Development of AChE-positive neuronal projections from basal forebrain to cerebral cortex in organotypic tissue slice cultures

Abstract

Development of the innervation of the cerebral cortex by acetylcholinesterase (AChE)-stained basal forebrain neurons was studied in vitro using the roller tube technique. Slice cultures were maintained from 3 days to 4 weeks either in serum based medium or in chemically defined medium, each supplemented in some cases with nerve growth factor (NGF). The distribution of AChE and choline acetyltransferase (CAT)-containing neurons was investigated using histo- and immunocytochemical techniques. Slice cultures of basal forebrain revealed the presence of large and medium sized AChE-positive neurons. Within one week of cultivation, numerous AChE-labeled fibers could be seen growing out from the basal forebrain toward the cortex. After entering cortical tissue most of the afferent basal forebrain fibers projected either radially or obliquely into the cortical layers. Many afferent axons initially also travelled tangentially within the white matter, and turned then to grow into the cortical layers. Cerebral cortex tissue maintained a coarse laminar organization. Ramifications of basal forebrain fibers were visible within the subplate region, the deep and superficial cortical layers, and within the marginal zone; greatest density occured in the subplate region and in marginal zones. Many of these processes exhibited branching patterns markedly similar to those observed during cortical development in vivo. Cortex slices placed with the pial surface adjacent to the basal forebrain revealed AChE-stained fibers that entered the cortical tissue through the marginal surface and gave off ramifications within the superficial layers and, less frequently, the deeper cortical layers. CAT-immunostaining revealed labeled cell bodies and neurites only in the basal forebrain, not in the cortex tissue. Control experiments with co-cultures of basal forebrain and cerebellum slices showed no AChE-positive fiber ingrowth into the cerebellum tissue. The results of these studies demonstrate that basal forebrain projections to cerebral cortex in vitro appear similar to the projections that develop in vivo, and indicate that organotypic co-cultures provide a valuable model for studies of developing cortical afferents.