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Uptake of L-DOPA by the cells of organotypic culture from chick-embryo brain hemispheres
Acta histochem. Ed. 60, S. 32-40 (1977)
Polish Academy of Sciences, Institute of Pharmacology, Krakow, Poland
Uptake of L-DOPA by the cells of organotypic culture from chick-embryo brain hemispheres By
JOZEF KALUZA
and
ZOFIA KOWALSKA
With 8 figures (Received February ll, 1977)
Summary With the use of the histofluorescence method investigations were performed on L-DOPA uptake by the cells of an organotypic culture from chick-embryo brain hemispheres and newborn rats. It has been found that in chick-embryos between the 8th to llth day of development and 5 to 14 days in culture in vitro, with a concentration of 0.1 mg/lOml of L-DOPA applied to the culture, intense fluorescence in cell membranes, perinuclear region and sometimes in the whole cell body and the initial parts of the cell processes can be observed. It is supposed that in the stage of development of chick-embryos and newborn rats under study neural elements and glial cells take up the aminoacid applied. The authors suggest that the uptake of L-DOPA proceeds rather through the uptake II mechanism.
The growth and morphological differentiation of brain hemispheres neurons was a subject of investigations of several authors SENSENBRENNER et aL (1971), LUDUENA (1973), KALUZA and KOWALSKA (1975). Independent of the morphological, further studies concerned with functional maturation of the neurons were performed. HERMETET et aL (1970) have shown in neurons of sympathetic ganglia from chick embryos methylcholinesterase activity, which was growing parallel to the maturation of the cells. GOLDEN (1975) while studing the development of central nervous system structures, which eventually should containe biogenic amines, found in brain slices of mouse, that in the striatum the fluorescence appeared at 13th gestational day_ In the hypothalamus the axon terminals were visible at least on the 19th gestational day_ In most catecholamine containing structures the fluorescence appeared between the 13th and 14th gestational day with some differences in the intensity of fluorescence, dependent on the anatomical structure observed. GOLDEN (1975) could not find any serotoninergic axon terminals with the applied histofluorescence technique. Eo (1973) while studing cells from the neural crest of chick-embryo in early stages of development [stages 4 to 15 ace. to HAMBURGER and HAMILTON (1951)] - found catecholamine containing cells in explants taken from about 1.5 day old embryos and after 3 days of growth in vitro. Among other observations Eo (1973) mentions his unpublished once, which he presented as follows: "with the present culture method, no CA containing cells could be demonstrated in explants made from brain
Uptake of L-DOPA
33
chick embryos incubated for 3 to 5 days"_ We have failed to show CA containing neurons in organotypic and dispersed culture in vitro of brain hemispheres at the stage 35 to 40 acc_ to HAMBURGER and HAMILTON (1951) that is in the 9 to 12 day of development_ Exogenous noradrenalin and dopamine added to 4 days old cultures did not cause any kind of fluorescence_ The 4th day of culture seemed to us the optimal since on plasma clot, calf serum and chick embryo extract using PARKER's fluid as a medium we have obtained the best growth_ After application of exogenous substance we did not observe fluorescence of the investigated tissue in vitro_ However isolated, mature neurons can take up exogenous noradrenalin_ BONDAREFF and GRIFFITH (1972) have isolated neurons from the Nucleus reticularis pontis caudalis of the rat and exposed them 5 min to noradrenaline in concentrations from 0_5 mg/ml to 2 mg/mL After the histochemical procedure of FALCK et aL (1963) they obtained fluorescence in the pericarya of the neurons_ The authors suggest in the discussion, that the uptake observed is rather of uptake II mechanism, which operates extra-neuronally at relatively