Falck to the Rescue

Luckily, Hillarp’s closest and most loyal friend was Bengt Falck (Figure 6). He had mentored Falck for nine years and had the utmost confidence in his talent as a histochemist and in his integrity. He simply asked Falck in mid-1959 shortly after announcing his intention to move if he would continue the project they had begun together in Lund: to devise a reliable method to make amine-containing cells visible under a microscope. Naturally, Falck agreed. He was happy to be asked.

The Falck-Hillarp Relationship was Special

Having spent a decade working day after day in the same office-laboratory space, their desks facing one another, Hillarp and Falck trusted each other implicitly. Except for Nils-Åke’s refusal to discuss any aspect of his early life, they could discuss any subject, including politics, on which they agreed. Their views were moderately left of center. Åke was not as open with strangers, though – he was basically an extremely shy man. When strangers came to visit the laboratory, Hillarp usually vanished while the outgoing Falck spoke with them about histochemistry.

Hillarp was kind and generous. When he made his casual agreement with Falck both tacitly understood that the one was not asking the other to be his slave. Falck would be doing the hard, meticulous histochemistry, and if he cared to publish some of it as his, it would be all right with Åke. It was no secret between them that Åke would be receiving funds from the Swedish Medical Research Council for specific missions.(1) As long as Åke’s name appeared as author on a few creditable articles and his position in the department of pharmacology in Gothenburg was made clear, it would be good enough to satisfy the Council that it had invested its money in him advantageously.

Falck put the Gothenburg department’s name on every article he drafted for publication whether or not Hillarp was a co-author, as a kindness to Carlsson even though he knew him only superficially. He knew that the more often the Gothenburg department’s name appeared on publications, the more likely all its applications for grant support would receive serious consideration, and money for research was scarce.

He had got to know Arvid Carlsson superficially when he was in the nearby pharmacology department at Lund and never got to know him better when he was in Gothenburg. Most of what he knew about him came from his association with Åke Bertler and Evald Rosengren, who were research fellows in pharmacology. Bertler and Rosengren would visit Falck in the histology department and tell him they envied his luck to be associated with Hillarp.

Trusting Falck’s ability to be discreet, they vented their unease working where they did and the reasons for it. A cardinal problem was the way Arvid doled out their modest stipends. He was in the habit of paying them monthly, sometimes late. (2) It was so unlike the habit of any other department head they knew about, that they – sensitive Rosengren especially – feared him. He imagined, correctly or not, that “the boss” was letting them know that if he thought they failed to work hard, he might not retain them. Shy Rosengren was invaluable to the department for his talent in biochemical pharmacology. If his story is credible, there is strong tendency to think that his department head was too single-mindedly focussed on the production of his laboratory; that he was apparently inattentive to emotions of those who worked under him. The more extroverted Bertler, who liked to joke and mimic, was less intimidated than Rosengren. Ill as he was with terminal heart failure 200 miles away in Linköping, he sent word through his wife that confirmed Rosengren’s predicament. Bertler minced no words: He sized the boss up as cold, calculating and ruthless.

In 2005, I met with Evald Rosengren at his home in Lund where he lived with a sister. We sat next to a colorful flower garden in his back yard and chatted for more than an hour. He was a very polite, refined man, who speaks English well, but slowly and thoughtfully. In the course of conversation, which I directed with soft-spoken questions, he repeated first-hand what I had previously learned second-hand from Professor Falck. He repeated his habit of renewing his and Bertler’s appointments monthly. He repeated that this was highly unusual practice for a department head, that nearly all renewed the appointments and their stipends every six months or annually. Rosengren said that he had always feared Arvid Carlsson because he was cold, distant, spoke only about laboratory matters, and never made pleasant small talk or asked him about himself.

It was perfect opportunity to ask what I had been curious about for years: why Professor Carlsson’s name did not appear with Åke Bertler’s and his as a co-author of an extremely important article that appeared on January 15, 1959 in the prominent Swiss medical journal Experientia. He explained it unhesitatingly:

He and Bertler were intrigued by studies of a highly respected neuroscientist Marthe Vogt that led her to conclude (1954) (Vogt, M., J. Physiol.: 123:451) that the concentrations of noradrenaline vary throughout the brain. They were also aware that in 1957 British scientists (Montagu, and her mentor Weil-Malherbe) found that dopamine, the immediate precursor of noradrenaline, is present in whole brain. Therefore, they deemed it important to see if dopamine’s concentration in the brain was uniform or variable and suggested to their director A. Carlsson that this would be a useful study to undertake. They were disappointed to hear him reply that it sounded to him like a waste of time – “a foolish idea”. They felt differently, especially after consulting another, somewhat senior member of the pharmacology department, Dr. Gunnar Hollunger.

Hollunger allegedly agreed wholeheartedly with them, encouraged them to carry out the study themselves, and advised them to have a witness, who could testify that the work was theirs alone. And so they did. Carlsson observed them working but never interfered or asked what they were doing.

To their amazement, after studying brains of seven species of laboratory animals, they found dopamine in concentrations of the same order of magnitude as noradrenaline’s. More surprising was that very little noradrenaline was present where the dopamine concentration was high. The dopamine “is predominantly localized in the corpus striatum,” an anatomical component of the little understood “basal ganglia”. Bertler and Rosengren’s broad knowledge of neuroscience and clinical medicine allowed them to include the following in their January 1959 article, which was based on work completed in 1958:

The distribution of dopamine in the brain… indicates that it may have a function of its own and [is]… an intermediate in the formation of noradrenaline and adrenaline. The results favour the assumption that dopamine is concerned with the function of the corpus striatum and thus with the control of motor function.“ [That is, control of normal muscle activity].

A common clinical observation supported their conclusion. The drug reserpine that was in wide use throughout the world in the late 1950s and 1960s for the treatment of high blood pressure in humans was widely known to have a serious side effect. It caused symptoms that mimic Parkinson’s disease, a cruel illness marked in its advanced stage by a paucity of muscle movements. In 1956 Bein described what was known of the effects of reserpine. (Bein, H.J. Pharmacol. Rev. 8:435–483, 1956 ). In the same year Holzbauer and Vogt in Edinburgh (their paper was received by the Journal of Neurochemistry on November 26, 1955) described depletion of noradrenaline in the cat hypothalamus (where the concentration of this monoamine was normally present in highest concentrations). P. Holtz, H. Balzer et al. in Frankfurt am Main, mentioned en passant in a paper received by the Archives of experimental pathology and pharmacology (Arch. exper. Path. u. Pharmakol. Vol. 231: p. 340 on June 26, 1957) that administration of dopa shortens the length of anesthesia in dogs when they had been pretreated with reserpine, indicating that they were well aware that dopamine had been depleted from the animals’ brains by reserpine. 1957 Montagu reported what was obviously dopamine in brains of rats and other animals (Montagu, K.A. Catechol compounds in rat tissues and in brains of different animals. Nature 180:244–245, 1957. It is the first reported discovery of this monamine in brain). Her laboratory director Weill-Malherbe quickly confirmed her observation. (Weil-Malherbe, H., and A.D. Bone. Intracellular Distribution of Catecholamines in the Brain. Nature 180:1050–1051, 1957.)

Independently and practically simultaneously the fact that reserpine releases dopamine from brain was reported by Weill-Malherbe and Bone in Nature 181;1474, 1958 and by Carlsson, Lindqvist, Magnuson and Waldeck in Science 127: 471, 1958. The most significant consequence of Bertler and Rosengren’s 1957 work and their January 1958 publication of it was its coming to the attention of Dr. Oleh Hornykiewicz in the physiology department at the University of Vienna. He was working at the time on dopamine metabolism (Holzer, G. And O. Hornykiewicz. Dopamine (hydroxytyramine) metabolism in the rat brain. (Arch exp. Path. Pharmak. 237; 27–33, 1959) and had worked with dopa between 1954 and 1956 when he collaborated with two Viennese neurologists prior to a 1957 Fellowship in Oxford. (Hornykiewicz, O. and G. Niebauer. The polyphenol oxidase in human blood serum. Arch. Exp. Pathol. Pharmakol. 218:448–456, 1963.

They were searching for the cause of muscle tremor in Wilson’s disease, a rare abnormality of basal ganglia. Eventually it was found that patients with the disease have an abnormal copper-binding protein in their blood (Gastager, H., O. Hornykiewicz and H. Tschabitscher. Copper and polyphenol oxidase in the blood serum in cases with clinically manifest and subclinical forms of hepatolenticular degeneration [Wilson’s disease]. Wien Zeitschr. Nervenheilkd. 9:312–319, 1954).

