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Rita Levi-Montalcini: The story of an uncommon intellect and spirit

2013; Elsevier BV; Volume: 252; Linguagem: Inglês

10.1016/j.neuroscience.2013.06.055

1873-7544

Moses V. Chao, Antonino Cattaneo, William C. Mobley,

Italian Fascism and Post-war Society

The breath of life gave out before her ideas. One hundred and three years was enough time for Rita Levi-Montalcini to transform our understanding of the nervous system and intracellular communication, but not enough to fully define her genius. Raised in affluence, disenfranchised by fascism, nurtured by an accepting and supportive cadre of mentors and colleagues in Italy and the US, Rita's life speaks of the power of curiosity, intuition, imagination, as well as an abiding concern for the less fortunate. Hers is a life to be honored – a story worthy of telling. Indeed, it is a story with chapters yet to be written and discoveries yet to be made. Born to an upper-middle class Jewish family in Torino, the family environment played a fundamental role in Rita's life and is beautifully described in Rita's autobiography "In praise of imperfection" (Levi-Montalcini, 1988Levi-Montalcini R. In praise of imperfection. Basic Books, Inc, New York1988Google Scholar) and in her letters. In her own words, "I was brought up in an environment that, though not permissive, was brimming with affection and never troubled by disagreements between my mother and my father. The possible negative influence of having been born and raised in a Victorian climate, unsuited to my natural tendencies, was mitigated by my mother's complete acceptance of the role prescribed for women during the reign of Queen Victoria and the first two decades of this century. The absence of complexes, a remarkable tenacity in following the path I believe to be right, and a way of underestimating the obstacles standing between me and what I want to accomplish – a trait I believe I inherited from my father – have helped me enormously in facing the difficult years in life." Together with her twin sister Paola, who was to become a very talented and renowned artist, Rita had an older brother (Gino, who became one of the most prominent Italian architects of last mid-century) (Fig. 1) and sister (Anna). Rita's somewhat conflicted relationship with her autocratic father, a very active engineer and entrepreneur who brought her up as a "freethinker", is epitomized by the dedication that opens her autobiography "To Paola and to the memory of our father whom she adored while he lived and whom I loved and worshiped after his death". Despite the fact that Rita and Paola had demonstrated outstanding aptitude for study in primary school, their father decided that they should attend a girl's high school, from which there was no possibility of going onto the university. After finishing high school, Paola's manifest artistic aptitudes enabled her to immediately join the atelier of Felice Casorati, an artist painter of international fame. In contrast to Paola's smooth transition to the next step in her career, for Rita defining the next step was difficult. She struggled with the question of what to make of her life. What was clear is that she felt no desire for a married life with children. Eventually her decision was to undertake medical studies. This necessitated both her father's approval and undergoing the studies needed to take the admission exams. Rita succeeded in both tasks and finally enrolled in Medicine at the University of Torino in 1930. This was a turning point in Rita's life and, as one can see now, in the future of neuroscience. Rita looked beyond difficulties to see the new possibilities and meaning that can come through setbacks and disappointment. She states, "I was grateful to my father for having kept me back from the university for so long. If I had studied classics after middle school, as had been my wish, I would certainly have enrolled in philosophy, a subject demanding strong powers of logical thought which, to my misfortune, I lack". Rita speaks about her early days in neuroscience in The Saga of the Nerve Growth Factor (Levi-Montalcini, 1997Levi-Montalcini R (1997) The saga of the nerve growth factor: preliminary studies, discovery, further development; World Scientific Pub Co Inc., Singapore; World Scientific Series in 20th Century Biology (Book 3).Google Scholar). Beginning in the early 1930s, while a medical student in the University of Turin, she studied with Giuseppe Levi, a neurohistologist who had mastered the method of Camillo Golgi developed in 1873 for staining neurons so as to enable the visualization of individual neurons and their expansive elaborate processes. In so doing, she entered a growing new discipline, that of neuroembryology, made possible by the establishment of the neuron doctrine by Cajal and His (in the 1880s and 90s), of Harrison's convincing proof (1907) that dendrites and axons originate from neuron cell bodies, and of Sherrington's discovery of the synapse (1897). Very significant was Roux's approach to embryological studies, using manipulation to define relationships between different embryonic tissues, thus moving from description to deciphering cause–effect relationships (Hamburger, 1980Hamburger V. Trophic interactions in neurogenesis: a personal historical account.Annu Rev Neurosci. 