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Pattern formation in the mammalian forebrain: patch neurons from the rat striatum selectively reassociate in vitro
Developmental Brain Research, 47 (1989) 137-142 Elsevier BRD 60284
137
Short Communications
Pattern formation in the mammalian forebrain: patch neurons from the rat striatum selectively reassociate in vitro Leslie A. Krushel, Joe A. Connolly and Derek van der Kooy Neurobiology Research Group, Department of Anatomy, Universityof Toronto, Toronto, Ont. (Canada) (Accepted 21 June 1988) Key words: Development; Thymidine; Compartment; Tissue culture; Adhesion
Mechanisms involved in the developmental organization of the rat striatum were investigated in vitro. The neurons of the patch and matrix compartments were preferentially labeled in vivo with a [3H]thymidine injection on embryonic day (E) 13 or 18, respectively. Two or 7 days later the striatum was removed, dissociated into a single cell suspension and plated on a collagen-coated substrate. After 5 days in culture the neurons had migrated into aggregates. Within an individual aggregate, neurons labeled on El3 tended to clump together, whereas neurons labeled on El8 were randomly dispersed. Comparing between aggregates, [aH]thymidine-labeled El3 cells were located in aggregates containing numerous other labeled El3 cells, whereas [3H]thymidinelabeled El8 cells were dispersed randomly between aggregates. These results suggest that early born striatal neurons (primarily patch cells) selectively associate with each other, and that this process may be crucial to the developmental compartmentalization of the rat striatum. The mammalian striatum contains morphologically homogeneous neurons 6'13 that can be compartmentalized on the basis of their different phenotypes into the patch and matrix compartments ~°'19. Each compartment is identified by differences in neurotransmitters, neurotransmitter receptors, and neuroanatomical connections ~9. The development of the striatum also occurs in a compartmentalized manner ~s. Neurons destined to be patch cells in the rat leave the mitotic cycle early in gestation, peaking at E l 3 Is. Matrix cells are generated later, peaking at El8, and eventually comprise 80-85% of the rat striatum 16. In the face of this massive wave of migrating matrix cells during development, we hypothesized that the multicellular patch aggregates may form through the selective adhesion of patch cells to each other. This patch cell adhesion would exclude the matrix cells and consequently compartmentalize the developing striatum. One approach to study the role of selective cell association in striatal compartmentalization involves investigating the striatum's ability to organize itself in vitro. If the embryonic
striatum is removed, dissociated into single cells, and allowed to reaggregate, will the patch cells selectively reaggregate with each other? Three culture protocols (Fig. 2A) were utilized to assess the ability of the striatal compartments to reorganize in vitro. Timed-pregnant Wistar albino rats were injected with 1 mCi of [3H]thymidine (spec. act. 52 mCi/mmol) on E l 3 or E l 8 to primarily label the patch or matrix cells, respectively 18. Two or 7 days later (El5 or E20) the fetuses were removed. These delays allowed for the dilution of the [3H]thymidine in the cells continuing to divide, and permitted heavy labeling of only the ventricular cells becoming postmitotic on the day of the [3H]thymidine injection 18. This created 3 experimental conditions (Fig. 2A): cells labeled with [3H]thymidine on El3 (primarily patch cells) and cultured 2 (E13i/E15c) or 7 (E13i/E20c) days later, and cells labeled with [3H]thymidine on E18 (primarily matrix cells) and cultured two days later (E18i/E20c). Removing the E13 labeled tissue at different ages was done to examine the ability of
Correspondence: L. Krushel, Department of Anatomy, Medical Sciences Building, University of Toronto, Toronto, Ont. M5S 1A8, Canada. 0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B,V. (Biomedical Division)