In the course of experimenting with this protein Hornykiewicz found that it combines with and destroys dopa as well as the dopamine. (Hornykiewicz and Niebauer, 1954). Therefore, when Hornykiewicz read Bertler and Rosengren’s report, its content struck an authority on dopamine and basal ganglia. He knew that diseased basal ganglia can play a role in diseases of muscle movement, and was experienced with both dopa and its derivative dopamine. He immediately switched his research interest from rat brains to human brains and led the pack in showing beyond doubt that Parkinson’s disease in humans is due to a lack of dopamine in their basal ganglia. In an extraordinarily dramatic way he then demonstrated the almost immediate alleviation of symptoms in long suffering victims of Parkinson’s disease when he injected dopa into their bloodstreams.

The achievement was wholly on a par with the discovery of insulin and penicillin.

Dr. Rosengren and I continued to talk. He told me that he stayed on in the pharmacology department long after his colleague and collaborator Bertler decided to leave – about two or three years after Carlsson left for Gothenburg. Bertler was more clinically oriented than he and wanted to become a clinical pharmacologist. He had become so respected for his work on dopamine and was so good at recounting the history of its discovery in the brain that he had no trouble gaining a full professorship at the University of Linköping as a clinical pharmacologist. Bertler’s salary greatly improved when he made the move. The University paid part of it and the remainder came from grants bestowed by the Swedish Medical Research Council.

The Annual Review of Pharmacology, a highly respected journal, wrote Bertler and Rosengren, asking them to write a historical review of their work with dopamine. Bertler was asked to read it in Milan at an international pharmacology congress. Rosengren said it was an honest and factual review but shortly after its presentation the Medical Research Council suddenly cut off their research grants to Bertler.

Asked why he thought this happened, Rosengren shook his head, parted his clightly clasped hands and answered that it was beyond his comprehension since Bertler was an honest, intelligent and successful scientist. He could only guess that there was a force or forces antagonistic to Bertler at a high level. It happened so soon after the Milan conference that he tended to think that there was someone who pressured on Council who may have been displeased with Bertler’s presentation.

Rosengren did not suffer as Bertler did, but by this time he was working his way up the academic ladder at Lund and had reliable sources of research grants. In any case, money was not as important to him as it was to Bertler because he was not married, had no children to support and lived with his sister. She was trained as a biochemist and worked in the biochemistry department.

Before Bertler died of disease less than a year before this interview with Rosengren, it was possible to communicate with him by email. He lacked the energy to answer himself, leaving it to his wife. Her messages, transmitted to me in telephone calls, confirmed all details that Rosengren recounted.

The Rosengren-Bertler information led me to question Professor Falck about his own relationship with Arvid Carlsson. He had been at Lund for ten years while Carlsson was an associate professor in the nearby department of pharmacology. Falck reported that Carlsson was detached; he could not remember ever being invited into his office in Lund. In fact, Falck could not remember ever being in Carlsson’s office in Gothenburg except once. It was when he visited Hillarp in January 1961. Carlsson had very little to say. He was cool and aloof.

“Didn’t he ask how your work was going? Weren’t you there in January 1960 to show Hillarp the slides showing that you were on the way to success with the method? That was an important occasion, wasn’t it”

“Yes, I know; but Carlsson said very little. I imagine he said a few words, but nothing of any importance.”

Portrait of Bengt Falck

Professor Falck, a tall goodlooking man with a quick smile, is the sprout of a distinguished family from Malmoe where his father was a City Judge, and grandfathers and great-grandfathers on both sides had been legal scholars, judges and well-known doctors in Scania, the most southwestern section of Sweden. During World War II the large home where he was raised in Malmoe required the services of live-in domestic help. It was a busy home – a meeting place for intellectual people who were virulently anti-Nazi. (Until 1943 most thoughtful Swedish people feared that Hitler would be victorious but they became more optimistic after the Allied powers’ victories at el Alamein and Stalingrad in 1943).

Educated at the ancient University of Lund, as his father and paternal grandfather had been, Bengt Falck grew up to be a refined, worldly and tolerant man, an excellent conversationalist with a finely honed sense of humor. He seemed young for a man of seventy-eight. He has an idealistic streak in him, which must have been even more fervid when he was in his mid-thirties, his age when he took control of the histochemistry laboratory as Hillarp’s successor in January 1960.

As a young scientist he was admittedly a romantic – he pursued science for the sheer joy of it without thought of earning kudos or prizes and it was his absolute belief that everyone else who was a scientist felt similarly. It is not hard to believe, therefore, that his sense of “team spirit” was strong.

It dawned on this interviewer during conversation with Falck that his and Hillarp’s upbringings could not have been more different. In spite of it they shared the same values. Their immediate families were close, too. In summers they rented the same beach house, their wives and children were close, they enjoyed weekly foursomes of bridge. Family relations were so close that Bengt’s wife once jokingly asked him why they even bothered to have a home of their own.

It is interesting that Falck admires Hillarp for his intelligence and his virtues. You have to wonder if Hillarp’s admiration of Falck was influenced by appreciation that he had as his closest friend a very bright patrician who shared his idealism, even his political views, yet was down-to-earth and never for a moment condescending. Nils-Åke Bengtsson, who changed his name to Hillarp, son of an ironmonger and sometime evangelical minister from nondescript Hässleholm, and Bengt Olof Torsten Falck, son of a judge from sophisticated Malmoe: they complemented one another almost perfectly.

The Years 1960-1962

Once Åke was in Gothenburg 125 miles north of Lund they remained in touch by telephone. In 1960 Hillarp would from time to time take an early train to Lund for a short day-visit. He wanted to be sure that Falck had all the equipment and ancillary materials he needed. He also wanted to speak with his former technician about laboratory tests left over from studies of his own, that she was finishing up. On one of these visits he asked if she would mind considering herself Dr. Falck’s full-time technician. She was happy to; she liked Dr. Falck.

Hillarp’s irregular visits became less and less frequent. When I expressed curiosity about how frequent they had become by the end of 1960, Falck picked up his telephone and rang up his former laboratory technician. Between her guess and his they occurred irregularly, at most about once every six to eight weeks.

I was inquisitive about the contents of their telephone conversations. Falck explained that a large part of them was just friendly talk, an opportunity to keep up with each other’s lives and family. But I wanted to know if the calls were useful to him.

“Of course they were,” Professor Falck informed me. “They were always inspiring. Hillarp was a highly gifted scientist.”

“Did he ever offer an innovative idea?”

“He was very inspiring.”

“I can understand that; but did he ever offer a bright idea that made you change the direction of whatever you were doing?”

“Well, just off hand I can’t think of any but it was always inspiring to speak with him. We exchanged ideas and discussed various experimental possibilities. Hillarp was an encouraging friend and an outstanding scientist, so our telephone conversations meant a lot to me. ”

As hard as I tried and as hard as Professor Falck tried, I could never find out if the conversations were ever anything more than “inspiring.” Weeks later, when we were continuing to “chat” by email, I learned that those telephone calls that I had assumed were very frequent were less common than I originally thought. They peaked in frequency and intensity in March or April 1961, as we will discover.

Here is What Really Happened

Early Studies of Amines

When the Parisian physiologist Alfred Vulpian (1826-87) announced in 1856 at a meeting of the Collège de France that adrenal medulla tissue forms colors when it is exposed to oxidant chemicals, he put no special name to the phenomenon. In 1865, when the German microscopist Henle (1809-1885) found that chromic acid, a very strong oxidant, combines with the medullary cells to form a dark pigment, he called the reaction “chromaffin” and the discolored cells “chromaffin cells.”

In 1910, in London, Sir Henry Dale’s chemist colleague A.J. Ewins examined colored derivatives of catecholamines: adrenochrome and noradrenochrome. They are pink compounds that appear at once when either is oxidized or in an alkaline solution. After Ewins’s work, study of the chemistry of adrenaline and noradrenaline derivatives attracted continuing interest.

When noradrenochrome is exposed to an oxidant in an alkaline solution, it develops green fluorescence (Paget 1930; cited by Loewi in his 1935 Nobel Lecture). In 1948-50 chemists Ehrlén and Lund identified Ehrlén’s fluorophore derived from adrenaline, determined its structure and did the same with the fluorophore of noradrenaline, namely, trihydroxyindole (THI):

Figure 1. Noradrenolutin or Trihydoxyindole (THI)

Figure 1. Noradrenolutin or Trihydoxyindole (THI)

Intriguing Characteristics of Fluorescence

Fluorescence is an intriguing phenomenon. Substances with fluorophore properties consist of molecules with atoms so structured that when light of a specific wavelength, usually in the ultraviolet range, strikes them (incident light), an electron in a particular orbital suddenly gains energy and vibrates more vigorously. (Electrons spin about an atomic nucleus in orbits somewhat as planets spin about the sun. Their vibrations are reminiscent of Brownian motion.) The increased energy state is very short-lived; when the excited electron returns to its prior state, the energy it took from the incident light is emitted in the form of heat plus light of a longer wavelength (less energy) than the excitation light. The intensity of emitted light can be measured; the excitation it causes in a photoelectric cell is proportional to the concentration of the chemical being quantified.