high -amine concentrations_ The negative results we have obtained with exogenous noradrenaline, added to tissue culture on the 4th day of growth, have led to us to consider the application of catecholamine precursor L-DOPA_ From investigations of HAKANSON et aL (1971) and BJORKLUND et aL (1972) it can be concluded, that the 3,4-dihydroxyphenylalanine fluorize after a treatment of tissue as described for catecholamines_ According to the above cited results, we could expect, that exogenous L-DOPA, if taken up by the cells in culture can be detected by the fluorescence technic as elaborated by FALCK et aL (1963)_
Materials and methods The tissue culture of neurons and glial cells have been obtained from chick embryos and newborn rats_ The tissue for the culture was taken from chick embryos of the Legho·rn species in the age of 7 to 12 days and newborn Wistar rats_ After dissection of the skull the brains were taken out in toto and transferred to cold TYRODE'S fluid 4°C_ In TYRODE'S fluid bath the leptomeninx were removed: in chick embryos together with the chorioid plexus_ Thereafter the hemispheres were thrown aside and the cerebral cortex isolated from the basal ganglia_ As much as possible the white matter was removed_ The cerebral cortex was cut on very small fragments of ± 1 mm2 _ The prepared material was devided in 2 parts_ One part of the material as explantation was transferred on gla8s-slides covered with plasma, the other part was smoothly blended on bolting cloth and suspended in chilled TYRODE'S fluid_ The obtained cell suspension was spread on glass-slides covered with plasma_ Both types of culture - explantations and dispersed culture - were covered with embryo extract diluted with TYRODE'S fluid in a relation of 1: 10_ As a medium PARKER'S fluid 199 enriched with calf serum (10 % of the total volume of medium) and embryo extract 0.3 % of the total volume of medium was used. The medium was changed usually every 5 to 7 days and if necessary in shorter time periods. The growth of living culture was observed in a plancton-microscope by the use of phase-contrast. The number of tissue cultures used in the described experiments; the time of action and concentration of L-DOPA applied to the culture is given in table 1. After the experiments have been performed the tissue culture were prepared for further observations were made, according to the method of FALCK et a1. by use of the fluorescence microscopc of Zeiss (Jena) with BG 12 as excitation filter and OG1 as selective filter. The lamp HBO 200 was the source of wave emission. The microphotographs were done using a Trix-Kodak film with exposition time 30 sec to 1.5 min. The film was de"eloped according to the standard prescription.
+
Results In experiments performed we determined the optimal time of fluorescence intensity after L-DOPA administration_ We have found 10 min after application of this compound to the medium as the best_ The photograms illustrate the most intense 3
Acta histochem_ Bd. 60
34
J.
KALUZA
and Z.
KOWALSKA
Fig. 1-4. Crude culturo from cerebral cortex of a 9-day chick embryo. Multiprocessed glial cells and neuroblasts. Plancton-microscope. X 120.
Uptake of L-DOPA
3·
35
36
J.
.....
. . .
••
~
~,
•••• •
•
T
~
..\• .., ..t..'. ....
.•
~
~ ......
~
,
~" (
,.
and Z.
.
," ...., ..". • tt-e -(- : . ~~.' ., . .
,-
KALUZA
-
....
•
4'\. I
•
•
.,
,
\.... ;z. • •.
KOWALSKA
~.
~ >.Ct" •
'':.:! .•
. . ". ..., . . , .. ..." .
..
,
•t "
etI}~
• •
. . ,...If ."· ..''.,. 1'" .......•
.
~
....
11. • . . , ... .
•
.,.~
!'C~,
•
,
• ,... .~
• ,".. . : .
-=....
\~ I-::' . :; .·.4~. _ ...~ . ~ -
.
••
','
"]!I
-'
•
. . .. .
-
Fig. 5, 6. Intense fluorescence in all cells in the growth zone after L·DOPA treatment. Fluorescence microscope. X 120.
-Uptake of L-DOPA
37
Fig. 7, 8. Intense fluorescence after L-DOPA treatment in cell membrane, cytoplasm and cell processes. In some cells diffuse fluorescence in the whole cell body. Fluorescence microscope. X 160.
38
.J.
KALUZA
and Z.
KOWALSKA
Table I.
No.
