J. Anat. (1985), 142, pp. 73-83 73With 29 figuresPrinted in Great Britain

Anatomical localisation of neuromelanin in the brainsof the frog and tadpole. Ultrastructural comparison

of neuromelanin with other melanins*

MILENA KEMALI AND DOMENICO GIOFFRE

Istituto di Cibernetica del CNR, 80072 Arco Felice (Naples), Italy

(Accepted 10 January 1985)

INTRODUCTION

Neuromelanin is a dark pigment which occurs in some groups of neurons ofvertebrate nervous systems. This neural pigment owes its name to its similarity incolour and histochemical reactions to cutaneous and other melanins. However,neuromelanin differs from melanin pigment occurring elsewhere in its mode offormation (Foley & Baxter, 1958), its chemical composition (Lillie & Yamada, 1960)and its ultrastructure (Moses, Ganote, Beaver & Schuffman, 1966).Mammals have neuromelanin occurring in a few areas of the nervous system,

mainly in the 'substantia nigra' and in the 'locus coeruleus' which are located inthe brain stem. Although these nuclei have been widely studied with respect to theirfunctions and pathological changes, the reasons for their cells being pigmented areobscure.Amphibians have a large quantity of neuromelanin distributed throughout their

central nervous system (Adler, 1939). Such neuromelanin granules are similar inmany respects to horseradish peroxidase granules and may represent a source ofpossible errors in the interpretation of neuronal connections (Mensah & Finger,1975) when this enzyme is used as an intra-axonal transport tracer.In the present paper, the localisation of neuromelanin has been studied in the

central nervous system of the frog Rana esculenta and its distribution in tadpolesat various stages of development.The aim of this work is to define the exact sites where neuromelanin granules occur

in the brain of a low vertebrate and to try to give a phylogenetic and ontogeneticmeaning to their distribution. In addition, the ultrastructural differences betweenneuromelanin and melanin occurring elsewhere in the frog have been defined.

MATERIAL AND METHODS

The frogs and tadpoles used in the present study belong to the species Ranaesculenta. Besides three frogs whose brains were processed histologically unstained,specimens were utilised which had been treated for functional neuroanatomicalpurposes with the cobalt tracing technique (Kemali & de Santis, 1983); it was noticedthat this chemical procedure emphasised the presence of melanin granules.The frog tadpoles were raised in the authors' laboratory following the procedure

described by Gioffre (1976). As a general reference for developmental stages,* Presented in part at the Fifth International Meeting of the International Society of Developmental

Neuroscience, Chieti (Italy), June 1984.

MILENA KEMALI AND DOMENICO GIOFFRE

chronological Tables of the development of Rana esculenta (Manelli & Margheritora,1961) were used. However, since the times of development indicated by these Tablesdid not correspond exactly to the stages seen in this study, the length of the tadpoles(from head to tip of the tail) was used in preference. Tadpoles 5, 7, 8, 9, 11-5, 17,22-5, 31-5, 35-5, 43 and 45 mm long were used.

Frogs and tadpoles were anaesthetised with methanesulphonate (MS 222, Sandoz)1: 3000. The three frogs whose brains were not stained at all and the tadpoles werefixed in 10% formalin, embedded in paraffin and serial sections cut at a thickness of20.and 15 ,um respectively. Tadpoles were then stained according to the Nissl method.In the case of the last three tadpole stages (the longest), the brain was dissected outfrom the body before it was processed for histological procedures.The brains of the cobalt-treated frogs were sectioned at a thickness of 25 ,um in

a cryostat (Reichert-Jung, Mod. 27) and the slide-mounted sections were intensifiedaccording to the procedure described by L'azar, Toth, Csank & Kicliter (1983).Lightly counterstained sections were used in the present study. Sections were cuthorizontally and transversely. Photographs in bright and dark field and withNomarski optics were taken with a Zeiss photomicroscope.

Three adult frogs were fixed by perfusion for electron microscopy. The fixativeused was a mixture of 1 % glutaraldehyde and 1 % formaldehyde in phosphate buffer,pH 7.4. Pieces dissected from the diencephalon, mesencephalic tegmentum, retinaand skin were postfixed in 2% osmium tetroxide and block stained by uranylacetate. The pieces were then dehydrated, embedded in Spurr and cut by an ultra-microtome (Reichert, ultrotome III).