To determine the concentration of a fluorescent substance, the desired wavelengths of incident and emitted ultraviolet light are chosen by rotating quartz prizms with calibrated dials. The substance is in solution in a small receptacle of pure quartz. (Fortunately, ultraviolet light passes through pure quartz glass.) Accordingly, the assay apparatus is known as a photofluorimeter. In the mid-1950s at the National Institutes of Health (NIH) in Bethesda, Maryland, Dr. Robert Bowman, who was an inveterate tinker, built one of the first machines for assay of fluorescent substances. He let various NIH scientists test his fluorimeter as he improved it. (3) It became available as an “Aminco-Bowman spectrophotofluorimeter” and was used in laboratories throughout the world.

The Search for Monoamines in Tissues

In 1954 Dr. Marthe Vogt, a German emigrée pharmacologist in the UK, (4) isolated noradrenaline from animal brain. It would be of utmost value to find exactly what cells in the brain and what nerve cells elsewhere contain it. Professor Harold Burn at Oxford soon complicated von Euler’s noradrenaline transmission concept with a curious proposal: that noradrenaline is released peripherally not by terminal nerve fibrils but by clusters of cells situatedq close to them. He published this proposition in 1960. Therefore, if it were possible to establish that noradrenaline is located in high concentration in the terminal axons of nerves, Euler’s concept would be vastly strengthened.

In 1959 Falck and Hillarp published a paper describing their first joint attempts to localize catecholamines in brain tissue. By this time there had been not only Vogt’s 1954 article; she published another in 1957. Katherine Montagu in England examined extracts of whole brains in 1957 and reported finding both noradrenaline and dopamine in comparable amounts. In that same year Weil-Malherbe and Bone described monoamines in the central nervous system; Sana et al. followed in 1958 in Japan. The distribution of dopamine in brain was the subject of the extremely important early 1958 paper by Åke Bertler and Evald Rosengren in Lund. They showed that the dopamine was heavily concentrated in the region of the basal ganglia.

Hillarp and Falck’s Early Work in 1959

Although Hillarp and Falck spent 1958 considering how to use histochemical methods to precisely localize the sites of noradrenaline and dopamine in brain, they began their quest with a method that did not involve making amines fluorescent. Falck, Hillarp and Torp (1959) found evidence suggesting that dopamine might be stored in a previously unknown type of chromaffin cell (5) in ruminants. Employing the same histochemical method, they looked for these cells in rats’ brains. Their results were instructive – neither the noradrenaline nor the dopamine in brain tissue is located in chromaffin cells.

Developing a Fluorescence Method

Professor Olavi Eränkö in Helsinki published a notable article in Endocrinology 1955 (57: 363–368) with the title Histochemistry of noradrenaline in the adrenal medulla of rats and mice. He was the first to describe making adrenal medulla tissue fluorescent by exposing it to formaldehyde vapor. The article motivated Hillarp and Falck to develop a fluorescence method with formaldehyde to pinpoint the locations of catecholamines in all neural tissues.

The literature contains a plethora of information about the potential for making amines fluorescent. One need only read Iversen’s excellent 1967 monograph (Chapter 4B) to learn that as early as 1918 Otto Loewi observed the fluorescence of adrenaline in alkaline solutions. Hillarp and Falck knew of Ehrlén’s published method of converting catecholamines with an oxidizing solution of iodine to form adrenochrome and then applying ammonia to convert it to fluorescent adrenolutin (trihydroxyindole or THI). They knew too that formaldehyde preservation (and hardening) of some amine-containing tissues can produce fluorescent substances; it was widely known to histochemists.

Hillarp and Falck in Helsinki

In November 1959 Hillarp and Falck visited Helsinki where Hillarp presented a paper at a meeting of the Finnish Endocrinology Society on storage and release of adrenaline and noradrenaline from adrenal medulla cells. The meeting’s chairman was Olavi Eränkö, a lone pioneer in attempts to make these two monoamines fluorescent with formaldehyde. In 1952 Eränkö had noticed that when he “fixed” (hardened) adrenal medulla tissue in formaldehyde solution a faint green fluorescence often became visible. In 1955, histochemical study convinced him that the fluorescence was due to reaction of formaldehyde with adrenaline.

It fell to Falck and Torp early in 1960, after Hillarp had left for Gothenburg, to make ultrathin frozen sections of several tissues including the adrenal medulla, treat them with iodine and then ammonia in the fashion of Ehrlén, freeze-dry them and examine them with a fluorescence microscope. The medullary cells took on an intense fluorescence but in tissues supplied with adrenergic nerves: heart, iris, salivary gland, blood vessels – they could see no intact terminal axons. Their conjecture was that the amine in exceedingly fine axons had been dissolved by the watery agents they used, causing a “diffusion” problem. (Their results looked like modern art.)

This first unsuccessful attempt at making single axons fluorescent was not discouraging, though. Methods other than Ehrlén’s were available. In fact, while trying the Ehrlén method, Falck had already begun studies with formaldehyde vapor precisely because the chat with Eränkö had inspired him and Hillarp.

Falck and Torp’s Studies with Adrenal Medullary Tissue

Application of gaseous formaldehyde to sections of adrenal medulla caused a marvelous degree of fluorescence. However, adrenal medullary cells are clumped together and have no axons. It was not possible to distinguish individual cells under the microscope and Falck realized that the formaldehyde gas contained too much water vapor. As with the earlier Ehrlén stain there was “diffusion.”

Falck gave careful thought to how he might lessen the concentration of water vapor that was contaminating the gaseous formaldehyde set free from heated solutions. Early in 1960 he hit upon the idea of using a crystalline polymer of formaldehyde, namely, paraformaldehyde. When he heated the polymer, formaldehyde vapor was released and he was sometimes able to make the noradrenaline in axons fairly well visible.

Much encouraged, he improved the method by drying the crystals with a technique well known to elementary chemists. Concentrated sulfuric acid takes up water vapor readily. When the crystals were enclosed in a container in which there was also an open beaker of sulfuric acid, much of the water in them was removed. However, excessive drying vitiated the power of the heated polymer to cause fluorescence.

Heating partially dried crystalline paraformaldehyde improved results dramatically. In January 1961 Falck and Torp had for all intents come close to perfecting the method. The volume of their work suddenly increased and their old freeze-dryer suffered a mechanical breakdown in February. His technician caught her hand under a loosened belt and suffered a fractured finger. Then and there, Falck decided to build a new and more capacious freeze-dryer. He had to build one; the capacity of commercially available models was inadequate.

His gift for invention was remarkable. He bought inexpensive fixtures and fittings that had been manufactured for uses unrelated to freeze-dryers and used them to construct an excellent, big one. He worked on the project whenever he found spare time, slowly improving it as he continued to study a variety of tissues from a variety of species in the animal kingdom. He considered it finished in July 1962.

Falck and Torp made certain that the fluorescence in nerve fibers was limited to noradrenaline: when they took measures to cause degeneration of the fibers shown by von Euler and others to release noradrenaline, the fluorescence did not appear.

The Falck and Torp Publications

During 1961 Falck prepared two papers for publication. The first dealt with studies of adrenal medulla cells; the second with noradrenaline-containing terminal axons. He sent each in turn to Hillarp who suggested a few changes and, true to their tacit understanding, blessed their authorship by Falck and Torp. He knew that they had independently done every bit of the histochemical work and that Hillarp’s name need not categorically be included as an author of every one of Falck and Torp’s publications.

Hillarp needed only some published evidence of meritorious research to satisfy the Council’s main expectation of him, to study the intracellular localization and functions of tissue amines. The Council got its money’s worth in 1962 with fat Supplements to Acta physiologica scandinavica, a journal with worldwide readership.