The age of the chick embryos [days]
Days of cuI- Concentration of ture growth L-DOPA
+
Time of action of L-DOPA
Evaluation of fluorescence intensity
0.1 mg/l0 mi 1.0 mg/W ml
10' 5' 10'
10' 15' 10' 15' 10' 10' 15' 10'
strong fluorescence prolonged time of action enhances the unspecific fluorescence yellow-greenish fluorescence in cell bodies and cell processes yellow-greenish fluorescence in cell bodies and cell processes fluorescence in cells weak fluorescence in cell processes on cytoplasm fluorescence medium in cell bodies and weak in cell processes intense yellow-green fluorescence between 10' and 15' no differ· ence in fluorescence was observed strong fluorescence. - Between 10' and 15' no difference in fluorescence was observed Prolonged action of L-DOPA enhances the unspecific fluorescence
I. 2.
8 8
5 (I) 5 (2)
3.
8
14 (2)
1.0 mg/W mi
4.
9
7 (2)
1.0 mg/l0 ml
5. 6.
10 10
7 (1) 9 (2)
0.1 mg/l0 ml 0.1 mg/l0 ml
7.
10
5 (1)
0.01 mg/l0 ml
8.
11
4 (2)
0.1 mg/l0 ml
9.
11
5 (2)
0.1 mg/W mi
10.
11
8 (2)
1.0 mg/l0 ml
10' 15'
II.
new born rat 11 (4)
1.3 mg/l0 ml
12.
new born rat 11 (6)
0.1 mg/l0 ml
15' 30' 1h 24 h 5' 10' 15' 30' 6h 24 h
10' 15' 10' 15'
The most specific, adherent to cells and their processes was visible after 10'-15' of LDOPA action
*) The number of cultures studied is given in brackets.
fluorescence in the cells. According to the morphological criteria these cells have been identified as glial cells. Only few of them show some feature which may correspond to these which may be indicative of unmature neural cells. As may be seen on photomicrogram there is an intense fluorescence in all of the cells independent of their morphology. The topography seems to be very characteristic. We found the fluorescence mainly localized in the cell membran. The other kind of fluorescence was diffuse and had an appearance of flooding granuls localized inside of the cell body or on his outer surface, what is difficult to differentiate by the method used. The most intense fluorescence we have observed in cells of the growth-zone. Additionally intense
Uptake of L-DOPA
39
fluorescence was visible probably in those cells in the tissue-culture, of which almost the whole cells surface, or eventually a part of the surface was elevated over the plasma clot and exposed to the action of medium and substances which were in medium dissolved. Weaker fluorescence was observed in cell processes, what is visible on photomicrograms. Tissue fragments in the matrix do not fluorize and the fluorescence was limited to cellular elements. Discussion According to the investigation of IVERSEN (1974) it was accepted that only catecholaminergic neurons take lip exogenous catecholamines. It seemed possible that neurocytes in embryo brain can show the same properties as the brains in vivo. According to the experiments of GOLDEN (1973), in which brains of embryos in various ontogenetic development phases were used some of the neurocytes contain catecholamines as has been shown with the histofluorescence method but some other neurocytes do not contain catecholamines. Bo (1973) has demonstrated that on a particular number of cells only as small part of them contains catecholamines. The other cells pertain in this respect negative. Further experiments performed have shown that the mechanism of catecholamines uptake operates under certain conditions and that the dose applied is of basic importance for the uptake mechanism. High catecholamine concentration of exogenous catecholamines leads to an unspecific re-uptake described as uptake II mechanism. According to IVERSEN (1974) the uptake II is less Na-dependent and shows no stereochemical selectivity for Na. BONDAREFF and GRIFFITH (1972) stated that uptake II is operative at high catecholamine concentration and is probably connected with membrane function of the cell. In our investigations on tissue culture in vitro from chick embryo and newborn rat we observed that the application of L-DOPA in the dose as indicated in Table 1 induce fluorescence in all the cell in