138 patch cells to reaggregate before (El5) and after (E20) the first evidence of compartmentalization is seen in vivo l°. The E13i/E20c culture also controls for removal of the E18i/E20c tissue at a late embryonic date. The striata were dissected out, minced into ~<1 mm 2 pieces and placed into Puck's glucose. The tissue was then centrifuged at 3000 rpm for 3 rain and the pellet transferred into growth medium 17 consisting of 80% high glucose D-MEM, 10% fetal bovine serum, 10% horse serum (Gibco), supplemented with 5/~g/ml insulin, 10,000 units/ml penicillin, 10 mg/ml streptomycin, and 200 ~tM L-glutamine (Sigma). The tissue was triturated with pipets of decreasing bore size into a single cell suspension and plated at a density of approximately 1 x 10 6 cells per 35 mm dish. The cells were grown in 2.5 ml of medium which was replaced on the third day. Immediately after seeding, all dishes were examined to insure the complete dissociation of the tissue into single cells. The cells were plated on acid-washed, carbon-coated coverslips. A 500 ~tl drop of diluted collagen (Gibco) had been previously spread and air-dried on each coverslip. After 5 days in culture cells which adhered to the coverslips were rinsed with PBS and fixed with 4% paraforrnaldehyde in PBS (pH 7.4). The tissue was then defatted, dried and dipped in Kodak NTB2 nuclear track emulsion. After a 6-18 week exposure, the emulsion was developed with Kodak D-19 at 18 °C and fixed in 25% sodium thiosulfate at room temperature. The tissue was then dehydrated and counterstained with Thionin or Cresyl violet. Immediately after seeding, the vast majority of the cells adhered individually to the coverslip (Fig. 1A). After the first day in vitro the ceils sprouted processes and small aggregates of cells had formed (Fig. 1B). These cell aggregates, interconnected by axon fascicles, gradually enlarged and attained their largest diameter approximately 5 days after plating (Fig. 1C), A similar time course of aggregation was seen after both El5 and E20 plating. The growth of the aggregates was generally attributable to the migration of neurons and glia s and to the further division of the glial cells. Continued division of neuronal precursors in vitro is limited 13"1m4 and is inhibited by inclusion of serum in the medium 3'4. The aggregates consisted of a closely clustered group
Fig. 1. Photomicrographs of striatal monolayer cultures from initial seeding to the formation of aggregates (A-C). After seeding, the cells are dispersed (A), but soon (l.5 days in culture) begin to sprout processes and migrate into small interconnected aggregates (B), which reach their largest diameter after 5 clays in culture (C). Calibration bar in (C! represents 100 t~m (A,B), or 225 um (C).
139 of neurons on a bed of glia. Few neurons outside these aggregates survived, and the areas outside the aggregates were dominated by astrocytes a n d fibroblasts 2°. These observations were based on phase-contrast microscopy, as well as immunocytochemistry employing antibodies to the glial-fibrillary acidic protein, and the 68 k D a neurofilament protein (data not shown). Many of the aggregates in each of the culture protocols contained both [3H]thymidine-labeled and unlabeled neurons. We considered these neuronal aggregates to represent 'mini-striatums', consisting
~l-Thy
of both patch and matrix neurons. To assess reaggregation patterns of the labeled cells within these reaggregates, photomicrographs were taken of twenty randomly selected, similar-sized aggregates (which contained [3H]thymidine labeled cells) for each culture condition (Fig. 2 B - D ) . Prints obtained from the negatives were enlarged equally and the distance between a [3H]thymidine-labeled cell and its nearest labeled neighbor was measured. A cell was considered labeled with [3H]thymidine if it possessed a minimum of 5-10 times more silver grains than the background level over neighboring
B
Culture
E131 E15c 3H-Thy
Culture
E18J E20e ~4-Vhy
Culturo
E13i E20c
I , E13
I , E15
I
, I E18
, I E20
,
I
,
I
IFag
Fig. 2. The temporal protocol for labeling and culturing striatal neurons (A) and photomicrographs of the subsequent aggregates (B-D). Primarily patch ceils are labeled with an in vivo [3H]thymidine injection on E13, while matrix cells are primarily labeled with [3H]thymidine on E18 TM. After 5 days in culture, cells that were labeled (some are arrowed) with [3H]thymidine (clusters of black grains) on E13 (B - - E13i/E15c, C - - E13i/E20c) are located close to one another within an aggregate; whereas cells labeled with [3H]thymidine (some are arrowed) on E18 (D - - E18i/E20c) are more evenly dispersed within an aggregate. Calibration bar represents 50/~m.
