4 36 YELLOW PIGMENT I N NERVE CELLS

MICROSPECTROGRAPHIC STUDIES ON THE YELLOW PIGMENT IN NERVE CELLS

BY HOLGER HYDBN AND Bo LINDSTR~M Received 28th September, 1950

The results from a study with ultra-violet and X-ray microspectrography, fluorescence spectrography and cytochemical tests of the yellow pigment in human nerve cells are reported. This pigment, lipofuscin, is found in the cytoplasm of the nerve cell and increases in quantity with rising age.

Pentose nucleoproteins are a physiological constituent of all big nerve cells. They occur in great amounts in the cytoplasm and in smaller amounts in the nucle~lus .~-~ A production and consumption of the pentose nucleoproteins in the nerve cells has been shown to be a phe- nomenon accompanying adequate stimulation of the neuron.s, 6 e

The occurrence of the yellow pigment in the cytoplasm of human nerve cells thus makes a study of its composition a prerequisite for further investigations.

1 Brachet, C.R. Soc. B i d , 1940, 133-1#, 88. a Brachet, Enzymologdu, 1941, 10, 87.

Hyddn (Landstr6m), Caspersson and Wohlfart, 2. mikr. umt. Forsch., 1941, 49. Hydh, Nmd. Med., 1941, 4, 3048.

5 Hyddn, Nord. Med., 1942, I, 144. 6 HydBn, Acta Physiol. Scand., 1943, Suppl. 17.

Gersh and Bodian, J. Cell Corn#. Physiol., 1943, 21, 253. 6 H y d h and Hamberger, A d a oto-laryng., 1945, Suppl. 61.

Hyddn and Hamberger, Actu oto-laryng., 1949. Suppl. 75. lo H y d h and Hamberger, A d a oto-luvyng., 1949. Suppl. 75.

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HOLGER HYDEN AND BO LINDSTROM 43 7 Cytological observations were early made on the incidence and fre-

quency of the yellow pigment in the uerve cells. Between the age of 7 and 30 years only a limited number of nerve cells in the spinal cord and in the medulla oblongata contain yellow pigment whereas between the ages of 40 and 80 years it is present in nearly all the nerve cells in this area."-'* The substance can occupy the greater part of the cytoplasm, the nucleus then being displaced towards the periphery of the cell. Its distribution within different parts of the nervous system has also been de~cribed.1~~ l6

The lipofuscin contains a lipid part which is dissolved with organic solvents and another part which stains with basic dyes.18*1s The dye is not bound to the lipid-soluble part. The yellow pigment takes up basic dyes at a pH greater than 5-5. On this observation an elective staining procedure has been based.20 The stained part of the yellow pigment is insoluble in acids, alkali and alcohol-ether. The colour remains after treatment with potassium permangate, sodium hydrogen sulphite and hydrogen peroxide.13~ lB, 22

The yellow pigment in the nerve cells has frequently been interpreted as a slag product, impairing the function.'** ** Its occurrence at an early age and its large quantity in the cell on the other hand are against such an interpretation.16

Experimental Material and Methods.-Anterior horn cells from the cervical intumes-

cences of the spinal cord from individuals of varying ages, between 16 and 89 years, were studied. The material was taken 3 to 18 hours post mortem and fixed in liquid air accotding to the Altmann-Gersh freezing-drying method or in 10 yo formaldehyde solution or in Carnoy's solution. Frozen sections of unfixed material were taken fcr fluorescence spectrography. To remove paraffin, chIoroform was used which was folIowed by absolute alcohol. The section was immersed in glycerine for a t least 2 hr. Before the ultra-videt measurements suitable cells were dissected free from adjoining tissue on one side to obtain a blank. Sections for fluorescence spectrography were only covered by glycerine and the cover slip (ir treated with chloroform and alcohol or with methanol- chloroform.

ABSORPTION SPECTRA were taken in the ultra-violet range and the visible part of the spectrum in various parts of the cells, using the apparatus constructed by Caspersson.2* The cells were selected by means of photography a t 2570 A in the ultra-violet microscope constructed according to Kiihler.

