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Myelination in developing cultured newborn rat cerebellum inhibited by 5-bromodeoxyuridine
Preliminary notes 455
material. The existence of pinocytosis in these cells has been contested for a long time; however, in recent years several authors have shown that lymphocytes were able to phagocytize small particles such as peroxidase and ferritin [6, 71 in vivo, as well as mycoplasma and thorotrast in vitro [13]. We thank Professor M Be& for continuous interest in this work. We are indebted to Mrs G Polini for capable technical assistance. This work was supported by research grants DGRST no. 72.7.0298 and CNRS, ATP No. 6017.
References 1. Avrameas, S, Compt rend acad sci 270 (1970)
2205. 2. Bernhard, W & Avrameas, S, Exptl cell res 64 (1971) 232. 3. Frye, L D & Edidin, M, J cell sci 7 (1970) 319. 4. Goldstein, I J, So, L L, Young, Y & Callies, Q C, J immunol 103 (1969) 291. 5. Graham, R C & Kamovsky, M J, J histochem cytochem 14 (1966) 291. 6. Han, S S &Johnson, A G, Science 153 (1966) 176. 7. Koszewski, B J, Emerick, C W & Dicus, D R, Blood 12 (1957) 559. 8. Leon, M A &Powell, A E, J reticul endothel sot 5 (1968) 35. 9. Powell, A E &Leon, M A, Exptl cell res 62 (1970) 315. 10. Singer, S J & Nicolson, G L, Science 175 (1972) 720. Il. Smith, S W & Hollers, J C, J reticul endothel sot 8 (1970) 458. 12. Tanaka, Y, Blood 35 (1970) 793. 13. Zucker-Franklin, D, Davidson, M & Thomas, L, J exptl med 124 (1966) 533. Received July 31, 1972
Myelination in developing cultured newborn rat cerebellum inhibited by 5-bromodeoxyuridine LINDA YOUNKIN and D. SILBERBERG, Department of Neurology, 424 Johnson Pavilion, University of Pennsylvania School of Medicine, Philadelphia, Pa 19104, lJSA
Summary Incorporation of 5-bromodeoxyuridine (BUdR) into developing newborn rat cerebellum organ cultures inhibited formation of myelin without apparent effect
on morphological maturation. Inhibition of the appearance of myelin, which usually begins on l&l 1 days in vitro (DIV), occurred only when BUdR was present during’6 and 7 DIV, thus suggesting a ‘critical cell division on those days for the differentiating oligodendrocytes that will produce myelin.
Explanted pieces of newborn rat cerebellum maintained in organ culture exhibit morphological and biochemical maturation similar to that which occurs in vivo [l-5]. One of the most obvious results of the differentiation of this nervous tissue is the myelination of axons. Myelin, a compacted, specialized membrane which is wrapped around axons, is produced by some of the oligodendrocytes in this tissue which contains many cell types [6]. These oligodendrocytes go through an as yet undetermined number of cell divisions and a progressive differentiation from precursor cells to mature oligodendrocytes [7-91. The time in this sequenceof differentiation when the oligodendrocytes become determined as myelin-producers is not yet clear. We report that Sbromodeoxyuridine (BUdR), at concentrations known to inhibit the development of tissue-specific functions in other differentiating tissues [IO-161, inhibits myelination in the developing cerebellum. The specific effect of BUdR on differentiating cells appears to depend on the level of development the tissue has reached when BUdR is added [lO-141. It is generally agreed that BUdR-induced inhibition is a result of the incorporation of BUdR into DNA in place of thymidine during DNA synthesis [lO-161. The mechanism by which this substitution inhibits cell differentiation is still unknown. BUdR has been used to describe the time of the final precursor cell division in cell populations that ceasedivision upon differentiation [IO-121 and to locate the time of a regulatory event that determines the cell-specific function in continually-dividing differentiated populations [13, 141. As a first step in analysing the kinetics of oligodendrocyte Exptl Cell Res 76 (1973)
456
Preliminary notes
0
l( 80
20
0 10-e
10-5
10-a
Fin. I. Abscissa:
BUdR dose (moles); ordinate: Y, of-cultures with myelinated axons. 0 i 13; 0, 15 div. Myelination observed at 13 and 15 DIV as a function of BUdR dose. BUdR was in the culture medium from explant and in each bi-weekly feeding. Each point on the curves represents a mean of at least 8 cultures; 1.5 x lo-& M BUdR and control points are means of 32 cultures each. These curves represent the same cultures at two observation periods and indicate the progressive appearance of myelin in these cultures.