1980; 3: 269-278Crossref PubMed Scopus (45) Google Scholar). Rita notes the paucity of tools available to study the developing nervous system. But they were more than adequate to define fundamental aspects of development. Of great importance also was her ability to interpret the results of experiments without constraints imposed by earlier schools of thought. A singular example is the interpretation of the role that peripheral tissues have on their sources of innervation. An increasingly popular experimental preparation was the developing chick embryo in which the extirpation of a limb resulted in a dramatic reduction in the number of the motor neurons and sensory neurons that under normal conditions supplied the limb. Viktor Hamburger, a student of Spemann, the Nobelist honored in 1935 for his discovery of the 'organizer-induction hypothesis, interpreted the result as due to failure to obtain from the extirpated target a 'message' moved retrogradely in axons to recruit undifferentiated cells to become neurons in the number needed to adequately supply the target (Hamburger, 1980Hamburger V. Trophic interactions in neurogenesis: a personal historical account.Annu Rev Neurosci. 1980; 3: 269-278Crossref PubMed Scopus (45) Google Scholar). The idea of recruiting neurons from among undifferentiated cells fits well the predictions of the Spemann hypothesis. Published in 1934, Rita read the paper for several years. On the basis of her own studies, she came to a very different conclusion than Hamburger. The studies that Rita pursued under the mentorship of Levi in Turin in the 1930s are noteworthy not only for the strategy employed, and the impressive insights derived, but also for the extraordinary conditions under which many of them were performed. Before and after receiving the MD in 1936, Rita pursued studies inspired by Cajal's approach which emphasized the utility of examining successive stages of nervous system development. In retrospect, this choice would prove extraordinarily important because it emphasized a stage-by-stage investigation in which she surveyed the emergence of the neural elements that would make up the mature nervous system. Rita's early career was soon threatened by her expulsion from the university in 1938. She was a Jew, a non-Aryan, and was thereby deprived by the Mussolini government of all civil rights, including access to public schools and universities. Her remarkable reaction, demonstrating an indomitable spirit, was to create a small laboratory in her bedroom. She comments that the 'forced restriction' which prevented access to many experimental resources, was in fact fortuitous. It forced her to focus on the work, using the tools of the neurohistologist that were available to investigate an important question, the role that peripheral tissues had on innervating spinal ganglia. Continuing the link with Levi, she reinvestigated the results of limb extirpation in embryos sacrificed at 6 h intervals following surgery. The detailed studies of each of the stages in the response to limb extirpation pointed not to a change in proliferation of sensory neurons but rather to their failed survival and atrophy on the operated side. The observations gave evidence that the regressive changes began when the axons of neurons reached the surgical zone and failed to find their cellular targets. Thus, the findings ruled out an effect of extirpation on decreased recruitment of sensory neurons and pointed instead to increased degeneration and death. Published before the end of WWII (Levi-Montalcini and Levi, 1944Levi-Montalcini R, Levi G (1944) Correlazioni nello sviluppo tra varie parti del sistema nervoso. Pontificia Academia Scientiarum. Commentationes VIII:527–568.Google Scholar) under the auspices of the Pontificia Academia Scientarium, the work was soon discovered by Hamburger. Reflecting many years later on their very different interpretation of very similar findings, he wrote "I had sent a reprint of my publication on the wing bud extirpation experiments to Professor Guiseppe Levi, a distinguished neuroscientist and Director of the Anatomy Department of the University of Turin in Italy. He had given it to Dr. Rita Levi-Montalcini, who was an investigator in his laboratory. She stated later that she had been much impressed by the striking effects of the operation on the spinal centers as well as my detailed analysis, but she had serious doubts about my explanation of the hypoplasia in terms of the recruitment hypothesis. Dr. Levi-Montalcini had a hunch that the absence of the limb had resulted in a regression of differentiated neurons, and she set out immediately to test her hypothesis by doing daily cell counts in spinal ganglia… I think that she first had this intuition when she read my paper and that it took firm hold in her mind. From then on she had no doubt that this was the correct explanation of the reduced size of the nerve centers" (Hamburger, 1992Hamburger V. History of the discovery of neuronal death in embryos.J Neurobiol. 