OPTICAL SYSTEM.-Zeiss quartz monochromates corrected for 2570 A and for 2750 A. Objective, 2.5 mm., numerical aperture 0.85, O C U Z U ~ , x 10. Condenser, numerical aperture 0.65. Within the visible parts of the spectrum the optical system used was a Zeiss apochromate, numerical aperture 1-25 and compensation ocular x 10.

For a study of the ash content of the cells, micro-incineration was carried out a t 5 m o C for I hr., after which the cells were photographed in the dark field. Sections were digested with ribonuclease, prepared according to Kuni tz method and recrystallized in alcohol three times, in a solution containing

This does not remove the yellow substance.

l1 Obersteiner, Arb. Neurol. Instit. Wiener Univer., 1903. 10, 245. l2 Miihlmann, Verk. dtsh. path. Ges., 1g00-I,. 3-4, 148. l3 Muhlmann, Arch. mikrosko?. Anat. Entwzcklungsgeschichte, 1901, 58, 231. l4 Pilcz, Arb. Anat. Pl;ysiol., 1895, 3, 123. 16 Altschul, Virchow's Arch. path. Anat., 1938, 301, 273. l6 Altschul, J . Comp. Neur., 1943, 78, 45. 17 Wolf and Pappenheimer, J . Neuropath. Expt. Neurol., 1945, 4, 402. l8 Hueck, Bietr. path. Amat. nllgem. Path., 1912, 54, 68. Is Bethe and Fluck, 2. Zellforsch. mikroskop. Anat., 1937-38, 27, 211. 2o Volkmann, 2. wissensch. Mikr. mikr. Technik., 1932, 49, 457.

22 Brahn and Schmidtmann, Virchow's Arch. path. Anat., 1920, 227, 137. 23 Hett, 2. Altersforch., 1941-4, 3-4, 174. 24 Caspersson, J . Roy. Microsc. SOC., 1940, 60, 8.

Oberndorfer, Zentr ges. Neurol. Psych., 1921, 26, I .

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438 YELLOW PIGMENT IN NERVE CELLS 0.29 mg. of ribonuclease/ml. (pH 6.5). Microspectrography was carried out before and after digestion.

FLUORESCENCE SPEcTRoGRAPHY.-EXciting radiation was obtained from an arc lamp using glass filters U.G. I and B.G. 3. The radiation obtained in the ultra-violet region was limited to approximately 310-370 mp.

Microscope optics.-Zeiss achromate numerical aperture 0.65, Quartz condenser with numerical aperture 1-2 and 0.6 respectively. On the place of the ocular a Jena filter GGg.

The spectra were taken on Ilford HP 3 plates. A Zeiss quartz Q12 spectro- graph was used. The image of the object was focused in the plane of the entrance slit, the magnification being 135 times. The minimum slit width was 0.05 mm.

MASS DETERMINATIONS BY X-RAYS according to Engstrom and Lindstrom’s method zS was carried out for the purpose of studying the distribution of the dry substance in parts of the nerve cells containing yellow pigment as com- pared with those parts of the cells containing pentose nucleic acids and proteins. The method is based on the fact that the absorption of suitably selected con- tinuous X-radiation is proportional to the total amount of substance per surface unit in the histological section. In our determinations we used radiation gener- ated at a tension of 3000 volts and filtered thrcugh a gp thick aluminium foil, the wavelength then being approximately 8 A. No element present in large quantities in the tissue has any disturbing absorption peak in this range.

Thick sections 6 to 10 p of the cervical intumescence from a human spinal cord, fixed in 10 yo formaldehyde solution, was photographed on a Lippmann film together with a reference system. This consisted of a wedge of collodion foils in steps approximately 0.1 p thick. The film allows effective magnification up to 400 times. After developing in an alkaline-hydroquinone developer, the picture of the tissue and the reference system was photographed in a micro- camera a t a magnification of 400 times. The density on the photographic plate within parts of the nerve cells containing yellow pigment or pentose nucleo- proteins was measured with a Kipp and Zonen microphotometer.

Since both control cells and cells containing yellow pigment occurred in the same section and were photographed together with the reference system, those sources of error were eliminatcd that could be caused by differences in the density curve cf the films and by the developing and fixation processes.