Falck and Torp accumulated a vast amount of data beginning late December 1960 and January 1961 to fill the two manuscripts. The first of the Falck and Torp papers – A Fluorescence Method for Histochemical Demonstration of Noradrenalin in the Adrenal Medulla – was submitted to a translator and then to Medicina Experimentalis, a Swiss-American journal, on September 19th, 1961, which published within the year. They sent the second – New Evidence for the Localization of Noradrenalin in the Adrenergic Nerve Terminals – first to Hillarp to ask if he had editorial advice, then mailed it to a translator in November, and submitted it to the same Swiss-American journal in mid-December of 1961. This paper reported results of fluorescence studies of hundreds of organ specimens from a variety of animals, clear evidence that the studies must have begun early in 1961. The publisher began its Christmas-New Year vacation early, found it in their mailbox on January 4th, 1962 and printed it within the same year. The paper is historically important because it was proof that they completed their fluorescence method early in 1961. Falck and Torp were the first to see glowing intraneural monoamines that were obviously chemical transmitter substances. They revolutionized neuroanatomical and neurophysiologic research.

Accordingly, a statement in the “scientific” literature that fluorescent nerve terminals were “at last” observed on “August 26, 1961.” is incorrect. A professor at the University of Gothenburg made this allegation in 1995 in a speech at a Wenner-Gren Symposium. It was published in 1996. (6) Sadly, the untruth is repeated in a University of Lund archival volume purporting to contain the official history of the background and development of a BioMedical Center in Falck’s own medical faculty: BMC’s rötter, Lund 2001 [H.Westling, Ed.] pp.57-70. Unless corrected, the volume will live in posterity and in the history of the distinguished University of Lund, established 1666. The allegation comes not only from the the above cited publication alone but from an earlier one as well by the same author and by Carlsson (1986).(7)

In the 1995 article mention of Falck and Torp’s 1962 publication is conspicuously in the text or in the appended list of references. In the 1986 article Making Visible the Invisible there is no mention of Falck and Torp’s work in the text; it is buried instead in a reference to it in the bibliography. The practical concealment of the reference lessened the need to retract the assertion that the world first saw fluorescent axons on August 26th, 1961. It is a gravely serious matter to find published articles that seem to have to make Bengt Falck invisible.

When these tactics came up for discussion during the 2003 interview with Falck, I asked why he took no action when he became aware of them. If, in his opinion they distorted history and his place in it, he should have objected. He answered that he genuinely believed that concrete proof in the form of his and Torp’s publications would overcome their dishonesty. He believed that truth always wins out in the long run. He said that he realized how naïve he had been only when he discovered that a persons in high academic positions were gulled by these articles.

For the first time, I gently remarked that the flurry of prizes he received and the praise von Euler and Hillarp showered on him must have made him realize that he might one day be a candidate for a Nobel Prize. He seemed embarrassed even to hear this but with considerable reluctance admitted that he was aware of occasional rumors that his name had come up “but probably not seriously” in conclaves of the Nobel Scientific Committee. “It’s hard for me to take that seriously,” he added.

Falck’s Recognition; Hillarp and Carlsson’s Dependence on Him

Back in 1960-63 Falck was certainly pleased by his success in the laboratory but never lost his strong sense of team loyalty. In addition to the Department of Histology in Lund he added the Department of Pharmacology in Gothenburg as a site of origin of every paper he turned out whether or not Hillarp was an author. Hillarp, as pointed out, needed to be an author only on some of Falck’s articles to satisfy the expectations of the Swedish Medical Research Council. Any and all mentions of the department in Gothenburg certainly benefited Carlsson. It aided his applications for research grant money from University and other Foundations in Sweden as well as the United States, including the National Institutes of Health.

Falck readily acceded to Hillarp’s insistence on a prevalent European tradition that authors be listed alphabetically. More often than not Falck benefited because F came before the first letter of the last names of nearly all his coworkers. On three major papers Carlsson’s name was on Hillarp and Falck’s work. Many scientists elsewhere, were led to believe that someone other than Falck performed the hard histochemical work in these cases. Carlsson was not a histochemist; and by his open admission (see page) had no adequate histochemistry equipment in his department.

It is obvious that Hillarp and Carlsson were dependent on Falck’s continuing collaboration – the renewal of his academic appointment at Lund was essential to them. On March 8, 1962 Carlsson, who held an imposing title: Professor and Chair of the Department of Pharmacology at the University of Gothenburg, wrote to the authorities at Lund who would decide whether to renew Falck’s appointment as an untenured Assistant Professor. He and Hillarp wanted to ensure that they were fully aware of his expertise and importance. Carlsson wrote on his departmental stationery:

It seems to me essential to emphasize that Falck as well has played an important role in these studies that Hillarp and I [sic] have undertaken. When Falck became a member of this research team, he had already provided evidence of his originality in fundamental research with studies of the ovary. He must accordingly be considered as richly deserving as us older members of the team; indeed more so, for he has been responsible for an even greater share of the research accomplishments than we [have been].

Carlsson spoke the truth here. At a later juncture he may have wished he had not.

Falck becomes a tenured professor

A year later (1963) the committee on promotions at the University of Lund considered whether Bengt Falck at last deserved a tenured professorship. It was a formal process in which experts from other universities were asked their written opinions. By this time Hillarp had left Gothenburg and had been Professor of Histology at Stockholm’s Karolinska Institute for a year. Lund sought his written opinion of Falck’s qualification. These opinions are not confidential; they are available in Lund’s University Library. Hillarp’s opinion is too lengthy to repeat in toto but its highlights can be related.

Aside from repeating the Carlsson quote of the previous year, Hillarp reviewed Falck’s career from the time he became associated with Lund’s Department of Histology in 1949 when he was a medical student. Between 1949 and 1951 he was a novice, learning basic histology methods. In 1952 he received his first university appointment as an Assistant.

The complexity of the project for a Doctorate that occupied him between 1952 and 1958 cannot be overstated. Nor can the unusual nature of his findings. He discovered that manufacture of estrogen in the ovary involves two different hormone-producing cell types. One makes a testosterone-like substance and transfers it to the other, which converts it to estrogen. Textbooks still refer to this phenomenon as “Falck’s Two-Cell Synthesis.” He developed expertise in cell transplantation procedures, which impressed Hillarp, who averred that he had “paved an entirely new way” to do ovarian research.

By 1957 he began to find time to work directly with Hillarp and a new medical student novice Alf Torp on what appeared to be a variant form of chromaffin cell in ruminants. In September 1959 Falck defended his Doctoral Dissertation and received his M.D. Degree. He was thirty-two years old.

Hillarp was a mature forty-three in 1959; he had had ten years to observe and evaluate Bengt Falck’s potential and was certain that he wanted him to remain with him in the Department of Histology. The 1950s were exciting years for all the biological sciences because molecular biology had come of age. There were particularly talented men and women in every field, and faculties of good universities sought them for leadership positions. It is no wonder that Lund wanted to be certain that Bengt Falck was one of them.

Hillarp based his opinion of Falck’s merit on the contents of ten of the 29 works he had published to date on the histochemistry of monoamines, that is, adrenaline (A), noradrenaline (NA), dopamine (DA), and serotonin (better known in the science world as 5-HT.)

  1. He attributed to Falck “the first histochemical fluorescence method for cellular visualization of NA and A with a sensitivity of quite another order than earlier methods reported by others.”
  2. “Falck and Torp [were] the first to directly demonstrate that the adrenergic transmitter is accumulated in sympathetic nerve terminals.” [sic]
  3. “Without a doubt, Falck has [by this “sensational” method] made the most important contribution to neurohistology since Golgi’s and Cajal’s era.”
  4. Hillarp continued: “Professor U. von Euler, one of the most distinguished scientists within this field, has characterized this [fluorescence] achievement as one of the most important in neuroscience during the last twenty years. . .Without a doubt Falck has provided the most important methodologic contribution to neurohistology since the era of Golgi and Cajal. . . it is now possible to demonstrate cerebral monoamines on the cellular level, which has fundamental importance for the understanding of the role of these amines in the Central Nervous System.”

Indeed it was Bengt Falck’s histochemistry that first confirmed Hornykiewicz’s earlier understanding of the cause of Parkinson’s disease by demonstrating the normal presence of dopamine in basal ganglia. The dopamine was localized in the case of the caudate nucleus within submicroscopic structures belonging to terminal axons. Falck discovered this (1962) within two years of Ehringer and Hornykiewicz’s epoch-making study (1960) that there is profound loss of dopamine in the putamen and other basal ganglia of patients with Parkinson’s disease.

Bengt Olof Torsten Falck became a tenured Associate Professor at the University of Lund on February 1, 1964. His elevation to full professorship took place in 1970 after a more perfunctory consideration by the Committee on Promotions.