140 cells within an aggregate. This heavy [3H]thymidine labeling was restricted to a subpopulation of neurons as only neurons leave the mitotic cycle at the time of our [3H]thymidine injections 9'18. O u r in vitro results c o n c u r r e d with this as glia and o t h e r non-neuronal cells p r o d u c e d only b a c k g r o u n d levels of silver grains. Quantitative m e a s u r e m e n t s revealed that regardless of the age p l a t e d ( E l 5 or E20) neurons labeled with [3H]thymidine on E l 3 were consistently found to be close t o g e t h e r within the reaggregates (Fig. 3). That is, the E l 3 labeled cells formed small patches, as o p p o s e d to the neurons labeled on E l 8
which were m o r e evenly spaced throughout each aggregate. O v e r two-thirds of the E l 3 labeled cells were within 7 / t m ( a p p r o x i m a t e l y one cell body) of a neighboring labeled cell, while only one third of the E l 8 labeled cells were distributed in this manner. To d e t e r m i n e if the distribution of [3H]thymidine labeled neurons was r a n d o m within an aggregate, the nearest-neighbor statistical analysis was applied 8. L a b e l e d cells in 80% (16/20) of the E13i/E15c and in 75% (15/20) of the E13i/E20c aggregates exhibited a n o n - r a n d o m distribution within each aggregate ( P < 0.01). Alternatively, only 15% (3/20) of the E t 8 i /
• E13i/E15c [ ] E13i/E20c r"l E18i/E20c 60 0
[ i
40
|
I
0 .o
E ¢~ m
20
7
14
21
28
3S
B s t m ~ to the nmrNt IJsded ce. (tam)
Fig. 3. The distances between each [3H]thymidine labeled cell and its nearest [aH]thymidine labeled neighbor were measured in 20 aggregates for each of the 3 experimental conditions. The measurements from each aggregate were then grouped into equidistant bins and expressed as a percentage of all the nearest neighbor measurements for that aggregate. Data represent means :1: S.E.M. for the 20 aggregates in each experimental condition. El3 labeled cells are located closer to other El3 labeled cells regardless of age cultured (El3i/E15c and E13i/E20e), than are El8 labeled cells to other El8 labeled cells. The total number of [~H]tlaymidine labeled cells examined in each condition was E13i/E15c, n = 179; E13i/E20c, n = 105; E18i/E20c, n = 158.
141 E20c aggregates contained labeled cells that displayed a significant non-random distribution. The distribution of El3 labeled cells was not only segregated within an aggregate but between individual aggregates as well. A large majority of the El3 labeled cells (90% - - E13i/E15c, 83% - - E13i/E20c) were located in aggregates containing more than 3 labeled cells. However, in spite of the fact that labeled cell density throughout the plates was approximately equal between the E13i/E15c and E18i/ E20c cultures, only 65% of the E l 8 labeled cells were located in aggregates containing more than 3 labeled cells. This finding led to a corollary observation in a reanalysis of our cultures under a lightfield microscope: 31% (33/105) of the E13i/E15c aggregates examined did not contain any labeled cells, whereas individual cell aggregates in E18i/E20c cultures almost always (94/97) contained a minimum of one [3H]thymidine labeled cell. It should be noted that based on counts in the adult, the number of neurons generated at El8 is larger than at El3 TM. However, the total number of striatal cells increases between El3 and El8, maintaining roughly the same ratio of [3H]thymidine labeled cells to unlabeled cells in striata cultured at El5 and at E20. Since neuronal cell division is limited in vitro 1'7'n'14, we observed approximately similar densities of [3H]thymidine labeled cells in the E13i/E15c and E18i/E20c cultures. Striatal neurons leaving the mitotic cycle on El3, as opposed to El8, have a tendency to segregate into particular aggregates and within such aggregates clump together. This is observed after the striatum is dissociated and allowed to reaggregate on an artificial substrate. It is most likely that neuronal migration and interaction rather than cell division underlies this result, as continued neuronal division would have diluted the [3H]thymidine label within individual cells. The ability of [3H]thymidine-labeled El3 cells (primarily patch cells) to reaggregate with each other, implies that patch cells becoming postmitotic at the time of the [3H]thymidine injection on E13 possess a stronger attraction to each other, than to patch cells born at other times. Perhaps the amount of the currently unknown cell adhesion molecule that mediates patch cell reaggregation varies quantitatively with time since leaving the cell cycle. This