In order to control the cells after X-ray microphotography, the same sections were stained with toluidine blue. This permitted a direct correlation of the results of the density determination with the cytological picture. In order to determine the thickness of the section within the part of the cell to be measured particles in the upper and lower surface of the section were focused and the focal length determined.

OPTIcs.-Objective : apochromate 90, N.A. I -32. Compensating ocular x 10. Staining of the cells with toluidine blue was also performed after absorp- tion measurement in the ultra-violet and visible parts of the spectrum a t pH 4, and also a t pH greater than 5-5 with Gentian violet solution acccrding to Bethd’s and Volkmann’s methods.1S. 2 O Feulgen’s nucleal reaction was used to demon- strate the presence of desoxypentose nucleic acids.

Nerve Cells containing Pentose Nucleoproteins (Controls) .-The control material consisted of anterior horn cells from a youth aged 18. Control cells and those containing yellow pigment were present in the same section on X-ray microphotography (see the foregoing). A microscopic control was first made of the cells selected for measurement to ensure that they contained no stained substances.

ABSORPTION MEASUREMENTS-Motor anterior horn cells in man have been shown, by means of cytochemical methods, to contain considerable concentra- tions of pentose nucleic acids and protein substances. Quantitative determin- ations have shown a substantial content of nucleic acids, i.e. I to z yo.% 26 The substance containing nucleoproteins is distributed in more or less well-defined agglomerations in the cytoplasm and in the dendrites, corresponding to the distribution of the basophil Nissl substance. Desoxypentose nucleic acids are only found in scanty amounts in the motor nerve cells and are confined t o a small number of particles in the periphery of the nucleolus, comprising the so-called nucleolus-associated chromatin. No characteristic absorption maxima could be observed in the control material on measurements in the visible parts of the spectrum.

2ti Engstriim and Lindstrom, Biockim. Biophys. Acta, 1950. 4, 351. 26 Hyddn and Hartelius, Acta psyclziatv. neuvol., Suppl. 48, 1948.

A monochromator was used as the source of light.

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FIG. I .-Anterior horn cell from a human spinal cord. nucleic acids and proteins but no yellow pigment. mann film with an acceleration tension of 3000 volts.

The cell contains pentose Photographed on a Lipp-

The nucleolus and the nucleus have a considerable absorption compared with the nucleus.

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FIG. Z.-Anterior horn cell from a human spinal cord photographed a t 2570 A. The cytoplasm as a whole is characterized by a high absorption. Note that the absorbing substance of the right lower part of the cyto- plasm has a fine granular appearance. This area contains yellow pigment.

FIG. 3.-The same nerve cell as in Fig. 2, stained with toluidine blue a t pH 4. The right lower part of the cytoplasm which contains yellow pigment and absorbs strongly at 2570 A does not take the basic dye

groups.

FIG. 4.-The same nerve cell as in Fig. z and 3 micro - incinerated at 500° C and photographed in the dark field. In the right lower part of the cytoplasm containing yellow pigment the ash residue seems denser than in the remainder of the

cell.

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(4 (b) FIG. 6.-Anterior horn cell ccntaining yellow pigment from human spinal cord photographed a t 2570 (a) and after staining with toluidine blue at pH 4 (b )

FIG. 7.-Anterior horn cell from human spinal cord. Photographed on a Lipp- man film with an acceleration tension of 3000 volts. The area in the cytoplasm to the left of the left black line contains yellow pigment and stands out clearly

due to its high absorption capacity. Cf. the nerve cell in Fig. I.

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HOLGER HYDZN AND BO LINDSTROM 439 MASS DETERMINATIONS BY X-RAYs.-Examples are given in the following

of determinations of the total amount of substance in the nerve cells which contain no yellow pigment. The nucleolous stands out clearly in the cell in Fig. I by means of its more intense absorption compared with the remainder of the nucleus. A number of points in the nucleolus, the nucleus and the cyto- plasm were measured on Dhotometrical determinations. The relation between 2he total amount of substince in different parts cf two control cells with the quantity of substance in the cytoplasm as a unit is given below. They are taken at the tension of 3000 volts. Each single figure is the mean figure of about ten determinations.

