MELANIN PIGMENT IN THE CESTRAL NERVOUS SYSTEM O F VERTEBRATES

ALEXANDRA ADLER 1-eurological Zintt, Bostoit Ci ty Hospatal and Departnient of Neurology,

Haman1 llledzcal School, Bastoit, Massncl ius~t ts

TWO PLATES (SFVEN FIGURES)

INTRODYCTION

The ganglion cells of three centers of the human brain, the substaiitia nigra, the locus coeruleus, and some cells around the dorsal vagus nucleus, contain melanin pigment. The analogous regions are not pigmented in animals iiicluding the highest species of apes. No homologues of the substantia nigra and the locus coeruleus can be recognized below the reptilian level. I n man these centers a re not pigmented at birth. The pigmentation takes place only after the third year of life, ancl in the locus coeruleus earlier than in the sub- stantia nigra. The significance of the pigment is unknown.

Chromatopliores and certain ncurosensory cells a re known to contain melanin throughout the animal kingdom. The sig- nificance and function of those a re partly known (Parker, 'SO). But only in certain species of Amphibia could ganglion cells containing melanin be found. This has been described especially in frogs and toads by Scliarrer ( ' 3 5 ) .

To obtain additional information about the presence of melanin in the central iiervous system and especially in the nerve cells, a survey was made of the different species of vertebrates, adults, embryos, ancl larvae. The results of the study are given in this paper.

For many helpful suggestions and criticisms throughout the investigation the author is greatly iqdebted to Prof. Tracy J. Putnam. I also wish to express my gratitude to Prof. George

31.7

TIIh J O r R S A L O F COYP.4RATIVh IIEUROLOGY, YOL. 70, X O , 2

316 ALEXANDRA ADLER

B. Wislocki of the Department of Anatomy of Harvard Xtedi- cal School, who facilitated my work by the hospitality afforded me a t his institute, and to Prof. C. I?. Ariens Kappers of the Brain Research Institute and Dr. C. P. Raven of the Embryo- logical Institute, Amsterdam, for the privilege of studying their serial sections of amphibians during a short stay a t their institutes. To 51r. T. Barbour, Director, and Nr. A. Love- ridge, Curator of the Museum of Comparative Zoology, Cambridge, I am indebted for specimens of Protcus, Crypto- branchus, Negalobatrachus, Typhlotriton and Dermophis. Dr. Jean ROUX, Curator of the Zoological Department of the Museum of Natural History in Basel, Switzerland, kindly sent three specimens of Ichthyopliis which were collected by the Drs. P. and F. Sarasin.

METHODS

Specimens were taken from the brain and spinal cord of each animal. Sections of each species were studied unstained, and in a methyIene blue and neutral red stain. If pigment was present, the various well-known chemical and staining reactions were performed to determine its nature and espe- cially the presence of melanin. Certain possible errors have to be considered. Sometimes mitotic figures are present in such an amount as to give a black appearance in the unstained specimen. But they are easily stainable and are, unlike melanin, intranuclear. Other intranuclear inclusions were found in larval Necturus, which, by the use of polarized light and different staining reactions, proved to be crystals, a com- paratively rare occurrencc in the nuclei of nerve cells. Form- aldehyde precipitates do not have the typicaI shape, distribu- tion, and location of melanin granules. In some instances special stains had to he applied to determine more closely the kind of pigment, as for instance in two Sirens, where a Turn- bull’s blue reaction gave evidence of the presence of hemo- siderin, apparently caused by an accidental hemorrhage which left large amounts of :-ellowish crystals in the cytoplasm of various types of cells. I n my experience a light methylene

MELANIN I N T E E NERVOUS SYSTESC 317

blue stain covers pigment less than any other stain and there- fore can be best used together with unstained sections for the morphological study of pigment. Small amounts of pigment can be covered easily by other preparations, for instance, hematoxylin-eosin stains a i d can even be rendered completely invisible,

NATERIAL

Caecilians Derwophis g r ~ g o r i . There was no trace of melanin in the

nerve cells of brain and spinal cord. However, melanophores were found in the coniicctive tissue outside the central ner- vous system. This Caecilian does riot undergo a metamor- phosis.