predicts a hierarchy of adhesiveness among patch cells depending on neuronal birthdate. However, it is also conceivable that some of the unlabeled neurons observed among the El3 labeled clusters may be patch cells born before or after the labeled cells. The reaggregation of patch cells in vitro suggests, by active exclusion, that later born cells (mainly matrix cells) should also reaggregate. Birthdate must not influence this reaggregation as the [3H]thymidine labeled matrix neurons in the E18i/ E20c cultures do not clump within aggregates, although labeled patch cells do clump in the E13i/ E20c cultures in spite of a lower overall density of labeled neurons compared to the E18i/E20c cultures. It remains possible that a weak adhesiveness between matrix neurons labeled with [3H]thymidine is obscured by the large population of unlabeled matrix neurons. However, it is difficult to imagine that much adhesiveness occurs amongst matrix neurons in vivo, since the matrix neurons must migrate into the developing striatum over a period during which the already present and stationary patch cells dominate the total population. Any matrix cell adhesion would inhibit neuronal movement and thus would impair the massive matrix cell migration. It is more parsimonious to postulate that early born patch cell migration is inhibited by contact with and adhesiveness to other patch cells, thus creating 'patches' or 'tubes' as matrix cells migrate around. Selective cell adhesion presumably also plays a role in the in vivo development of the striatum. We reanalysed sections from our previously published data TM on the in vivo adult distribution of striatal neurons labeled embryonically with [3H]thymidine to ask if there was any persistent evidence of the embryonic adhesive events characterized here. Nearest-neighbor analyses (as described above) were performed on [3H]thymidine labeled cells within the patch (El3 [3H]thymidine labeled) or matrix (El8 [3H]thymidine labeled) compartments. The adult compartments were independently demonstrated by the distribution of opiate receptor binding in the same sections. El3 [3H]thymidine labeled cells were found to clump significantly within the patch compartment in 10/13 patches examined. In contrast, significant non-random association of El8 [3H]thymidine labeled cells was found in only 2/13 similar size striatal matrix regions examined. Amazingly,
142 the labeled patch n e u r o n s remain clumped in the adult (due either to a r e m n a n t of the embryonic association or to a persistent adhesiveness in the adult), in spite of their lower overall striatal densities compared to the unclumped, labeled matrix neurons. In conclusion, the selective adhesiveness of early born striatal cells (primarily patch cells) to one
after becoming postmitotic 12'~",v~. We suggest that the initial structural organization of the nervous system may depend not only on the correct migration of n e u r o n s but also on the immobility of other neurons. Selective adhesiveness of other early born neuronal populations may be a mechanism underlying the formation of functional c o m p a r t m e n t s throughout the b r a i n t 5
a n o t h e r may underlie the correct positioning of the striatal compartments during development. This may
Supported by a Medical Research Council of
enable the further phenotypic differentiation of the compartments. Striatal n e u r o n s are probably committed to their compartments before or very soon
Canada grants to D . v . d . K . and J . A . C . , and a Medical Research Council of C a n a d a Studentship to L.A.K.
1 Ahmed, Z. and Fellows, R.E., Determination of the birth date and proliferative state of dissociated cells from fetal rat brain, Dev. Brain Res., 37 (1987) 77-87. 2 Asou, H., Iwasaki, N., Hirano, S. and Dahl, D., Mitotic neuroblasts in dissociated cell cultures from embryonic rat cerebral hemispheres express neurofilament protein, Brain Res., 332 (1985) 355-357. 3 Barakat, I., Labourdette, G. and Sensenbrenner, M., Inhibitory effects of fetal calf serum on proliferation of chick neuroblasts in culture, Dev. Neurosci., 6 (1983/84) 169-183. 4 Barakat, I., Sensenbrenner, M. and Labourdette, G., Stimulation of chick neuroblast proliferation in culture by brain extracts, J. Neurosci. Res., 8 (1982) 303-314. 5 Barakat, I., Wittendorp-Rechenmann, E., Rechenmann, R.V. and Sensenbrenner, M., Influence of meningeal cells on the proliferation of neuroblasts in culture, Dev. Neurosci., 4 (1981) 363-372. 