Nerve Cells containing Yellow Pig-

Fig. 2 shows an anterior horn cell, un- ments .-ABSORPTION MEASUREMENTS -

TABLE I

Cytoplasm I Nucleus I Nucleolus

I I’

stgined and photographed at 2570 A. The cytoplasm absorbs strongly. In the right-hand part the absorbing substance is not localized to distinct agglomerations but is diffusely distributed within the cytoplasm. Fig. 5 demonstrates absorption spectra taken within the ultra-violet range and the visible parts of the spectrum to 1600 A at points in this area. A strong absorption maximum appears a t 2600 . The absorption is also strong a t 2800 A indicating the presence of heterocyclic and aromatic amino acids (tryptophane, phenyl alanine and tyrosine) in the protein present. That part of the cytoplasm in the same nerve cells frcim which these absorption spectra were taken could not be stained with toluidine blue a t pH 4 (Fig. 3).

FIG. 5.-Absorption spectra taken between 2400 to 4600 A in aieas of anterior horn cells containing yellow pigment from human spinal cord.

Fig. 6(a) and (b ) show another example. On the left of the figure is seen the cell photographed a t 2570 A. On the right i t is stained with toluidine blue. The area nevertheless stained well with toluidine blue a t pH greater than 6 or with Volkmann’s Gentian violet solution, and contained much yellow pigment. The murexide reaction gave negative result.

On micro-incineration, this part of the cell yieIded considerable ash (Fig. 4). A comparison with the remainder of the cytoplasm, wbich contains pentose nucleic acids and protein substances, showed that the ash had a substantially greater density in the area containing yellow pigment.

Experiments with digestion with ribonuclease revealed that it was not possible to remove that part of the cytoplasm containing yellow pigment with the help of the enzyme. The absorbing substance remained, and absorption spectra taken after digestion showed no differences in comparison with those taken .

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440 YELLOW PIGMENT IN NERVE CELLS

Pentose nucleic acids and proteins .

Yellow pigment .

before digestion. On the other hand, the pentose nucleic acids in the remainder of the cytoplasm were removed.

In view of the absorption maximum a t 2600 A and 3750 A and the yellow colour of the pigment, it was easy to assume a flavin component. The tests prescribed by Warburg and Christian 27 were therefore made on the sections. These writers demonstrated that on illumination in an alkaline solution the composition of the yellow ferment is changed and after acidification can be extracted by chloroform. Our experiments did not, however, result in a dis- appearance of the yellow pigment in the nerve cells.

FLUORESCENCE SPECTRUM.-The yellow pigment shows a yellowish fluores- cence which is not changed or diminished after treatment of the section by acids, alkalis, chloroform, chloroform-methanol or irradiation.

Fluorescence spectra were taken on frozen, unfixed cells containing yellow pigment and on cells after the treatment described above. The photographic plate was also exposed without the cell but with the adjoining tissue in focus and also without the section. Spectra show a moderately strong fluorescence band between 440-460 m p and another band between 530-560 mp.

MASS DETERMINATION BY X-RAYS.-In order to make a direct comparison, measurements were taken of the control cells and the cells rich in pigment lying in the same section. Fig. 7 shows an example of a nerve cell containing both yellow pigment and nucleoproteins, photographed on a Lippmann film with an acceleration tension of 3000 volts. The area t o the left of the left black line is seen to absorb particularly strongly. This contains yellow pigment. The right part of the cytoplasm on both sides of the black line contains pentose nucleic acids and proteins but no yellow pigment (see Table 11).

4-2 x I O - ~ I 1.3

5'5 x 10-6 9.8

TABLE I1 ~~

Cytoplasm containing: 1 Weight in ,ug.\pz Thickness in p 1% Dry Substance I I I I

37

56

Each determination is the mean figure of apprcximately 10 measurements. The results of these determinations thus show that the areas containing yellow pigment in the nerve cells have a considerably larger total quantity of dry sub- stance than corresponding areas in the nerve cells containing pentose nucleic acids and proteins but no yellow pigment. Approximately IOO measurements, made on nerve cells with and without yellow pigment, have uniformly shown the same difference in the total quantity of substance per unit volume. In the examples of measurements series given, the figures show that, on an average, the area containing yellow pigment had 50 yo more dry substance than corre- sponding areas containing nucleus acids and proteins.