Semithin sections, 1 I/m thick, from the mesencephalic tegmentum containingneurons bearing melanin granules were counterstained with toluidine blue andphotographed using a light microscope. Ultrathin sections were counterstained withlead citrate and observed on a Philips 400 electron microscope.

RESULTS

Light microscopyNeuromelanin has the property of being refractive in dark field (Barden, 1969)

and by this means it was easily identified, particularly in unstained sections. Sitesof distribution of neuromelanin were identified according to the Atlas of the FrogBrain (Kemali & Braitenberg, 1969).

FrogThe distribution of melanin-bearing neurons-in the adult frog is illustrated in

Figure 1 which shows schematically, in transverse sections, the various levels through-out the brain where neuromelanin was confined. Neuromelanin was presentbilaterally at the level of: (a) the telencephalic hemispheres, in the primordiumhippocampi; (b, c) the hypothalamus; (d, e) the mesencephalic tegmentum; (f, g, h)the rhombencephalic tegmentum; (i, j) the basal plate of the medulla oblongata.Neuromelanin granules were also present around the base of the third ventricle

(b, c, d), the cerebral aqueduct (e, f, g, h) and the fourth ventricle (i, j). Some melanin-bearing cells were distributed along the ventricle of the optic tectum (e) and in theepiphysis (b).

74

Neuromelanin in the frog brain

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Fig. 1. Drawings of transverse sections of the frog brain made by means of the camera lucida.The stippling shows the places where neuromelanin has been located. The section levels are:(a) primordium hippocampi; (b) epiphysis and ventral thalamus; (c) periventricular gray matterof hypothalamus; (d) rostral portion of mesencephalon; (e) middle portion of mesencephalon;(f) caudal portion of mesencephalon (nucleus isthmi); (g) rostral portion of cerebellum;(h) caudal portion of cerebellum; (i) rostral portion of medulla oblongata; (j) caudal portionof medulla oblongata.

Figures 2-9 illustrate sections in which melanin was seen with a light microscope.In Figures 2, 3 and 4, which are three images of the same zone of the diencephalonflanking the third ventricle, neuromelanin is shown in bright field, dark field andNomarski interferential phase respectively. These sections are from an unstainedbrain of an adult frog. In sections from frogs processed for the cobalt tracing tech-nique, neuromelanin was more readily apparent and was also evident when itoccurred in small quantities such as in the epiphysis (Fig. 5) or in structures such asthe choroid plexus of the third ventricle (Fig. 6). The neuromelanin granules weredistributed in the cytoplasm of the cell, sometimes assembled at one pole of theneuron, as shown in Figures 8 and 9 which are sections taken at the level of thehypothalamus and tegmentum of the mesencephalon respectively. The intracyto-plasmic granules were readily apparent in semithin sections of specimens fixed forelectron microscopy (Figs. 23, 24). Another feature emphasised by cobalt was theappearance of pial melanophores occurring in the cerebral meninx covering theepithalamic zone (Fig. 7).

75

MILENA KEMALI AND DOMENICO GIOFFRI

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12 13Figs. 10-13. Nissl-stained transverse sections of tadpoles of length 8, 11-5, 17 and 31-5 mmrespectively showing the pigment localised in the most external layer of the telencephalic hemi-sphere (Fig. 10); distributed massively in selected zones (habenulae: Fig. 11); hypothalamus(Figs. 12, 13). Figs. 10-11, x 110; Fig. 12x 70; Fig. 13, x50.