differentiation, we have used BUdR to locate the critical time for the oligodendrocytes’ determination as myelin-producing cells. Materials and Methods Sections of newborn rat cerebellum were cultured in the Maximow double-coverslip assembly as previously described 15. 171.At about IO-11 days in vitro (DIV) myelinated ‘axons began to appear. At the light microscope level (600 x ) myelinated axons in living cultures are readily distinguished from unmyelinated axons as highly refractile, smooth parallel lines. Each culture was examined neriodicallv. and all mvelinated axons were counted -as they appeared. When BUdR (CalBiochem) was included in the culture medium ultraviolet lights were not used in the sterile room, and the cultures were protected from daylight. At least 8 cultures, from different animals of the same litter, were used for each experimental variation. Tritiated BUdR (SH-BUdR, New England Nuclear, 26.1 Ci/mM spec. act.) was included in some cultures for autoradiography. At 15 DIV these cultures were fixed in glutaraldehyde (4%) and osmium tetroxide (1 %), dehydrated in alcohol and propylene oxide, and embedded in Araldite. Semi-thick sections (0.51.0 pm) were made on a Porter-Blum MT-I Microtome, dipped in Kodak Nuclear Track Emulsion NTB-2 (diluted 1 : 1 with water), exposed for 7-10 days, developed and stained with 0.5 % toluidine blue. Exptl Cell Res 76 (1973)
Results and Discussion Cerebellum cultures exposed to 1.5 x 1O-4 M BUdR from explant looked as healthy as the controls throughout the culture period except that few or no axons became myelinated. At 13 DIV 70 % of the control cultures were myelinated with a mean of 8 myelinated axons per culture, whereas only 8 % of the BUdR-treated cultures were myelinated with a mean of less than 1 myelinated axon per culture. At 15 DIV 100 Yb of the controls were myelinated with a mean of 48 myelinated axons per culture, and only 21 “/o of the BUdR cultures were myelinated with a mean of 2 myelinated axons per culture (fig. 1). There was some evidence of cell flattening and culture spreading as reported in other systems [lo, 11, 15, 161. BUdR-treated cultures were indistinguishable from controls when examined as toluidine blue-stained semi-thick sections with the light microscope. All types of cells apparently differentiated to the same degree as in the controls, including the small, darkly-stained oligodendrocytes ordinarily associated with myelin [18, 7-91. Thus it appears that BUdR may interfere with myelin production by oligodendrocytes without otherwise altering their morphological differentiation. However, the only reliable morphological feature which permits
identification
of a myelin-producing
to a oligodendrocyte is its connection myelinated axon. A range of BUdR doses was included in the culture
medium
from
explant
to determine
the lowest
concentration which inhibited myelin formation (fig. 1). Both 1.5 and 3.0 X 1O-4 M BUdR inhibited myelination significantly; 7.5 x 1O-5 and 1.2 x 1O-4 M BUdR caused partial inhibition of myelination. Lower doses of BUdR did not inhibit myelination; in fact, they appeared to increase the number of myelinated axons (see the 13 DIV
curve, fig. 1).
Preliminary notes 457 , 2 ( 0.04 11.7 ( 1.1 , 0.45
3
4
5
6
7
8
9
10
11
12
13
14
15 8
1 J
( 0.24
I
(0.15
F&&.