Hillarp’s praise was subsequently amplified by 1970’s Nobel Laureate Ulf von Euler, Professor and Chairman of the Department of Physiology and Pharmacology of the Karolinska Institute.

In a Foreword to a volume on Current Research on the Histochemistry and Function of Biogenic Amines containing the contents of a 14 January, 1977 Symposium in Lund to pay tribute to Bengt Falck on his 50th birthday, Professor von Euler enlarged upon the significance of his accomplishment.

“…if the fluorescence technique has supplied a secure foundation for the knowledge of the sympathetic nervous system, its importance for the elucidation of monoaminergic systems in the central nervous system may be regarded as more far-reaching. Although it was previously known that the brain contains noradrenaline, adrenaline and dopamine, the fluorescent technique made it possible to establish the localization and course of the fibre bundles…which use these transmitters.” [Falck, B. Cellular Localization of Monoamines. Progress in Brain Research. 1964; 8:28-44. Bertler A, Falck B, Gottfries CG, Ljungren L, Rosengren E. Some observations on adrenergic connections between mesencephalon and cerebral hemispheres. Acta Pharmacol Toxicol (Copenh): 21:283-9, 1964] [Bolding added]

von Euler undoubtedly was making reference to extensive work Falck did with brain tissue. In 1963 he gave a comprehensive lecture at a symposium in Galesburg, Illinois organized by a prominent biochemist Harold Himwich and his wife Williamina Himwich, a psychiatrist interested in chemical functions in the brain.

Falck, was one of the first invitees to lecture. He described his fluorescence method. Carlsson was there, listened to it, and offered no objection to the general understanding that Falck had perfected it.

In a section of the lecture entitled Fluorescent Structures in the Central Nervous System Falck described in detail and provided photographs of fluorescent monoamines he had found in hypothalamic nerve fibers. He detailed various types of fluorescent structures in the neurohypophysis. He continued with a description of varicose axons surrounding the infundibular arteries, in the walls of the third ventricle, and elongated masses of monoamine-containing fibers running from the mammillary region to the pons. The suprachiasmatic nucleus contains varicose fibers and many are located in the brain stem. He described finding dopamine in the caudate nucleus.

Among Falck’s studies of brain tissue in vertebrates, he was the first to visualize the entirety of a dopaminergic tract in birdbrain. In other labors He examined mollusc tissues: in cooperation with Professor Dahl of Lund, he visualized fluorescent monoaminergic varicose fibers in them.

Professor Falck’s full lecture in Illinois and many of his other accomplishments – not all – are found in; Falck, B. Cellular Localization of Monoamines. Progress in Brain Research 8: 28-44, 1964; in Bertler A, Falck B, Gottfries CG, Ljungren L, Rosengren E. Some observations on adrenergic connections between mesencephalon and cerebral hemispheres. Acta pharmacol toxicol (Copenh). 21: 283-9, 1964.

Bengt Falck personally supervised many histofluorescence studies at Lund; his list of publications is long.

In 1965 the Czech Academy of Sciences paid Dr. Falck recognition with the esteemed Purkinje Medal. von Euler was probably responsible for his receiving the Royal Swedish Academy of Sciences Medal, the Florman Prize, and the Axel Hirsch Award of the Karolinska Institute.

Unexpectedly, the Ernst Leitz Wetzlar company paid tribute to him in 1972 with a splendid gift: an elaborately equipped fluorescence microscope. It was the 800,000th microscope the company had produced since 1847. Leitz had honored only nine individuals in this in way in earlier years. Among these were Robert Koch, Paul Ehrlich and Albert Schweitzer.

Santiago Ramon y Cajal wrote many years ago that when a scientist receives recognition, he must accept the reality that there will be those who envy him in a malevolent way.

Intimation of Unpleasantness

There are people who, as we have already learned, and for reasons they alone know, wanted to depreciate Bengt Falck and Alf Torp’s accomplishment. For example, they maintained that, in reality, Hillarp’s so-called “Model Studies” performed in Gothenburg during 1961 paved the way for the development of Falck and Torp’s paraformaldehyde fluorescence method and that Hillarp controlled the histochemistry work at Lund by visiting it often and by telephoning instructions. Recollection by Falck’s retired technician Mrs. Kerstin Fogelström is that his friendly visits were irregular, took place at roughly six-week intervals, if that, and that from what she overheard, Drs. Falck and Hillarp had discussions that sounded to her like planning sessions. (Professor Falck contacted her in my presence by telephone.)

Hillarp’s visits did not increase in frequency even though he was shown in mid-January 1961 that Falck was able with promising reliability and uniformity to make terminal axons fluorescent in tissues from any organ supplied with terminal nerve fibers containing noradrenaline. There was no reason for a quick visit on his part at that time because Falck took it upon himself to gather up his slides and take a train to Gothenburg in January, to show them to Hillarp. Nils-Åke was pleased and undoubtedly informed Arvid Carlsson, the head of the department.

The Model Studies: Cause and Effect

Hillarp waited until March 1961 to take a train to Lund to view more of Falck’s slides. They sat down to discuss the nature and significance of his achievement. It did not take long for each to admit that neither knew the nature of the chemical reaction between formaldehyde and noradrenaline that caused production of a fluorescent product. Nor was the structure of the fluorescent product at all apparent. The matter was of some importance because the NIH group in Bethesda, whose members knew that formaldehyde would react in a test tube with monoamines of the serotonin type, were inclined to think that serotonin might be more important in nerve function than adrenaline and its cousins such as noradrenaline.

The subject obviously had to be studied. Hillarp was not an expert organic chemist but offered nevertheless to make a preliminary investigation when he returned to Gothenburg because it would require no histochemistry equipment. He did have a microscope fitted with filters that allowed detection of ultraviolet wavelengths.

Hillarp began his project in late March or sometime in April 1961, aided by Georg Thieme, a man with broad technical ingenuity. (Nearly every physiology/pharmacology department of any size employs a person of this sort. Their input is invaluable. Thieme had accompanied him from Lund.) Hillarp’s project was solidly prepared but time-consuming. It took about eight weeks to complete. Hillarp called them Model Studies.

In his usual generous way he included as authors anyone who contributed to it in the least. Falck was included because his work was the stimulus to undertake them. Hillarp burned the telephone lines discussing it with Falck. Falck’s input was needed because he alone knew the identity of every chemical he used to make axonal monoamines fluorescent.

Execution of the Model Studies

Hillarp dissolved eighteen amines in solutions of sugar, gelatin, protein (serum albumin) and gliadin, a plant protein, placed a drop of each mixture on a glass slide, exposed them to formaldehyde vapor, let them dry, and examined the spots with the fluorescence microscope.

The only spots that were fluorescent were those that had been dissolved and dried with serum albumin. It was not clear to him or Falck about why a “protein skin” around amine molecules enabled them to become fluorescent. The resulting paper by Falck, Hillarp, Thieme and Torp was influential because it established the basic conditions for making monoamines fluorescent by exposure to formaldehyde. The amine must have an -OH group on a carbon atom in a certain location and must lie within an animal protein skin.

Published in May 1962, the article had academic value for many laboratories including Lund’s but no practical value for the work that had been in progress in Lund since December-January 1961. After all, all axons have an animal protein constituent within them. It is now abundantly clear that Falck, laboring with a single professional co-worker, could never have accumulated as much data as he did in 1961, if he had been dependent on the Model Studies results to “know” how to proceed.

The structure of the fluorescent product was thought to be a quinoline or isoquinoline (Figure 2.), and this was confirmed when Hillarp consulted a German organic chemist H. Corrodi. Corrodi agreed that an isoquinoline is the critical product, which amounted to more academic information but was of no practical assistance to Falck. It was without doubt of educational interest and he appreciated it.

Fluorophore formation from noradrenaline (I) by formaldehyde (HCHO).

Figure 2. Fluorophore formation from noradrenaline (I) by formaldehyde (HCHO). First, a non-fluorescent substance tetrahydroisoquinoline (II) is formed. In a second step, two types of dehydrogenated isoquinolines (III) are formed; these are the eventual fluorophores. (Falck 1975)

The Significance of Varicose Terminal Axons

Falck and Torp’s results agreed completely with von Euler’s conceptions about the nature of the major chemical transmitter released by nerves that control blood pressure, activate glands, dilate the pupil, etc. That is, the fluorescence could be attributed to noradrenaline. In normal animals the brilliantly fluorescent, regularly spaced, little knobs on terminal axons corresponded perfectly with Cajal’s “varicosities” and Hillarp’s “beads”. As they peered at these intensely fluorescent structures densely filled with transmitter substance, they intuitively understood that these must also be the sites of release of the chemical that stimulates organ muscles, glands, and a vast variety of viscera.