Ichthyopltis glzctinoszis Lezcisi. The brain and spinal cord of two larvae and an adult specimen showed the same condition as Dermophis gregori. No ganglion cells containing melanin were found, Melanin containing chromatophores were pres- ent in other tissues. This Caecilian undergoes a metamor- phosis during which gills, present during the larval stage, are lost.

Perewdmmchs

These Amphibia are 'permanent larvae' which closely re- semble the larvae of other Amphibia but retain this larval characteristic during life without undergoing or only partly undergoing metamorphosis.

Specimens of Amphiuma means, young and adult, Megalo- batrachus (18 inches) , Cryptobrarichus (9 inches) , showed no trace of melanin in brain and spinal cord.

Necturzis punctatus. Brain and spinal cord of a complete series of specimens ranging from the earliest stages to 31 mm. in length did not show any melanin. One specimen, 60 mm. long, had scattered through the brain tissue minute black granules, which had the characteristics of melanin in staining reactions. They were, in many instances, located in the inter- stitial tissue and not within cellular elements. However, granules could also be seen, one 01' two in number, closely

318 ALEXANDRA ADLER

adjaceht to the nucleus of nerve cells. No melanin granules were found in nerve cells with large cytoplasm. Therefore, an exact localization of the minute granules was not possible in this one specimen. The brain of an older specimen, 95 mm. long, no longer showed melanin within the central nervous system.

Protezis anguineus. The available specimen, 5 inches long, had fine melanin granules in several ganglion cells, especially in the largest nerve cells of the medulla oblongata. About one in eight ganglion cells contains melanin. These pi,gment granules are not so closely adjacent to the nuclear membrane as is seen in several cells of other species (fig. 1).

In this species a metamorphosis of the skin can be induced by administering thyroid extract, while, with the exception of Cryptobranchus (Noble, ’31), the other perennibranchs are insensitive to thyroid extract. It is com- monly known that thyroid extract precipitates metamorphosis in certain Amphibia. A larval specimen, 18 inches long, was studied. The brain and spinal cord showed the presence of melanin granules. One melanin-containing ganglion cell could be seen in thirty-fire cells. The granules were coarser than in Protcus (fig. 2 ) .

AduEt Siren. The two specimens which were studied showed single melanin-containing ganglion cells. The number of those ganglion cells and of the melanin granules in every cell was less than in the sections of the larval specimen. The granules were most regularly observed in the largest nerve cells of the medulla oblongata (fig. 3 ) .

In these ‘permanent larvae’ the single black graiiules are always separated and well discernible, while, as d l be de- scribed later, the single granules conglomerate in amphibians with complete metaniorphosis along with their approaching maturity.

Tailed Amphibia with rneta?norpkosis Amblystoma punctatztm. A complete series of sections

through specimens ranging from 2 mm. to 35 mm. in length

Siren Zncertina.

MELANIX I N THE NERVOUS SYSTEM 319

was available. h dark brown pigment, which in chemical and staining reactions shows characteristics of melanin, is present in practically every cell of the brain and spinal cord of the younger animals. At 6 mm. in length so-called ‘pigment bodies’ appear, which are globular in shape. They are formed by the aggregation of single fine pigment granules. They are typically found first in the brain and in older specimens in the spinal cord as well. The pigment passes from the cells in the form of these pigment bodies when the animal grows older (fig. 6). The amount of piepent increases in this series until the animal reaches the length of 15 mm. after which it diminishes more and more. At that stage the legs have de- veloped while gills and swimming tail are still present.