1992; 23: 1116-1123Crossref PubMed Scopus (80) Google Scholar). To Hamburger's great credit, and to resolve the controversy, in 1947 he invited Rita to come to his laboratory at Washington University in St Louis (Fig. 2). Within 2 years, the two had confirmed that cell death was the cause of the reduced number of neurons innervating the extirpated limb (Hamburger and Levi-Montalcini, 1949Hamburger V. Levi-Montalcini R. Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions.J Exp Zool. 1949; 111: 457-501Crossref PubMed Scopus (663) Google Scholar). Reflecting many years later on their very different interpretation of the limb extirpation studies, Viktor wrote "to an experimental embryologist of the Spemann school the idea that we might be dealing with the death of neurons would have been hardly conceivable. …But the neurologist of the Levi school was not encumbered by the mindset of the experimental embryologist. Dr. Levi-Montalcini had no difficulty in imagining a loss of neurons during development" (Hamburger, 1992Hamburger V. History of the discovery of neuronal death in embryos.J Neurobiol. 1992; 23: 1116-1123Crossref PubMed Scopus (80) Google Scholar). These studies confirmed and then extended in a large body of work that in the developing nervous system the target powerfully regulates the life and death of young neurons, a phenomenon with widespread implications for the structure and function of the nervous system. This insight is owed largely to Rita, who pursued an intuition with enormous energy and attention to detail without bowing to conventional thinking. While cell death in embryos was well established as a defined and biologically regulated event, supported by impressive studies of Kallius, his students Ernst and Glucksmann and others (Hamburger, 1992Hamburger V. History of the discovery of neuronal death in embryos.J Neurobiol. 1992; 23: 1116-1123Crossref PubMed Scopus (80) Google Scholar), the collaboration in St Louis resulted in 1949 and 1950 in the first definitive evidence for naturally occurring developmental degeneration of neurons. In particular, Rita observed in the developing chick embryo the massive degeneration of preganglionic sympathetic neurons in the cervical region during a very well defined and narrow time window of development (Levi-Montalcini, 1950Levi-Montalcini R. The origin and development of the visceral system in the spinal cord of the chick embryo.J Morphol. 1950; 86: 253-283Crossref Scopus (143) Google Scholar). Though surprising in retrospect, given the experimental context for the studies at the time, Viktor commented in 1992 that it was "not surprising that normally occurring neuronal death was discovered in the context of experimentally induced neuronal death, that is, by a lengthy and circuitous process". In the jointly published 1949 paper, Rita and Viktor made what would turn out to be extraordinarily important observations (1) that extirpation of the limb created a degenerative process essentially identical to that which occurs naturally in ganglia supplying smaller targets – i.e. the cervical and thoracic regions; (2) that in both cases the target was responsible for regulating or maintaining the number and size of sensory neurons. As they stated: "In the experimental situation, the reduction of the peripheral area is definitely responsible for the process of degeneration. It is possible that the same mechanism operates in the case of the normal cervical and thoracic ganglia. This would imply that in the early stages cervical and thoracic VL cells" (i.e. large exteroceptive sensory neurons) "send out more neurites than the periphery can support…. The experiment of peripheral overloading of cervical or thoracic ganglia should show whether this idea is correct". In this statement they forecast the development of a field of study and predicted future discoveries in neurobiology. Importantly, the stage was set for the next important discovery. In studies to modify the size of the target of innervation, in 1948 Elmer Bueker published studies using a mouse sarcoma implanted into the body wall of chick embryos to replace a limb (Bueker, 1948Bueker E. Implantation of tumors in the hind limb field of the embryonic chick and the developmental response of the lumbosacral nervous system.Anat Rec. 1948; 102: 369-389Crossref PubMed Scopus (113) Google Scholar). It followed earlier studies in which he attempted to use a number of different tissues as surrogate for the missing limb. Noting that implanting complex tissues impacted the status of both sensory and motor neurons, he chose to use a well-defined, homogeneous tumor implant; mouse sarcoma 180 as well as other