A second Hillarp visit in 1961 at Falck’s Request

While completing their work with terminal axons of a variety of species and tissues (hundreds of specimens) Falck and Torp had begun to draft a second manuscript for the publication of their now immense amount of data on noradrenaline-containing axons in a variety of tissues of a variety of species. Falck wanted mutual agreement with Åke about new directions the studies should begin to take. It seemed to him senseless to delay collection of new data until after the manuscript was completed – he did not want to break his stride. Åke agreed and asked if the weekend of 26-27 August would be a suitable time. It seemed so to Falck and so Åke arrived in mid-morning Saturday the 26th. They met at the laboratory rather than at Falck’s country home because it was considerably closer to the train station and Åke had no car. (He never did drive a car.)

Falck asked if he would like to see his most recently obtained results. Hillarp said he had seen enough of his slides of fluorescent terminal axons in various tissues. Rather than go to the tedious trouble of perusing more, he suggested that they simply air-dry a rat’s iris, which would take 15 or 20 minutes and then Bengt could expose it to the hot dry paraformaldehyde vapor. He had not before worked with as simple a tissue as an iris or a mesentery, nor had Åke ever suggested it. Falck happily went along with the suggestion. They were truly pleased to see the congeries of terminal axons that Hillarp had years before stained with methylene blue. Hillarp was astonished at how much more colorful they were when they were treated with vapor from Falck’s heated paraformaldehyde and how much more conspicuous the “beads” seemed to be:

Iris of a rat

Figure 3. Iris of a rat. You see a network of varicose terminal axons. The closely juxtaposed varicosities are filled with a brilliantly green fluorophore derivative of noradrenaline x 600 (Falck, 1962)

Both men were in exceptionally good humor, mainly because they realized the tremendous, if not revolutionary, impact the fluorescence method could have on neuroscience. If Falck could make it work on brain tissue, it would be possible for them and others for generations to come to make sense of the brain’s interconnecting tracts of axons.

What better thing to do as they headed toward Falck’s old farmhouse for lunch than stop for a bottle of sparkling wine to drink a toast. It should now be clear, though, that any “scientific literature” that purports to say that August 26, 1961 is when the world first saw fluorescent nerve terminals is incorrect. Falck and Hillarp saw them in freeze dried specimens well before August 1961, viz., in January and March.

Other Publications in 1961-62

In 1962 Falck and Hillarp were occupied preparing two articles for publication as Supplements (No. 196 and 197) to Acta physiologica scandinavica. In this period Falck also made final touches to his freeze-dryer, tweaked it to enhance its operational ease and thereby permanently increased his capacity to handle a large number of specimens.

Contents of Supplement 196

Falck explored new ground when he learned that noradrenaline is present nearly everywhere in the upper brainstem where it is accumulated in fine varicose fibers. It was evident that noradrenaline is a synaptic transmitter in the central nervous system. Examination of the spinal cord led to similar conclusions.

It was interesting and new to learn that dopamine exists in high concentration in the region of the pituitary gland. The pituitary is often called the master gland of the body: its secretions orchestrate the functions of nearly all other endocrine glands. Falck performed all of the histochemical work in Lund. He added precision as well to the 1958 study of Bertler and Rosengren, who first discovered that dopamine is heavily concentrated in the basal ganglia, by showing that dopamine exists in “circumscribed and more or less continuous areas, including the amygdala (a basal ganglion) and in other cerebral ganglia. However the cell bodies of submicroscopic nerve fibers containing dopamine could not be seen; they were not sufficiently fluorescent. On the other hand, the caudate nucleus, another large basal ganglion, was highly fluorescent.

“All histochemical and pharmacological data strongly indicate that this fluorescence–mainly at least–is due to the dopamine present. The amine is probably localized to submicroscopic structures…” perhaps very many short fibers that form a felt-like border on the caudate nucleus.”

The 28 page Supplement No. 196 (1962) on cellular localization of brain monoamines is a classic of the mid-20th century. It confirmed the association of dopamine as a constituent of basal ganglia. The relationship between Parkinson’s disease and profound loss of dopamine in basal ganglia found by Ehringer and Hornykiewicz in 1960, and its replenishment with l-dopa by Birkmayer and Hornykiewicz in 1961 were dramatic antecedent discoveries. Hornykiewicz’s discovery was equal in importance to the discovery of insulin.

The relation between basal ganglion disease and Parkinsonism was established during the influenza pandemic of the 1920s. Many victims of the virus developed the typical tremor and body rigidity of the affliction described in 1817 by James Parkinson of Hoxton, England. Autopsies revealed lesions of the basal ganglia, presumably caused by the virus. Throughout the 1950’s, the drug reserpine was widely used to treat hypertension and doctors discovered that overdosed patients developed Parkinson-like signs and symptoms. The ill effect was common knowledge to clinicians. As early as 1955 Holzbauer and Vogt showed that reserpine depleted monoamines in the brains and adrenal glands of dogs.

Contents of Supplement 197

Falck wrote a monograph with the genteel title Observations on the Possibilities of the Cellular Localization of Monoamines by a Fluorescence Method (1962). In 25 pages he expanded on the truism that many problems in understanding adrenergic mechanisms depend for their solution on accurately localizing them at the cellular level. Stating simply that his laboratory at Lund had experimented with several methods for localizing catecholamines, he wrote that the most promising of them is based on the principle that amines can be transformed into intensely fluorescent isoquinoline derivatives by reaction with formaldehyde.

Approximately fifteen beautiful black and white microphotographs of what he had seen in color with his fluorescence microscope please the eye. The extraordinarily dense plexus of terminal axons surrounding arterioles and a venule are astounding.

Other Related Work

Falck described unpublished work of a preliminary nature that he was performing with J. Häggendal. They examined frog heart muscle. In these cold-blooded creatures the adrenergic transmitter is adrenaline, not noradrenaline; they visualized the adrenaline in frog sympathetic terminal axons. The work was completed and published by Falck, Häggendal and Ch. Owman in 1963. Important reference is also made to the studies carried out in 1962 on molluscs. Falck found time before undertaking the search for the location of dopamine in mammalian brains to collaborate among others with Professor E. Dahl of the Zoology department at Lund and with Margit Lindqvist, a skillful laboratory assistant in Gothenburg, on a study of monoamines in mollusc neurons.

At a point where noradrenaline alone had been found in mammalian brains, a report was published with H. Welsh and M. Moorehead (J. Neurochem. 6: 146, 1960), describing 5-HT in the nervous elements of molluscs. Always inquisitive, cerebral ganglia of snails underwent careful examination with the fluorescence microscope. The study revealed facts that “prove almost conclusively that 5-HT and a catecholamine, probably dopamine, are localized in mollusc neurons.” The histologic pattern and intraneuronal distribution of the amines in molluscs closely resembled that of adrenergic neurons studied in vertebrates. Indeed, Lindqvist’s fluorophotometric assay confirmed the presence of dopamine in the cerebral ganglia of the snails.

Falck’s Apparatus

It might be assumed that the development and perfection of a technique would have been sufficient expectation of any seasoned researcher with a single professional colleague, Alf Torp, during 1959-62. It is understandable that during the 1961 period Falck became increasingly dissatisfied with the adequacy of his equipment for handling large amounts of work. One need only read the Falck and Torp articles, the two monographs and the mollusc study to appreciate the number of specimens that needed to be processed. Examination of even a small piece of tissue can yield hundreds of adjacent sections that must be observed and followed sequentially under the lens of a microscope to follow the course of a small nerve or a tract in the brain.

Bengt Falck’s freeze-dryer is shown below. He planned and built it resourcefully, using parts that were never intentionally manufactured for use in a freeze dryer.

aparatus

Figure 4. Falck's self-designed, self-constructed Freeze Dryer. Ironically, it was a cause of bad blood

Hillarp Moves to the Karolinska Institute in Stockholm

In late August 1962 Hillarp moved his family to Stockholm and went to work as its new Professor of Histology. Annica Dahlström wrote about the department he inherited and Hillarp’s first inspection visit in April of the year. [8]

“The professor who had been in charge of it for years had been “far from inspiring. All microscopes were locked away under plastic hoods. Somebody wanting to use a microscope had to ask the professor for the key and then relate in detail why the use of a microscope was necessary.” She was considering quitting the department until Hillarp came through on his first inspection tour in April 1962. It was “like a hurricane sweeping through the old department, blowing away dust and stiff authoritarian reign and introducing a strange new feeling of enthusiasm into the building.”