the tissue culture including glial cells. It can be that the uptake of exogenous L-DOPA proceeds on the basis of uptake II. We observed the most intense fluorescence in cell membrane and in the vincinity of the nucleus. Histofluorescence studies of MALMFORS (1965) indicated that catecholamines applied in high doses according to IVERSEN over 40 pg!ml) are taken up from the whole cell. Also the initial fragments of the cell processes show some fluorescence visible on microphotographs. Taking into account the results obtained it seems to us that in tissue culture of chick embryo brain and newborn rats unspecific uptake mechanism opperates in anatomical structures, which in tissue in situ do not show such properties. Despite the fact that L-DOPA is only an aminoacid it can be to some extand an indicator of uptake mechanism in various cells of the central nervous system related to catecholamine metabolism. As has been shown by INCHIOZA et al. (1969) L-DOPA was found in various organs of the mammals. It can be that L-DOPA penetrates much easier into the embryonic brain cell than the catecholamines. Very intense fluorescence in the cell membrane may be indicative that some part of the compound is accumulated in the cell membrane. In the cell the fluorescence is concentrated in the central portion and it is supposed to be the perinuclear region. This would be in agreement with the observation of MALMFORS (1965). We do not know whether or not this fluorescence is connected with cellular organells. According to our observation we can only say that in tissue culture in vitro, cells from embryonic brain hemisphere beeing in a certain stage of ontogenetic development show uptake properties of L-DOPA and that this uptake can be of uptake II mechanism.
Literature BJORKLUND, A., FALCK, B., and OWMAN, OH., Fluorescence microscopic and microspectrofluorometric techniques for cellular localisation and characterisation of biogenic amines. The thyroid and biogenic amines. In: RALL and KOPIN, chapter 2, pp. 318-368 (1972). Amsterdam: NorthHolland Publishing Oomp.
40
J. KALUZA and Z. KOWALSKA, Uptake of L·DOPA
Bo, B., The production of catecholamine.containing cells in vitro by young chick embryos studied by the histochemical fluorescence method. J. Anatomy llS, 119-131 (1973). BONDAREFF, W., and GRIFFITH, D. L., Uptake of Norepinephrine by Isolated Neurons of Rat Pontine Reticular Formation. Z. Zellforsch. 123,295-304 (1972). FALCK, B., HILLARP, N. A., THIEME, G., and TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. 10, 348-354 (1962). GOLDEN, G. S., Prenatal Development of the Biogenic Amine System of the Mouse Brain. Develop. Bio!. 33, 300-311 (1973). HAKANSON, R., LJOBERG, A. K., and SUNDLER, F., Formaldehyde induced fluorescence of peptides I with N-terminal 3,4-Dihydroxyphenylalanine. Histochemie 28, 367-371 (1971). HAMBURGER, V., HAMILTON, H. L .• A series of normal stages in the development of the chick embryo. J. Morph. 88, 49-86 (1951). HERMETET, J. C., TRESKA, J., and MANDEL, P., Histochemical study of isolated neurons in culture from chick embryo sympathetic ganglia. Histochemie 22,177-186 (1970). INCHIOZA, M. A., and RODRIQUEZ, J. B., Distribution of epinephrin oxidasing enzyme in mammalian tissue. Biochem. Pharm. 18, 2032-2035 (1969). IVERSEN, L. L., Uptake for neurotransmitter amines. Biochem. Pharm. 23, 1927-1935 (1974). KALUZA, J., and KOWALSKA, Z., Differentiated growth of brain hemispheric neurocytes in organized (organotypic) and dispersed cultures in vitro. Folia Bio!. 23, 224-229 (1975). LUDUENA, M. A., Nerve cell differentiation in vitro. Develop. Bio!. 33, 268-284 (1973). MALMFORS, T., Studies on adrenergic nerves. Acta Physio!. Scand. 64, Supp!. 248 (1965). SENSENBRENNER, M., BOOHER, J., and MANDEL, P., Cultivation and growth of dissociated neurons from chick embryo cerebral cortex in the presence of different substrates. Z. Zellforsch. ll7, 559-569 (1971). Address: Dr. J OZEF KALUZA, Department of Neurology, Section Neuropathology, u!. Botaniczna 3, Krakow, Poland.