Tailless Arnphibia with metamorphosis The central nervous systems of all the various types of

frogs and toads have a great deal of melanin pigment. Speci- mens of Rana clamitans, Rana viridescens, Bufo lentiginosus, Bombinator, Rhinoderma darwini were available. In the larval stage, practically every cell contains melanin. This is analogous to the finding in tailed amphibians. At first, the granules are fine and later become more coarse. The amount first increases and then diminishes after metamorphosis has taken place. No formation of ‘pigment bodies’ occurs. The piepent conglomerates in coarse particles after leaving the cell and is found in this stage in large amounts around the vessels (fig. 5). The amount of pigment varies in different species. Serial sections made of Rana clamitans tadpoles, before their metamorphosis, 3 months old (in this species metamorphosis is complete after 1 year as a rule) showed only one or two very fine melanin granules in each cell, their size being about equal to those found in the medium-sized Xecturus (fig. 4). Other embryonic species of Rana, for instance Rana viridescens, have several black granules in each cell in earliest stages.

Special reference should be made to the so-called Rohon- Beard cells, transient giant ganglion cells at the dorsal periph-

320 ALEXANDRA ADLER

ery of the spinal cord of most amphibians and fish. Coghill ( ’14) has shown that these cells form an intramedullary sub- stitute of the later spinal ganglia in lower vertebrates. Wherever pigment can be found in the spinal cord, the Rohon- Beard cells usually contain the greatest amount of it (fig. 7) . There is no pigment in the Rohon-Beard cells of those animals whose central nervous systems do not contain melanin. I t is interesting to observe that the development of melanin in these transient cells takes a different course from that in the other nerve cells. In Amblystoma no single pigment bodies are formed in these cells while they are present in the other cells. I n these giant ganglion cells melanin forms about six coarse granules before their complete degeneration and dis- appearance. In Bufo lentiginosus the pigment of the giant ganglion cells becomes finely granulated and diminishes while they degenerate. The nucleus becomes scarcely visible and homogenous. Frequently, at the final stages, there remain only melanin granules without any cell to be seen. Occasion- ally there is only a nucleus without cytoplasm or pigment. In Bombinator the Rohon-Beard cells take on a brownish hya- linization before they disappear.

I n Rana viridescens, Bombinator and Bufo lentiginosus the giant ganglion cells disappear at the time when the second pair of legs develops. In Amblystoma punctatum the Rohon- Beard cells can be seen when both pair of legs are present while the external gills still exist. No older specimen was available in this series.

Pigmentless Amphib ia Typhlotriton is a blind cave salamander, the skin of which

appears white and histologically contains very little pigment, like the skin of other underground Amphibia, such as Proteus and Typhlomolge. But Typhlotriton is the only pigmentless amphibian which undergoes a typical metamorphosis. In this animal no melanin could be found in the brain and spinal cord, while, as described above, it is present in the ‘pigmentless’ perennibranchial Proteus.

MELANIN IN THE NERVOUS SYSTEM 321

F is Ji wit 7~ met a m o r p k o s i s

Particular attention mas paid to those fisli that undergo a metamorphosis. Serial sections of larval Lepidosteus osseus and Amia calva were available. They, like other fish and the remaining classes of vertebrates, did not show any melanin in their nerre cells.

M a ~ z While in Amphibia the melanin is distributed through the

whole central nervous system, in man it is sharply localized in three centers, the substantia nigra, locus coeruleus, and the dorsal vagus nucleus. As in the Amphibia, the granules are at first fine and their amount and size increase as the indi- vidual grows older. While in Amphibia the amount dimin- ishes after completion of metamorphosis, the course has not been stated definitely in man. Pilcz (1895) found that the melanin pigment increases until the age of about twenty and remains stationary from then on. Scharrer (’35) mentions that the pigmeiit diminishes during the senium. However, with my series of brains of individuals at a few days of age, 1 year, 4 years, 18 years, 20 years, 30 years, 40 years, 50 years, and 68 years of age respectively, the brains of the 68-year-old man and woman in the series contained by far the greatest amount of melanin pigment in the substantia nigra. It was present in about equal amounts in the brains of the individuals 18 to 50 years of age, of course being absent in the brain of the newborn infant and of the 1-year-old child. Only a few fine granules were present in the brain of the 4-year-old child. It is conceivable that the quantity of melanin varies for differ- ent periods of life, as well as for different individuals.