Hillarp found himself with five students ranging in age from 21 to 29, of whom Dahlström was the youngest. Five others soon joined them. The new professor fully intended to exploit the fluorescence method, supposedly taught it to them, taught them the anatomy of the autonomic nervous system and divided his Monoamine Club into small groups, each to specialize. Fuxe and Dahlström were to study localization of monoamines in brain [by the fluorescence method]. Others were assigned to study peripheral nerves, the physiology of the sympathetic nervous system and to learn electron microscopy.

The trouble with Dahlström’s description is that it omits an important fact. There was no histochemistry equipment in the department when Hillarp took over. He noticed this during his April inspection and thought it would be easy to correct by inviting Bengt Falck to join him and bring his equipment with him. He was particularly anxious for him to bring his large freeze-dryer along.

Hillarp ran into unexpected difficulty when Falck politely turned his close friend down. He sincerely appreciated the offer but he was born and raised in Scania. He lived on a charming rural farm in the village of Tirup near Lund, where he and his wife happily raised children, bees and a few crops. He did not wish to leave this life, thought it would wound his children, and his wife was also adamantly unwilling to move to the big city of Stockholm.

Hillarp was bitterly disappointed, tried hard to persuade Mrs. Falck to change her mind and, for the first time ever, exchanged a few brief, harsh words with his old friend. He implied that Falck was letting him down, but he soon relented and their friendship remained as it had been. Falck understood his predicament and promised to help him in any way he could short of moving to Stockholm.

Whether Dahlström was aware of Hillarp’s frustrated attempt to lure Falck from Lund to Stockholm is unknown.. In the event, she did not report Falck’s friendly offer to enable Åke to have a freeze-dryer and other necessary equipment built in his department. Instead, she added to the paragraph above the following sentence:

“Not unexpectedly, because rivalry among scientists can be heated, the team worked in spirited competition with Falck’s group in Lund and Carlsson’s in Göteborg.”

“Spirited Competition?”

It is entirely possible that Annica Dahlström thought she was involved in “spirited competition” because she was very young and inexperienced in 1960-65 and could not have known what had happened in Lund during that period. Yet, approximately thirty-five years later she misrepresented events that took place during those years. It is logical to ask who might have filled her mind with these misrepresentations.

Hillarp initially appointed her and a fellow student, twenty-six year-old Kjell Fuxe, to be the team to learn the fluorescence method but there was no apparatus and, as it turned out, no adequate expertise even in the person of Hillarp, to set them on the path.

Falck’s Helping Hand to Hillarp

In mid-August 1962, in response to Hillarp’s telephoned request, Falck generously invited his student Kjell Fuxe to Lund whenever he was able to come. He would gladly show him how to handle his freeze-dryer and how to make monoamines (noradrenaline, adrenaline, dopamine and 5-HT) fluorescent by exposing tissue sections to vapors of heated paraformaldehyde.

Falck went further. When Hillarp asked about the structure of his new, big freeze-dryer, and added that he would like to have Georg Thieme build one like it in his Stockholm department, Falck immediately offered his help. He would instruct Fuxe not only how to use the freeze dryer; he would teach him about the structure of the apparatus and how he had been able to assemble it from items that can be bought inexpensively, modified, and be used to make a facsimile of it.

To avoid waste of time, he would order two hard-to-find items right away from a Lund trading company to expedite their delivery. Falck followed through without delay and the items arrived in Lund from England on or about September 21, and he had his Lund glassblowers modify them. He then sent them to Hillarp as gifts to allow Thieme an early start.

Fuxe was a serious student. He arrived in October and worked earnestly. In May 2003 Professor Falck’s senior technician, who lives in retirement near Lund, provided testimony by telephone that perfectly confirmed Falck’s recollection of Fuxe’s visit: “. . . the young man came to learn.” The technician went out of her way for several days and had taken pains, as had Falck, to teach him as much as they could, explaining how the freeze dryer was constructed of a miscellany of parts and how it worked.

1962 had been a productive year. Falck and Torp published their classic New Evidence for the Localization of Noradrenaline in the Adrenergic Nerve Terminals and prepared numerous other articles that involved the fluorescence method. Falck completed construction of his large freeze dryer in July and it was working well.

 

packslip

Figure 5. Packing slip accompanying parts secured by Falck for Hillarp's department in Stockholm.

1965: Fuxe Earns his Doctorate

Two years and four months later Fuxe presented and defended his doctoral thesis Evidence for the Existence of Monoamine-Containing Neurons in the Central Nervous System (April 26, 1965). It chanced that Falck read a long paper to the Royal Physiographic Society in Lund six weeks beforehand on February 10th and published it shortly afterward (Falck and Owman). Their monograph described essentially every detail of the fluorescence method for demonstrating biogenic monoamines in cells as Falck and Torp developed it between 1960-62. That is, it described neither more than less than Kjell Fuxe had learned in Lund.

In introductory pages of his thesis and in a following footnote, Fuxe provided a brief recapitulation of events leading to the development of the fluorescence method and the source of the techniques he used in his research.

Whether he wrote all of it or someone helped him is not known. It could not have been Hillarp, who died of cancer six weeks earlier, in mid-March. In any case, Fuxe began where bad taste leaves off. He wrote that the method for making axonal monoamines visibly fluorescent with formaldehyde vapor had been developed primarily by Hillarp when he was in Gothenburg, which was impossible because the necessary histochemistry apparatus did not exist in Gothenburg. Arvid Carlsson, the group’s scientific mentor after Hillarp had become seriously ill, openly admitted. (See page 42.) [Italics added]

Fuxe continued:

“Later on Falck (1962) applied [Hillarp’s] method also on freeze dried tissues…”

On the next page Fuxe inserted a long footnote:

The following description of the method [the one Fuxe used] is based on a comprehensive summary of the relevant data on the method published in an article on central monoamine neurons by Hillarp, Fuxe and Dahlström (Symposium on Release of Biogenic Amines, February 1965, in press (sic)). This section of the article was written by Hillarp already in the early autumn of 1964, and is in turn a summary of an extensive manuscript on the histochemical fluorescence method which Hillarp wrote during the summer 1964. The summary was communicated to the members of the group in September 1964. The description (p.6) by Falck and Owman (Acta Universitatis Lundensis, Sectio II 1965 No.7, Lund 1965) is evidently based on this summary. [Italics added].

In sum, Fuxe asserted for the record that the fluorescence method that he and his teammate Dahlström used to trace the course of monoamine-containing brain axons was based on an unpublished source. He claimed that the method(s) was based on a “summary” circulated by Hillarp to his students in September 1964. How Falck and Owman might have used an unpublished “summary” to write the most detailed description to date of the fluorescence method is beyond comprehension.

Nevertheless, one must not summarily accuse Kjell Fuxe, a young doctoral candidate, of insolence or ingratitude. One must consider the history Fuxe was certainly aware of and whatever history he may not have been aware of when the now public figure Arvid Carlsson replaced the moribund Nils-Åke Hillarp as the students’ supervisor.

Fuxe had undoubtedly read that Falck and Torp wrote the first paper describing the successful use of the fluorescence method on axons. Falck completed it no later than November 1961 because the printer noted on its first page that it was in his mail on January 4, 1962. It is possible that Fuxe did not know that the Falck method had been functioning in December 1960 and that Falck took a train to Gothenburg in January 1961 to show Nils-Åke Hillarp the first fluorescent axons. However, as an eager, ambitious student, Fuxe almost certainly read Falck’s good description of the method that he published in 1962 as a Supplement to Acta physiologica scandinavica. It is Supplement 196, which is less detailed than the Owman and Falck publication of February 10, 1965.

Fuxe also may not have known that Falck received an advance copy of his dissertation at a much later date than is customary and therefore was unable to attend its presentation and defense – his calendar was already too full. Instead, on April 24, 1965, two days before Fuxe presented it, Falck wrote to the Faculty of the Karolinska Institute to ask how a candidate for a Doctoral Degree could have written so distorted a history of the methodology that made his dissertation possible. Doctoral Dissertations, when they are approved, are always published and thereby carry the seal of approval of distinguished academic authorities.

The Faculty charged Associate Professor Lars Gyllensten, who was second in command in the Department of Histology, to ask Professor Arvid Carlsson for an explanation of Fuxe’s distorted history of the fluorescence method. If anyone knew why Fuxe had written what he did, it would (besides Gyllensten) have been Carlsson, if they had been properly performing their duties as academic supervisors.