6 Bishop, G.A., Chang, H.T. and Kitai, S.T., Morphological and physiological properties of neostriatal neurons: an intracellular horseradish peroxidase study in the rat, Neuroscience, 7 (1982) 179-191. 7 Boss, B.D., Gozes, I. and Cowan, W.M., The survival of dentate gyrus neurons in dissociated culture, Dev. Brain Res., 36 (1987) 199-218. 8 Clark, P.J. and Evans, F.C., Distance to nearest neighbor as a measure of spatial relationships in populations, Ecology, 35 (1954) 445-453. 9 Das, G.D., Gliogenesis and ependymogenesis during embryonic development of the rat, J. Neurol. Sci., 43 (1979) 193-204. 10 Fishell, G. and Van der Kooy, D., Pattern formation in the striatum: developmental changes in the distribution of striatonigral neurons, J. Neurosci., 7 (1987) 1969-1978. 11 Gensburger, C., Labourdette, G. and Sensenbrenner, M.,
Brain basic fibroblast growth factor stimulates the proliferation of rat neuronal precursor cells in vitro, FEBS Lett., 217 (1987) 1-5. 12 Johnston, J.G., Boyd, S.R. and Van der Kooy, D.. Compartmentalization of the embryonic striatum after intraocular transplantation, Dev. Brain Res., 33 (1987) 310-314. 13 Kemp, J.M. and Powell, T.P.S., The structure of the caudate nucleus of the cat: light and electron microscopy, Phil. Trans. R. Soc. Ser. B., 262 (1971) 383-401. 14 Kriegstein, A. and Dichter, M.A., Neuron generation in dissociated cell cultures from fetal rat cerebral cortex, Brain Res., 295 (1984) 184-189. 15 Krushel, L.A. and Van der Kooy, D., Selective in vitro reassociation of early versus late postmitotic neurons from the rat forebrain, Soc. Neurosci. Abstr., 13 (1987) 1114. 16 Lanca, A.J., Boyd, S.R., Kolb, B. and Van der Kooy, D., The development of a patchy organization of the rat striatum, Dev. Brain Res., 27 (1986) 1-10. 17 Sotelo, J., Gibbs, C.J., Jr., Gajdusek, C., Hock Toh, B. and Wurth, M., Method for preparing cultures of central neurons: cytochemieal and immunochemical studies, Proc. Natl. Acad. Sci. U.S.A., 77 (1980) 653-657. 18 Van der Kooy, D. and Fishell, G., Neuronal birthdate underlies the development of striatal compartments, Brain Res., 401 (1987) 155-161. 19 Van der Kooy, D., Fishell, G., Krushel, L.A. and Johnston, J.G., The development of striatal compartments: from proliferation to patches. In M.B. Carpenter and A. Jayaraman (Eds.), The Basal Ganglia I1, Plenum Press, New York, 1987, pp. 81-98. 20 Victorov, I.V. and Krukoff, T.L., Patterns of reaggregation and formation of linear aggregate chains in collagen-well cultures of dissociated mouse brain and spinal cord cells, Brain Res.. 198 (1980) 167-182.
137
Short Communications
Pattern formation in the mammalian forebrain: patch neurons from the rat striatum selectively reassociate in vitro Leslie A. Krushel, Joe A. Connolly and Derek van der Kooy Neurobiology Research Group, Department of Anatomy, Universityof Toronto, Toronto, Ont. (Canada) (Accepted 21 June 1988) Key words: Development; Thymidine; Compartment; Tissue culture; Adhesion
Mechanisms involved in the developmental organization of the rat striatum were investigated in vitro. The neurons of the patch and matrix compartments were preferentially labeled in vivo with a [3H]thymidine injection on embryonic day (E) 13 or 18, respectively. Two or 7 days later the striatum was removed, dissociated into a single cell suspension and plated on a collagen-coated substrate. After 5 days in culture the neurons had migrated into aggregates. Within an individual aggregate, neurons labeled on El3 tended to clump together, whereas neurons labeled on El8 were randomly dispersed. Comparing between aggregates, [aH]thymidine-labeled El3 cells were located in aggregates containing numerous other labeled El3 cells, whereas [3H]thymidinelabeled El8 cells were dispersed randomly between aggregates. These results suggest that early born striatal neurons (primarily patch cells) selectively associate with each other, and that this process may be crucial to the developmental compartmentalization of the rat striatum. The mammalian striatum contains morphologically homogeneous neurons 6'13 that can be compartmentalized on the basis of their different phenotypes into the patch and matrix compartments ~°'19. Each compartment is identified by differences in neurotransmitters, neurotransmitter receptors, and neuroanatomical connections ~9. The development of the striatum also occurs in a compartmentalized manner ~s. Neurons destined to be patch cells in the rat leave the mitotic cycle early in gestation, peaking at E l 3 Is. Matrix cells are generated later, peaking at El8, and eventually comprise 80-85% of the rat striatum 16. In the face of this massive wave of migrating matrix cells during development, we hypothesized that the multicellular patch aggregates may form through the selective adhesion of patch cells to each other. This patch cell adhesion would exclude the matrix cells and consequently compartmentalize the developing striatum. One approach to study the role of selective cell association in striatal compartmentalization involves investigating the striatum's ability to organize itself in vitro. If the embryonic