TadpoleThe distribution of melanin granules in tadpoles varied according to the stage of

development. In tadpoles 5 and 7 mm in length, the pigment was distributed incolumns along the full extension of the neural tube from the ependyma throughoutthe gray matter. When the tadpole was 8 mm long, the pigment was no longervisible in the ependyma, but was localised in the most external cellular layer (Fig. 10).In tadpoles 9 mm and more long, the pigment tended to be distributed massively in

Figs. 2-4. Three transverse unstained serial sections of the frog brain showing neuromelaningranules at the level of the periventricular gray matter of the hypothalamus photographed withthe light microscope. Fig. 2. Bright field. Fig. 3. Dark field. Fig. 4. Nomarski interferentialcontrast. x 100.Fig. 5. Neuromelanin in cells of the epiphysis. Transverse unstained section. x 300.Fig. 6. Neuromelanin at the level of the choroid plexus of the epithalamus. Transverse unstainedsection. x 300.Fig. 7. Melanophore in the pia mater on the epithalamus of a frog processed for the cobalttracing technique. x 340.Figs. 8-9. Transverse sections of a cobalt-processed frog showing neuromelanin contained inneurons of the periventricular hypothalamic gray matter and of the mesencephalic tegmentumrespectively. x 400.

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17 18Fig. 14. Nissl-stained transverse section ofa tadpole 22.5 mm long showing the massive distribu-tion of the pigment. x 120.Figs. 15-18. Nissl-stained transverse sections of tadpoles 35-5 mm long. The pigment is con-fined to delimited zones of the periventricular grey matter of the hypothalamus in Figs. 15-16,x 90 and x 100 respectively; to the tegmentum of the mesencephalon at the level of the inter-peduncular nucleus in Fig. 17, x 110; to the rhombencephalon at the level of the raphe nucleiin Fig. 18, x 110.

selected zones. This is apparent in Figures 11, 12 and 13 which are from tadpoles11 5, 17 and 31L5 mm long respectively.Figure 14 shows the massive distribution of pigment within the periventricular

gray matter in a tadpole 22 5 mm long. As development proceeded (Figs. 15, 16;35F5mm tadpole) the pigment was confined to delimited zones of the periventriculargray matter. The decrease in quantity of the pigment and its tendency to be aggre-gated in well defined nuclei was apparent also at more caudal levels of the brains oftadpoles of the same length. This is shown in Figure 17, a section at the level of theinterpeduncular nucleus, and Figure 18, a section of the rhombencephalic teg-mentum at the level of the raphe nuclei which in amphibia are poorly defined asreticular nuclei (Ariens Kappers, Huber & Crosby, 1960).

78

Neuromelanin in the frog brain 79

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45 mm long. It was interesting to compare the large amount of melanin in thehabenular nuclei of tadpoles11c 5 mm long (Fig. 11) to the exiguous distribution inthe same nuclei in tadpoles 45 mm long (Fig. 21).The characteristic common to tadpole melanin was that it presented as dark spots

where no granules were discernible, contrary to the appearance of melanin in theadult frog.

Electron microscopyPigment granules of retina, skin and pia mater of the epithalamus were ex-

amined electron microscopically to compare their ultrastructural details with thoseof neuromelanin.

Melanin granules as seen with the electron microscope varied in shape accordingto the site under examination. Neuronal pigment granules appeared as if formed bya granular matrix encircled by linear arrays, in a cytoplasm where lipid globulesoccurred (Fig. 25). On the other hand, melanin granules in the retina appeared cigarshaped within the finger-like processes of the pigment epithelial cells protrudingbetween photoreceptors (Fig. 26), with a longitudinal striation (Fig. 29). They were

spherical in pia mater covering the epithalamic region of the diencephalon (Fig. 28)and oval ns imelanophore processes in the epidermal zone of the skin (Fig. 27).

spherical in pia matr covering the epithlamic region of the iencephalon (Fig. 28and oval in melanophore processes in the epidermal zone of the skin (Fig. 27).,.,.." ,

MILENA KEMALI AND DOMENICO GIOFFRE

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Neuromelanin in the frog brainThe feature common to pigment granules in skin, retina and pia mater was that

they all showed a limiting membrane and little detectable substructure except for thestriation of retinal melanin.