I
, 0.03
Abscissa:
DIV;
ordinate:
BUdR
, Lo.o*
,
~0.06 , 0.16 , 0.63 , 1.5 , 1.2
, 1.7 10.42 10.42 (0.53 (0.49 , 0.38 , 0.04
, I
Results of experiments designed to prevent BUdR inhibition with excess thymidine and to localize 3H-BUdR incorporation agreed with the generally recognized idea that the site of BUdR action is the DNA. Thymidine, at 2, 5 or 7.5 times the concentration of BUdR, when added to cultures simultaneously explanted in 1.5 X lo-* M BUdR, prevented the inhibition of myelination. Thymidine at twice the BUdR concentration allowed most of the cultures to myelinate, but the mean number of myelinated axons per culture remained low, equivalent to inhibited cultures. The higher doses of thymidine permitted control levels of myelination in the presence of BUdR. Autoradiographs of sections of cultures exposed to 1 ,Ki 3H-BUdR/ml from 7 to 15 DIV in the presence of 1.5 x 1O-4 M BUdR showed most of the label in cell nuclei. Some cells, usually in necrotic area of the cultures, had cytoplasmic label. Labelled chromosomes were also seen. Many of the labelled cells were identifiable as oligodendrocytes; as expected, granule cells were also labelled. The large neurons, which complete DNA synthesis and division before birth [19, 201, contained no label, even though Purkinje cells reportedly synthesize
’ 1 ’ ’ ’ ’
Myelination of 15 DIV cultures that have been exposed to BUdR for various lengths of time. The numbers on the bars depicting the BUdR (1.5 x 1O-4 M) pulses are the ratios of the mean number of myelinated axons per BUdR culture to the mean number of myelinated axons per control culture (control cultures are therefore 1.0 and inhibited culture ratios are less than 1.0). At least 8 cultures were used for each BUdR pulse; 36 cultures were exposed to continuous BUdR.
DNA and become tetraploid sometime after birth [21]. Pulses of BUdR were used to determine that time in development in vitro when BUdR most effectively inhibits myelination. The cultures were always washed when the medium was changed. Myelination was inhibited only when BUdR was present in the cultures during 6 and 7 DIV (fig. 2). Continuous exposure to BUdR, a pulse of BUdR from explant to 7 DIV, a pulse of BUdR from 5 or 6 DIV to 15 DlV, and a pulse of BUdR from 6-8 DIV all covered this ‘critical’ time and inhibited myelination. Pulses of BUdR that did not cover this time period completely or were near to it resulted in partial inhibition of myelination. Pulses of BUdR distant from this critical time period seemed to increase myelination (fig. 2). If current suggestions are correct that BUdR causes inhibition of cell-specific functions only after being incorporated into DNA [IO-163, it may then be assumed that at approx. 6-7 DIV in these cerebellum cultures there is an important cell division occurring in the oligodendrocytes that will later produce myelin. It is not yet clear whether this is the only postnatal division of these cells, or whether it is one of several divisions and is the Exptl Cell Res 76 (1973)
458
Preliminary notes
one during which the cells becomedetermined as myelin-producers. Since myelin does not appear until 10-l 1 DIV, these oligodendrocytes, determined as myelin-producers on 6-7 DIV, appear to go through a 4-5 day ‘maturation’ period. However, this maturation period is probably an overestimate since myelin is not visible with the light microscope until it has reached a 3-4 lamellae thickness around the axon [22]. The presence of morphologically differentiated oligodendrocytes in BUdR-inhibited cultures leaves open the question of the exact stage in myelinogenesis which is being affected by BUdR. Developing neurons probably contribute to the process of myelination. Only when the kinetics of oligodendrocyte differentiation has been described can cellular interactions between neurons and oligodendrocytes be adequately defined.
15. Abbott, J & Holtzer, H, Proc natl acad sci US 59 (1968) 1144. 16. Mayne, R, Sanger, J W & Holtzer, H, Dev biol 25 (1971) 547. 17. Bornstein, M B & Murray, M R, 3 biophys biochem cytol 4 (1958) 499. 18. Raine, C S & Bronstein, M B, J neuropath exptl neurol 29 (1970) 552. 19. Miale, I L & Sidman, R L, Exptl neurol 4 (1961) 311 20. .&man. J. Handbook of neurochemistrv (ed A Lajtha): Structural neurochemistry, vol. ’ 2, D. 137. Plenum Press. New York (1969). 21. Lentz, R D & Lapham, L W, J neurochem 16 (1969) 379. 22. Murray, M R, Cells and tissues in culturemethods, biology and physiology (ed E N Wilmer) vol. 11, p. 373. Academic Press, London (1965).
This work was supported by US PHS grantsNS-08197 (Dr Silberberg) and NS-05273 (Dr Younkin). Dr Younkin was a post-doctoral fellow in the Institute of Neurological Sciences at the University of Pennsylvania at the time of this work. We thank Doris Politz and Susan Dorfman for technical assistance.