Polish Academy of Sciences, Institute of Pharmacology, Krakow, Poland
Uptake of L-DOPA by the cells of organotypic culture from chick-embryo brain hemispheres By
JOZEF KALUZA
and
ZOFIA KOWALSKA
With 8 figures (Received February ll, 1977)
Summary With the use of the histofluorescence method investigations were performed on L-DOPA uptake by the cells of an organotypic culture from chick-embryo brain hemispheres and newborn rats. It has been found that in chick-embryos between the 8th to llth day of development and 5 to 14 days in culture in vitro, with a concentration of 0.1 mg/lOml of L-DOPA applied to the culture, intense fluorescence in cell membranes, perinuclear region and sometimes in the whole cell body and the initial parts of the cell processes can be observed. It is supposed that in the stage of development of chick-embryos and newborn rats under study neural elements and glial cells take up the aminoacid applied. The authors suggest that the uptake of L-DOPA proceeds rather through the uptake II mechanism.
The growth and morphological differentiation of brain hemispheres neurons was a subject of investigations of several authors SENSENBRENNER et aL (1971), LUDUENA (1973), KALUZA and KOWALSKA (1975). Independent of the morphological, further studies concerned with functional maturation of the neurons were performed. HERMETET et aL (1970) have shown in neurons of sympathetic ganglia from chick embryos methylcholinesterase activity, which was growing parallel to the maturation of the cells. GOLDEN (1975) while studing the development of central nervous system structures, which eventually should containe biogenic amines, found in brain slices of mouse, that in the striatum the fluorescence appeared at 13th gestational day_ In the hypothalamus the axon terminals were visible at least on the 19th gestational day_ In most catecholamine containing structures the fluorescence appeared between the 13th and 14th gestational day with some differences in the intensity of fluorescence, dependent on the anatomical structure observed. GOLDEN (1975) could not find any serotoninergic axon terminals with the applied histofluorescence technique. Eo (1973) while studing cells from the neural crest of chick-embryo in early stages of development [stages 4 to 15 ace. to HAMBURGER and HAMILTON (1951)] - found catecholamine containing cells in explants taken from about 1.5 day old embryos and after 3 days of growth in vitro. Among other observations Eo (1973) mentions his unpublished once, which he presented as follows: "with the present culture method, no CA containing cells could be demonstrated in explants made from brain
Uptake of L-DOPA
33
chick embryos incubated for 3 to 5 days"_ We have failed to show CA containing neurons in organotypic and dispersed culture in vitro of brain hemispheres at the stage 35 to 40 acc_ to HAMBURGER and HAMILTON (1951) that is in the 9 to 12 day of development_ Exogenous noradrenalin and dopamine added to 4 days old cultures did not cause any kind of fluorescence_ The 4th day of culture seemed to us the optimal since on plasma clot, calf serum and chick embryo extract using PARKER's fluid as a medium we have obtained the best growth_ After application of exogenous substance we did not observe fluorescence of the investigated tissue in vitro_ However isolated, mature neurons can take up exogenous noradrenalin_ BONDAREFF and GRIFFITH (1972) have isolated neurons from the Nucleus reticularis pontis caudalis of the rat and exposed them 5 min to noradrenaline in concentrations from 0_5 mg/ml to 2 mg/mL After the histochemical procedure of FALCK et aL (1963) they obtained fluorescence in the pericarya of the neurons_ The authors suggest in the discussion, that the uptake observed is rather of uptake II mechanism, which operates extra-neuronally at relatively high -amine concentrations_ The negative results we have obtained with exogenous noradrenaline, added to tissue culture on the 4th day of growth, have led to us to consider the application of catecholamine precursor L-DOPA_ From investigations of HAKANSON et aL (1971) and BJORKLUND et aL (1972) it can be concluded, that the 3,4-dihydroxyphenylalanine fluorize after a treatment of tissue as described for catecholamines_ According to the above cited results, we could expect, that exogenous L-DOPA, if taken up by the cells in culture can be detected by the fluorescence technic as elaborated by FALCK et aL (1963)_
