Developmental Brain Research, 47 (1989) 137-142 137 Elsevier BRD 60284 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]thymidine- labeled 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 morphologi- cally homogeneous neurons 6'13 that can be compart- mentalized on the basis of their different phenotypes into the patch and matrix compartments ~°'19. Each compartment is identified by differences in neuro- transmitters, neurotransmitter receptors, and neuro- anatomical 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 El3 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 hypo- thesized 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 compart- mentalization 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 selec- tively 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 El3 or El8 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 experimen- tal 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 (pri- marily 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)