R. W. LEU, A. W. L. F. EDDLESTON, R. A. GOOD and J. W. HADDEN, Department of Pediatrics and Pathology, University of Minnesota Hospitals, Minneapolis, Minn. 55455, USA
Received July 31, 1972 Revised version received October 9, 1972
Paradoxical effects of ouabain on the migration of peritoneal and alveolar macrophages
Modulation of leukocyte migration by immunological factors has received considerable attention in recent years. Among the References factors thought to influence the migration of 1. Ross. L L. Bornstein. M B & Lehrer, G M. J cell biol 14’(1962) 19. ’ leukocytes are a variety of chemotactic factors 2. Seil. F J & Hemdon. R M, J cell biol 45 (1970) for macrophages, polymorphonuclear leuko212: 3. Wolf, M, J camp neurol 140 (1970) 281. cytes, and eosinophilic leukocytes. Of partic4. Lehrer, G M, Bomstein, M B, Weiss, C, Furman, ular interest to us is a lymphocyte-produced M & Lichtman, C, Exptl neurol 27 (1970) 410. 5. Silberbere. D H. Beniamins. J. Herschkowitz, factor termed macrophage migration inN & McKhann, G M, j neurochem 19 (1972) 11: hibitory factor (MIF) which we have recently 6. Bunge, R P, Physiol rev 48 (1968) 197. 7. Vaughn, J E, 2 zellforsch 94 (1969) 293. shown to operate through a surface receptor 8. Mori, S & LeBlond, C P, J camp neurol 139 mechanism [I]. Interest in the mechanism (1970) 1. 9. Manuelidis, L & Manuelidis, E E, Acta neuropath whereby surface active effector molecules 18 (1971) 193. might regulate cell movement prompted us to 10. Stockdale, F, Okazaki, K, Nameroff, M & Holtzer, H, Science 146 (1964) 533. study the effects of ouabain, a cardiac glyco11. Bischoff, R & Holtzer, H, J cell biol 44 (1970) side, on macrophage migration. 134. 12. Turkington, R W, Majumder, G C & Riddle, M, J biol them 246 (1971) 1814. 13. Muira, Y & Wilt, F H, J cell biol 48 (1971) 523. 14. Gontcharoff, M & Mazia, D, Exptl cell res 46 (1967) 315. ExptI Cell Res 76 (1973)
Materials and Methods Guinea pig alveolar and peritoneal macrophages were obtained and their migration assayed by the capillary
material. The existence of pinocytosis in these cells has been contested for a long time; however, in recent years several authors have shown that lymphocytes were able to phagocytize small particles such as peroxidase and ferritin [6, 71 in vivo, as well as mycoplasma and thorotrast in vitro [13]. We thank Professor M Be& for continuous interest in this work. We are indebted to Mrs G Polini for capable technical assistance. This work was supported by research grants DGRST no. 72.7.0298 and CNRS, ATP No. 6017.
References 1. Avrameas, S, Compt rend acad sci 270 (1970)
2205. 2. Bernhard, W & Avrameas, S, Exptl cell res 64 (1971) 232. 3. Frye, L D & Edidin, M, J cell sci 7 (1970) 319. 4. Goldstein, I J, So, L L, Young, Y & Callies, Q C, J immunol 103 (1969) 291. 5. Graham, R C & Kamovsky, M J, J histochem cytochem 14 (1966) 291. 6. Han, S S &Johnson, A G, Science 153 (1966) 176. 7. Koszewski, B J, Emerick, C W & Dicus, D R, Blood 12 (1957) 559. 8. Leon, M A &Powell, A E, J reticul endothel sot 5 (1968) 35. 9. Powell, A E &Leon, M A, Exptl cell res 62 (1970) 315. 10. Singer, S J & Nicolson, G L, Science 175 (1972) 720. Il. Smith, S W & Hollers, J C, J reticul endothel sot 8 (1970) 458. 12. Tanaka, Y, Blood 35 (1970) 793. 13. Zucker-Franklin, D, Davidson, M & Thomas, L, J exptl med 124 (1966) 533. Received July 31, 1972
Myelination in developing cultured newborn rat cerebellum inhibited by 5-bromodeoxyuridine LINDA YOUNKIN and D. SILBERBERG, Department of Neurology, 424 Johnson Pavilion, University of Pennsylvania School of Medicine, Philadelphia, Pa 19104, lJSA
Summary Incorporation of 5-bromodeoxyuridine (BUdR) into developing newborn rat cerebellum organ cultures inhibited formation of myelin without apparent effect
on morphological maturation. Inhibition of the appearance of myelin, which usually begins on l&l 1 days in vitro (DIV), occurred only when BUdR was present during’6 and 7 DIV, thus suggesting a ‘critical cell division on those days for the differentiating oligodendrocytes that will produce myelin.