tumors were tested. The results showed that only the sarcoma grew well and had a significant effect. The most interesting finding was the observation that successful tumor implantation and growth resulted in the enlargement of sensory ganglia whose axons entered the tumor. Cell number was much less affected than size and there was no apparent effect on the survival of motor neurons. He concluded that the tumor acted through its inherent physicochemical properties and mechanics of growth to impact sensory neurons (Bueker, 1948Bueker E. Implantation of tumors in the hind limb field of the embryonic chick and the developmental response of the lumbosacral nervous system.Anat Rec. 1948; 102: 369-389Crossref PubMed Scopus (113) Google Scholar). The findings fit well to the conception that a target of a certain size and specific properties would support the survival of neurons. Rita and Viktor soon repeated the studies and appreciated to a much greater extent the dramatic changes induced. Using implants of mouse sarcoma 180 or 37, the same rigorous and detailed approach to examining changes over time was most revealing. Rita discovered that sensory and sympathetic ganglia participated in the innervation and showed both hypertrophy and hyperplasia, but that motor neurons neither sent axons to the tumor nor shared in the changes seen in sensory and sympathetic neurons. The effect on sensory neurons was specific for the M-D population – neurons that we now know convey nociceptive information. Furthermore, the effects were far more dramatic than that seen with transplanted limbs and showed that direct contact of axons with tumor cells was not present. The 1951 publication with Viktor thus defined the impact of the tumors and pointed to the existence of a growth promoting agent delivered retrogradely by axons to neuronal cell bodies (Levi-Montalcini and Hamburger, 1951Levi-Montalcini R. Hamburger V. Selective growth-stimulating effects of mouse sarcoma on the sensory and sympathetic nervous system of the chick embryo.J Exp Zool. 1951; 116: 321-361Crossref PubMed Scopus (605) Google Scholar). But the real turning point in the analysis, one that would presage and hasten the discovery of how cells communicate with one another, was Rita's observation of the anomalous innervation of nontumor tissues. As she comments: "it dawned on me that the tumor effect was different from that of normal embryonic tissues in that the tumor acted by releasing a growth factor of unknown nature rather than by making available to the nerve fibers a larger-than-usual field of innervation. This hypothesis…became a certitude with me long before I obtained supporting evidence in its favor." Her comment in the Saga of the Nerve Growth Factor shows again her ability to imagine a novel explanation for a finding and pursue it with energy and passion. Asked by one of us many years later when she first knew that the findings forecasted a fundamental change in thinking about the nervous system, she answered (paraphrased) "when I saw that axons were entering the lumen of veins, in structures never seen before, I understood the ability of the factor to break the normal rules of biology". She discovered that even neurons distant from the tumor were affected, a response presumed to be mediated by access to the factor through the circulation. The speculated diffusibility of the nerve growth factor was demonstrated to be true in studies using tumor implants onto the chick chorioallantoic membrane, studies that demonstrated again the remarkable but specific growth promoting activity released from the tumor (Levi-Montalcini and Hamburger, 1953Levi-Montalcini R. Hamburger V. A diffusible agent of mouse sarcoma, producing hyperneurotization of viscera in the chick embryo.J Exp Zool. 1953; 123: 233-288Crossref Scopus (225) Google Scholar). It is understandable at that time that Rita and Victor could not differentiate between an enriched source of an endogenous factor or one induced only in a pathological setting – i.e. to decipher whether what they were observing was an exaggeration of a normal or a tumor-induced result. What was evident was that a powerful biological signal had been discovered. The hunt was on for the factor, but there were three hurdles to surmount: a reliable assay, a source abundant enough for purification, and the biochemical expertise to complete the task. Rita took the leading role in the search. The first was addressed when Rita traveled to Rio de Janeiro to learn from Hertha Meyer a method that could be used to examine directly the response to the sarcoma-secreted factor. Explantation of DRG or sympathetic ganglia into semisolid media allowed her to examine neurite outgrowth. The inclusion of sarcoma tissue placed at a distance of 1–2 mm resulted within 24 h in a spectacular outgrowth of neurites, a by-now classical signature of NGF action (Levi-Montalcini et al., 1954Levi-Montalcini R. Meyer H. Hamburger V. In vitro experiments on the effects of mouse sarcomas 180 and 37 on