striatum is removed, dissociated into single cells, and allowed to reaggregate, will the patch cells selectively reaggregate with each other? Three culture protocols (Fig. 2A) were utilized to assess the ability of the striatal compartments to reorganize in vitro. Timed-pregnant Wistar albino rats were injected with 1 mCi of [3H]thymidine (spec. act. 52 mCi/mmol) on E l 3 or E l 8 to primarily label the patch or matrix cells, respectively 18. Two or 7 days later (El5 or E20) the fetuses were removed. These delays allowed for the dilution of the [3H]thymidine in the cells continuing to divide, and permitted heavy labeling of only the ventricular cells becoming postmitotic on the day of the [3H]thymidine injection 18. This created 3 experimental conditions (Fig. 2A): cells labeled with [3H]thymidine on El3 (primarily patch cells) and cultured 2 (E13i/E15c) or 7 (E13i/E20c) days later, and cells labeled with [3H]thymidine on E18 (primarily matrix cells) and cultured two days later (E18i/E20c). Removing the E13 labeled tissue at different ages was done to examine the ability of
Correspondence: L. Krushel, Department of Anatomy, Medical Sciences Building, University of Toronto, Toronto, Ont. M5S 1A8, Canada. 0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B,V. (Biomedical Division)
138 patch cells to reaggregate before (El5) and after (E20) the first evidence of compartmentalization is seen in vivo l°. The E13i/E20c culture also controls for removal of the E18i/E20c tissue at a late embryonic date. The striata were dissected out, minced into ~<1 mm 2 pieces and placed into Puck's glucose. The tissue was then centrifuged at 3000 rpm for 3 rain and the pellet transferred into growth medium 17 consisting of 80% high glucose D-MEM, 10% fetal bovine serum, 10% horse serum (Gibco), supplemented with 5/~g/ml insulin, 10,000 units/ml penicillin, 10 mg/ml streptomycin, and 200 ~tM L-glutamine (Sigma). The tissue was triturated with pipets of decreasing bore size into a single cell suspension and plated at a density of approximately 1 x 10 6 cells per 35 mm dish. The cells were grown in 2.5 ml of medium which was replaced on the third day. Immediately after seeding, all dishes were examined to insure the complete dissociation of the tissue into single cells. The cells were plated on acid-washed, carbon-coated coverslips. A 500 ~tl drop of diluted collagen (Gibco) had been previously spread and air-dried on each coverslip. After 5 days in culture cells which adhered to the coverslips were rinsed with PBS and fixed with 4% paraforrnaldehyde in PBS (pH 7.4). The tissue was then defatted, dried and dipped in Kodak NTB2 nuclear track emulsion. After a 6-18 week exposure, the emulsion was developed with Kodak D-19 at 18 °C and fixed in 25% sodium thiosulfate at room temperature. The tissue was then dehydrated and counterstained with Thionin or Cresyl violet. Immediately after seeding, the vast majority of the cells adhered individually to the coverslip (Fig. 1A). After the first day in vitro the ceils sprouted processes and small aggregates of cells had formed (Fig. 1B). These cell aggregates, interconnected by axon fascicles, gradually enlarged and attained their largest diameter approximately 5 days after plating (Fig. 1C), A similar time course of aggregation was seen after both El5 and E20 plating. The growth of the aggregates was generally attributable to the migration of neurons and glia s and to the further division of the glial cells. Continued division of neuronal precursors in vitro is limited 13"1m4 and is inhibited by inclusion of serum in the medium 3'4. The aggregates consisted of a closely clustered group
Fig. 1. Photomicrographs of striatal monolayer cultures from initial seeding to the formation of aggregates (A-C). After seeding, the cells are dispersed (A), but soon (l.5 days in culture) begin to sprout processes and migrate into small interconnected aggregates (B), which reach their largest diameter after 5 clays in culture (C). Calibration bar in (C! represents 100 t~m (A,B), or 225 um (C).