Discussion A survey of studies of the yellow pigment by optical methods reveals

(i) absorption bands a t 2600 and 3750 A, (ii) fluorescence bands between 440-460 mp and between 530-560 m p , around 50 % larger quantity of dry substance than areas containing pentose nucleoproteins.

The absorption maximum at 2600 A did not disappear after digestion with ribonuclease whereas the pentose nucleic acids present in the nerve cells could be removed by the enzyme. The specific band can be explained either by assuming the presence of substances other than nucleic acids or by assuming pentose nucleic acids in a chemical configuration which cannot be attacked by ribonuclease. The absorption data together with the fluorescence data suggest flavins or pterins.

27 Warburg and Christian, Naturwiss., 1932, 20, 980.

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HOLGER HYDEN AND BO LINDSTROM 44 1

The position of the absorption and the fluorescence bands with ab- sorption at 260 mp to 375 mp and emission at 450 mp and 550 mp in- dicates the presence of a complex chromophore. Flavins, e.g. ribo- flavins can be excluded. The data obtained indicate more a substance belonging to the pterins. Standard spectra of pterins in solution, both xanthopterin and leucopterin, show two strong bands between 435 mp and 546 mp.zs As the fluorescence spectrum of a pterin differs according to whether it is solid or in solution further data are required. The data obtained are, however, in favour of pterins as components of the yellow pigment.

The determinations by mass determinations by X-rays showed that the areas containing yellow pigment in the nerve cells had a considerably higher dry weight than areas containing pentose nucleoproteins. The difference was on an average 50 yo. The difference in density of ash residue shown by micro-incineration was therefore in this case a true one.

Regarding the biological aspect of the results obtained the following reflections may be made. The data given show that the amount of pentose nucleic acids in the anterior horn cells decreases with rising age. The space occupied by them appears to be replaced by the complex yellow pigment. A partial change in the chemical structure of the nerve cells therefore takes place with increasing age.

Since this reconstruction of the nerve cell in rising age is so consider- able a substantial redfiction of function could be expected if the yellow pigment were a slag product due to the importance of the nucleic acids for the neuronal function. The reduction in motor function accompanying increasing age is not, however, proportional to the considerable chemical reconstruction of the nerve cell. The experiments reported thus are against the interpretation of the yellow pigment as a slag product.

From a general biological point of view it can be said that following the embryological period of growth and differentiation there occurs a further chemical differentiation of the nerve cell under the adult stage, apparently with maintained function.

Summary .-The yellow pigment lipofuscin, that is present in human nerve cells and increases in quantity with increasing age, is studied with ultra-violet, X-ray and fluorescence microspectrography, digestion with ribonuclease and micro-incineration. Absorption maxima are found at 2600 and 2800 A indicating the presence of organic nitrogenous bases and protein substances. The strongly absorbing substance at 2600 A cannot be removed by ribonuclease. Another absorption maximum is found at 3750 A. and between 5300 to 5600 A. The presence of flavins can be excluded. Data obtained indicate some possibility that the yellow substance belongs to pterins.

The mass determinations by X-rays show that the areas in the nerve cells that contain the yellow pigment have around 50 yo greater quantity of dry substance than the areas containing pentose nucleoproteins. A partial chemical reconstruction of human nerve cells seems to take place with increasing age. The findings are not in favour of the view that the yellow pigment is a slag product.

Fluorescence bands are found between 4400 to 4600

The present investigation has been carried out with the aid of grants from Stiftelsen Therese and Johan Anderssons Minne and from the Swedish Medical Research Council.

Department of Histology, Medical School,

Goteborg C., Sweden.

Department for Cell Research, Karolinska Institutet,

Stockholm 60, Sweden.

28 Jacobson and Simpson, Biochem. J., 1946, 40, 3.

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