Explanted pieces of newborn rat cerebellum maintained in organ culture exhibit morphological and biochemical maturation similar to that which occurs in vivo [l-5]. One of the most obvious results of the differentiation of this nervous tissue is the myelination of axons. Myelin, a compacted, specialized membrane which is wrapped around axons, is produced by some of the oligodendrocytes in this tissue which contains many cell types [6]. These oligodendrocytes go through an as yet undetermined number of cell divisions and a progressive differentiation from precursor cells to mature oligodendrocytes [7-91. The time in this sequenceof differentiation when the oligodendrocytes become determined as myelin-producers is not yet clear. We report that Sbromodeoxyuridine (BUdR), at concentrations known to inhibit the development of tissue-specific functions in other differentiating tissues [IO-161, inhibits myelination in the developing cerebellum. The specific effect of BUdR on differentiating cells appears to depend on the level of development the tissue has reached when BUdR is added [lO-141. It is generally agreed that BUdR-induced inhibition is a result of the incorporation of BUdR into DNA in place of thymidine during DNA synthesis [lO-161. The mechanism by which this substitution inhibits cell differentiation is still unknown. BUdR has been used to describe the time of the final precursor cell division in cell populations that ceasedivision upon differentiation [IO-121 and to locate the time of a regulatory event that determines the cell-specific function in continually-dividing differentiated populations [13, 141. As a first step in analysing the kinetics of oligodendrocyte Exptl Cell Res 76 (1973)
456
Preliminary notes
0
l( 80
20
0 10-e
10-5
10-a
Fin. I. Abscissa:
BUdR dose (moles); ordinate: Y, of-cultures with myelinated axons. 0 i 13; 0, 15 div. Myelination observed at 13 and 15 DIV as a function of BUdR dose. BUdR was in the culture medium from explant and in each bi-weekly feeding. Each point on the curves represents a mean of at least 8 cultures; 1.5 x lo-& M BUdR and control points are means of 32 cultures each. These curves represent the same cultures at two observation periods and indicate the progressive appearance of myelin in these cultures.
differentiation, we have used BUdR to locate the critical time for the oligodendrocytes’ determination as myelin-producing cells. Materials and Methods Sections of newborn rat cerebellum were cultured in the Maximow double-coverslip assembly as previously described 15. 171.At about IO-11 days in vitro (DIV) myelinated ‘axons began to appear. At the light microscope level (600 x ) myelinated axons in living cultures are readily distinguished from unmyelinated axons as highly refractile, smooth parallel lines. Each culture was examined neriodicallv. and all mvelinated axons were counted -as they appeared. When BUdR (CalBiochem) was included in the culture medium ultraviolet lights were not used in the sterile room, and the cultures were protected from daylight. At least 8 cultures, from different animals of the same litter, were used for each experimental variation. Tritiated BUdR (SH-BUdR, New England Nuclear, 26.1 Ci/mM spec. act.) was included in some cultures for autoradiography. At 15 DIV these cultures were fixed in glutaraldehyde (4%) and osmium tetroxide (1 %), dehydrated in alcohol and propylene oxide, and embedded in Araldite. Semi-thick sections (0.51.0 pm) were made on a Porter-Blum MT-I Microtome, dipped in Kodak Nuclear Track Emulsion NTB-2 (diluted 1 : 1 with water), exposed for 7-10 days, developed and stained with 0.5 % toluidine blue. Exptl Cell Res 76 (1973)
Results and Discussion Cerebellum cultures exposed to 1.5 x 1O-4 M BUdR from explant looked as healthy as the controls throughout the culture period except that few or no axons became myelinated. At 13 DIV 70 % of the control cultures were myelinated with a mean of 8 myelinated axons per culture, whereas only 8 % of the BUdR-treated cultures were myelinated with a mean of less than 1 myelinated axon per culture. At 15 DIV 100 Yb of the controls were myelinated with a mean of 48 myelinated axons per culture, and only 21 “/o of the BUdR cultures were myelinated with a mean of 2 myelinated axons per culture (fig. 1). There was some evidence of cell flattening and culture spreading as reported in other systems [lo, 11, 15, 161. BUdR-treated cultures were indistinguishable from controls when examined as toluidine blue-stained semi-thick sections with the light microscope. All types of cells apparently differentiated to the same degree as in the controls, including the small, darkly-stained oligodendrocytes ordinarily associated with myelin [18, 7-91. Thus it appears that BUdR may interfere with myelin production by oligodendrocytes without otherwise altering their morphological differentiation. However, the only reliable morphological feature which permits
identification
of a myelin-producing
to a oligodendrocyte is its connection myelinated axon. A range of BUdR doses was included in the culture
medium
from
explant
to determine
the lowest
concentration which inhibited myelin formation (fig. 1). Both 1.5 and 3.0 X 1O-4 M BUdR inhibited myelination significantly; 7.5 x 1O-5 and 1.2 x 1O-4 M BUdR caused partial inhibition of myelination. Lower doses of BUdR did not inhibit myelination; in fact, they appeared to increase the number of myelinated axons (see the 13 DIV
curve, fig. 1).