139 of neurons on a bed of glia. Few neurons outside these aggregates survived, and the areas outside the aggregates were dominated by astrocytes a n d fibroblasts 2°. These observations were based on phase-contrast microscopy, as well as immunocytochemistry employing antibodies to the glial-fibrillary acidic protein, and the 68 k D a neurofilament protein (data not shown). Many of the aggregates in each of the culture protocols contained both [3H]thymidine-labeled and unlabeled neurons. We considered these neuronal aggregates to represent 'mini-striatums', consisting
~l-Thy
of both patch and matrix neurons. To assess reaggregation patterns of the labeled cells within these reaggregates, photomicrographs were taken of twenty randomly selected, similar-sized aggregates (which contained [3H]thymidine labeled cells) for each culture condition (Fig. 2 B - D ) . Prints obtained from the negatives were enlarged equally and the distance between a [3H]thymidine-labeled cell and its nearest labeled neighbor was measured. A cell was considered labeled with [3H]thymidine if it possessed a minimum of 5-10 times more silver grains than the background level over neighboring
B
Culture
E131 E15c 3H-Thy
Culture
E18J E20e ~4-Vhy
Culturo
E13i E20c
I , E13
I , E15
I
, I E18
, I E20
,
I
,
I
IFag
Fig. 2. The temporal protocol for labeling and culturing striatal neurons (A) and photomicrographs of the subsequent aggregates (B-D). Primarily patch ceils are labeled with an in vivo [3H]thymidine injection on E13, while matrix cells are primarily labeled with [3H]thymidine on E18 TM. After 5 days in culture, cells that were labeled (some are arrowed) with [3H]thymidine (clusters of black grains) on E13 (B - - E13i/E15c, C - - E13i/E20c) are located close to one another within an aggregate; whereas cells labeled with [3H]thymidine (some are arrowed) on E18 (D - - E18i/E20c) are more evenly dispersed within an aggregate. Calibration bar represents 50/~m.
140 cells within an aggregate. This heavy [3H]thymidine labeling was restricted to a subpopulation of neurons as only neurons leave the mitotic cycle at the time of our [3H]thymidine injections 9'18. O u r in vitro results c o n c u r r e d with this as glia and o t h e r non-neuronal cells p r o d u c e d only b a c k g r o u n d levels of silver grains. Quantitative m e a s u r e m e n t s revealed that regardless of the age p l a t e d ( E l 5 or E20) neurons labeled with [3H]thymidine on E l 3 were consistently found to be close t o g e t h e r within the reaggregates (Fig. 3). That is, the E l 3 labeled cells formed small patches, as o p p o s e d to the neurons labeled on E l 8
which were m o r e evenly spaced throughout each aggregate. O v e r two-thirds of the E l 3 labeled cells were within 7 / t m ( a p p r o x i m a t e l y one cell body) of a neighboring labeled cell, while only one third of the E l 8 labeled cells were distributed in this manner. To d e t e r m i n e if the distribution of [3H]thymidine labeled neurons was r a n d o m within an aggregate, the nearest-neighbor statistical analysis was applied 8. L a b e l e d cells in 80% (16/20) of the E13i/E15c and in 75% (15/20) of the E13i/E20c aggregates exhibited a n o n - r a n d o m distribution within each aggregate ( P < 0.01). Alternatively, only 15% (3/20) of the E t 8 i /
• E13i/E15c [ ] E13i/E20c r"l E18i/E20c 60 0
[ i
40
|
I
0 .o
E ¢~ m
20
7
14
21
28
3S
B s t m ~ to the nmrNt IJsded ce. (tam)
Fig. 3. The distances between each [3H]thymidine labeled cell and its nearest [aH]thymidine labeled neighbor were measured in 20 aggregates for each of the 3 experimental conditions. The measurements from each aggregate were then grouped into equidistant bins and expressed as a percentage of all the nearest neighbor measurements for that aggregate. Data represent means :1: S.E.M. for the 20 aggregates in each experimental condition. El3 labeled cells are located closer to other El3 labeled cells regardless of age cultured (El3i/E15c and E13i/E20e), than are El8 labeled cells to other El8 labeled cells. The total number of [~H]tlaymidine labeled cells examined in each condition was E13i/E15c, n = 179; E13i/E20c, n = 105; E18i/E20c, n = 158.