the spinal and sympathetic ganglia of the chick embryo.Cancer Res. 1954; 14: 49-57PubMed Google Scholar). Cultures in which sarcomas were absent, or in which control tissues were included, failed to show the effect. With the assay in hand, Rita returned to St Louis to continue the search, now with the assistance of Stanley Cohen, a biochemist, to address the second hurdle. The culture system allowed them to confirm that the growth effect was present in cell-free homogenates of the sarcomas first grown in the chick embryo (Cohen et al., 1954Cohen S. Levi-Montalcini R. Hamburger V. A nerve growth-stimulating factor isolated from sarcomas 37 and 180.Proc Natl Acad Sci U S A. 1954; 40: 1014-1018Crossref PubMed Google Scholar). Most of the activity was contained in the microsomal/nucleic acid fraction. The Nerve Growth Factor (NGF), albeit in crude form, was thus identified. This part of Rita's story is interesting in the good fortune that guided the work. In an attempt to determine what role could be attributed to the nucleic acid within the partially purified fraction, the team used snake venom as a source of phosphodiesterase. The surprising, and indeed serendipitous, result was the presence within the venom of an even richer source of a factor with the same properties of that made by the sarcomas (Cohen and Levi-Montalcini, 1956Cohen S. Levi-Montalcini R. A nerve growth-stimulating factor isolated from snake venom.Natl Acad Sci U S A. 1956; 42: 571-574Crossref PubMed Google Scholar). The purification protocol produced a preparation that was a protein with a specific activity 1000 times that in the sarcoma samples. The question of whether the two factors were the same was not yet resolved, but identical biological effects were detected in vitro and in vivo, and very similar biochemical properties, pointed to this possibility (Cohen and Levi-Montalcini, 1956Cohen S. Levi-Montalcini R. A nerve growth-stimulating factor isolated from snake venom.Natl Acad Sci U S A. 1956; 42: 571-574Crossref PubMed Google Scholar). Surmounting the last obstacle, NGF was soon discovered in the male mouse submaxillary gland, a finding suggested by the similar functions of this gland and its snake counterpart (Levi-Montalcini and Cohen, 1960Levi-Montalcini R. Cohen S. Effects of the extract of the mouse submaxillary salivary glands on the sympathetic system of mammals.Ann N Y Acad Sci. 1960; 85: 324-341PubMed Google Scholar). Later studies using more sophisticated tools demonstrated that the snake venom and mouse factors were indeed closely related. Significantly, this source of NGF provided the amounts needed for large-scale purification for structural studies, for those to investigate biological effects in mice and rats, and eventually for cloning the gene for NGF. In retrospect it is remarkable that in the face of what we now know are a host of neurotrophic factors acting on many different preparations, that Rita and Stanley found what appears to have been the same factor, NGF, in a tumor followed by snake venom followed by the mouse submaxillary gland. Good fortune, yes, but well deserved because at every step in the journey rigorous comparison was made of the magnitude and specificity of the biological effect and eventually the biochemistry of the factors. The dramatic effects demonstrated by the NGF proteins isolated from the various sources did not obviate the question as to what physiological role, if any, NGF plays. An extraordinarily important set of studies demonstrated that endogenous NGF was essential to the normal survival and differentiation of sympathetic neurons. Injection into newborn mice of antisera to NGF dramatically reduced sympathetic ganglion size and neuronal number (Levi-Montalcini and Cohen, 1960Levi-Montalcini R. Cohen S. Effects of the extract of the mouse submaxillary salivary glands on the sympathetic system of mammals.Ann N Y Acad Sci. 1960; 85: 324-341PubMed Google Scholar; Levi-Montalcini and Booker, 1960Levi-Montalcini R. Booker B. Destruction of the sympathetic ganglia in mammals by an antiserum to a nerve-growth protein.Proc Natl Acad Sci U S A. 1960; 46: 384-391Crossref PubMed Google Scholar). Cautious in her interpretation, Rita refrained from making the link that now seems so clear. Apparently influenced by the discovery of NGF or NGF-like activity in blood, and the existence of large quantities of NGF in the submaxillary gland of the mouse which at that time was thought possibly to take up and store NGF, she considered the possibility that a circulating source might be responsible or that indirect effects of the antiserum mediated by an immune response might be responsible. Indeed it took some time for the field to test and embrace the 'neurotrophic factor hypothesis' which states that the survival and differentiation of neurons are due to the presence of limited quantities of neurotrophic factors present at targets of innervation. Though Rita and her colleagues provided a very powerful body of data to support the hypothesis, and