It would be of great interest to investigate the brain of an albino. The finding of melanin in the central nervous system of Proteus, the ‘pigmentless’ amphibian, and, on the other ha‘nd, its absence in the pigmentless Typhlotriton, raises the question whether or not human albinos have melanin in the centers of the brain normally pigmented.

322 ALEXANDRA ADLER

I>ISC'CSSION

Tlw sigiiificance of melanin pigment

Fuerth and Schneider ( '02) traced the formation of melanin back to the action of autolytic ferments on products of de- composition of protein, particularly tyrosin. They sliowecl that this fermentation caused darkening of insect blood and blackening of various plant juices when exposed to the air. A similar process can be traced in the brown, red, and black coloring of fading leaves. Florentine ( '34) expressed an opinion that black pigment may be the expression of a col- loidal and pigmentary degeneration of certain nerve cells. He based this upon the finding of melanin in ganglion cells of the preoptic nucleus of the toad. This was disputed by Scharrer ('35) who pointed out the fact that dark pi,ment can be found diffusely in frogs' and toads' brains although the cells show no signs of degeneration. Narinesco ('09) attributes 'un certain r d e functionel' to the melanin pi,ment of the central nervous system. Far i s ('24) stated that in Aniblystoma differentiation areas a re areas of more abundant p i p e n t which increases as the differentiation proceeds. Pro- liferation is not accompanied by an increase of pigmentation.

There is 110 parallelism between the presence of melanin in ganglion cells and that in melanophores or sensory cells although it1 is of the same or of similar chemical structure. TVhenever melanin-containing ganglion cells can be found in the central nervous system, it is present also in other organs or in connective tissue cells of the animal. This is true also for Proteus, the ' pigmentless,' white salamander, some organs of which have even great amounts of black pigment. This condition is not reversible since plenty of melanin may be present i n melanophores or sensory cells without the presence of melanin in nerve cells. Another difference is shown by the fact that while melanin in the ganglion cells diminishes and disappears after metamorphosis, it increases in amount or remains unchanged after metamorphosis in connective tissue melanophores and in sensory cells. Melanin is not an

MELANIN IX THE SEBVOTJS SYSTEM 333

ingredient essential f o r metamoi*phosis, since some nrocleles (Typhlot ritoii, Eurycea) and C’aecilians with metamorphosis (Tchthpopliis) lack black pigment in their central 11~1*vous system. 111 the ‘perinanelit larvae’ it is completely ahsent in Ampliiuma, Cryptobraiiclins a id hlegalobatrachas. The nerve cells of Siren and Proteus contain melanin, but f a r less of it than the other ampliibinns with completcl metamorphosis.

These facts raise the question wlietlier tlie amount of pig- ment in Xmpliibia may be taken as an indication of the degree of maturity of a species in phylogenesis. The following table compares tlie amourits of pigment with the degree of meta- morphosis listiiig those with the smallest amount of pigment and lowest degree of metamorphosis on top. The degree of metaInorpliosis in perennihranchs is quoted according to Noble’s (’31) statement which is hased upon the most obvious changes of metamorphosis. The table shows no parallel bc- tween amount of pigment and degree of metamorphosis in pereiiiiibrarichs phylogenctically.