Preliminary notes 457 , 2 ( 0.04 11.7 ( 1.1 , 0.45
3
4
5
6
7
8
9
10
11
12
13
14
15 8
1 J
( 0.24
I
(0.15
F&&.
I
, 0.03
Abscissa:
DIV;
ordinate:
BUdR
, Lo.o*
,
~0.06 , 0.16 , 0.63 , 1.5 , 1.2
, 1.7 10.42 10.42 (0.53 (0.49 , 0.38 , 0.04
, I
Results of experiments designed to prevent BUdR inhibition with excess thymidine and to localize 3H-BUdR incorporation agreed with the generally recognized idea that the site of BUdR action is the DNA. Thymidine, at 2, 5 or 7.5 times the concentration of BUdR, when added to cultures simultaneously explanted in 1.5 X lo-* M BUdR, prevented the inhibition of myelination. Thymidine at twice the BUdR concentration allowed most of the cultures to myelinate, but the mean number of myelinated axons per culture remained low, equivalent to inhibited cultures. The higher doses of thymidine permitted control levels of myelination in the presence of BUdR. Autoradiographs of sections of cultures exposed to 1 ,Ki 3H-BUdR/ml from 7 to 15 DIV in the presence of 1.5 x 1O-4 M BUdR showed most of the label in cell nuclei. Some cells, usually in necrotic area of the cultures, had cytoplasmic label. Labelled chromosomes were also seen. Many of the labelled cells were identifiable as oligodendrocytes; as expected, granule cells were also labelled. The large neurons, which complete DNA synthesis and division before birth [19, 201, contained no label, even though Purkinje cells reportedly synthesize
’ 1 ’ ’ ’ ’
Myelination of 15 DIV cultures that have been exposed to BUdR for various lengths of time. The numbers on the bars depicting the BUdR (1.5 x 1O-4 M) pulses are the ratios of the mean number of myelinated axons per BUdR culture to the mean number of myelinated axons per control culture (control cultures are therefore 1.0 and inhibited culture ratios are less than 1.0). At least 8 cultures were used for each BUdR pulse; 36 cultures were exposed to continuous BUdR.
DNA and become tetraploid sometime after birth [21]. Pulses of BUdR were used to determine that time in development in vitro when BUdR most effectively inhibits myelination. The cultures were always washed when the medium was changed. Myelination was inhibited only when BUdR was present in the cultures during 6 and 7 DIV (fig. 2). Continuous exposure to BUdR, a pulse of BUdR from explant to 7 DIV, a pulse of BUdR from 5 or 6 DIV to 15 DlV, and a pulse of BUdR from 6-8 DIV all covered this ‘critical’ time and inhibited myelination. Pulses of BUdR that did not cover this time period completely or were near to it resulted in partial inhibition of myelination. Pulses of BUdR distant from this critical time period seemed to increase myelination (fig. 2). If current suggestions are correct that BUdR causes inhibition of cell-specific functions only after being incorporated into DNA [IO-163, it may then be assumed that at approx. 6-7 DIV in these cerebellum cultures there is an important cell division occurring in the oligodendrocytes that will later produce myelin. It is not yet clear whether this is the only postnatal division of these cells, or whether it is one of several divisions and is the Exptl Cell Res 76 (1973)
458
Preliminary notes
one during which the cells becomedetermined as myelin-producers. Since myelin does not appear until 10-l 1 DIV, these oligodendrocytes, determined as myelin-producers on 6-7 DIV, appear to go through a 4-5 day ‘maturation’ period. However, this maturation period is probably an overestimate since myelin is not visible with the light microscope until it has reached a 3-4 lamellae thickness around the axon [22]. The presence of morphologically differentiated oligodendrocytes in BUdR-inhibited cultures leaves open the question of the exact stage in myelinogenesis which is being affected by BUdR. Developing neurons probably contribute to the process of myelination. Only when the kinetics of oligodendrocyte differentiation has been described can cellular interactions between neurons and oligodendrocytes be adequately defined.