141 E20c aggregates contained labeled cells that displayed a significant non-random distribution. The distribution of El3 labeled cells was not only segregated within an aggregate but between individual aggregates as well. A large majority of the El3 labeled cells (90% - - E13i/E15c, 83% - - E13i/E20c) were located in aggregates containing more than 3 labeled cells. However, in spite of the fact that labeled cell density throughout the plates was approximately equal between the E13i/E15c and E18i/ E20c cultures, only 65% of the E l 8 labeled cells were located in aggregates containing more than 3 labeled cells. This finding led to a corollary observation in a reanalysis of our cultures under a lightfield microscope: 31% (33/105) of the E13i/E15c aggregates examined did not contain any labeled cells, whereas individual cell aggregates in E18i/E20c cultures almost always (94/97) contained a minimum of one [3H]thymidine labeled cell. It should be noted that based on counts in the adult, the number of neurons generated at El8 is larger than at El3 TM. However, the total number of striatal cells increases between El3 and El8, maintaining roughly the same ratio of [3H]thymidine labeled cells to unlabeled cells in striata cultured at El5 and at E20. Since neuronal cell division is limited in vitro 1'7'n'14, we observed approximately similar densities of [3H]thymidine labeled cells in the E13i/E15c and E18i/E20c cultures. Striatal neurons leaving the mitotic cycle on El3, as opposed to El8, have a tendency to segregate into particular aggregates and within such aggregates clump together. This is observed after the striatum is dissociated and allowed to reaggregate on an artificial substrate. It is most likely that neuronal migration and interaction rather than cell division underlies this result, as continued neuronal division would have diluted the [3H]thymidine label within individual cells. The ability of [3H]thymidine-labeled El3 cells (primarily patch cells) to reaggregate with each other, implies that patch cells becoming postmitotic at the time of the [3H]thymidine injection on E13 possess a stronger attraction to each other, than to patch cells born at other times. Perhaps the amount of the currently unknown cell adhesion molecule that mediates patch cell reaggregation varies quantitatively with time since leaving the cell cycle. This
predicts a hierarchy of adhesiveness among patch cells depending on neuronal birthdate. However, it is also conceivable that some of the unlabeled neurons observed among the El3 labeled clusters may be patch cells born before or after the labeled cells. The reaggregation of patch cells in vitro suggests, by active exclusion, that later born cells (mainly matrix cells) should also reaggregate. Birthdate must not influence this reaggregation as the [3H]thymidine labeled matrix neurons in the E18i/ E20c cultures do not clump within aggregates, although labeled patch cells do clump in the E13i/ E20c cultures in spite of a lower overall density of labeled neurons compared to the E18i/E20c cultures. It remains possible that a weak adhesiveness between matrix neurons labeled with [3H]thymidine is obscured by the large population of unlabeled matrix neurons. However, it is difficult to imagine that much adhesiveness occurs amongst matrix neurons in vivo, since the matrix neurons must migrate into the developing striatum over a period during which the already present and stationary patch cells dominate the total population. Any matrix cell adhesion would inhibit neuronal movement and thus would impair the massive matrix cell migration. It is more parsimonious to postulate that early born patch cell migration is inhibited by contact with and adhesiveness to other patch cells, thus creating 'patches' or 'tubes' as matrix cells migrate around. Selective cell adhesion presumably also plays a role in the in vivo development of the striatum. We reanalysed sections from our previously published data TM on the in vivo adult distribution of striatal neurons labeled embryonically with [3H]thymidine to ask if there was any persistent evidence of the embryonic adhesive events characterized here. Nearest-neighbor analyses (as described above) were performed on [3H]thymidine labeled cells within the patch (El3 [3H]thymidine labeled) or matrix (El8 [3H]thymidine labeled) compartments. The adult compartments were independently demonstrated by the distribution of opiate receptor binding in the same sections. El3 [3H]thymidine labeled cells were found to clump significantly within the patch compartment in 10/13 patches examined. In contrast, significant non-random association of El8 [3H]thymidine labeled cells was found in only 2/13 similar size striatal matrix regions examined. Amazingly,
142 the labeled patch n e u r o n s remain clumped in the adult (due either to a r e m n a n t of the embryonic association or to a persistent adhesiveness in the adult), in spite of their lower overall striatal densities compared to the unclumped, labeled matrix neurons. In conclusion, the selective adhesiveness of early born striatal cells (primarily patch cells) to one
after becoming postmitotic 12'~",v~. We suggest that the initial structural organization of the nervous system may depend not only on the correct migration of n e u r o n s but also on the immobility of other neurons. Selective adhesiveness of other early born neuronal populations may be a mechanism underlying the formation of functional c o m p a r t m e n t s throughout the b r a i n t 5
a n o t h e r may underlie the correct positioning of the striatal compartments during development. This may
Supported by a Medical Research Council of
enable the further phenotypic differentiation of the compartments. Striatal n e u r o n s are probably committed to their compartments before or very soon
Canada grants to D . v . d . K . and J . A . C . , and a Medical Research Council of C a n a d a Studentship to L.A.K.
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