TAR1,k; 1

Perennib, n w k ~

Dvgrep of r t i r f n n m r p / ~ o r ~ u

Siren Megalohatrachus, Cryptol)raiictrus, Aiuphiuiun

Proteus, Nerturus (Necturus) Crypt obranr. hus Siren Megalobatrachus Proteus

I i n o i i n t of p i g m e n t

(pigment is a h n i t )

Frogs and toads, the amphibians with the most complete metamorphosis, have tlie grea test amount of pigment in their ceiitral nervous system. Their metamorphosis can be, as is known, precipitated by administering thyroid extract. X metamorphic change of the skin oiily brought about by ad- ministration of thyroxin, has been described in Siren. This is a fact, but cannot be used to prove coriclusively that the presence or the amount of pigment iii anipliibiaris is entirely or predomiriaiitly rt@ated bj- the action of the thyroid gland.

THE JOURNAL O F POMPARATTVG NIlUXOLOGY, VOL 70, NO. 2

324 ALEXSNDRA ADLER

These findings show that rrielaiiiri certaiiily is not an c‘ssen- tial irigreclieiit for metamorphosis. However, since it regu- larly increases in amount till the height of metamorphosis is reached and tlieii decreases arid disappears, it can he con- sidered as a by-pi-ocluct 01% waste product of metabolic proc- esses of cells during tlie differentiation and alteration wliicb take place dnring metamorphosis.

Tn mail melanin is sharply localized in three centers of the central nervous system. Kotliiiig is knowii about tlie function of the locus coeruleus either in mail o r in otlier aiiimals, and oiie cannot determine the possible fuiiction of the few pig- meiited cells around the vegetative vagus nucleus. Tlie sub- stantia 1iigi.a is a part of the extrapyiamidal system which has, as i s kiio\vii, a greater fuiictioual value iii children of early age arid animals tliari in adult inaii. In other animals a large portion of the cortex can be removed without definite impair- ment of motor fuiictioii which is carried or1 by the estrapyrami- dal system. Tlie humaii baby, which lias heen called ‘ thalamus- pallidnm being’ learns to use tlie new system, thc pyramidal system, only when well along in its development. These coil- ditions raisc the question as to wliether the melaiiiri formation in the substantia nigm iii adult mail maj- he a11 indication of the c>liangiiig fuiictioii of tliis estrapjximidal center as com- pared to the infant and the animal where the center has no melanin pigment. The occurreiice of gradual increase and then diminution of melanin in the central riervous system of certain species of Amphibia during and after meturnoiyliosis suggests a compi’isoii of these fiiicliiigs.

SUMhlLIRT

1. Abuiiclaiit amouiits of melaiiiii can he fouiid in the gang- lion caclls of tlie central iiervous systems of tailless (frogs and toads) arid tailed (Amblystonia) Amphibia during and shortly after metamorphosis. L4mpliibia which do not metamorphose have either no black pigment ( ClryptobraiichusY Amphiuma, hlegalobati*achus) or (Siren, Proteus) smaller amouiits of i t than dmpliibia with coriiplcte metamoiyliosis.

2. Some Plcthoclon ticlac ( Typhlotritou, Eui*ycea) and Cae- ciliaris have no melaiiiii pigment iii tlie iierve cells regardless of whether they metamoi-phose o r midergo a direct develop- ment.

3. ‘l’hese findings show that nielaiiiii is not an essential iii- gredient for metamorphosis. In all available series of am- phibiaiis in which melanin coiitaining ganglion cells are preseiit, tlie amount of melanin increased till the height of metamorphosis was reached and then dimiiiishcd gradually or disappeared completely. It can therefore he eoiisiclered as a bv- p r odnc t o r waste product of met a ho li sm a id cliff e rent ia - tion of cells during incomplete o r complete metamoqhosis. 4. The gradual development of melanin in the siibstantia

nigra of adult man, which is absent in the corresponding cen- ter of infants and of animals, shows ccbrtain similarities with the process observed in Ampliibia. In this connection atten- tion is called to tlic fact that the substantia nigra is a part of the extrapyramidal system which lias a changing functional value in adult mail as compared to childreii and animals.