Materials and methods The tissue culture of neurons and glial cells have been obtained from chick embryos and newborn rats_ The tissue for the culture was taken from chick embryos of the Legho·rn species in the age of 7 to 12 days and newborn Wistar rats_ After dissection of the skull the brains were taken out in toto and transferred to cold TYRODE'S fluid 4°C_ In TYRODE'S fluid bath the leptomeninx were removed: in chick embryos together with the chorioid plexus_ Thereafter the hemispheres were thrown aside and the cerebral cortex isolated from the basal ganglia_ As much as possible the white matter was removed_ The cerebral cortex was cut on very small fragments of ± 1 mm2 _ The prepared material was devided in 2 parts_ One part of the material as explantation was transferred on gla8s-slides covered with plasma, the other part was smoothly blended on bolting cloth and suspended in chilled TYRODE'S fluid_ The obtained cell suspension was spread on glass-slides covered with plasma_ Both types of culture - explantations and dispersed culture - were covered with embryo extract diluted with TYRODE'S fluid in a relation of 1: 10_ As a medium PARKER'S fluid 199 enriched with calf serum (10 % of the total volume of medium) and embryo extract 0.3 % of the total volume of medium was used. The medium was changed usually every 5 to 7 days and if necessary in shorter time periods. The growth of living culture was observed in a plancton-microscope by the use of phase-contrast. The number of tissue cultures used in the described experiments; the time of action and concentration of L-DOPA applied to the culture is given in table 1. After the experiments have been performed the tissue culture were prepared for further observations were made, according to the method of FALCK et a1. by use of the fluorescence microscopc of Zeiss (Jena) with BG 12 as excitation filter and OG1 as selective filter. The lamp HBO 200 was the source of wave emission. The microphotographs were done using a Trix-Kodak film with exposition time 30 sec to 1.5 min. The film was de"eloped according to the standard prescription.
+
Results In experiments performed we determined the optimal time of fluorescence intensity after L-DOPA administration_ We have found 10 min after application of this compound to the medium as the best_ The photograms illustrate the most intense 3
Acta histochem_ Bd. 60
34
J.
KALUZA
and Z.
KOWALSKA
Fig. 1-4. Crude culturo from cerebral cortex of a 9-day chick embryo. Multiprocessed glial cells and neuroblasts. Plancton-microscope. X 120.
Uptake of L-DOPA
3·
35
36
J.
.....
. . .
••
~
~,
•••• •
•
T
~
..\• .., ..t..'. ....
.•
~
~ ......
~
,
~" (
,.
and Z.
.
," ...., ..". • tt-e -(- : . ~~.' ., . .
,-
KALUZA
-
....
•
4'\. I
•
•
.,
,
\.... ;z. • •.
KOWALSKA
~.
~ >.Ct" •
'':.:! .•
. . ". ..., . . , .. ..." .
..
,
•t "
etI}~
• •
. . ,...If ."· ..''.,. 1'" .......•
.
~
....
11. • . . , ... .
•
.,.~
!'C~,
•
,
• ,... .~
• ,".. . : .
-=....
\~ I-::' . :; .·.4~. _ ...~ . ~ -
.
••
','
"]!I
-'
•
. . .. .
-
Fig. 5, 6. Intense fluorescence in all cells in the growth zone after L·DOPA treatment. Fluorescence microscope. X 120.
-Uptake of L-DOPA
37
Fig. 7, 8. Intense fluorescence after L-DOPA treatment in cell membrane, cytoplasm and cell processes. In some cells diffuse fluorescence in the whole cell body. Fluorescence microscope. X 160.
38
.J.
KALUZA
and Z.
KOWALSKA
Table I.
No.