Carlsson’s less then two-page response to the faculty is on a Gothenburg Department of Pharmacology letterhead and is dated 3 May 1965. The postal system must have been extraordinarily efficient in Sweden because it is also stamped as “received” on 3 May 1965. It is in Swedish, and it does not answer the faculty’s question. Instead, it is an ad hominem attack on Falck and contains inaccuracies. Its translation follows:

The representative of histology, Associate Professor Lars Gyllensten has asked me to provide the Faculty with my opinion on certain parts of an official letter of 24 April 1965 from Associate Professor Bengt Falck to the faculty. The description Fuxe presents on pages 7-8 of his thesis with respect to the development of the histochemical method is perfectly correct. In March 1961, when Hillarp was working in this department [Gothenburg] together with G. Thieme, they discovered and worked out the optimal conditions for the formation of fluorescent products of catechol- and indoleamines after exposing them to formaldehyde vapour in a dried protein film. The time was then ripe [sic] to apply the method to tissues. Since the department lacked the necessary histological equipment, Hillarp contacted his colleague, then Assistant Professor Falck in the department of histology at Lund, and they agreed to carry out the experiments jointly. On Hillarp’s proposal they first used air-dried stretch-preparations of rat mesentery [sic] and iris. Thanks to Hillarp and Thieme’s carefully performed model experiments, Hillarp and Falck were able by the end of September or beginning of October 1961, after less than a day’s work in Lund [sic] to demonstrate fluorescent structures in such stretch preparations having exactly the same appearance as Hillarp’s autonomic ground plexus. [Hillarp’s 1946 Doctoral Dissertation. The term “same appearance” is an exaggeration.] To apply the method on other tissues than these thin stretched preparations, a special procedure had to be developed in which tissues were freeze-dried, exposed to formaldehyde and then embedded and sectioned. This task turned out to be far from simple, calling for great technical skill and inventiveness. An essential part [sic] of the task was carried out by Falck in a meritorious way. Probably it was not unimportant that Hillarp took part by telephone and numerous visits to the department in Lund as the work progressed. Hillarp was now very concerned that Falck have full benefit from his achievement. The risk that a younger coworker’s contribution to the work of a team may be depreciated is considerable, as is well known, and there were strong reasons to believe that Falck would not be an exception. It is against this background that the following facts must be taken into account: – Falck and his collaborator Torp are co-authors of the publication reporting the above-mentioned model experiments.
– The above-mentioned experiments with these stretch – preparations were not published separately – reference to these “previous experiments (Falck and Hillarp, to be published)” together with a summary of a portion of the results is nonetheless described in Falck, Acta physiol. scand. 56, Suppl. 197, 1962 p.9. [This is false. There was no “stretch preparation” of mesentery.]
– Falck and Torp, and Falck, respectively, published the use of the method on [hundreds of various] peripheral tissues by themselves, including demonstration, among other things, of nerve terminals. Yet, as a symbol of the intimate cooperation of the department of histology in Lund with that of pharmacology in Gothenburg, it is stated [on the frontispiece of both publications] that they were issued from both Lund and Gothenburg. Arrangements of this kind can be disputed from various aspects but are probably inevitable in real life. That Hillarp here, as in so many other similar situations, showed a unique generosity is obvious. The present arrangement can, however, taking into consideration the above stated circumstances, not be considered unreasonable. It should be borne in mind thatFalck has not only made an excellent contribution in the preparation of the methodology but has also been responsible for the greater part of the practical carrying through of the mutual projects, for example concerning monoamines the central nervous system. The paper on monoamines [Supp.196] in the brain was considered a mutual project for Falck, Hillarp and me, the undersigned. Gothenburg May 3, 1965. Arvid Carlsson, professor

Regarding the allegation that Supplement 196 to Acta physiologica scandinavica was “considered a mutual project” Falck recalls no such agreement or any participation by Carlsson in the laboratory. He agrees, however, that Carlsson’s chemical suggestions transmitted to him through Hillarp helped him be certain that the fluorescence he found in basal ganglia represented dopamine, not noradrenaline. Hillarp drafted the paper in Gothenburg, added Carlsson’s name and sent it to Falck for comments. Falck had no objections, and the paper was sent to the publisher. Carlsson’s reference to it in the May 3 letter to the Karolinska Faculty is gratuitous and defensive.

In 1958 Carlsson became more interested in dopamine than he had been in serotonin in 1956-7. It was late in 1957 when his research assistants Åke Bertler and Evald Rosengren asked if he thought it would be useful to see where dopamine is located in the brain. and he said it would not be useful. (Rosengren, personal communication, September 2005.) Bertler and Rosengren made the study anyway on the advice of a somewhat more senior member of the pharmacology at Lund who thought it important to carry it our. Their results were vitally important.. Publication of their immediately stimulated the brilliant work of Hornykiewicz in Vienna.

Mitigating information that must be considered.

Falck and Hillarp were together trying near the beginning of 1964 to compile a publishable history of the method. They met twice, conferred by telephone and succeeded in drawing up a tentative outline of the points they thought they ought to cover. Unfortunately, they never finished their dissertation because Hillarp’s malignancy progressed too rapidly. He became too sick to continue.

Falck offered to try to finish it by the deadline set by Pharmacological Reviews, and to include Hillarp as an author. Hillarp, as in the past, would not accept credit for what he had not done himself. He expressed his appreciation but declined the offer. When Christer Owman joined Falck, they took up the project, edited the accumulated odds and ends of the draft, added to it, and produced the polished monograph that was published in February 1965. It is entirely possible that a partial outline (“summary”?) of the proposed contents, which of course was never published, was found in Hillarp’s desk after his death.

Fuxe should not be discredited because he was ambitious. In his younger years he may have been rash or susceptible to authoritarian suggestion. A lot of us were like him when we were medical students. The Fuxe-Dahlström team used the Falck (or Falck and Torp) method well – Fuxe was unquestionably a bright young man. He learned quickly, including enough about the construction of Falck’s freeze dryer to facilitate Thieme’s help in constructing a near facsimile of it.

By Dahlström’s published admission, the two had more trouble at first than Falck did with humidity and diffusion. When they fixed it, they worked hard and used the method to produce a large amount of good neuroanatomical data for which they deserve credit even if they did not invent the method they exploited. There is no compelling reason to believe that Fuxe (or Dahlström) knew, prior to Hillarp’s sad, fast fade from the scene, about Falck and Hillarp’s failed attempt to write a comprehensive history of the fluorescence method.

Perhaps a mellowed mature Fuxe will one day tell historians the reason he put such depreciatory text and so inflammatory a footnote in the introduction to an otherwise excellent dual dissertation. In the scheme of things it may be more useful if this presentation succeeds in modifying Dahlström’s second-hand view if events in the very early 1960s in Lund. Those skewed views have influenced the editor of the above-mentioned “BMC’s rötter”. It would be a simple matter for him to insert a corrective addendum to what he has written about the history of the fluorescence method at the University of Lund. If he would do that, it will be of far greater historical worth than trying to “settle scores.”

Bengt Falck and Nils-Åke Hillarp in Tirup 1963

Figure 6. Bengt Falck and Nils-Åke Hillarp in Tirup 1963


Footnotes:

1. Dahlström, A. Nils-Åke Hillarp: A Great Scientist. In Molecular Mechanisms of Neuronal Communication. Pergamon, 19956.

2. It was customary, Falck told me, for department heads to appoint research fellows for twelve, or at least six months, which provided them with a sense of security.

3. Personal communication from Dr. Julius Axelrod, who used it.

4. Vogt’s parents were neuropathologists and devoted communists. On Lenin’s death in 1924, Stalin asked Oskar Vogt to Moscow to examine Lenin’s brain for special features Vogt reported finding large cells that accounted for his brilliance. This was self-defensive nonsense. In 1933 Hitler wanted to kill Oskar and Cécile Vogt. His right wing supporter Bertha Krupp, who inherited control of the giant Krupp works, forbade it. Bertha, after whom the big German cannon (Big Bertha) of WWI was named, provided them with a home and laboratory in the Schwarzwald where they safely studied neuropathology and wrote monographs during WWII. Marthe went to England.

5. Unlike other chromaffin cells they stained with a red dye.

6. Dahlström, A. Nils-Åke Hillarp: A Great Scientist. In Molecular Mechanisms of Neuronal Communication. Pergamon, 1996.

7. Annica Dahlström and Arvid Carlsson, Making visible the invisible (Recollections of the first experiences with the histochemical fluorescence method for visualization of tissue monoamines) in: Discoveries in Pharmacology, Volume 3: Pharmacological Methods, Receptors & Chemotherapy MJ. Parnham & ]. Bruinvels, eds. © Elsevier Science Publishers B.V. (Biomedical Division) 1986.