indeed many of the experiments critical to support it, it was not until the 1980s that the concept took hold firmly and only in the 1990s that important additional pieces or evidence were forthcoming. For example, in studies initiated by Hendry, Thoenen, Schwab and colleagues it was shown that NGF was retrogradely transported in axons from a peripheral target of innervation to neuron cell bodies (Hendry et al., 1974Hendry I.A. Stoeckel K. Thoenen H. et al.The retrograde axonal transport of nerve growth factor.Brain Res. 1974; 68: 103-121Crossref PubMed Scopus (441) Google Scholar, Stoeckel and Thoenen, 1975Stoeckel K. Thoenen H. Retrograde axonal transport of nerve growth factor: specificity and biological importance.Brain Res. 1975; 85: 337-341Crossref PubMed Scopus (77) Google Scholar). In another important series of studies, Hendry and others showed that naturally occurring death among sympathetic neurons was prevented by delivering exogenous NGF (Hendry and Campbell, 1976Hendry I.A. Campbell J. Morphometric analysis of rat superior cervical ganglion after axotomy and nerve growth factor treatment.J Neurocytol. 1976; 5: 351-360Crossref PubMed Scopus (241) Google Scholar). The suggestion that the same would prove true for sensory neurons (Mobley et al., 1977Mobley W.C. Server A.C. Ishii D.N. Riopelle R.J. Shooter EM (1977) Nerve growth factor (first of three parts).N Engl J Med. 1977; 297: 1096-1104Crossref PubMed Scopus (88) Google Scholar) was demonstrated to be the case in 1981 by Hamburger and colleagues (Hamburger et al., 1981Hamburger V. Brunso-Bechtold J.K. Yip J.W. Neuronal death in the spinal ganglia of the chick embryo and its reduction by nerve growth factor.J Neurosci. 1981; 1: 60-71Crossref PubMed Google Scholar). Thus, by the 1980s, the evidence that NGF acted to prevent the death and enhance the maturation of postmitotic neurons was strong. But showing that tissues are rate limiting in their production of NGF required the molecular biological tools and studies carried out in the 1990s. These tools and the work of many laboratories demonstrated the important role of the target in creating the competition predicted by Rita and Viktor so many years before (Cowan, 2001Cowan W.M. Viktor Hamburger and Rita Levi-Montalcini: the path to the discovery of nerve growth factor.Annu Rev Neurosci. 2001; 24: 551-600Crossref PubMed Scopus (116) Google Scholar). Indeed, one wonders if we might yet be without a clear view of the life and death of neurons without the bold, intuition-driven and rigorous approach that Rita employed. Building on Rita's discoveries, during the last 50 years we have gained immensely exciting insights into NGF's many cellular effects on responsive neurons, its structure and the structure and function of its receptors, and the means by which axons retrogradely transmit signals. In part this was due to many other investigators successfully following the path that Rita and Stanley Cohen took to identifying NGF in the 1950s. However, it took decades until other neurotropic factors were purified and characterized. Indeed, the quest to find new trophic factors, particularly those that acted upon the CNS, proved to be frustrating. The effort was stymied for many years, due to false starts, negative data and the lack of suitable neuroanatomical and biochemical assays like the ones that Rita had established. A breakthrough came when new axonal tracing methods with 125I-NGF showed that while the majority of CNS neurons did not take up NGF, including those in the locus coeruleus, substantia nigra and hippocampus (Schwab et al., 1979Schwab M.E. Otten U. Agid Y. Thoenen H. Nerve growth factor (NGF) in the rat CNS: absence of specific retrograde axonal transport and tyrosine hydroxylase induction in locus coeruleus and substantia nigra.Brain Res. 1979; 168: 473-483Crossref PubMed Scopus (328) Google Scholar), a small population of cholinergic neurons projecting from the basal forebrain to the hippocampus and cortex was heavily labeled. This observation suggested that NGF exerted trophic effects upon CNS cholinergic neurons, a prediction validated in a number of laboratories over the next several years (Gnahn et al., 1983Gnahn H. Hefti F. Heumann R. Schwab M.E. Thoenen H. NGF-mediated increase of choline acetyltransferase (ChAT) in the neonatal rat forebrain: evidence for a physiological role of NGF in the brain?.Brain Res. 1983; 285: 45-52Crossref PubMed Scopus (582) Google Scholar, Mobley et al., 1985Mobley W.C. Rutkowski J.L. Tennekoon G.I. Buchanan K. Johnston M.V. Choline acetyltransferase activity in striatum of neonatal rats increased by nerve growth factor.Science. 1985; 229: 284-287Crossref PubMed Scopus (396) Google Scholar, Martínez et al., 1985Martínez H.J. Dreyfus C.F. Jonakait G.M. Black I.B. Nerve growth factor promotes cholinergic development in brain striatal cultures.Proc Natl Acad Sci U S A. 1985; 82: 7777-7781Crossref PubMed Scopus (193) Google Scholar, Williams et al., 1986Williams L.R. Varon S. Peterson G.M. W