The age of the chick embryos [days]
Days of cuI- Concentration of ture growth L-DOPA
+
Time of action of L-DOPA
Evaluation of fluorescence intensity
0.1 mg/l0 mi 1.0 mg/W ml
10' 5' 10'
10' 15' 10' 15' 10' 10' 15' 10'
strong fluorescence prolonged time of action enhances the unspecific fluorescence yellow-greenish fluorescence in cell bodies and cell processes yellow-greenish fluorescence in cell bodies and cell processes fluorescence in cells weak fluorescence in cell processes on cytoplasm fluorescence medium in cell bodies and weak in cell processes intense yellow-green fluorescence between 10' and 15' no differ· ence in fluorescence was observed strong fluorescence. - Between 10' and 15' no difference in fluorescence was observed Prolonged action of L-DOPA enhances the unspecific fluorescence
I. 2.
8 8
5 (I) 5 (2)
3.
8
14 (2)
1.0 mg/W mi
4.
9
7 (2)
1.0 mg/l0 ml
5. 6.
10 10
7 (1) 9 (2)
0.1 mg/l0 ml 0.1 mg/l0 ml
7.
10
5 (1)
0.01 mg/l0 ml
8.
11
4 (2)
0.1 mg/l0 ml
9.
11
5 (2)
0.1 mg/W mi
10.
11
8 (2)
1.0 mg/l0 ml
10' 15'
II.
new born rat 11 (4)
1.3 mg/l0 ml
12.
new born rat 11 (6)
0.1 mg/l0 ml
15' 30' 1h 24 h 5' 10' 15' 30' 6h 24 h
10' 15' 10' 15'
The most specific, adherent to cells and their processes was visible after 10'-15' of LDOPA action
*) The number of cultures studied is given in brackets.
fluorescence in the cells. According to the morphological criteria these cells have been identified as glial cells. Only few of them show some feature which may correspond to these which may be indicative of unmature neural cells. As may be seen on photomicrogram there is an intense fluorescence in all of the cells independent of their morphology. The topography seems to be very characteristic. We found the fluorescence mainly localized in the cell membran. The other kind of fluorescence was diffuse and had an appearance of flooding granuls localized inside of the cell body or on his outer surface, what is difficult to differentiate by the method used. The most intense fluorescence we have observed in cells of the growth-zone. Additionally intense
Uptake of L-DOPA
39
fluorescence was visible probably in those cells in the tissue-culture, of which almost the whole cells surface, or eventually a part of the surface was elevated over the plasma clot and exposed to the action of medium and substances which were in medium dissolved. Weaker fluorescence was observed in cell processes, what is visible on photomicrograms. Tissue fragments in the matrix do not fluorize and the fluorescence was limited to cellular elements. Discussion According to the investigation of IVERSEN (1974) it was accepted that only catecholaminergic neurons take lip exogenous catecholamines. It seemed possible that neurocytes in embryo brain can show the same properties as the brains in vivo. According to the experiments of GOLDEN (1973), in which brains of embryos in various ontogenetic development phases were used some of the neurocytes contain catecholamines as has been shown with the histofluorescence method but some other neurocytes do not contain catecholamines. Bo (1973) has demonstrated that on a particular number of cells only as small part of them contains catecholamines. The other cells pertain in this respect negative. Further experiments performed have shown that the mechanism of catecholamines uptake operates under certain conditions and that the dose applied is of basic importance for the uptake mechanism. High catecholamine concentration of exogenous catecholamines leads to an unspecific re-uptake described as uptake II mechanism. According to IVERSEN (1974) the uptake II is less Na-dependent and shows no stereochemical selectivity for Na. BONDAREFF and GRIFFITH (1972) stated that uptake II is operative at high catecholamine concentration and is probably connected with membrane function of the cell. In our investigations on tissue culture in vitro from chick embryo and newborn rat we observed that the application of L-DOPA in the dose as indicated in Table 1 induce fluorescence in all the cell in the tissue culture including glial cells. It can be that the uptake of exogenous L-DOPA proceeds on the basis of uptake II. We observed the most intense fluorescence in cell membrane and in the vincinity of the nucleus. Histofluorescence studies of MALMFORS (1965) indicated that catecholamines applied in high doses according to IVERSEN over 40 pg!ml) are taken up from the whole cell. Also the initial fragments of the cell processes show some fluorescence visible on microphotographs. Taking into account the results obtained it seems to us that in tissue culture of chick embryo brain and newborn rats unspecific uptake mechanism opperates in anatomical structures, which in tissue in situ do not show such properties. Despite the fact that L-DOPA is only an aminoacid it can be to some extand an indicator of uptake mechanism in various cells of the central nervous system related to catecholamine metabolism. As has been shown by INCHIOZA et al. (1969) L-DOPA was found in various organs of the mammals. It can be that L-DOPA penetrates much easier into the embryonic brain cell than the catecholamines. Very intense fluorescence in the cell membrane may be indicative that some part of the compound is accumulated in the cell membrane. In the cell the fluorescence is concentrated in the central portion and it is supposed to be the perinuclear region. This would be in agreement with the observation of MALMFORS (1965). We do not know whether or not this fluorescence is connected with cellular organells. According to our observation we can only say that in tissue culture in vitro, cells from embryonic brain hemisphere beeing in a certain stage of ontogenetic development show uptake properties of L-DOPA and that this uptake can be of uptake II mechanism.