DISCUSSION

Melanin pigment is reported to be distributed through the whole central nervoussystem of amphibia (Adler, 1939). However, the present results demonstrate that asin the central nervous system of mammals, this pigment, classified as neuromelaninon the basis of its refractiveness when observed in dark field, is confined in the adultfrog to selected neuronal structures.On the other hand, in the frog brain, the sites where neuromelanin occurs are very

numerous (Fig. 1) when compared to the brains of mammals where the best knownpigmented nuclei are the substantia nigra and the locus coeruleus.The wide distribution of neuromelanin in the frog brain might represent a primi-

tive feature which undergoes a re-distribution during phylogenesis. It has beenpostulated that neuromelanin is a product of chemical evolution in the brain(Marsden, 1965 a, b) and that it occurs in phylogenetically old brain stem nucleiwhich have greatly diminished functions in higher mammals.Apart from a few exceptions, for example, the primordium hippocampi and the

epithalamus, neuromelanin in the frog appears to be distributed in the ventralportion of the neuraxis. In general, the neuraxis can be subdivided into two zones,a ventral and a dorsal, in connection with motor and sensitive functions respectively.It seems then that neuromelanin in the frog is located in regions correlated with theefferent portion of the nervous system.The type of transmitter used by the melanin-bearing cells may be of importance

since the occurrence of the pigment in the mammalian substantia nigra and locuscoeruleus has been correlated with the presence of catecholamines which are inter-mediates in the biosynthesis of melanin.

Variations in the distribution of pigment in tadpoles according to their develop-mental stage agrees with the observation that in certain anura primary melanin (ofovarian origin) is widely distributed in the early embryo (Hughes, 1963). Later indevelopment, the pigment becomes confined to selected zones in the brain; thishappens fairly early during ontogenesis since it occurs in tadpoles 35.5 mm long.According to Hughes (1963), the neurons derived from the oldest neuroblasts may

remain pigmented by dilution of primary melanin through mitosis and cell degenera-

Figs. 23-34. Semithin sections of the frog mesencephalic tegmentum showing two types ofneurons (fusiform and multipolar respectively) containing granules of pigment within theircytoplasm. Fig. 23, x 500; Fig. 24, x 600.Fig. 25. Electron micrograph of the frog tegmentum mesencephali containing neuromelanin.The pigment granules appear to be formed by a coarse matrix with linear arrays in a cytoplasmwhere lipid globules also occur. Vesicles with a dense core are also visible. x 30400.Fig. 26. Melanin granules in a pigment epithelium process close to a photoreceptor in the frogretina. x 20000.Fig. 27. Round and oval melanin granules in a melanophore process of the frog skin. 56000.Fig. 28. Round melanin granules occurring in the pia mater covering the epithalamus of thefrog brain. x 8400.Fig. 29. Longitudinal striation of the elongated melanin granules of the frog retina. x 37400.

81

MILENA KEMALI AND DOMENICO GIOFFRE

tion. However, the presence in tadpoles of melanin in well defined nuclei, whichpersist not only in the brain of the adult frog but which in some cases are found inhomologous zones in higher vertebrates, favours the hypothesis that the presence ofmelanin-pigmented neurons in certain nuclei is probably due to their chemical andmetabolic evolution during ontogenesis and to their functional specialisation duringdevelopment rather than to the origin of the cells. The map of distribution ofmelanin might thus coincide with the distribution of its intermediates.The data obtained by electron microscopy favour the assumption that neuro-

melanin is a pigment separate and distinct from other melanins. Not only the pig-ment-bearing cells but also the granules themselves are different in neuronal andnon-neuronal melanins.With regard to pigment-bearing types of cells, neuromelanin in neurons is mainly

found in the cytoplasm of the cell bodies. Non-neuronal melanin occurs in other typesof specialised cells such as the melanophores of the meninges and of the skin and thepigmented epithelial cells of the retina. In the two latter cell types, the granulesaggregate or disperse along the cell processes according to changes in the state of theenvironment as, for example, illumination conditions. In neurons, modification inneuromelanin content occurs in certain pathological conditions, for example, inParkinson's disease.With regard to the granules, the electron microscope shows profound ultra-

structural differences between neuromelanin and the other melanins, as shown inthis study, confirming differences in their chemical composition and origin. This isin accordance with ultrastructural data obtained in mammalian melanins (Moses,Ganote, Beaver & Schuffman, 1966).