15. Abbott, J & Holtzer, H, Proc natl acad sci US 59 (1968) 1144. 16. Mayne, R, Sanger, J W & Holtzer, H, Dev biol 25 (1971) 547. 17. Bornstein, M B & Murray, M R, 3 biophys biochem cytol 4 (1958) 499. 18. Raine, C S & Bronstein, M B, J neuropath exptl neurol 29 (1970) 552. 19. Miale, I L & Sidman, R L, Exptl neurol 4 (1961) 311 20. .&man. J. Handbook of neurochemistrv (ed A Lajtha): Structural neurochemistry, vol. ’ 2, D. 137. Plenum Press. New York (1969). 21. Lentz, R D & Lapham, L W, J neurochem 16 (1969) 379. 22. Murray, M R, Cells and tissues in culturemethods, biology and physiology (ed E N Wilmer) vol. 11, p. 373. Academic Press, London (1965).
This work was supported by US PHS grantsNS-08197 (Dr Silberberg) and NS-05273 (Dr Younkin). Dr Younkin was a post-doctoral fellow in the Institute of Neurological Sciences at the University of Pennsylvania at the time of this work. We thank Doris Politz and Susan Dorfman for technical assistance.
R. W. LEU, A. W. L. F. EDDLESTON, R. A. GOOD and J. W. HADDEN, Department of Pediatrics and Pathology, University of Minnesota Hospitals, Minneapolis, Minn. 55455, USA
Received July 31, 1972 Revised version received October 9, 1972
Paradoxical effects of ouabain on the migration of peritoneal and alveolar macrophages
Modulation of leukocyte migration by immunological factors has received considerable attention in recent years. Among the References factors thought to influence the migration of 1. Ross. L L. Bornstein. M B & Lehrer, G M. J cell biol 14’(1962) 19. ’ leukocytes are a variety of chemotactic factors 2. Seil. F J & Hemdon. R M, J cell biol 45 (1970) for macrophages, polymorphonuclear leuko212: 3. Wolf, M, J camp neurol 140 (1970) 281. cytes, and eosinophilic leukocytes. Of partic4. Lehrer, G M, Bomstein, M B, Weiss, C, Furman, ular interest to us is a lymphocyte-produced M & Lichtman, C, Exptl neurol 27 (1970) 410. 5. Silberbere. D H. Beniamins. J. Herschkowitz, factor termed macrophage migration inN & McKhann, G M, j neurochem 19 (1972) 11: hibitory factor (MIF) which we have recently 6. Bunge, R P, Physiol rev 48 (1968) 197. 7. Vaughn, J E, 2 zellforsch 94 (1969) 293. shown to operate through a surface receptor 8. Mori, S & LeBlond, C P, J camp neurol 139 mechanism [I]. Interest in the mechanism (1970) 1. 9. Manuelidis, L & Manuelidis, E E, Acta neuropath whereby surface active effector molecules 18 (1971) 193. might regulate cell movement prompted us to 10. Stockdale, F, Okazaki, K, Nameroff, M & Holtzer, H, Science 146 (1964) 533. study the effects of ouabain, a cardiac glyco11. Bischoff, R & Holtzer, H, J cell biol 44 (1970) side, on macrophage migration. 134. 12. Turkington, R W, Majumder, G C & Riddle, M, J biol them 246 (1971) 1814. 13. Muira, Y & Wilt, F H, J cell biol 48 (1971) 523. 14. Gontcharoff, M & Mazia, D, Exptl cell res 46 (1967) 315. ExptI Cell Res 76 (1973)
Materials and Methods Guinea pig alveolar and peritoneal macrophages were obtained and their migration assayed by the capillary