Literature BJORKLUND, A., FALCK, B., and OWMAN, OH., Fluorescence microscopic and microspectrofluorometric techniques for cellular localisation and characterisation of biogenic amines. The thyroid and biogenic amines. In: RALL and KOPIN, chapter 2, pp. 318-368 (1972). Amsterdam: NorthHolland Publishing Oomp.
40
J. KALUZA and Z. KOWALSKA, Uptake of L·DOPA
Bo, B., The production of catecholamine.containing cells in vitro by young chick embryos studied by the histochemical fluorescence method. J. Anatomy llS, 119-131 (1973). BONDAREFF, W., and GRIFFITH, D. L., Uptake of Norepinephrine by Isolated Neurons of Rat Pontine Reticular Formation. Z. Zellforsch. 123,295-304 (1972). FALCK, B., HILLARP, N. A., THIEME, G., and TORP, A., Fluorescence of catecholamines and related compounds condensed with formaldehyde. J. Histochem. 10, 348-354 (1962). GOLDEN, G. S., Prenatal Development of the Biogenic Amine System of the Mouse Brain. Develop. Bio!. 33, 300-311 (1973). HAKANSON, R., LJOBERG, A. K., and SUNDLER, F., Formaldehyde induced fluorescence of peptides I with N-terminal 3,4-Dihydroxyphenylalanine. Histochemie 28, 367-371 (1971). HAMBURGER, V., HAMILTON, H. L .• A series of normal stages in the development of the chick embryo. J. Morph. 88, 49-86 (1951). HERMETET, J. C., TRESKA, J., and MANDEL, P., Histochemical study of isolated neurons in culture from chick embryo sympathetic ganglia. Histochemie 22,177-186 (1970). INCHIOZA, M. A., and RODRIQUEZ, J. B., Distribution of epinephrin oxidasing enzyme in mammalian tissue. Biochem. Pharm. 18, 2032-2035 (1969). IVERSEN, L. L., Uptake for neurotransmitter amines. Biochem. Pharm. 23, 1927-1935 (1974). KALUZA, J., and KOWALSKA, Z., Differentiated growth of brain hemispheric neurocytes in organized (organotypic) and dispersed cultures in vitro. Folia Bio!. 23, 224-229 (1975). LUDUENA, M. A., Nerve cell differentiation in vitro. Develop. Bio!. 33, 268-284 (1973). MALMFORS, T., Studies on adrenergic nerves. Acta Physio!. Scand. 64, Supp!. 248 (1965). SENSENBRENNER, M., BOOHER, J., and MANDEL, P., Cultivation and growth of dissociated neurons from chick embryo cerebral cortex in the presence of different substrates. Z. Zellforsch. ll7, 559-569 (1971). Address: Dr. J OZEF KALUZA, Department of Neurology, Section Neuropathology, u!. Botaniczna 3, Krakow, Poland.