LITEXATCXE CITE11

COGHILL, G . E. 1914 Correlated anatomical and pli: siologieal studies of the J. Conip. Keur., vol. 24,

FARIS, H. S. 1924 A study of pigment in embryos of Anrhlystonia. Siiat. Ree.,

FLORR~NTINE, P. L’excrPtion de la ecllloide pituitaire chez le crapaud (Bufo

FUERTH, 0. V., AND H. sCHNIII1)h.R ITeher tierisehe Tarosinasen uiid ihre Beitr. z. Chem. u. Pathol., Ed. I,

growth of the nervous system of Amphihia . pp. 160-232.

vol. 27, pp. 63-76. 1934

vulgaris).

Beeiehungen zur Pigineiitbildung. s. 229-242.

Compt. rend. Soe. de Biol., T. 117, pp. 18. i - lXR. 1903

MARINESCO, G. 1909 La Cellule Nerveuse. Ootarc Doin et Fils, Paris. NORLE, G. K. 1931 The Biology of the Amphibin. McGrav Hill Book Company,

NOHLF, G . K., AND (:. J. FARRTS 1929 A metnnioipliic change produced iii

P A R K E R , G. H. 1930 Cbroinatopho~cs. Biol. Rev., vol. 5, pp. 59-90. PILCZ, A. Xeitrag zur Lellie r 0 1 i der t ’ ig i i ie i i teut~~i~kl i in~ in der Xerleii-

Arb. a. d. Wiener Neurologisclien Institut, Bd. 3, S. 123-139. SCIIARRER, E. 1935 Ubcr das Pigment iln Ampliibienyeliirn. Zool. Am., Bd.

New York aiid London.

Crrptobranchus by thyroid solutions.

1895 zellr.

Annt. Ree., vol. 42, p. 59.

709, 8. 304-307.

1’LSTi.: 1

ESPLSSATIGP; OF’ FIGEBICS

I’hotoiiiicro~ral,Iis. Tlic arrows in figures 1, 3 and 1 point toward riielaiiiii granules.

1 Gaiiglion cell i n iiicdulla oblongata of I’rotens anguineus. Twelve fine melanin granules are visible in t,lic cytoplasm. X G O O .

2 Cells in telenccptialoii of ktrral Siren Incertinn. Kote two gniiglion cells with several coarse black grnnules adjoining the nuclear menibrauc. linstaincd speeimrn. X 1000.

3 Ganglion cell in medulla ohlongata of adiilt Siren lacertina with nine melanin graiiulrs in tlic cgtoplnsm. They are coarser t,liaii in Protens. S iss l stain. x (i00. 4 Epmd;vniel cclls aroiiiid criitrel caiial of ~ p i n a l cord in Rann. claniitans, age

about 3 months, 3 inclics Irngtli, hcforp mctnmorpliosis. Thr arrows point toward singlr fine melanin granules. 1-Tem:rtoxylin stain. x 1100.

hiclanin pigment in large amounts around vessels.

Kiss1 stain.

5 TTnstained section of torus semicircularis in adult Rana riridcscens. x 400.

PLATE 2

FSPLANATICN OF FIGURES

A (‘ross section througli the spinal cord of larrnl Anihlystoma puiictatuiii (18 nnn. length. IJrgs are growing, gills and sw-iniiiiing tail still present). ‘Pigment bodies ’ :I re formed h j eonglomeration of single pigment granules before the pigment leaves tlir cell in tlie maturing aninial. Veningeal ehroniatophores a t tlie dorsal periphery. X 330.

Cross section tlirougli the spinal cord of 1:irral Bufo Irntiginosus (16 mni. lrngtli, before the appearance of second pair of legs). Laige :~inonnts of melanin pigment conglonierations, particularly ~vithiii the ventral horn cells. A single giant ganglion cell ( Kohon-Beard cell) a t the dorsal midline contains the largest amount of melanin. Boras c:irniine orange grcen stain. x 330.

A l i m rhocliinral orange green stain. 7

328

PLATE 2