In conclusion, several structural features are revealed by light and electronmicroscopy, but the functional meaning of the occurrence of neuromelanin withinthe cytoplasm of selected groups of neurons remains to be elucidated. However, asalready suggested (Kemali, 1984), the frog brain might represent a convenientmodel for neuromelanin studies and, in addition, the retina might serve as a modelfor neuroactive drug trials. The administration of these drugs induces changes in thepattern of melanin migration (pigment screening) in the retina of the frog (Kemali,Milici & Kemali, 1983) and such a dynamic phenomenon may represent an advan-tageous tool for pharmacological studies.

SUMMARY

The localisation of neuromelanin was studied in the central nervous system of theadult frog, Rana esculenta, and its distributiQn in tadpoles at various stages ofdevelopment.

In tadpoles the distribution of neuromelanin was different according to the stageof development but quite early in ontogenesis the pigment was found to be confinedto the same zones as in the adult frog. These sites were mainly located in the ventralpart of the neuraxis.

It is hypothesised that the presence of melanin-bearing neurons in selected nucleimight be due to their chemical and metabolic evolution during ontogenesis and totheir functional specialisation during development.The electron microscope has shown marked ultrastructural differences between

neuromelanin and other melanins.

82

Neuromelanin in the frog brain 83The authors would like to thank the Naples Stazione Zoologica for the use of

their Philips 400 electron microscope and Mr G. Dafnis for technical assistance withthe instrument.

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ARIENs KAPPERS, C. U., HUBER, C. G. & CROSBY, E. C. (1960). The Comparative Anatomy of the NervousSystem of Veretebrates including Man. New York: Hafner Publishing Co.

BARDEN, H. (1969). The histochemical relationship of neuromelanin and lipofuscin. Journal of Neuro-pathology and Experimental Neurology 28, 419-441.

FOLEY, J. M. & BAXTER, D. (1958). On the nature of the pigment granules in the cells of the locuscoeruleus and substantia nigra. Journal of Neuropathology and Experimental Neurology 17, 586-598.

GIOFFRE, D. (1976). Un acquario per la Rana. Aquarium 6, 393-396.HUGHES, A. (1963). On the labelling of larval neurones by melanin of ovarian origin in certain Anura.

Journal of Anatomy 97, 217-224.KEMALI, M. (1984). Distribution and ultrastructure of pigment producing cells in the frog brain and

retina. The brain: a convenient model for neuromelanin; the retina: a device for neuroactive drugstrial. Proceedings of the Vth European Workshop on Melanin Pigmentation, Marseille, France, p. 2.

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KEMALI, M., MILICI, N. & KEMALI, D. (1983). Modification of the pigment screening of the frog retinafollowing administration of neuroactive drugs. Experimental Eye Research 37, 493-498.

LAZAR, GY., TOTH, P., CSANK, GY. & KICLrrER, E. (1983). Morphology and location of tectal projectionneurons in frog: a study with HRP and cobalt-filling. Journal of Comparative Neurology 215, 108-120.

LILLIE, R. D. & YAMADA, H. (1960). Histochemical studies on the neuromelanins. Okajimas folia ana-tomica japonica 36, 155-163.

MANELLI, H. & MARGARITORA, F. (1961). Tavole cronologiche dello sviluppo di Rana esculenta.Rendiconti Accademia Nazionale dei XL. XII, 1-15 +Tavole I-xv.

MARSDEN, C. D. (1965a). The development of pigmentation and enzyme activity in the nucleus sub-stantia nigra of the cat. Journal ofAnatomy 99, 175-180.

MARSDEN, C. D. (1965 b). Hypothesis: brain pigment and its relation to brain catecholamine. Lancet IIn,475-476.

MENSAH, P. & FINGER, T. (1975). Neuromelanin: a source of possible error in HRP material. BrainResearch 98, 183-188.

MOSES, H. L., GANOTE, C. E., BEAVER, D. L. & SCHUFFMAN, S. S. (1966). Light and electron microscopicstudies of pigment in human and Rhesus monkey substantia nigra and locus coeruleus. AnatomicalRecord 155, 167-184.